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Volume 8
Issue 5
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J. Fungi, Volume 8, Issue 5 (May 2022) – 132 articles

Cover Story (view full-size image): Yeasts need a lot of potassium to grow. The main K+ uptake system Trk1 has been shown to exist in low- or high-affinity modes according to the K+ availability, but when and how the affinity changes remains unknown. Here, we characterize the Trk1 kinetic parameters under various conditions and find that Trk1’s KT and Vmax change gradually. This gliding adjustment is rapid and controlled by changes in intracellular potassium content and membrane potential. The introduction of mutations to four specific structural segments (P-helices) of Trk1 showed the importance of two P-helices for the transporter’s transition to a high-affinity state, as well as the importance of the other two for proper Trk1 folding and activity at the plasma membrane. View this paper
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15 pages, 2261 KiB  
Article
Peel Diffusion and Antifungal Efficacy of Different Fungicides in Pear Fruit: Structure-Diffusion-Activity Relationships
by Gui-Yang Zhu, Ying Chen, Su-Yan Wang, Xin-Chi Shi, Daniela D. Herrera-Balandrano, Victor Polo and Pedro Laborda
J. Fungi 2022, 8(5), 547; https://doi.org/10.3390/jof8050547 - 23 May 2022
Cited by 11 | Viewed by 2305
Abstract
Fungal pathogens can invade not only the fruit peel but also the outer part of the fruit mesocarp, limiting the efficacy of fungicides. In this study, the relationships between fungicide structure, diffusion capacity and in vivo efficacy were evaluated for the first time. [...] Read more.
Fungal pathogens can invade not only the fruit peel but also the outer part of the fruit mesocarp, limiting the efficacy of fungicides. In this study, the relationships between fungicide structure, diffusion capacity and in vivo efficacy were evaluated for the first time. The diffusion capacity from pear peel to mesocarp of 11 antifungal compounds, including p-aminobenzoic acid, carbendazim, difenoconazole, dipicolinic acid, flusilazole, gentamicin, kojic acid, prochloraz, quinolinic acid, thiophanate methyl and thiram was screened. The obtained results indicated that size and especially polarity were negatively correlated with the diffusion capacity. Although some antifungal compounds, such as prochloraz and carbendazim, were completely degraded after a few days in peel and mesocarp, other compounds, such as p-aminobenzoic acid and kojic acid, showed high stability. When applying the antifungal compounds at the EC50 concentrations, it was observed that the compounds with high diffusion capacity showed higher in vivo antifungal activity against Alternaria alternata than compounds with low diffusion capacity. In contrast, there was no relationship between stability and in vivo efficacy. Collectively, the obtained results indicated that the diffusion capacity plays an important role in the efficacy of fungicides for the control of pear fruit diseases. Full article
(This article belongs to the Section Fungal Pathogenesis and Disease Control)
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Figure 1
<p>Density functional theory (DFT; M06-2X(PCM)/6-311G(d,p)) optimized structures for the studied fungicides. (<b>A</b>) <span class="html-italic">p</span>-Aminobenzoic acid. (<b>B</b>) Carbendazim. (<b>C</b>) Difenoconazole. (<b>D</b>) Dipicolinic acid. (<b>E</b>) Flusilazole. (<b>F</b>) Gentamicin. (<b>G</b>) Kojic acid. (<b>H</b>) Prochloraz. (<b>I</b>) Quinolinic acid. (<b>J</b>) Thiophanate methyl. (<b>K</b>) Thiram. All molecules are shown at the same scale to allow size comparisons.</p> Full article ">Figure 2
<p>Linear regression analyses. (<b>A</b>) Correlation between volume, polarity and diffusion. (<b>B</b>) Correlation between diffusion and in vivo antifungal activity. Linear regression analyses were performed using SPSS software version 16.0.</p> Full article ">Figure 3
<p>Stability of studied fungicides in pear peel and mesocarp. (<b>A</b>) <span class="html-italic">p</span>-Aminobenzoic acid. (<b>B</b>) Carbendazim. (<b>C</b>) Difenoconazole. (<b>D</b>) Dipicolinic acid. (<b>E</b>) Flusilazole. (<b>F</b>) Gentamicin. (<b>G</b>) Kojic acid. (<b>H</b>) Prochloraz. (<b>I</b>) Quinolinic acid. (<b>J</b>) Thiophanate methyl. (<b>K</b>) Thiram.</p> Full article ">Figure 4
<p>Images of the symptoms caused by <span class="html-italic">Alternaria alternata</span> on pear fruit after application of <span class="html-italic">p</span>-aminobenzoic acid, carbendazim, difenoconazole, dipicolinic acid, flusilazole, gentamicin, kojic acid, prochloraz, quinolinic acid, thiophanate methyl and thiram. The fungicides were applied at the EC<sub>50</sub> concentration. The control experiment was carried out in the absence of fungicides.</p> Full article ">
8 pages, 435 KiB  
Article
Effect of Household Laundering, Heat Drying, and Freezing on the Survival of Dermatophyte Conidia
by Mohammad Akhoundi, Jade Nasrallah, Anthony Marteau, Dahlia Chebbah, Arezki Izri and Sophie Brun
J. Fungi 2022, 8(5), 546; https://doi.org/10.3390/jof8050546 - 23 May 2022
Cited by 6 | Viewed by 3123
Abstract
Dermatomycoses are one of the most common dermatological infectious diseases. Dermatophytoses, such as tinea pedis (athlete’s foot) in adults and tinea capitis in children, are the most prevalent fungal diseases caused by dermatophytes. The transmission of anthropophilic dermatophytoses occurs almost exclusively through indirect [...] Read more.
Dermatomycoses are one of the most common dermatological infectious diseases. Dermatophytoses, such as tinea pedis (athlete’s foot) in adults and tinea capitis in children, are the most prevalent fungal diseases caused by dermatophytes. The transmission of anthropophilic dermatophytoses occurs almost exclusively through indirect contact with patient-contaminated belongings or environments and, subsequently, facilitates the spread of the infection to others. Hygienic measures were demonstrated to have an important role in removing or reducing the fungal burden. Herein, we evaluated the effectiveness of physical-based methods of laundering, heat drying, and freezing in the elimination of Trichophyton tonsurans, T. rubrum, and T. interdigitale conidia in diverse temperatures and time spectra. Based on our findings, laundering at 60 °C was effective for removing the dermatophyte conidia from contaminated linens. On the contrary, heat drying using domestic or laundromat machines; freezing at −20 °C for 24 h, 48 h, or one week; and direct heat exposure at 60 °C for 10, 30, or 90 min were unable to kill the dermatophytes. These results can be helpful for clinicians, staff of children’s communities, and hygiene practitioners for implementing control management strategies against dermatophytoses caused by mentioned dermatophyte species. Full article
(This article belongs to the Special Issue Epidemiology and Pathogenesis of Dermatophytes)
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<p>(<b>A</b>) Temperature courses of the laundering program at 60 °C for 100 min using domestic machine; (<b>B</b>) temperature variations during heat drying performed using laundromat.</p> Full article ">
17 pages, 1803 KiB  
Article
Exposure to Essential and Toxic Elements via Consumption of Agaricaceae, Amanitaceae, Boletaceae, and Russulaceae Mushrooms from Southern Spain and Northern Morocco
by Marta Barea-Sepúlveda, Estrella Espada-Bellido, Marta Ferreiro-González, Hassan Bouziane, José Gerardo López-Castillo, Miguel Palma and Gerardo F. Barbero
J. Fungi 2022, 8(5), 545; https://doi.org/10.3390/jof8050545 - 23 May 2022
Cited by 9 | Viewed by 2461
Abstract
The demand and interest in mushrooms, both cultivated and wild, has increased among consumers in recent years due to a better understanding of the benefits of this food. However, the ability of wild edible mushrooms to accumulate essential and toxic elements is well [...] Read more.
The demand and interest in mushrooms, both cultivated and wild, has increased among consumers in recent years due to a better understanding of the benefits of this food. However, the ability of wild edible mushrooms to accumulate essential and toxic elements is well documented. In this study, a total of eight metallic elements and metalloids (chromium (Cr), arsenic (As), cadmium (Cd), mercury (Hg), lead (Pb), copper (Cu), zinc (Zn), and selenium (Se)) were determined by ICP-MS in five wild edible mushroom species (Agaricus silvicola, Amanita caesarea, Boletus aereus, Boletus edulis, and Russula cyanoxantha) collected in southern Spain and northern Morocco. Overall, Zn was found to be the predominant element among the studied species, followed by Cu and Se. The multivariate analysis suggested that considerable differences exist in the uptake of the essential and toxic elements determined, linked to species-intrinsic factors. Furthermore, the highest Estimated Daily Intake of Metals (EDIM) values obtained were observed for Zn. The Health Risk Index (HRI) assessment for all the mushroom species studied showed a Hg-related cause of concern due to the frequent consumption of around 300 g of fresh mushrooms per day during the mushrooming season. Full article
(This article belongs to the Special Issue Heavy Metals in Mushrooms)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Graphical combination of the resulting HCA dendrograms with a heatmap to identify clustering trend patterns among the studied mushroom species based on the content of the eight elements determined.</p> Full article ">Figure 2
<p>(<b>A</b>) Score obtained for PC1 and PC2 for all the samples (n = 16); (<b>B</b>) Loadings obtained in PC1 and PC2.</p> Full article ">Figure 3
<p>Bar charts of the Health Risk Index (HRI) result according to metallic elements and metalloids determined for all the wild edible mushroom species studied: (<b>A</b>) Cr HRIs; (<b>B</b>) As HRIs; (<b>C</b>) Cd HRIs; (<b>D</b>) Hg HRIs; (<b>E</b>) Pb HRIs; (<b>F</b>) Cu HRIs; (<b>G</b>) Zn HRIs; (<b>H</b>) Se HRIs. The HRI limit has been represented with the help of a vertical black line in the bar charts.</p> Full article ">
19 pages, 782 KiB  
Article
In Vitro Characterization and Identification of Potential Probiotic Yeasts Isolated from Fermented Dairy and Non-Dairy Food Products
by Nadia S. Alkalbani, Tareq M. Osaili, Anas A. Al-Nabulsi, Reyad S. Obaid, Amin N. Olaimat, Shao-Quan Liu and Mutamed M. Ayyash
J. Fungi 2022, 8(5), 544; https://doi.org/10.3390/jof8050544 - 23 May 2022
Cited by 10 | Viewed by 3180
Abstract
This study is about the isolation of yeast from fermented dairy and non-dairy products as well as the characterization of their survival in in vitro digestion conditions and tolerance to bile salts. Promising strains were selected to further investigate their probiotic properties, including [...] Read more.
This study is about the isolation of yeast from fermented dairy and non-dairy products as well as the characterization of their survival in in vitro digestion conditions and tolerance to bile salts. Promising strains were selected to further investigate their probiotic properties, including cell surface properties (autoaggregation, hydrophobicity and coaggregation), physiological properties (adhesion to the HT-29 cell line and cholesterol lowering), antimicrobial activities, bile salt hydrolysis, exopolysaccharide (EPS) producing capability, heat resistance and resistance to six antibiotics. The selected yeast isolates demonstrated remarkable survivability in an acidic environment. The reduction caused by in vitro digestion conditions ranged from 0.7 to 2.1 Log10. Bile salt tolerance increased with the extension in the incubation period, which ranged from 69.2% to 91.1% after 24 h. The ability of the 12 selected isolates to remove cholesterol varied from 41.6% to 96.5%, and all yeast strains exhibited a capability to hydrolyse screened bile salts. All the selected isolates exhibited heat resistance, hydrophobicity, strong coaggregation, autoaggregation after 24 h, robust antimicrobial activity and EPS production. The ability to adhere to the HT-29 cell line was within an average of 6.3 Log10 CFU/mL after 2 h. Based on ITS/5.8S ribosomal DNA sequencing, 12 yeast isolates were identified as 1 strain for each Candidaalbicans and Saccharomyces cerevisiae and 10 strains for Pichia kudriavzevii. Full article
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<p>Boxplot summarizing the survival rate (%) of the 105 yeast isolates under pH 2.5 for 2 h at 37 °C. Bullets represent outliners.</p> Full article ">Figure 2
<p>Neighbour-joining phylogenetic tree based on ITS/5.8S ribosomal DNA. The numbers in parentheses are accession numbers of the identified sequences from the GenBank. The filled circles are the reference strains from NCBI.</p> Full article ">
10 pages, 1464 KiB  
Article
Cytochalasans from the Endophytic Fungus Phomopsis sp. shj2 and Their Antimigratory Activities
by Bing-Chao Yan, Wei-Guang Wang, Ling-Mei Kong, Jian-Wei Tang, Xue Du, Yan Li and Pema-Tenzin Puno
J. Fungi 2022, 8(5), 543; https://doi.org/10.3390/jof8050543 - 23 May 2022
Cited by 4 | Viewed by 1983
Abstract
Cytochalasans from the endophytic fungi featured structure diversity. Our previous study has disclosed that cytochalasans from the endophytic fungus Phomopsis sp. shj2 exhibited an antimigratory effect. Further chemical investigation on Phomopsis sp. shj2 has led to the discovery of seven new cytochalasans ( [...] Read more.
Cytochalasans from the endophytic fungi featured structure diversity. Our previous study has disclosed that cytochalasans from the endophytic fungus Phomopsis sp. shj2 exhibited an antimigratory effect. Further chemical investigation on Phomopsis sp. shj2 has led to the discovery of seven new cytochalasans (1–7), together with four known ones. Their structures were elucidated through extensive spectroscopic data interpretation and single-crystal X-ray diffraction analysis. Compounds 1–3 and 8–11 exhibited antimigratory effects against MDA-MB-231 in vitro with IC50 values in the range of 1.01−10.42 μM. Full article
(This article belongs to the Special Issue Secondary Metabolites from Fungi—in Honour of Prof. Dr. Ji-Kai Liu's 60th Birthday)
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<p>Structures of compounds <b>1</b>–<b>11</b>.</p> Full article ">Figure 2
<p>Key HMBC (red arrows) and <sup>1</sup>H-<sup>1</sup>H COSY (blue bold) correlations of compounds <b>1</b>–<b>7</b>.</p> Full article ">Figure 3
<p>Key ROESY correlations of compounds <b>1</b>–<b>7</b>.</p> Full article ">Figure 4
<p>X-ray crystallographic structures of compounds <b>1</b> and <b>3</b>.</p> Full article ">
16 pages, 1580 KiB  
Article
Co-Occurrence Patterns of Ustilago nuda and Pyrenophora graminea and Fungicide Contribution to Yield Gain in Barley under Fluctuating Climatic Conditions in Serbia
by Radivoje Jevtić, Vesna Župunski, Mirjana Lalošević, Ljiljana Brbaklić and Branka Orbović
J. Fungi 2022, 8(5), 542; https://doi.org/10.3390/jof8050542 - 23 May 2022
Cited by 1 | Viewed by 1924
Abstract
The utilization of production systems with reduced chemical input renewed the interest in Ustilago nuda and Pyrenophora graminea. The investigations of seed fungicide treatments are more related to their efficacy than to their contribution to yield gain. The data were collected from [...] Read more.
The utilization of production systems with reduced chemical input renewed the interest in Ustilago nuda and Pyrenophora graminea. The investigations of seed fungicide treatments are more related to their efficacy than to their contribution to yield gain. The data were collected from research and development trials on fungicide efficacy against U. nuda and P. graminea conducted from 2014 to 2020 in Serbia. Partial least squares, multiple stepwise regression and best subset regression were used for statistical modeling. The total number of plants infected with U. nuda and P. graminea per plot differed significantly in the seven-year period. Shifts in the predominance of one pathogen over the other were also shown. Temperature, total rainfall and relative humidity in flowering time (p < 0.001) influenced the occurrence of both pathogens. The strongest impact on yield loss was observed for temperature in the phenological phases of leaf development (p = 0.014), temperature in flowering time (p < 0.001) and total number of plants infected with U. nuda and P. graminea per plot (p < 0.001). Our results indicated that regression models consisting of both biotic and abiotic factors were more precise in estimating regression coefficients. Neither fungicidal treatment had a stable contribution to yield gain in the seven-year period. Full article
(This article belongs to the Special Issue Plant Fungal Pathogenesis 2022)
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<p>Joined occurrence of <span class="html-italic">U. nuda</span> and <span class="html-italic">P. graminea</span> in seed-untreated plots in 2014–2020. Means that do not share a letter are significantly different.</p> Full article ">Figure 2
<p>Partial least square coefficient plot of climatic factors influencing yield loss of variety Krajišnik in the period 2014–2020; (1) Total rainfall in November, (2) Total rainfall in December, (3) T in November, (4) T in December, (5) Total rainfall in January, (6) Total rainfall in February, (7) Total rainfall in March, (8) Total rainfall in April, (9) Total rainfall in May, (10) T in January, (11) T in February, (12) T in March, (13) T in April, (14) T in May.</p> Full article ">Figure 3
<p>Relationship between <span class="html-italic">U. nuda</span> and <span class="html-italic">P. graminea</span> occurrence in seed-treated and seed-untreated plots and yield gain of variety Krajišnik in the period 2014–2020.</p> Full article ">Figure 4
<p>Regression analysis of the relationship between yield gain and pathogen pressure in the period 2014–2020: (<b>a</b>) Relationship between yield gain and total number of plants infected with <span class="html-italic">P. graminea</span> in the seed-untreated plot; and (<b>b</b>) relationship between yield gain and total number of plants infected with <span class="html-italic">U. nuda</span> in the seed-untreated plot.</p> Full article ">Figure 5
<p>Fungicide efficacy against <span class="html-italic">U. nuda</span> (<b>a</b>) and <span class="html-italic">P. graminea</span> (<b>b</b>) in the period 2014–2020; T2—RAXIL S 040 FS; T3—VITAVAX 200 FF; T4—MANKOGAL S; T5—VIBRANCE DUO; T6—RANCONA TRIO; T7—CERTICOR 050 FS; T8—CELEST EXTRA 050 FS; T9—DIVIDEND EXTREME 115 FS (A12532C); T10—CELEST TOP 312.5 FS; T11—YUNTA QUATTRO; T12—VIAL TRUST FS; T13—LAMARDOR FS 400; T14—LAMARDOR FS 400 + GAUCHO 600 FS.</p> Full article ">
13 pages, 764 KiB  
Article
Association between Following the ESCMID Guidelines for the Management of Candidemia and Mortality: A Retrospective Cohort Study
by Charles Maurille, Julie Bonhomme, Anaïs R. Briant, Jean-Jacques Parienti, Renaud Verdon and Anna Lucie Fournier
J. Fungi 2022, 8(5), 541; https://doi.org/10.3390/jof8050541 - 23 May 2022
Cited by 3 | Viewed by 2271
Abstract
Objectives: The objective of this study was to evaluate the association between ESCMID adherence and 30-day mortality in candidemia. Methods: We performed a retrospective cohort study in two French tertiary-care hospitals. All patients with at least one positive blood culture (BC) for Candida [...] Read more.
Objectives: The objective of this study was to evaluate the association between ESCMID adherence and 30-day mortality in candidemia. Methods: We performed a retrospective cohort study in two French tertiary-care hospitals. All patients with at least one positive blood culture (BC) for Candida spp. between January 2013 and December 2019 were included. An adherent case was defined as a candidemia case for which the treatment fulfilled a bundle of defined criteria based on the latest ESCMID recommendations. We explored factors associated with adherence to ESCMID recommendations in an unadjusted model, and we used a propensity score method to address potential channeling biases with regard to 30-day mortality. Results: During the study period, 165 cases of candidemia were included. Among the ESCMID criteria, funduscopic examination was not performed in 45% and neither was echocardiography in 31%, while the ESCMID criteria were fully implemented in 44 cases (27%). In the propensity score analysis, the all-cause 30-day mortality rate was significantly lower among adherent cases (3.4/36.6, 9%) than among nonadherent cases (42.4/119.5, 36%) (OR = 5.3 95% CI [1.6–17.1]). Conclusions: In our study, adherence to the bundle of criteria for candidemia management was associated with increased survival, supporting additional efforts to implement these recommendations. Full article
(This article belongs to the Topic Infectious Diseases)
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<p>Point-by-point adherence with ESCMID recommendations.</p> Full article ">Figure A1
<p>Distribution of weighting in pseudo-population according to ESCMID recommendations adherence.</p> Full article ">Figure A2
<p>Multivariate analysis of factors associated with ESCMID nonadherence. Abbreviations: Department 1, medical ICU; Department 2, surgical ICU; Department 3, medical; Department 4, surgical; IDC, infectious disease consultation; LOS, length of stay.</p> Full article ">
13 pages, 3792 KiB  
Review
Molecular Genetics of Anthracnose Resistance in Maize
by Wendi Ma, Xinying Gao, Tongling Han, Magaji Tukur Mohammed, Jun Yang, Junqiang Ding, Wensheng Zhao, You-Liang Peng and Vijai Bhadauria
J. Fungi 2022, 8(5), 540; https://doi.org/10.3390/jof8050540 - 23 May 2022
Cited by 7 | Viewed by 3090
Abstract
Maize (Zea mays), also called corn, is one of the top three staple food crops worldwide and is also utilized as feed (e.g., feed grain and silage) and a source of biofuel (e.g., bioethanol). Maize production is hampered by a myriad [...] Read more.
Maize (Zea mays), also called corn, is one of the top three staple food crops worldwide and is also utilized as feed (e.g., feed grain and silage) and a source of biofuel (e.g., bioethanol). Maize production is hampered by a myriad of factors, including although not limited to fungal diseases, which reduce grain yield and downgrade kernel quality. One such disease is anthracnose leaf blight and stalk rot (ALB and ASR) caused by the hemibiotrophic fungal pathogen Colletotrichum graminicola. The pathogen deploys a biphasic infection strategy to colonize susceptible maize genotypes, comprising latent (symptomless) biotrophic and destructive (symptomatic) necrotrophic phases. However, the resistant maize genotypes restrict the C. graminicola infection and in planta fungal proliferation during the biotrophic phase of the infection. Some studies on the inheritance of ASR resistance in the populations derived from biparental resistant and susceptible genotypes reveal that anthracnose is likely a gene-for-gene disease in which the resistant maize genotypes and C. graminicola recognize each other by their matching pairs of nucleotide-binding leucine-rich repeat resistance (NLR) proteins (whose coding genes are localized in disease QTL) and effectors (1–2 effectors/NLR) during the biotrophic phase of infection. The Z. mays genome encodes approximately 144 NLRs, two of which, RCg1 and RCg1b, located on chromosome 4, were cloned and functionally validated for their role in ASR resistance. Here, we discuss the genetic architecture of anthracnose resistance in the resistant maize genotypes, i.e., disease QTL and underlying resistance genes. In addition, this review also highlights the disease cycle of C. graminicola and molecular factors (e.g., virulence/pathogenicity factors such as effectors and secondary metabolites) that contribute to the pathogen’s virulence on maize. A detailed understanding of molecular genetics underlying the maize—C. graminicola interaction will help devise effective management strategies against ALB and ASR. Full article
(This article belongs to the Section Fungal Cell Biology, Metabolism and Physiology)
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<p>Symptoms caused by <span class="html-italic">Colletotrichum graminicola</span> on the susceptible <span class="html-italic">Zea mays</span> inbred line B73. (<b>A</b>) Gray to brown oval necrotic lesions on the B73 leaves are the typical symptom of anthracnose leaf blight. The blight lesions contain dot-like black structures called microsclerotia, which serve as the primary source of inoculum in the next growing season. (<b>B</b>) Discoloration of the pith (rotting pith) is the typical symptom of anthracnose stalk rot. The rotten pith also leads to bleaching of the upper part of the maize plants (top dieback). (<b>C</b>) Heathy pith.</p> Full article ">Figure 2
<p>Global distribution of anthracnose leaf blight (ALB) and anthracnose stalk rot (ASR).</p> Full article ">Figure 3
<p>Circos plot exemplifying the distribution of 144 <span class="html-italic">NLRs</span> (encoding nucleotide-binding leucine-rich repeat resistance proteins) and QTL (conferring resistance to anthracnose leaf blight and stalk rot) in the <span class="html-italic">Zea mays</span> B73 genome. The outer track shows the <span class="html-italic">Z. mays</span> ideogram, comprising ten chromosomes (Chr1 through Chr10). The middle track consists of five circular ticks (6 <span class="html-italic"><span class="underline">NLRs</span></span>/tick); the bars inside the track exhibit the frequency distribution of <span class="html-italic">NLRs</span> on the chromosomes. The inner track indicates the location of QTL based on their flanking marker positions listed in <a href="#jof-08-00540-t001" class="html-table">Table 1</a>. The B73 genome lacks the QTL <span class="html-italic">qRCg1</span> controlling resistance to anthracnose stalk rot; hence, its location on the B73 genome is relative to the marker UMC15a.</p> Full article ">
12 pages, 3748 KiB  
Article
Physiological Response of Saccharomyces cerevisiae to Silver Stress
by Janelle R. Robinson, Omoanghe S. Isikhuemhen, Felicia N. Anike and Kiran Subedi
J. Fungi 2022, 8(5), 539; https://doi.org/10.3390/jof8050539 - 22 May 2022
Cited by 1 | Viewed by 2312
Abstract
Silver nanoparticle (AgNP) production and their use as antimicrobial agents is a current area of active research. Biosynthesis is the most sustainable production method, and fungi have become candidates of interest in AgNP production. However, investigations into the physiological responses of fungi due [...] Read more.
Silver nanoparticle (AgNP) production and their use as antimicrobial agents is a current area of active research. Biosynthesis is the most sustainable production method, and fungi have become candidates of interest in AgNP production. However, investigations into the physiological responses of fungi due to silver exposure are scanty. This present work utilized two strains of Saccharomyces cerevisiae (one used in commercial fermentation and a naturally occurring strain) to determine the physiological consequences of their transient exposure to AgNO3. The assessments were based on studies involving growth curves, minimal inhibitory concentration assays, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging, and inductively coupled plasma optical emission spectroscopy (ICP-OES). Results indicated (a) the capability of S. cerevisiae to produce silver nanoparticles, even at elevated levels of exposure; (b) strain origin had no significant impact on S. cerevisiae physiological response to AgNO3; and (c) coexposure to copper and silver significantly increased intracellular copper, silver, and calcium in treated yeast cells. In addition, electron microscopy and ICP-OES results revealed that both strains internalized silver after exposure, resulting in the shrunken and distorted physical appearance visible on SEM micrographs of treated cells. Though a promising candidate for AgNPs biosynthesis, this study analyzed the effects of transient silver exposure on S. cerevisiae growth physiology and morphology. Full article
(This article belongs to the Section Fungal Cell Biology, Metabolism and Physiology)
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<p>Replica plating for the selection of naturally sensitive isolates. (<b>a</b>) Single colonies from nonselective media impressed onto velvet. (<b>b</b>) Selective media containing silver received the impression from the velvet. (<b>c</b>) Incubation of selective media determined NS isolates on nonselective media.</p> Full article ">Figure 2
<p>Minimal inhibitory concentration of AgNO<sub>3</sub> for NS commercial (<span class="html-italic">n</span> = 10) and naïve (<span class="html-italic">n</span> = 10) yeast. The red asterisk represents AgNO<sub>3</sub> concentrations that resulted in significantly less (<span class="html-italic">p</span> &lt; 0.05) growth when compared to the control. The red box represents growth that occurred at the MIC. Error bars represent percentage error (5%).</p> Full article ">Figure 3
<p>Growth curves for NS commercial (<span class="html-italic">n</span> = 24) and naïve (<span class="html-italic">n</span> = 24) yeast exposed to 0 mg/L AgNO<sub>3</sub> and the sub-MIC (22.5 mg/L AgNO<sub>3</sub>). Error bars represent percentage error (5%).</p> Full article ">Figure 4
<p>SEM micrographs of NS commercial and NS naïve yeast at 10,000× magnification. (<b>a</b>) and (<b>c</b>) are NS commercial and NS naïve yeast exposed to 0 mg/L AgNO<sub>3</sub>, respectively. (<b>b</b>,<b>d</b>) are NS commercial and NS naïve yeast exposed to 22.5 mg/L AgNO<sub>3</sub>, respectively.</p> Full article ">Figure 5
<p>SEM micrographs of NS commercial (<b>a</b>) and NS naïve (<b>b</b>) yeast exposed to the MIC. The red asterisks indicate cells that do not have a drastic physical change and resemble controls.</p> Full article ">Figure 6
<p>TEM micrographs of NS commercial yeast at 14,000× magnification. (<b>a</b>) depicts yeast exposed to 0 mg/L AgNO<sub>3</sub>. Both (<b>b</b>,<b>c</b>) depict yeast exposed to 22.5 mg/L AgNO<sub>3</sub>. The dark spots enclosed in a red circle in (<b>b</b>,<b>c</b>) are silver nanoparticles formed due to AgNO<sub>3</sub> exposure. (<b>a1</b>,<b>b1</b>,<b>c1</b>) are the EDS spectrums that correspond to the image directly above them.</p> Full article ">Figure 7
<p>ICP-OES results of NS commercial (<span class="html-italic">n</span> = 5) and NS naïve (<span class="html-italic">n</span> = 5) yeast. Error bars represent percentage error (5%).</p> Full article ">
17 pages, 3240 KiB  
Article
Spontaneous Suppressors against Debilitating Transmembrane Mutants of CaMdr1 Disclose Novel Interdomain Communication via Signature Motifs of the Major Facilitator Superfamily
by Suman Sharma, Atanu Banerjee, Alexis Moreno, Archana Kumari Redhu, Pierre Falson and Rajendra Prasad
J. Fungi 2022, 8(5), 538; https://doi.org/10.3390/jof8050538 - 22 May 2022
Viewed by 1806
Abstract
The Major Facilitator Superfamily (MFS) drug:H+ antiporter CaMdr1, from Candida albicans, is responsible for the efflux of structurally diverse antifungals. MFS members share a common fold of 12–14 transmembrane helices (TMHs) forming two N- and C-domains. Each domain is arranged [...] Read more.
The Major Facilitator Superfamily (MFS) drug:H+ antiporter CaMdr1, from Candida albicans, is responsible for the efflux of structurally diverse antifungals. MFS members share a common fold of 12–14 transmembrane helices (TMHs) forming two N- and C-domains. Each domain is arranged in a pseudo-symmetric fold of two tandems of 3-TMHs that alternatively expose the drug-binding site towards the inside or the outside of the yeast to promote drug binding and release. MFS proteins show great diversity in primary structure and few conserved signature motifs, each thought to have a common function in the superfamily, although not yet clearly established. Here, we provide new information on these motifs by having screened a library of 64 drug transport-deficient mutants and their corresponding suppressors spontaneously addressing the deficiency. We found that five strains recovered the drug-resistance capacity by expressing CaMdr1 with a secondary mutation. The pairs of debilitating/rescuing residues are distributed either in the same TMH (T127ATMH1- > G140DTMH1) or 3-TMHs repeat (F216ATMH4- > G260ATMH5), at the hinge of 3-TMHs repeats tandems (R184ATMH3- > D235HTMH4, L480ATMH10- > A435TTMH9), and finally between the N- and C-domains (G230ATMH4- > P528HTMH12). Remarkably, most of these mutants belong to the different signature motifs, highlighting a mechanistic role and interplay thought to be conserved among MFS proteins. Results also point to the specific role of TMH11 in the interplay between the N- and C-domains in the inward- to outward-open conformational transition. Full article
(This article belongs to the Section Fungal Cell Biology, Metabolism and Physiology)
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<p>Cell localization and drug resistance profile of primary alanine mutants and their corresponding suppressor mutants. (<b>A</b>) <span class="html-italic">Ca</span>Mdr1 suppressor mutants localized by confocal microscopy. (<b>B</b>) Drug resistance heat map and MIC<sub>80</sub> values for the corresponding strains. A 2-fold dilution was applied to 8 mg/L CHX and 4-NQO and 32 mg/L FLC. (<b>C</b>) 5-fold serial dilution spot assays of the same strains done on solid YEPD medium, with either 0.15 mg/L CHX, 0.15 mg/L 4-NQO, or 0.8 mg/L FLC added. Data were collected after 48 h of incubation at 30 °C from 3 independent experiments.</p> Full article ">Figure 2
<p>Localization of the couples of primary debilitating and secondary rescuing transport mutants of <span class="html-italic">Ca</span>Mdr1 with respect to MFS Signature motifs and internal structural repeats. 3D model of <span class="html-italic">Ca</span>Mdr1 in inward-facing conformation displayed with respect to the four structural repeats (I–IV) [<a href="#B28-jof-08-00538" class="html-bibr">28</a>] and the conserved A, B, C, D2, and G signature motifs of the proton-dependent multidrug efflux systems [<a href="#B30-jof-08-00538" class="html-bibr">30</a>]. See <a href="#app1-jof-08-00538" class="html-app">Figure S2</a> and <a href="#jof-08-00538-f003" class="html-fig">Figure 3</a> for details. Primary debilitating (circles) and secondary rescuing (stars) mutants are shown in surface and sticks and indicated by the same color for each couple. Conserved motifs are shown in mesh form.</p> Full article ">Figure 3
<p>Position of conserved MFS Signature motifs and location of primary mutant and secondary suppressor couples in the inward- and outward-facing models of <span class="html-italic">Ca</span>Mdr1. (<b>A</b>) <b>Left</b>, inward-facing conformation based on GlpT sequence [<a href="#B34-jof-08-00538" class="html-bibr">34</a>], optimized with Modeller. <b>Right</b>, outward-facing conformation based on YajR crystal structure [<a href="#B21-jof-08-00538" class="html-bibr">21</a>]. Models are displayed in solid cartoon (Pymol v2.5.0), colored from the N-(blue) to the C-ends (red). Signature motifs are defined in <a href="#jof-08-00538-f002" class="html-fig">Figure 2</a>. (<b>B</b>) Front and side views of the inward- and outward-facing models with position of primary debilitating (circles) and secondary restoring (stars) mutants. Residues are colored by couples. Residues R215, Y378, and F497 are discussed in the text. Blue and red dotted lines indicate cytoplasmic and extracellular membrane limits as defined by the PPM server (<a href="https://opm.phar.umich.edu/ppm_server" target="_blank">https://opm.phar.umich.edu/ppm_server</a> accessed on 28 January 2021).</p> Full article ">Figure 4
<p>Short– and long–range interactions involving positions 230, 378, 497, and 528. (<b>A</b>). Cartoon representation of inward- and outward-facing models of <span class="html-italic">Ca</span>Mdr1 with membrane limits. Settings are as in <a href="#jof-08-00538-f003" class="html-fig">Figure 3</a>. TMH7, 11, and 12 have been partially masked. (<b>B</b>). MIC<sub>80</sub> values as described in <a href="#jof-08-00538-f001" class="html-fig">Figure 1</a>. Anisomycin (ANI, 10 mg/L) and cerulenin (CER, 4 mg/L) have been added to the screen. (<b>C</b>). Nile red (NR) accumulation assays in host AD1-8u-, WT <span class="html-italic">Ca</span>Mdr1-GFP, and variants. NR accumulation in host strain was set to 100%. Results are the mean of three independent cultures. All single mutants were compared with WT strain while double mutants were compared with their corresponding primary single mutants. Differences were considered statistically significant when <span class="html-italic">p</span> &lt; 0.05 (* signifies <span class="html-italic">p</span> value ≤ 0.05, ** signifies <span class="html-italic">p</span> value ≤ 0.01, and *** signifies <span class="html-italic">p</span> value ≤ 0.001).</p> Full article ">
30 pages, 2519 KiB  
Review
Saprolegniosis in Amphibians: An Integrated Overview of a Fluffy Killer Disease
by Sara Costa and Isabel Lopes
J. Fungi 2022, 8(5), 537; https://doi.org/10.3390/jof8050537 - 22 May 2022
Cited by 4 | Viewed by 3718
Abstract
Amphibians constitute the class of vertebrates with the highest proportion of threatened species, with infectious diseases being considered among the greatest causes for their worldwide decline. Aquatic oomycetes, known as “water molds”, are fungus-like microorganisms that are ubiquitous in freshwater ecosystems and are [...] Read more.
Amphibians constitute the class of vertebrates with the highest proportion of threatened species, with infectious diseases being considered among the greatest causes for their worldwide decline. Aquatic oomycetes, known as “water molds”, are fungus-like microorganisms that are ubiquitous in freshwater ecosystems and are capable of causing disease in a broad range of amphibian hosts. Various species of Achlya sp., Leptolegnia sp., Aphanomyces sp., and mainly, Saprolegnia sp., are responsible for mass die-offs in the early developmental stages of a wide range of amphibian populations through a disease known as saprolegniosis, aka, molding or a “Saprolegnia-like infection”. In this context, the main objective of the present review was to bring together updated information about saprolegniosis in amphibians to integrate existing knowledge, identify current knowledge gaps, and suggest future directions within the saprolegniosis–amphibian research field. Based on the available literature and data, an integrated and critical interpretation of the results is discussed. Furthermore, the occurrence of saprolegniosis in natural and laboratory contexts and the factors that influence both pathogen incidence and host susceptibility are also addressed. The focus of this work was the species Saprolegnia sp., due to its ecological importance on amphibian population dynamics and due to the fact that this is the most reported genera to be associated with saprolegniosis in amphibians. In addition, integrated emerging therapies, and their potential application to treat saprolegniosis in amphibians, were evaluated, and future actions are suggested. Full article
(This article belongs to the Special Issue Epidemic Mycoses Devastating Wild Animal Populations)
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<p>Maximum likelihood phylogram depicting phylogenetic relationships inferred among 105 isolates, based on combined analysis of ITS DNA sequence data. The terminal number corresponds to the bibliographic reference whose sequence belongs in <a href="#jof-08-00537-t001" class="html-table">Table 1</a>. Bootstrap values (&gt;50%) are presented to the right of each node.</p> Full article ">Figure 2
<p>Map showing the geographic distribution of saprolegniosis reports in wild populations. Please see more information in <a href="#jof-08-00537-t001" class="html-table">Table 1</a>. Numbers correspond to article reference: (1) Bragg and Bragg [<a href="#B71-jof-08-00537" class="html-bibr">71</a>]; (2) Bragg [<a href="#B56-jof-08-00537" class="html-bibr">56</a>]; (3) Strijbosch [<a href="#B21-jof-08-00537" class="html-bibr">21</a>]; (4) Leuven et al. [<a href="#B22-jof-08-00537" class="html-bibr">22</a>]; (5) Banks &amp; Beebee [<a href="#B23-jof-08-00537" class="html-bibr">23</a>]; (6) Beattie et al. [<a href="#B69-jof-08-00537" class="html-bibr">69</a>]; (7) Blaustein et al. [<a href="#B43-jof-08-00537" class="html-bibr">43</a>]; (8) Williamson &amp; Bull [<a href="#B52-jof-08-00537" class="html-bibr">52</a>]; (9) Kiesecker &amp; Blaustein [<a href="#B44-jof-08-00537" class="html-bibr">44</a>]; (10) Berger et al. [<a href="#B51-jof-08-00537" class="html-bibr">51</a>]; (11) Kiesecker et al. [<a href="#B14-jof-08-00537" class="html-bibr">14</a>]; (12) Gomez-Mestre et al. [<a href="#B41-jof-08-00537" class="html-bibr">41</a>]; (13) Johnson et al.[<a href="#B31-jof-08-00537" class="html-bibr">31</a>]; (14) Petrisko et al. [<a href="#B61-jof-08-00537" class="html-bibr">61</a>]; (15) Ruthig [<a href="#B68-jof-08-00537" class="html-bibr">68</a>]; (16) Kim et al. [<a href="#B67-jof-08-00537" class="html-bibr">67</a>]; (17) Fernández-Benéitez et al. [<a href="#B48-jof-08-00537" class="html-bibr">48</a>]; (18) Sagvik et al. [<a href="#B63-jof-08-00537" class="html-bibr">63</a>,<a href="#B64-jof-08-00537" class="html-bibr">64</a>]; (19) Ruthig [<a href="#B60-jof-08-00537" class="html-bibr">60</a>]; (20) Uller et al. [<a href="#B65-jof-08-00537" class="html-bibr">65</a>]; (21) Karraker &amp; Ruthig [<a href="#B66-jof-08-00537" class="html-bibr">66</a>]; (22) Blackburn et al. [<a href="#B24-jof-08-00537" class="html-bibr">24</a>]; (23) Prada-Salcedo et al. [<a href="#B39-jof-08-00537" class="html-bibr">39</a>]; (24) Fernández-Benéitez et al. [<a href="#B49-jof-08-00537" class="html-bibr">49</a>]; (25) Ault et al. [<a href="#B46-jof-08-00537" class="html-bibr">46</a>]; (26) Ruthig and Provost-Javier [<a href="#B57-jof-08-00537" class="html-bibr">57</a>]; (27) Perotti et al. [<a href="#B59-jof-08-00537" class="html-bibr">59</a>]; (28) Croshaw [<a href="#B75-jof-08-00537" class="html-bibr">75</a>]; (29) Muir [<a href="#B70-jof-08-00537" class="html-bibr">70</a>]; (30) Urban et al. [<a href="#B74-jof-08-00537" class="html-bibr">74</a>]; (31) Groffen et al. [<a href="#B50-jof-08-00537" class="html-bibr">50</a>]; (32) Sadinski, Gallant, and Cleaver [<a href="#B38-jof-08-00537" class="html-bibr">38</a>]; (33) Costa et al. in prep. Maps made with QGIS. Location information available on <a href="#app1-jof-08-00537" class="html-app">Table S2</a>.</p> Full article ">Figure 3
<p>Scheme illustrating the sexual and asexual lifecycles of <span class="html-italic">Saprolegnia</span> spp. with the identification of the development stages able to cause infection in amphibians (host infection).</p> Full article ">
11 pages, 1853 KiB  
Article
Pulmonary Sporotrichosis Caused by Sporothrix brasiliensis: A 22-Year, Single-Center, Retrospective Cohort Study
by Vivian Fichman, Caroline Graça Mota-Damasceno, Anna Carolina Procópio-Azevedo, Fernando Almeida-Silva, Priscila Marques de Macedo, Denise Machado Medeiros, Guis Saint-Martin Astacio, Rosely Maria Zancopé-Oliveira, Rodrigo Almeida-Paes, Dayvison Francis Saraiva Freitas and Maria Clara Gutierrez-Galhardo
J. Fungi 2022, 8(5), 536; https://doi.org/10.3390/jof8050536 - 21 May 2022
Cited by 10 | Viewed by 2954
Abstract
Pulmonary sporotrichosis is a rare condition. It can present as a primary pulmonary disease, resulting from direct Sporothrix species (spp). conidia inhalation, or as part of multifocal sporotrichosis with multiple organ involvement, mainly in immunocompromised patients. This study aimed to describe the sociodemographic [...] Read more.
Pulmonary sporotrichosis is a rare condition. It can present as a primary pulmonary disease, resulting from direct Sporothrix species (spp). conidia inhalation, or as part of multifocal sporotrichosis with multiple organ involvement, mainly in immunocompromised patients. This study aimed to describe the sociodemographic and epidemiological characteristics and clinical course of patients with positive cultures for Sporothrix spp. from pulmonary specimens (sputum and/or bronchoalveolar lavage) at a reference center in an area hyperendemic for zoonotic sporotrichosis. The clinical records of these patients were reviewed. Fourteen patients were included, and Sporothrix brasiliensis was identified in all cases. Disseminated sporotrichosis was the clinical presentation in 92.9% of cases, and primary pulmonary sporotrichosis accounted for 7.1%. Comorbidities included human immunodeficiency virus infection (78.6%), alcoholism (71.4%), and chronic obstructive pulmonary disease (14.3%). Treatment with amphotericin B followed by itraconazole was the preferred regimen and was prescribed in 92.9% of cases. Sporotrichosis-related death occurred in 42.9% while 35.7% of patients were cured. In five cases there was a probable contamination from upper airway lesions. Despite the significant increase in sporotrichosis cases, pulmonary sporotrichosis remains rare. The treatment of disseminated sporotrichosis is typically difficult. Prompt diagnosis and identification of all affected organs are crucial for better prognosis. Full article
(This article belongs to the Special Issue Sporothrix and Sporotrichosis 2.0)
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<p>Primary pulmonary sporotrichosis in a 71-year-old man (Case 1). (<b>A</b>) Topogram depicts lung cavitary lesions. (<b>B</b>–<b>D</b>) Axial nonenhanced chest computed tomography images show extensive thick-walled cavities with irregular margins in upper lobes. The cavitary lesion is more predominant in the left upper lobe, associated with thick septations and pulmonary volume loss.</p> Full article ">Figure 2
<p>Disseminated sporotrichosis in a 25-year-old man (Case 4). Multiple papular and nodular-ulcerative facial lesions, with aerodigestive tract impairment. Involvement of the nasal mucosa and palate is shown.</p> Full article ">Figure 3
<p>Nonenhanced chest computed tomography (CT) of a 20-year-old woman with disseminated sporotrichosis and lung lesions, at three time points (Case 14). (<b>A</b>) During treatment for confirmed pulmonary tuberculosis, CT shows no lung cavities. (<b>B</b>) Post-treatment for tuberculosis and recent diagnosis of sporotrichosis, CT shows a thick-walled cavity in the right upper lobe, with isolation of <span class="html-italic">Sporothrix</span> spp. from sputum. (<b>C</b>) Resolution of the cavity during sporotrichosis treatment.</p> Full article ">Figure 4
<p>Nonenhanced chest computed tomography of a 63-year-old man with disseminated sporotrichosis and lung lesions, at four moments (Case 2). (<b>A</b>,<b>E</b>) At the beginning of treatment for sporotrichosis. (<b>B</b>,<b>F</b>) Six months of treatment. (<b>C</b>,<b>G</b>) At 30 months of treatment. (<b>D</b>,<b>H</b>) At 36 months of treatment. Among many alterations, there is a thick-walled cavity with irregular margins in the right lower lobe, associated with architectural distortion, fibrotic opacities, and traction bronchiectasis. There is also bronchiectasis in the apicoposterior segment of the left upper lobe, some filled by fluid density material, suggestive of mucous plugging. Areas of pleural thickening in the upper third of the lungs. Images A to D and E to H are similar sections over time.</p> Full article ">
18 pages, 2794 KiB  
Article
Efficacy of Inhaled N-Chlorotaurine in a Mouse Model of Lichtheimia corymbifera and Aspergillus fumigatus Pneumonia
by Cornelia Speth, Günter Rambach, Andrea Windisch, Magdalena Neurauter, Hans Maier and Markus Nagl
J. Fungi 2022, 8(5), 535; https://doi.org/10.3390/jof8050535 - 20 May 2022
Cited by 3 | Viewed by 2086
Abstract
N-chlorotaurine (NCT) can be used topically as a well-tolerated anti-infective at different body sites. The aim of this study was to investigate the efficacy of inhaled NCT in a mouse model of fungal pneumonia. Specific pathogen-free female C57BL/6JRj seven-week-old mice were immune-suppressed with [...] Read more.
N-chlorotaurine (NCT) can be used topically as a well-tolerated anti-infective at different body sites. The aim of this study was to investigate the efficacy of inhaled NCT in a mouse model of fungal pneumonia. Specific pathogen-free female C57BL/6JRj seven-week-old mice were immune-suppressed with cyclophosphamide. After 4 days, the mice were inoculated intranasally with 1.5 × 10E7 spores of Lichtheimia corymbifera or 1.0 × 10E7 spores of Aspergillus fumigatus. They were randomized and treated three times daily for 10 min with aerosolized 1% NCT or 0.9% sodium chloride starting 1 h after the inoculation. The mice were observed for survival for two weeks, and fungal load, blood inflammation parameters, bronchoalveolar lavage, and histology of organs were evaluated upon their death or at the end of this period. Inhalations were well-tolerated. After challenge with L. corymbifera, seven out of the nine mice (77.8%) survived for 15 days in the test group, which was in strong contrast to one out of the nine mice (11.1%) in the control group (p = 0.0049). The count of colony-forming units in the homogenized lung tissues came to 1.60 (1.30; 1.99; median, quartiles) log10 in the test group and to 4.26 (2.17; 4.53) log10 in the control group (p = 0.0032). Body weight and temperature, white blood count, and haptoglobin significantly improved with NCT treatment. With A. fumigatus, all the mice except for one in the test group died within 4 days without a significant difference from the control group. Inhaled NCT applied early demonstrated a highly significant curative effect in L. corymbifera pneumonia, while this could not be shown in A. fumigatus pneumonia, probably due to a too high inoculum. Nevertheless, this study for the first time disclosed efficacy of NCT in pneumonia in vivo. Full article
(This article belongs to the Section Fungal Pathogenesis and Disease Control)
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<p>Survival curves of the immunosuppressed mice after mold lung infection by nasal inoculation with 1.0 × 10<sup>7</sup> spores of <span class="html-italic">Aspergillus fumigatus</span> or 1.5 × 10<sup>7</sup> spores of <span class="html-italic">Lichtheimia corymbifera</span> and treatment with inhaled 1% NCT or 0.9% NaCl (<span class="html-italic">n</span> = 9 each). The control mice that did not receive fungi but cyclophosphamide and inhalations with NCT or saline are also shown and survived except for one mouse in the saline group that lost weight massively and one mouse in the NCT group that accidentally died after blood-taking; <span class="html-italic">p</span> = 0.0049 between NCT and NaCl for <span class="html-italic">L. corymbifera</span>; <span class="html-italic">p</span> = 0.568 between NCT and NaCl for <span class="html-italic">A. fumigatus</span>; <span class="html-italic">p</span> = 0.587 between the controls without fungi and NCT with <span class="html-italic">L. corymbifera</span> (logrank Mantel–Cox test each). All the mice treated with inhalations of NCT or saline without cyclophosphamide (<span class="html-italic">n</span> = 9 each) survived (not shown). CP, cyclophosphamide; A22, <span class="html-italic">A. fumigatus</span>; LC9, <span class="html-italic">L. corymbifera</span>.</p> Full article ">Figure 2
<p>Pathogen counts in the lung of the mice challenged with <span class="html-italic">L. corymbifera</span>. Fungal (<b>a</b>) and bacterial (<b>b</b>) counts in the homogenized lung of the mice euthanized on day 2 and of the mice monitored for survival for 15 days. Scatter dot plots with medians and quartiles, Mann–Whitney test.</p> Full article ">Figure 3
<p>Organ weight per body weight × 1000 at time of death or euthanasia compared to the baseline for the lung (<b>a</b>) and the spleen (<b>b</b>). Scatter dot plots with the medians and quartiles (<span class="html-italic">n</span> = 4–9); ** <span class="html-italic">p</span> &lt; 0.01 versus plain NaCl (Kruskal-Wallis test); <span class="html-italic">p</span>-values &lt; 0.1 between each test and control group are shown (Mann–Whitney test).</p> Full article ">Figure 3 Cont.
<p>Organ weight per body weight × 1000 at time of death or euthanasia compared to the baseline for the lung (<b>a</b>) and the spleen (<b>b</b>). Scatter dot plots with the medians and quartiles (<span class="html-italic">n</span> = 4–9); ** <span class="html-italic">p</span> &lt; 0.01 versus plain NaCl (Kruskal-Wallis test); <span class="html-italic">p</span>-values &lt; 0.1 between each test and control group are shown (Mann–Whitney test).</p> Full article ">Figure 4
<p>Percentage of body weight at the time of death or euthanasia compared to the baseline. Scatter dot plots with the medians and quartiles (<span class="html-italic">n</span> = 8–9). The absolute weight loss in grams as the mean values and SD is indicated below the panels; ** <span class="html-italic">p</span> &lt; 0.01 versus plain NaCl (Kruskal–Wallis test); <span class="html-italic">p</span>-values &lt; 0.1 between each test and control group are shown (Mann–Whitney test). Of note, the two mice with the lowest weight in group 6 (<span class="html-italic">L. corymbifera</span>, NCT) were the only ones who died in this group before the endpoint of 15 days.</p> Full article ">Figure 5
<p>White blood cell count on day 2. Scatter dot plots with the medians and quartiles (<span class="html-italic">n</span> = 9–14). Both the plain NCT group and the plain NaCl groups were highly significantly different from all the other groups (** <span class="html-italic">p</span> &lt; 0.01, Kruskal–Wallis test). Further <span class="html-italic">p</span>-values are indicated (Mann–Whitney test).</p> Full article ">Figure 6
<p>Bronchoalveolar lavage. Samples of BAL of the indicated groups. Normal alveolar macrophages in the immunosuppressed mice that inhaled NaCl or NCT. Inflammation and scattered hyphae in samples from the mice inoculated with molds in addition. Magnification: 100× in the upper panels, 400× in the middle and lower panels.</p> Full article ">Figure 7
<p>Histology. Acute inflammation in lung samples of the mice infected with <span class="html-italic">L. corymbifera</span> (upper panels) and <span class="html-italic">A. fumigatus</span> (lower panels) on day 3. Conidia of <span class="html-italic">Lichtheimia</span> (arrows) and massive germination of <span class="html-italic">Aspergillus</span>. Magnification: 100× in the left panels, 400× in the right ones.</p> Full article ">
14 pages, 2974 KiB  
Article
MAPK CcSakA of the HOG Pathway Is Involved in Stipe Elongation during Fruiting Body Development in Coprinopsis cinerea
by Jing Zhao, Jing Yuan, Yating Chen, Yu Wang, Jing Chen, Jingjing Bi, Linna Lyu, Cigang Yu, Sheng Yuan and Zhonghua Liu
J. Fungi 2022, 8(5), 534; https://doi.org/10.3390/jof8050534 - 20 May 2022
Cited by 5 | Viewed by 1966
Abstract
Mitogen-activated protein kinase (MAPK) pathways, such as the high-osmolarity glycerol mitogen-activated protein kinase (HOG) pathway, are evolutionarily conserved signaling modules responsible for transmitting environmental stress signals in eukaryotic organisms. Here, we identified the MAPK homologue in the HOG pathway of Coprinopsis cinerea, which [...] Read more.
Mitogen-activated protein kinase (MAPK) pathways, such as the high-osmolarity glycerol mitogen-activated protein kinase (HOG) pathway, are evolutionarily conserved signaling modules responsible for transmitting environmental stress signals in eukaryotic organisms. Here, we identified the MAPK homologue in the HOG pathway of Coprinopsis cinerea, which was named CcSakA. Furthermore, during the development of the fruiting body, CcSakA was phosphorylated in the fast elongating apical part of the stipe, which meant that CcSakA was activated in the apical elongating stipe region of the fruiting body. The knockdown of CcSakA resulted in a shorter stipe of the fruiting body compared to the control strain, and the expression of phosphomimicking mutant CcSakA led to a longer stipe of the fruiting body compared to the control strain. The chitinase CcChiE1, which plays a key role during stipe elongation, was downregulated in the CcSakA knockdown strains and upregulated in the CcSakA phosphomimicking mutant strains. The results indicated that CcSakA participated in the elongation of stipes in the fruiting body development of C. cinerea by regulating the expression of CcChiE1. Analysis of the H2O2 concentration in different parts of the stipe showed that the oxidative stress in the elongating part of the stipe was higher than those in the non-elongating part. The results indicated that CcSakA of the HOG pathway may be activated by oxidative stress. Our results demonstrated that the HOG pathway transmits stress signals and regulates the expression of CcChiE1 during fruiting body development in C. cinerea. Full article
(This article belongs to the Section Fungal Genomics, Genetics and Molecular Biology)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>(<b>A</b>) The <span class="html-italic">CcsakA</span> gene encodes a putative homologue of the MAPK of the HOG pathway in <span class="html-italic">C. cinerea</span>. The amino acid sequence of CcSakA is aligned with <span class="html-italic">S. cerevisiae</span> Hog1 [<a href="#B40-jof-08-00534" class="html-bibr">40</a>], <span class="html-italic">S. pombe</span> Spc1 [<a href="#B41-jof-08-00534" class="html-bibr">41</a>], <span class="html-italic">A. fumigatus</span> SakA [<a href="#B39-jof-08-00534" class="html-bibr">39</a>], <span class="html-italic">P. grisea</span> Osm1 [<a href="#B42-jof-08-00534" class="html-bibr">42</a>], and <span class="html-italic">T. marneffei</span> SakA [<a href="#B43-jof-08-00534" class="html-bibr">43</a>]. Conserved TGY phosphorylation sites found in the stress Hog1/Spc1/p38 MAPK family are marked with blue boxes. (<b>B</b>) The relative mRNA expression level of <span class="html-italic">sakA</span> in the different stipe regions during the development of <span class="html-italic">C. cinerea</span> fruiting bodies. (<b>C</b>) Western blotting of the phosphorylation of CcSakA in the extracts from different stipe regions during the development of <span class="html-italic">C. cinerea</span> fruiting bodies. Phosphorylated CcSakA was detected using anti-phospho-p38 MAPK antibody. Levels of β-tubulin were used to demonstrate equal protein loading.</p> Full article ">Figure 2
<p>Construction and phenotypes of CcSakA gene-silenced strains. (<b>A</b>) Schematic representation of plasmids pCcExp (<b>A1</b>), pCc<span class="html-italic">pab-1</span> (<b>A2</b>), and pCC-SakAi (<b>A3</b>). The arrows below the plasmids indicate the primers for genomic PCR. (<b>B</b>) The relative mRNA expression level of <span class="html-italic">sakA</span> in mock transformants (circle) and SakAi transformants (triangle). (<b>C</b>) Growing fruiting bodies of the three representatives of the mock transformants and SakAi transformants at different time points. (<b>D</b>) The stipe lengths of the fruiting bodies of four mock transformants (circle, <span class="html-italic">n</span> = 109 fruiting bodies) and eight SakAi transformants (square, <span class="html-italic">n</span> = 124 fruiting bodies) with at least three repeats of each transformant at K + 0 (<b>D1</b>), K + 2 (<b>D2</b>), K + 4 (<b>D3</b>), and K + 6 (<b>D4</b>).</p> Full article ">Figure 3
<p>Construction and phenotypes of CcSakA phosphomimicking mutant strains. (<b>A</b>) Schematic representation of plasmids pCcExp (<b>A1</b>), pCc<span class="html-italic">pab-1</span> (<b>A2</b>), and pCC-SakA<sup>T170E+Y172D</sup> (<b>A3</b>). The arrows below the plasmids indicate the primers for genomic PCR. (<b>B</b>) Schematic diagram of SakA phosphorylation site mutations. (<b>C</b>) Growing fruiting bodies of the three representatives of the mock transformants and SakAm transformants at different time points. (<b>D</b>) The stipe lengths of the fruiting bodies of four mock transformants (circle, <span class="html-italic">n</span> = 123 fruiting bodies) and eight SakAm transformants (square, <span class="html-italic">n</span> = 131 fruiting bodies) with at least three repeats of each transformant at K + 0 (<b>D1</b>), K + 2 (<b>D2</b>), K + 4 (<b>D3</b>), and K + 6 (<b>D4</b>).</p> Full article ">Figure 4
<p>qRT–PCR analysis of the expression level of chitinase CcChiE1 in CcSakA gene silencing (SakAi) transformants (<b>A</b>) and CcSakA phosphomimicking mutant (SakAm) transformants (<b>B</b>) compared with mock transformants. A β-tubulin gene was used to standardize the mRNA level. Four mock transformants (circle), eight SakAi transformants (square in (<b>A</b>)), and eight SakAm transformants (square in (<b>B</b>)) were analyzed with at least three repeats of each transformant. Data are presented as the mean and standard error. Tests for significance were performed by a t-test in Microsoft Excel 2010.</p> Full article ">Figure 5
<p>The osmolality (<b>A</b>) and H<sub>2</sub>O<sub>2</sub> concentration (<b>B</b>) in the fast elongating apical part, the slow elongating median part, and the nonelongating basal part of the stipe of <span class="html-italic">C. cinerea</span>. Data are presented as the mean and standard error of three biological replicates (<span class="html-italic">n</span> = 6). The same letters indicate no significant difference (<span class="html-italic">p</span> &gt; 0.05), and different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05) by Duncan’s test. The intracellular ROS imaging of the different regions of the stipe (<b>C</b>). The fluorescence signals of the fluorescent intracellular ROS probe DCFH-DA were observed and Bar = 50 μm. The fluorescence intensity (<b>D</b>) was analyzed by using ImageJ 1.51 (<span class="html-italic">n</span> = 9). The same letters indicate no significant difference (<span class="html-italic">p</span> &gt; 0.05), and different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05) by Duncan’s test.</p> Full article ">
12 pages, 671 KiB  
Article
CYP51 Mutations in the Fusarium solani Species Complex: First Clue to Understand the Low Susceptibility to Azoles of the Genus Fusarium
by Pierre Vermeulen, Arnaud Gruez, Anne-Lyse Babin, Jean-Pol Frippiat, Marie Machouart and Anne Debourgogne
J. Fungi 2022, 8(5), 533; https://doi.org/10.3390/jof8050533 - 20 May 2022
Cited by 5 | Viewed by 2364
Abstract
Members of Fusarium solani species complex (FSSC) are cosmopolitan filamentous fungi responsible for invasive fungal infections in immunocompromised patients. Despite the treatment recommendations, many strains show reduced sensitivity to voriconazole. The objective of this work was to investigate the potential relationship between azole [...] Read more.
Members of Fusarium solani species complex (FSSC) are cosmopolitan filamentous fungi responsible for invasive fungal infections in immunocompromised patients. Despite the treatment recommendations, many strains show reduced sensitivity to voriconazole. The objective of this work was to investigate the potential relationship between azole susceptibility and mutations in CYP51 protein sequences. Minimal inhibitory concentrations (MICs) for azole antifungals have been determined using the CLSI (Clinical and Laboratory Standards Institute) microdilution method on a panel of clinical and environmental strains. CYP51A, CYP51B and CYP51C genes for each strain have been sequenced using the Sanger method. Amino acid substitutions described in multiple azole-resistant Aspergillus fumigatus (mtrAf) strains have been sought and compared with other Fusarium complexes’ strains. Our results show that FSSC exhibit point mutations similar to those described in mtrAf. Protein sequence alignments of CYP51A, CYP51B and CYP51C have highlighted different profiles based on sequence similarity. A link between voriconazole MICs and protein sequences was observed, suggesting that these mutations could be an explanation for the intrinsic azole resistance in the genus Fusarium. Thus, this innovative approach provided clues to understand low azole susceptibility in FSSC and may contribute to improving the treatment of FSSC infection. Full article
(This article belongs to the Special Issue Clinically Relevant Fusarium Species)
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<p>Calculated model of FSSC CYP51A protein. Point mutations shared by all FSSC strains and known to be associated with azole resistance in <span class="html-italic">A. fumigatus</span> are indicated by the amino acid letter and position. Amino acid L218 interacts with F75, and both are present at the entry of the substrate channel (yellow: heme; blue: itraconazole; purple: F75; red: L218, corresponding to the channel entry for the substrate).</p> Full article ">
28 pages, 7556 KiB  
Article
Taxonomic and Phylogenetic Characterizations Reveal Four New Species, Two New Asexual Morph Reports, and Six New Country Records of Bambusicolous Roussoella from China
by Dong-Qin Dai, Nalin N. Wijayawardene, Monika C. Dayarathne, Jaturong Kumla, Li-Su Han, Gui-Qing Zhang, Xian Zhang, Ting-Ting Zhang and Huan-Huan Chen
J. Fungi 2022, 8(5), 532; https://doi.org/10.3390/jof8050532 - 20 May 2022
Cited by 10 | Viewed by 2330
Abstract
During the ongoing investigation of bambusicolous ascomycetous fungi in Yunnan, China, 24 specimens belonging to the family Roussoellaceae were collected and identified based on morphological features and phylogenetic support. Maximum-likelihood (ML) analyses and Bayesian analyses were generated based on the combined data set [...] Read more.
During the ongoing investigation of bambusicolous ascomycetous fungi in Yunnan, China, 24 specimens belonging to the family Roussoellaceae were collected and identified based on morphological features and phylogenetic support. Maximum-likelihood (ML) analyses and Bayesian analyses were generated based on the combined data set of ITS, LSU, tef1, and rpb2 loci. The phylogenetic analyses revealed four novel lineages in Roussoella s. str.; thus, we introduced four new species viz., Roussoella multiloculate sp. nov., R. papillate sp. nov., R. sinensis sp. nov., and R. uniloculata sp. nov. Their morphological characters were compared with the known Roussoella taxa, which lack sequence data in the GenBank. Asexual morphs of R. kunmingensis and R. padinae were recorded from dead bamboo culms in China (from the natural substrates) for the first time. Neoroussoella bambusae, Roussoella japanensis, R. nitidula, R. padinae, R. scabrispora, and R. tuberculate were also reported as the first records from China. All new taxa are described and illustrated in detail. Plates are provided for new reports. Full article
(This article belongs to the Special Issue Holarctic Fungi: Taxonomy, Phylogeny, Ecology, Nutrition Interactions and Distribution)
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Figure 1
<p>Phylogenetic tree from the best scoring of the RAxML analysis based on combined ITS, LSU, <span class="html-italic">rpb2</span> and <span class="html-italic">tef1</span> loci is rooted to <span class="html-italic">Torula herbarum</span> (CBS 111855) and <span class="html-italic">T. hollandica</span> (CBS 220.69). Bootstrap values for maximum likelihood (MLBP) and Bayesian posterior probabilities (BYPP) equal to or greater than 50% and 0.80, respectively, are given at the branches. The newly generated sequences are marked with asterisk “★” and ex-type strains are indicated in bold. Bar = 0.1 expected number of nucleotide substitutions per site per branch.</p> Full article ">Figure 1 Cont.
<p>Phylogenetic tree from the best scoring of the RAxML analysis based on combined ITS, LSU, <span class="html-italic">rpb2</span> and <span class="html-italic">tef1</span> loci is rooted to <span class="html-italic">Torula herbarum</span> (CBS 111855) and <span class="html-italic">T. hollandica</span> (CBS 220.69). Bootstrap values for maximum likelihood (MLBP) and Bayesian posterior probabilities (BYPP) equal to or greater than 50% and 0.80, respectively, are given at the branches. The newly generated sequences are marked with asterisk “★” and ex-type strains are indicated in bold. Bar = 0.1 expected number of nucleotide substitutions per site per branch.</p> Full article ">Figure 2
<p><span class="html-italic">Neoroussoella bambusae</span> (GMB1291, new country record). (<b>a</b>) Bamboo specimen; (<b>b</b>) Black ascostromata on host surface; (<b>c</b>) Vertical section of ascostroma; (<b>d</b>) Cells of locules walls; (<b>e</b>) Pseudoparaphyses; (<b>f</b>–<b>i</b>) Asci; (<b>j</b>–<b>m</b>) Ascospores; (<b>n</b>) Ascospore in India ink; (<b>o</b>) Germinating ascospore; (<b>p</b>,<b>q</b>) Cultures on PDA from above and below. Scale bars: (<b>b</b>) = 150 μm, (<b>c</b>) = 100 μm, (<b>d</b>) = 50 μm, (<b>e</b>,<b>n</b>) = 10 μm, (<b>f</b>–<b>i</b>) = 20 μm, (<b>j</b>–<b>m</b>) = 5 μm, (<b>o</b>) = 15 μm.</p> Full article ">Figure 3
<p><span class="html-italic">Roussoella japanensis</span> (GMB1292, new country record). (<b>a</b>) Bamboo specimen; (<b>b</b>) black ascostromata on host surface; (<b>c</b>) vertical section of ascoma; (<b>d</b>) cells between locules; (<b>e</b>) pseudoparaphyses; (<b>f</b>–<b>j</b>) ascospores; (<b>k</b>) ascospore in India ink; (<b>l</b>) germinating ascospore; (<b>m</b>) cultures on PDA from below and above; (<b>n</b>–<b>s</b>) asci. Scale bars: (<b>b</b>) = 2 mm, (<b>c</b>) = 200 μm, (<b>d</b>) = 50 μm, (<b>e</b>–<b>j</b>) = 10 μm, (<b>k</b>,<b>l</b>) = 20 μm, (<b>n</b>–<b>s</b>) = 30 μm.</p> Full article ">Figure 4
<p><span class="html-italic">Roussoella kunmingensis</span> (GMB1203, sexual morph, (<b>a</b>,<b>c</b>,<b>e</b>,<b>g</b>,<b>h</b>–<b>q</b>); GMB1259, first report of asexual morph, (<b>b</b>,<b>d</b>,<b>f</b>,<b>r</b>–<b>y</b>)). (<b>a</b>,<b>b</b>) Bamboo specimens; (<b>c</b>) black ascostromata on host surface; (<b>d</b>) black conidiomata on host surface; (<b>e</b>) vertical section of ascostromata; (<b>f</b>) vertical section of conidioma; (<b>g</b>) pseudoparaphyses; (<b>h</b>–<b>k</b>) asci; (<b>l</b>–<b>n</b>) ascospores; (<b>o</b>,<b>p</b>) ascospores in India ink; (<b>q</b>) germinating ascospore; (<b>r</b>–<b>u</b>) conidiogenous cells contacting with conidia; (<b>v</b>,<b>w</b>) conidia; (<b>x</b>) germinating conidium; (<b>y</b>) cultures on PDA from above and below. Scale bars: (<b>c</b>) = 500 μm, (<b>d</b>) = 300 μm, (<b>e</b>) = 200 μm, (<b>f</b>) = 100 μm, (<b>g</b>–<b>x</b>) = 10 μm.</p> Full article ">Figure 5
<p><span class="html-italic">Roussoella multiloculate</span> (GMB1207, holotype). (<b>a</b>) Bamboo specimen; (<b>b</b>–<b>d</b>) black conidiomata on host surface; (<b>e</b>,<b>f</b>) vertical sections of conidiomata; (<b>g</b>–<b>i</b>) conidia attached to conidiogenous cells; (<b>j</b>,<b>k</b>) conidia. (<b>l</b>) germinating conidium. (<b>m</b>) cultures on PDA from above and below. Scale bars: (<b>b</b>–<b>d</b>) = 500 µm, (<b>e</b>,<b>f</b>) = 100 µm, (<b>g</b>–<b>l</b>) = 5 µm.</p> Full article ">Figure 6
<p><span class="html-italic">Roussoella nitidula</span> (GMB1270, new country record). (<b>a</b>) Bamboo specimen; (<b>b</b>) black ascostromata on host surface; (<b>c</b>) vertical sections of ascomata; (<b>d</b>) cells of locule wall. (<b>e</b>) pseudoparaphyses; (<b>f</b>–<b>j</b>) asci; (<b>k</b>–<b>n</b>) ascospores; (<b>o</b>) Germinating ascospore; (<b>p</b>) Cultures on PDA from above and below. Scale bars: (<b>b</b>) = 1 mm, (<b>c</b>) = 500 μm, (<b>d</b>) = 50 μm, (<b>e</b>–<b>j</b>) = 30 μm, (<b>k</b>–<b>n</b>) = 10 μm, (<b>o</b>) = 15 μm.</p> Full article ">Figure 7
<p><span class="html-italic">Roussoella papillate</span> (GMB129, holotype). (<b>a</b>) Bamboo specimen; (<b>b</b>) black ascostromata on host surface; (<b>c</b>) vertical section of ascoma; (<b>d</b>) cells of locule wall near the ostiole; (<b>e</b>) branching pseudoparaphyses; (<b>f</b>–<b>i</b>) asci; (<b>j</b>–<b>m</b>) ascospores; (<b>n</b>) germinating ascospore; (<b>o</b>,<b>p</b>) cultures on PDA from above and below. Scale bars: (<b>b</b>) = 500 µm, (<b>c</b>) = 150 µm, (<b>d</b>) = 50 µm, (<b>e</b>–<b>i</b>,<b>n</b>) = 30 µm, (<b>j</b>–<b>m</b>) = 15 µm.</p> Full article ">Figure 8
<p><span class="html-italic">Roussoella padinae</span> (GMB1320, first report of asexual morph, first record from terrestrial habitat and first record from China). (<b>a</b>) Bamboo specimen; (<b>b</b>) reddish-brown conidiomata on host surface; (<b>c</b>) vertical sections of conidiomata; (<b>d</b>) conidioma wall; (<b>e</b>–<b>h</b>) conidiogenous cells; (<b>i</b>) conidia; (<b>j</b>) germinating conidium; (<b>k</b>) cultures on PDA from above and below. Scale bars: (<b>b</b>) = 1 mm, (<b>c</b>) = 150 µm, (<b>d</b>) = 30 µm, (<b>e</b>–<b>h</b>) =15 µm, (<b>i</b>) = 5 µm, (<b>j</b>) = 10 µm.</p> Full article ">Figure 9
<p><span class="html-italic">Roussoella scabrispora</span> (GMB1286, new country record). (<b>a</b>) Bamboo specimen; (<b>b</b>) ascomata on bamboo host; (<b>c</b>) vertical section of ascoma; (<b>d</b>) peridium; (<b>e</b>) pseudoparaphyses; (<b>f</b>–<b>i</b>) ascospores; (<b>j</b>) germinating ascospore; (<b>k</b>–<b>n</b>) asci; (<b>o</b>,<b>p</b>) cultures on PDA from above and below. Scale bars: (<b>b</b>) = 1 mm, (<b>c</b>) = 500 μm, (<b>d</b>) = 100 μm, (<b>e</b>) = 30 μm, (<b>f</b>–<b>j</b>) = 15 μm, (<b>k</b>–<b>n</b>) = 50 μm.</p> Full article ">Figure 10
<p><span class="html-italic">Roussoella sinensis</span> (GMB1296, holotype). (<b>a</b>) Bamboo specimen; (<b>b</b>) black ascostromata showing black ostioles with openings on host surface; (<b>c</b>,<b>d</b>) vertical section of ascostromata; (<b>e</b>) cells of locule wall; (<b>f</b>) pseudoparaphyses; (<b>g</b>–<b>j</b>) ascospores; (<b>k</b>) germinating ascospore; (<b>l</b>–<b>o</b>) different developmental stages of asci; (<b>p</b>,<b>q</b>) cultures on PDA from above and below. Scale bars: (<b>c</b>,<b>d</b>) = 200 μm, (<b>e</b>) = 50 μm, (<b>f</b>) = 10 μm, (<b>g</b>–<b>k</b>) = 15 μm, (<b>l</b>–<b>o</b>) = 30 μm.</p> Full article ">Figure 11
<p><span class="html-italic">Roussoella tuberculata</span> (GMB1317, new country record). (<b>a</b>) Bamboo specimen; (<b>b</b>) black conidioma on host surface; (<b>c</b>) vertical sections of conidiomata; (<b>d</b>–<b>h</b>) conidiogenous cells and developing conidia; (<b>i</b>–<b>l</b>) conidia; m: germinating conidium; (<b>m</b>) germinating conidium; (<b>n</b>) cultures on PDA from above and below after two weeks; (<b>o</b>) cultures on PDA from above and below after four weeks. Scale bars: (<b>b</b>) = 500 µm, (<b>c</b>) = 200 µm, (<b>d</b>–<b>f</b>,<b>m</b>) = 15 µm, (<b>h</b>–<b>l</b>) = 10 µm.</p> Full article ">Figure 12
<p><span class="html-italic">Roussoella uniloculata</span> (GMB1288, holotype). (<b>a</b>) Bamboo specimen; (<b>b</b>) black ascostroma on host surface; (<b>c</b>) vertical section of ascomata; (<b>d</b>) pseudoparaphyses; (<b>e</b>–<b>h</b>) ascospores; (<b>i</b>) germinating ascospore; (<b>j</b>–<b>m</b>) asci; (<b>n</b>,<b>o</b>) cultures on PDA from above and below. Scale bars: (<b>b</b>) = 300 µm, (<b>c</b>) = 50 µm, (<b>d</b>,<b>j</b>–<b>m</b>) = 30 µm, (<b>e</b>–<b>i</b>) = 10 µm.</p> Full article ">
15 pages, 5298 KiB  
Article
Toolbox for Genetic Transformation of Non-Conventional Saccharomycotina Yeasts: High Efficiency Transformation of Yeasts Belonging to the Schwanniomyces Genus
by Angela Matanović, Kristian Arambašić, Bojan Žunar, Anamarija Štafa, Marina Svetec Miklenić, Božidar Šantek and Ivan-Krešimir Svetec
J. Fungi 2022, 8(5), 531; https://doi.org/10.3390/jof8050531 - 20 May 2022
Viewed by 2582
Abstract
Non-conventional yeasts are increasingly being investigated and used as producers in biotechnological processes which often offer advantages in comparison to traditional and well-established systems. Most biotechnologically interesting non-conventional yeasts belong to the Saccharomycotina subphylum, including those already in use (Pichia pastoris, Yarrowia [...] Read more.
Non-conventional yeasts are increasingly being investigated and used as producers in biotechnological processes which often offer advantages in comparison to traditional and well-established systems. Most biotechnologically interesting non-conventional yeasts belong to the Saccharomycotina subphylum, including those already in use (Pichia pastoris, Yarrowia lypolitica, etc.), as well as those that are promising but as yet insufficiently characterized. Moreover, for many of these yeasts the basic tools of genetic engineering needed for strain construction, including a procedure for efficient genetic transformation, heterologous protein expression and precise genetic modification, are lacking. The first aim of this study was to construct a set of integrative and replicative plasmids which can be used in various yeasts across the Saccharomycotina subphylum. Additionally, we demonstrate here that the electroporation procedure we developed earlier for transformation of B. bruxellensis can be applied in various yeasts which, together with the constructed plasmids, makes a solid starting point when approaching a transformation of yeasts form the Saccharomycotina subphylum. To provide a proof of principle, we successfully transformed three species from the Schwanniomyces genus (S. polymorphus var. polymorphus, S. polymorphus var. africanus and S. pseudopolymorphus) with high efficiencies (up to 8 × 103 in case of illegitimate integration of non-homologous linear DNA and up to 4.7 × 105 in case of replicative plasmid). For the latter two species this is the first reported genetic transformation. Moreover, we found that a plasmid carrying replication origin from Scheffersomyces stipitis can be used as a replicative plasmid for these three Schwanniomyces species. Full article
(This article belongs to the Special Issue Yeast Genetics 2021)
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Figure 1

Figure 1
<p>Construction of the plasmid series pRS50 (integrative plasmids) with a detailed list of intermediate cloning steps and relevant restriction sites. ble = original ble ORF from Klebsiella pneumoniae, EgTEFpromoter = promoter of the Eremothecium gossypii TEF gene, EgTEFterminator = terminator of the E. gossypii TEF gene, oBle = codon-optimized ble ORF encoding for resistance to phleomycin, oHyg = codon-optimized ORF encoding for resistance to hygromycin B, oKan = codon-optimized ORF encoding for resistance to G418, oNrs = codon-optimized ORF encoding for resistance to clonNAT, bla = resistance to ampicillin, ori = replication origin from the <span class="html-italic">E. coli</span> plasmid pBR322, lacZα = gene encoding α-peptide required for the blue-white screening.</p> Full article ">Figure 2
<p>Construction of the plasmid series pRS52, pRS53, pRS54, pRS55, and pRS56 (replicative plasmids, each carrying one yeast replication origin) with a detailed list of intermediate cloning steps and relevant restriction sites. 2µ ori, panARS, SsARS2, MgALS123, BbCEN2 = yeast replication origins, ScLEU2 = LEU2 gene from <span class="html-italic">Saccharomyces cerevisiae</span>, rop = gene encoding Rop protein in <span class="html-italic">E. coli</span>. Other labels are identical to those in <a href="#jof-08-00531-f001" class="html-fig">Figure 1</a>.</p> Full article ">Figure 3
<p>Schematic representation of integrative and replicative yeast plasmids constructed in this work and their names and main features. The pRS40 backbone carries sequences for selection and maintenance in <span class="html-italic">E. coli</span> (ori, bla) and other common vector features (<span class="html-italic">LacZ</span><span class="html-italic">α</span>, <span class="html-italic">f1 ori</span>), as well as TEF promoter (P<span class="html-italic"><sub>Eg</sub></span><sub>TEF</sub>) and terminator (T) from <span class="html-italic">Eremothecium gossypiii</span> for expression of antibiotic selectable markers in yeasts. Codon usage optimized markers conferring resistances to geneticin G418 (Kan<sup>R</sup>), hygromycin B (Hyg<sup>R</sup>), cloNAT (Nar<sup>R</sup>) or phleomycin (Phl<sup>R</sup>) were cloned under the regulation of the TEF promoter thus creating four different yeast integrative plasmids. Additionally, in each of these plasmids, the following origins of the replications were cloned: 2μ form <span class="html-italic">Saccharomycer cerevisiae</span>, panARS form <span class="html-italic">Kluyveromyces lactis</span>, <span class="html-italic">Ss</span>ARS2 from <span class="html-italic">Scheffersomyces stipitis</span>, <span class="html-italic">Mg</span>ALS123 form <span class="html-italic">Merozyma guilliermondii</span> or <span class="html-italic">Bb</span>CEN2 from <span class="html-italic">Brettanomyces bruxellensis</span>, thus creating a set of 20 replicative yeast plasmids.</p> Full article ">Figure 4
<p>Typical results of molecular analysis by Southern blotting of undigested (<b>A</b>) and digested (<b>B</b>) DNA isolated form transformants of <span class="html-italic">S. polymorphus var. africanus</span>.</p> Full article ">
15 pages, 2174 KiB  
Article
Sorbicillinoid Derivatives with the Radical Scavenging Activities from the Marine-Derived Fungus Acremonium chrysogenum C10
by Chengbao Duan, Shiyuan Wang, Ruiyun Huo, Erwei Li, Min Wang, Jinwei Ren, Yuanyuan Pan, Ling Liu and Gang Liu
J. Fungi 2022, 8(5), 530; https://doi.org/10.3390/jof8050530 - 20 May 2022
Cited by 8 | Viewed by 2295
Abstract
Sorbicillinoids are a class of structurally diverse hexaketide metabolites with good biological activities. To explore new structural sorbicillinoids and their bioactivities, the marine-derived fungus Acremonium chrysogenum C10 was studied. Three new sorbicillinoid derivatives, acresorbicillinols A–C (1–3), along with five [...] Read more.
Sorbicillinoids are a class of structurally diverse hexaketide metabolites with good biological activities. To explore new structural sorbicillinoids and their bioactivities, the marine-derived fungus Acremonium chrysogenum C10 was studied. Three new sorbicillinoid derivatives, acresorbicillinols A–C (1–3), along with five known ones, trichotetronine (4), trichodimerol (5), demethyltrichodimerol (6), trichopyrone (7) and oxosorbicillinol (8), were isolated. The structures of new sorbicillinoids were elucidated by analysis of nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy (HRESIMS). The absolute configurations of compounds 1–3 were determined by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra. Compound 3 exhibited a strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, with the IC50 value ranging from 11.53 ± 1.53 to 60.29 ± 6.28 μM in 24 h. Additionally, compounds 2 and 3 showed moderate activities against Staphylococcus aureus and Cryptococcus neoformans, with IC50 values of 86.93 ± 1.72 and 69.06 ± 10.50 μM, respectively. The boundary of sorbicillinoid biosynthetic gene cluster in A. chrysogenum was confirmed by transcriptional analysis, and the biosynthetic pathway of compounds 1–8 was also proposed. In summary, our results indicated that A. chrysogenum is an important reservoir of sorbicillinoid derivatives, and compound 3 has the potential for new natural agents in DPPH radical scavenging. Full article
(This article belongs to the Special Issue Secondary Metabolites from Fungi—in Honour of Prof. Dr. Ji-Kai Liu's 60th Birthday)
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<p>Structures of compounds <b>1</b>–<b>8</b>.</p> Full article ">Figure 2
<p>Key COSY and HMBC correlations of compounds <b>1</b>–<b>3</b>.</p> Full article ">Figure 3
<p>Key NOESY correlations of compounds <b>1</b>–<b>3</b>.</p> Full article ">Figure 4
<p>Calculated and experimental ECD spectra of compounds <b>1</b>–<b>3</b>.</p> Full article ">Figure 5
<p>DPPH radical scavenging activity of compound <b>3</b> and ascorbic acid as the positive control at 0.5, 1, 4, 6, 8 and 24 h.</p> Full article ">Figure 6
<p>(<b>A</b>) Organization of the sorbicillinoid biosynthetic gene cluster. FMO, FAD-dependent monooxygenase; PKS, polyketide synthase; TF, transcriptional factor; AM, auxiliary modifier; MFS, major facilitator superfamily transporter. (<b>B</b>) Transcriptional profiles of the <span class="html-italic">Acsor</span> genes during fermentation.</p> Full article ">Figure 7
<p>Proposed biosynthetic pathway of compounds <b>1</b>–<b>8</b>.</p> Full article ">
16 pages, 1508 KiB  
Article
The Search for Cryptic L-Rhamnosyltransferases on the Sporothrix schenckii Genome
by Héctor M. Mora-Montes, Karina García-Gutiérrez, Laura C. García-Carnero, Nancy E. Lozoya-Pérez and Jorge H. Ramirez-Prado
J. Fungi 2022, 8(5), 529; https://doi.org/10.3390/jof8050529 - 20 May 2022
Cited by 3 | Viewed by 2160
Abstract
The fungal cell wall is an attractive structure to look for new antifungal drug targets and for understanding the host-fungus interaction. Sporothrix schenckii is one of the main causative agents of both human and animal sporotrichosis and currently is the species most studied [...] Read more.
The fungal cell wall is an attractive structure to look for new antifungal drug targets and for understanding the host-fungus interaction. Sporothrix schenckii is one of the main causative agents of both human and animal sporotrichosis and currently is the species most studied of the Sporothrix genus. The cell wall of this organism has been previously analyzed, and rhamnoconjugates are signature molecules found on the surface of both mycelia and yeast-like cells. Similar to other reactions where sugars are covalently linked to other sugars, lipids, or proteins, the rhamnosylation process in this organism is expected to involve glycosyltransferases with the ability to transfer rhamnose from a sugar donor to the acceptor molecule, i.e., rhamnosyltransferases. However, no obvious rhamnosyltransferase has thus far been identified within the S. schenckii proteome or genome. Here, using a Hidden Markov Model profile strategy, we found within the S. schenckii genome five putative genes encoding for rhamnosyltransferases. Expression analyses indicated that only two of them, named RHT1 and RHT2, were significantly expressed in yeast-like cells and during interaction with the host. These two genes were heterologously expressed in Escherichia coli, and the purified recombinant proteins showed rhamnosyltransferase activity, dependent on the presence of UDP-rhamnose as a sugar donor. To the best of our knowledge, this is the first report about rhamnosyltransferases in S. schenckii. Full article
(This article belongs to the Special Issue Sporothrix and Sporotrichosis 2.0)
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<p>Analysis of gene expression. Cells were grown in YPD, BHI, Voguel, or PDB medium at 37 °C, and total RNA was extracted; cDNA was synthesized with oligo(dT) primer (20 mer) and quantified by RT-qPCR. Alternatively, <span class="html-italic">Galleria mellonella</span> larvae were inoculated with 1 × 10<sup>6</sup> yeast-like cells, and, after 72 h of interaction at 37 °C, fungal cells were retrieved and used for total RNA extraction. Expression levels were normalized using the gene encoding for the ribosomal protein L6 as the control and the fungal growth in YPD at 28 °C as reference conditions. Results are means ± SD of three independent experiments performed by duplicate. * <span class="html-italic">p</span> &lt; 0.05 when compared to the expression levels of the same gene in the other growing conditions.</p> Full article ">Figure 2
<p>Electrophoretic profile of recombinant Rht1 and Rht2 expressed in <span class="html-italic">Escherichia coli</span>. Bacteria were transformed with pCold-RHT1 (<b>A</b>), pCold-RHT2 (<b>B</b>), or empty pCold I (<b>C</b>) and grown under non-inducive conditions (lanes 1) or in inducing conditions (lanes 2, 0.1 M isopropyl-β-D-1-thiogalactopyranoside and 20 h at 20 °C). Protein samples were prepared from cultured cells and separated by denaturing SDS-PAGE. The recombinant proteins rRht1 (<b>D</b>) and rRht2 (<b>E</b>) were then subjected to purification by affinity chromatography using a cobalt-charged resin. Panel <b>A</b> corresponds to an electrophoretic analysis in 12% acrylamide gel, while gels of 10% acrylamide were used in panels (<b>B</b>–<b>E</b>).</p> Full article ">Figure 3
<p>Enzyme activity of recombinant Rht1. Reaction mixtures containing the purified enzyme (100 µg protein), 200 ng α-1,6-mannobiose, and 500 µM UDP-L-rhamnose were incubated at 37 °C. After the indicated times, samples were heated in boiling water and analyzed by HPAEC as described in the text. Panels (<b>A</b>,<b>C</b>,<b>E</b>) correspond to the incubation times of 0 min, 30 min, and 3 h, respectively. The enzyme product was treated with 1 U α-L-rhamnosidase for 60 min, and the monosaccharides released were analyzed by HPAEC. Panels (<b>B</b>,<b>D</b>,<b>F</b>) correspond to monosaccharides released from enzyme products obtained at the incubation times of 0 min, 30 min, and 3 h, respectively.</p> Full article ">Figure 4
<p>In silico structural analysis of <span class="html-italic">Sporothrix schenckii</span> Rht1 and Rht2 proteins. Alphafold2 predicted models for Rht1 (<b>A</b>) and Rht2 (<b>B</b>) and rendered on PyMol. Secondary structures are color-coded as follows: alpha helixes red, beta sheets yellow, coils green. Alignments (atoms superimposed) of the predicted 3D model to <span class="html-italic">Arabidopsis thaliana</span> rhamnosyltransferase UGT89C1: Rht1/UGT89C1 panel (<b>C</b>) and Rht2/UGT89C1 panel (<b>D</b>). Rht1 (<b>C</b>) and Rht2 (<b>D</b>) are colored according to secondary structure, while UGT89C1 is shown in cyan.</p> Full article ">
5 pages, 214 KiB  
Editorial
Special Issue “Signal Transductions in Fungi”
by Ulrich Kück
J. Fungi 2022, 8(5), 528; https://doi.org/10.3390/jof8050528 - 20 May 2022
Cited by 1 | Viewed by 1442
Abstract
In all living organisms, extracellular signals are translated into specific responses through signal transduction processes [...] Full article
(This article belongs to the Special Issue Signal Transductions in Fungi)
17 pages, 11872 KiB  
Article
Secretome Profiling by Proteogenomic Analysis Shows Species-Specific, Temperature-Dependent, and Putative Virulence Proteins of Pythium insidiosum
by Theerapong Krajaejun, Thidarat Rujirawat, Tassanee Lohnoo, Wanta Yingyong, Pattarana Sae-Chew, Onrapak Reamtong, Weerayuth Kittichotirat and Preecha Patumcharoenpol
J. Fungi 2022, 8(5), 527; https://doi.org/10.3390/jof8050527 - 20 May 2022
Cited by 3 | Viewed by 2155
Abstract
In contrast to most pathogenic oomycetes, which infect plants, Pythium insidiosum infects both humans and animals, causing a difficult-to-treat condition called pythiosis. Most patients undergo surgical removal of an affected organ, and advanced cases could be fetal. As a successful human/animal pathogen, P. [...] Read more.
In contrast to most pathogenic oomycetes, which infect plants, Pythium insidiosum infects both humans and animals, causing a difficult-to-treat condition called pythiosis. Most patients undergo surgical removal of an affected organ, and advanced cases could be fetal. As a successful human/animal pathogen, P. insidiosum must tolerate body temperature and develop some strategies to survive and cause pathology within hosts. One of the general pathogen strategies is virulence factor secretion. Here, we used proteogenomic analysis to profile and validate the secretome of P. insidiosum, in which its genome contains 14,962 predicted proteins. Shotgun LC–MS/MS analysis of P. insidiosum proteins prepared from liquid cultures incubated at 25 and 37 °C mapped 2980 genome-predicted proteins, 9.4% of which had a predicted signal peptide. P. insidiosum might employ an alternative secretory pathway, as 90.6% of the validated secretory/extracellular proteins lacked the signal peptide. A comparison of 20 oomycete genomes showed 69 P. insidiosum–specific secretory/extracellular proteins, and these may be responsible for the host-specific infection. The differential expression analysis revealed 14 markedly upregulated proteins (particularly cyclophilin and elicitin) at body temperature which could contribute to pathogen fitness and thermotolerance. Our search through a microbial virulence database matched 518 secretory/extracellular proteins, such as urease and chaperones (including heat shock proteins), that might play roles in P. insidiosum virulence. In conclusion, the identification of the secretome promoted a better understanding of P. insidiosum biology and pathogenesis. Cyclophilin, elicitin, chaperone, and urease are top-listed secreted/extracellular proteins with putative pathogenicity properties. Such advances could lead to developing measures for the efficient detection and treatment of pythiosis. Full article
(This article belongs to the Special Issue Novel, Emerging and Neglected Fungal Pathogens for Humans and Animals)
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<p>Venn diagram of the validated secretory/extracellular (<span class="html-italic">n</span> = 2980), cytosolic/intracellular (<span class="html-italic">n</span> = 4445), and SignalP-positive (<span class="html-italic">n</span> = 1208) proteins of <span class="html-italic">P. insidiosum</span>. CFA (culture filtrate antigens) and SABH (soluble antigens from broken hyphae) sets represent secretory/extracellular and cytosolic/intracellular proteins in origins, respectively. The SignalP set includes proteins that contain a signal peptide predicted by the SignalP software.</p> Full article ">Figure 2
<p>Classification of the <span class="html-italic">P. insidiosum</span> secretory/extracellular proteins based on the Clusters of Orthologous Groups of Proteins (COG) database. All 2980 validated secretory/extracellular proteins of <span class="html-italic">P. insidiosum</span> are allocated into 4 primary COG groups: (<b>i</b>) information storage and processing (<span class="html-italic">n</span> = 292; 9.8%; consisting of 5 subgroups); (<b>ii</b>) cellular processes and signaling (<span class="html-italic">n</span> = 450; 15.1%; 10 subgroups); (<b>iii</b>) metabolism (<span class="html-italic">n</span> = 424; 14.2%; 8 subgroups); and (<b>iv</b>) poorly characterized function (<span class="html-italic">n</span> = 1814; 60.9%; 2 subgroups). The box shows all 25 COG-defined functional subgroups.</p> Full article ">Figure 3
<p>Effect of the temperatures (25 vs. 37 °C) on the growth and the secretory/extracellular protein expression of <span class="html-italic">P. insidiosum</span>. The organism incubated at a different temperature shows (<b>A</b>) radial growth curve, (<b>B</b>) colony density, and (<b>C</b>) secretory/extracellular protein profile (demonstrated by SDS-PAGE analysis; molecular weight marker range, 17–180 kilodaltons) during the 7-day course. The microscopic features (branching hyphae) of the pathogen grown at either temperature condition were recorded by using an ECLIPSE Ci light microscope (Nikon, Tokyo, Japan; 200× magnification; B).</p> Full article ">Figure 4
<p>Functional domains present in the body temperature-upregulated proteins of <span class="html-italic">P. insidiosum</span>. The Web CD-search tool (<a href="https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi" target="_blank">https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi</a>; accessed on 1 March 2022) is used to detect the protein domain. Elicitin-like (<b>A</b>,<b>B</b>) and hypothetical (<b>C</b>,<b>D</b>,<b>F</b>,<b>G</b>) proteins contain 1 or 2 elicitin domains (green boxes). The hypothetical protein PINS01530002A lacks a defined functional domain (<b>E</b>). One hypothetical protein (PINS01630040A) harbors elicitin and kgd (orange box) domains (<b>G</b>). The blue boxes show protein IDs (as detailed in <a href="#jof-08-00527-t001" class="html-table">Table 1</a>). The black straight line demonstrates the relative protein length with numbers indicating the amino acid positions in each protein.</p> Full article ">Figure 5
<p>Proposed pathogenicity model of <span class="html-italic">P. insidiosum</span>. The pathogen secretes an array of virulence proteins to survive stress conditions and establish an infection inside the host. For example, on the left-hand side, a set of diverse elicitins released from <span class="html-italic">P. insidiosum</span> hyphae upon exposure to body temperature (37 °C) compete with the host in acquiring exogenous sterols (a major cell membrane component unable to synthesize by the pathogen) for microbial growth and fitness. On the right-hand side, the enzyme urease presented inside and outside the pathogen hydrolyzes urea (an amino acid breakdown product generated throughout the host body) to obtain carbamic acid (CH<sub>3</sub>NO<sub>2</sub>), carbonic acid (H<sub>2</sub>CO<sub>3</sub>), and ammonia (NH<sub>3</sub>). Ammonia could mediate several pathogenic features in the host, such as cell/tissue damage, increased local pH, Type-2 immunity polarization, impaired immune function (i.e., phagocytic activity), and disseminated infection.</p> Full article ">
15 pages, 3211 KiB  
Article
Sclerotinia sclerotiorum SsCut1 Modulates Virulence and Cutinase Activity
by Yingdi Gong, Yanping Fu, Jiatao Xie, Bo Li, Tao Chen, Yang Lin, Weidong Chen, Daohong Jiang and Jiasen Cheng
J. Fungi 2022, 8(5), 526; https://doi.org/10.3390/jof8050526 - 20 May 2022
Cited by 10 | Viewed by 2604
Abstract
The plant cuticle is one of the protective layers of the external surface of plant tissues. Plants use the cuticle layer to reduce water loss and resist pathogen infection. Fungi release cell wall-degrading enzymes to destroy the epidermis of plants to achieve the [...] Read more.
The plant cuticle is one of the protective layers of the external surface of plant tissues. Plants use the cuticle layer to reduce water loss and resist pathogen infection. Fungi release cell wall-degrading enzymes to destroy the epidermis of plants to achieve the purpose of infection. Sclerotinia sclerotiorum secretes a large amount of cutinase to disrupt the cuticle layer of plants during the infection process. In order to further understand the role of cutinase in the pathogenic process of S. sclerotiorum, the S. sclerotiorum cutinsae 1 (SsCut1) gene was cloned and analyzed. The protein SsCut1 contains the conserved cutinase domain and a fungal cellulose-binding domain. RT-qPCR results showed that the expression of SsCut1 was significantly upregulated during infection. Split-Marker recombination was utilized for the deletion of the SsCut1 gene, ΔSsCut1 mutants showed reduced cutinase activity and virulence, but the deletion of the SsCut1 gene had no effect on the growth rate, colony morphology, oxalic acid production, infection cushion formation and sclerotial development. Complementation with the wild-type SsCut1 allele restored the cutinase activity and virulence to the wild-type level. Interestingly, expression of SsCut1 in plants can trigger defense responses, but it also enhanced plant susceptibility to SsCut1 gene knock-out mutants. Taken together, our finding demonstrated that the SsCut1 gene promotes the virulence of S. sclerotiorum by enhancing its cutinase activity. Full article
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<p>Analysis of the cutinase protein SsCut1. (<b>A</b>) The evolutionary relationship of SsCut1 and its homologs from other fungi determined with the maximum-likelihood algorithm. Branch lengths are proportional to the average probability of change for characters on that branch. The phylogeny was constructed with Mega 6.0 using the neighbor-joining method (parameters: 1000 bootstraps). (<b>B</b>) The amino acid sequence alignment of SsCut1, SsCut, BcCutA and BcCutB. The black stars indicate the conserved GYSQG catalytic site. The rectangle box indicates the conserved CBM1 domain.</p> Full article ">Figure 2
<p>Expression patterns of <span class="html-italic">SsCut1</span> of <span class="html-italic">S. sclerotiorum</span> at different stages of <span class="html-italic">S. sclerotiorum</span>. (<b>A</b>) Expression patterns of the <span class="html-italic">SsCut1</span> in culture on PDA medium at 20 °C for 1–7 d (days). (<b>B</b>) Expression patterns of the <span class="html-italic">SsCut1</span> during the infection of <span class="html-italic">A. thaliana</span> at 20 °C for 0–24 h (hours). The <span class="html-italic">S. sclerotiorum β-tubulin</span> gene was used as an internal control to normalize the data. Error bars represent the standard error (<span class="html-italic">n</span> = 3).</p> Full article ">Figure 3
<p>SsCut1 has a functional signal peptide and is localized in the plant cell wall. (<b>A</b>) Validation of the secretion function of the SsCut1 signal peptide by the yeast secretion trap screen assay. Signal peptide of SsCut1 was fused in frame to the yeast invertase sequence in pSUC2 vector and expressed in YTK12 strains. The functional signal peptide of Avr1b was used as a positive control, while the YTK12 and pSUC2 empty plasmid was used as a negative control. (<b>B</b>) The invertase activity in TTC medium. TTC encounters raffinose breakdown products to produce triphenylformazan, which shows a red reaction to confirm that a functional signal peptide can cause sucrose invertase to be secreted. (<b>C</b>) Subcellular localization of SsCut1 in <span class="html-italic">N. benthamiana</span> epidermal cells. SsCut1-GFP localized in the plant cell wall. The fluorescence of GFP was monitored at 2 d post-agroinfiltration using confocal laser scanning microscopy. Bar = 20 µm.</p> Full article ">Figure 4
<p>The deletion of <span class="html-italic">SsCut1</span> has no significant effect on oxalate production, hypha morphology and infection cushions formation. (<b>A</b>) Qualitative determination of acid produced by the wild-type strain and <span class="html-italic">SsCut1</span> transformants on PDA medium containing 0.005% (<span class="html-italic">w</span>/<span class="html-italic">v</span>) bromophenol blue dye as a pH indicator. The presence of yellow indicates that acid was produced. Photographs were taken at 36 hpi. (<b>B</b>) In vitro hyphal development of the wild-type strain and <span class="html-italic">SsCut1</span> transformants. All strains were cultured on PDA medium for 36 hpi. Hyphal tips were observed under a dissecting microscope. Bars = 500 µm. (<b>C</b>) Infection cushions formation of wild-type strain and <span class="html-italic">SsCut1</span> transformants. Microscopic observation of infection cushions of wild-type strain and <span class="html-italic">SsCut1</span> transformants on onion epidermal cell layer after staining with trypan blue. Photographs were taken at 14 hpi. Bar = 20 µm.</p> Full article ">Figure 5
<p><span class="html-italic">SsCut1</span> knock-out mutants showing reduced virulence on the detached leaves of oilseed rape and <span class="html-italic">Arabidopsis</span> leaves. (<b>A</b>) Lesions formation on oilseed rape leaves inoculated with wild-type strain (1980) and <span class="html-italic">SsCut1</span> transformants, the photographs were taken at 48 h post-inoculation (hpi). (<b>B</b>) Statistical results of lesion area on oilseed rape leaves. (<b>C</b>) Lesions formation on <span class="html-italic">A. thaliana</span> leaves inoculated with wild-type strain and <span class="html-italic">SsCut1</span> transformants, the photographs were taken at 36 hpi. (<b>D</b>) Statistical results of lesion area on <span class="html-italic">A. thaliana</span> leaves. Bars indicate ± SE (<span class="html-italic">n</span> = 4). Statistical significance is indicated in the graph (one-way ANOVA): ** <span class="html-italic">p</span> &lt; 0.01, **** <span class="html-italic">p</span> &lt; 0.0001. The experiments were performed three times with similar results.</p> Full article ">Figure 6
<p>Cutinase activity of wild-type strain and <span class="html-italic">SsCut1</span> transformants. All strains were cultured on PDA medium. The hyphal of wild-type strain and <span class="html-italic">SsCut1</span> transformants was collected at 36 hpi. Cutinase activity levels were examined with the enzyme-linked immunosorbent assay (ELISA) method. Bars indicate ± SE. Statistical significance is indicated in the graph (one-way ANOVA): ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 7
<p>SsCut1 triggers plant defense responses and plays a role in the Sclerotinia–plant interaction. (<b>A</b>) SsCut1 promote flg22-triggered reactive oxygen species burst. The <span class="html-italic">N. benthamiana</span> with SsCut1 and empty vector were treated with 100 µg/mL flg22. Bars indicate ± SE. Error bars represent the SE from ten biological replicates. (<b>B</b>) Induction of defense response genes by SsCut1. SsCut1 induces <span class="html-italic">NbPR1</span> expression in <span class="html-italic">N. benthamiana.</span> Relative transcript accumulation of <span class="html-italic">NbPR1</span>, <span class="html-italic">NbNPR1</span>, <span class="html-italic">NbPDF1.2</span> genes determined by RT-qPCR. The transcript level of <span class="html-italic">NbEF1α</span> in <span class="html-italic">N. benthamiana</span> was used to normalize the expression levels in different samples. Error bars represent the SE from three replicates. (<b>C</b>) Expression of SsCut1 in <span class="html-italic">N. benthamiana</span> increases plant susceptibility to the <span class="html-italic">SsCut1</span> knock-out mutant Δ<span class="html-italic">SsCut1-3</span>. Leaves of <span class="html-italic">N. benthamiana</span> were agroinfiltrated with <span class="html-italic">Agrobacterium tumefaciens</span> containing empty vector or pCNF-SsCut1. The wild-type strain and <span class="html-italic">SsCut1</span> transformants were inoculated 48 h after agroinfiltration. Photographs were taken at 48 hpi. (<b>D</b>) Statistical results of lesion area on <span class="html-italic">N. benthamiana</span> leaves. In this experiment, four independent replicates were performed. Bars indicate ± SE. Statistical significance is indicated in the graph (one-way ANOVA): *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">
13 pages, 1733 KiB  
Article
Synergistic In Vitro Interaction of Isavuconazole and Isoquercitrin against Candida glabrata
by Petra V. Schwarz, Ilya Nikolskiy, Eric Dannaoui, Frank Sommer, Gert Bange and Patrick Schwarz
J. Fungi 2022, 8(5), 525; https://doi.org/10.3390/jof8050525 - 20 May 2022
Viewed by 1842
Abstract
In vitro interactions of broad-spectrum azole isavuconazole with flavonoid isoquercitrin were evaluated by a broth microdilution checkerboard technique based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference methodology for antifungal susceptibility testing against 60 Candida strains belonging to the species Candida [...] Read more.
In vitro interactions of broad-spectrum azole isavuconazole with flavonoid isoquercitrin were evaluated by a broth microdilution checkerboard technique based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference methodology for antifungal susceptibility testing against 60 Candida strains belonging to the species Candida albicans (n = 10), Candida glabrata (n = 30), Candida kefyr (n = 6), Candida krusei (n = 5), Candida parapsilosis (n = 4), and Candida tropicalis (n = 5). The results were analyzed with the fractional inhibitory concentration index and by response surface analysis based on the Bliss model. Synergy was found for all C. glabrata strains, when the results were interpreted by the fractional inhibitory concentration index, and for 60% of the strains when response surface analysis was used. Interaction for all other species was indifferent for all strains tested, whatever interpretation model used. Importantly, antagonistic interaction was never observed. Full article
(This article belongs to the Special Issue Antifungal Combinations in Fungal Infections)
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<p>Synergy distribution for the combination of isavuconazole with isoquercitrin against all <span class="html-italic">C. glabrata</span> strains tested. Always from the left to the right, first row: V2105272, V2105282, N2101711, V2105636, DSM 70614; second row: U2105834, V2105576, N2102530, U2106503, U2106602; third row: U2106664, U2106745, U2107113, U2107210, U2107214; fourth row: V2107409, N2102703, N2102712, N2102714, U2107517; fifth row: U2107630, U2107836, V2108007, V2108459, B2109750; last row: A2100553, U2107634, U2107796, U2108032, U2107634. The mode of interaction was defined based on the SUM-SYN-ANT metric. IND, indifference; SYN, synergy.</p> Full article ">
12 pages, 1522 KiB  
Article
Ustilago maydis Metabolic Characterization and Growth Quantification with a Genome-Scale Metabolic Model
by Ulf W. Liebal, Lena Ullmann, Christian Lieven, Philipp Kohl, Daniel Wibberg, Thiemo Zambanini and Lars M. Blank
J. Fungi 2022, 8(5), 524; https://doi.org/10.3390/jof8050524 - 20 May 2022
Cited by 7 | Viewed by 2793
Abstract
Ustilago maydis is an important plant pathogen that causes corn smut disease and serves as an effective biotechnological production host. The lack of a comprehensive metabolic overview hinders a full understanding of the organism’s environmental adaptation and a full use of its metabolic [...] Read more.
Ustilago maydis is an important plant pathogen that causes corn smut disease and serves as an effective biotechnological production host. The lack of a comprehensive metabolic overview hinders a full understanding of the organism’s environmental adaptation and a full use of its metabolic potential. Here, we report the first genome-scale metabolic model (GSMM) of Ustilago maydis (iUma22) for the simulation of metabolic activities. iUma22 was reconstructed from sequencing and annotation using PathwayTools, and the biomass equation was derived from literature values and from the codon composition. The final model contains over 25% annotated genes (6909) in the sequenced genome. Substrate utilization was corrected by BIOLOG phenotype arrays, and exponential batch cultivations were used to test growth predictions. The growth data revealed a decrease in glucose uptake rate with rising glucose concentration. A pangenome of four different U. maydis strains highlighted missing metabolic pathways in iUma22. The new model allows for studies of metabolic adaptations to different environmental niches as well as for biotechnological applications. Full article
(This article belongs to the Special Issue Smut Fungi 2.0)
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<p>Memote quality report of iUma22 with total score of 57%. The full HTML report is provided as <a href="#app1-jof-08-00524" class="html-app">Supplement 3</a>.</p> Full article ">Figure 2
<p>BIOLOG phenotype experiments with carbon sources from PM1 and PM2A. Growth was evaluated by OD 600 after 144 h for PM1 (<b>A</b>) and 288 h for PM2A (<b>B</b>) with a threshold of 0.4 a.u (black line with triangle), which excludes the normal distribution at low ODs representing no growth. Fifty-two substrates were correctly predicted to growth (true positive, green), and 128 were correctly assigned to nongrowth by iUma22 (true negative, yellow) in plates PM1 (<b>C</b>) and PM2A (<b>D</b>). Twelve substrates could not be balanced to enable growth in iUma22 (false negative). Results of PM1 and PM2A and an overview of the substrates on the plates are provided as <a href="#app1-jof-08-00524" class="html-app">Supplementary Materials</a>.</p> Full article ">Figure 3
<p>Growth characteristics of <span class="html-italic">U. maydis</span> glucose batch cultures from <a href="#jof-08-00524-t003" class="html-table">Table 3</a> and similarity to iUma22 predictions. (<b>A</b>) Seven batch experiments on glucose were analyzed to extract growth- and glucose-uptake rates. The linear least-squares correlation provides the biomass yield on glucose with 0.47 +/− 0.03 g<sub>CDW</sub>/g<sub>glc</sub>, and the interception of the x-axis provides the glucose maintenance uptake rate with 0.2 +/− 0.01 mmol/g<sub>CDW</sub>/h. The two inlet figures exemplify the growth rate estimation by a logistic Verhulst equation for growth (green) and linear substrate uptake (blue) for experiment ID ‘50glc’. (<b>B</b>) Glucose-uptake rate as a function of the initial glucose level indicating an inverse correlation. (<b>C</b>) Simulated and experimental growth rates, with optimal predictions represented by the black line. The individual growth rate data is provided in the <a href="#app1-jof-08-00524" class="html-app">Supplements</a>.</p> Full article ">Figure 4
<p>Comparison of enzymes in <span class="html-italic">U. maydis</span> strain pangenome and iUma22. (<b>A</b>) E.C.-annotated genes in strain 512 that are unique to 512 or shared with the other strains. (<b>B</b>) Coverage of the genes in iUma22 of E.C.-annotated genes in the pangenome identified by KAAS. <a href="#jof-08-00524-t004" class="html-table">Table 4</a> show the top five pathways with the most association for iUma22-unique, pangenome-unique, and their intersection. (<b>C</b>) The inositol phosphate metabolism contains 20 genes with the highest level of missing genes in iUma22 (<a href="#app1-jof-08-00524" class="html-app">Supplements</a>).</p> Full article ">
18 pages, 2124 KiB  
Article
Elevated Ozone Concentration and Nitrogen Addition Increase Poplar Rust Severity by Shifting the Phyllosphere Microbial Community
by Siqi Tao, Yunxia Zhang, Chengming Tian, Sébastien Duplessis and Naili Zhang
J. Fungi 2022, 8(5), 523; https://doi.org/10.3390/jof8050523 - 18 May 2022
Cited by 8 | Viewed by 2540
Abstract
Tropospheric ozone and nitrogen deposition are two major environmental pollutants. A great deal of research has focused on the negative impacts of elevated O3 and the complementary effect of soil N addition on the physiological properties of trees. However, it has been [...] Read more.
Tropospheric ozone and nitrogen deposition are two major environmental pollutants. A great deal of research has focused on the negative impacts of elevated O3 and the complementary effect of soil N addition on the physiological properties of trees. However, it has been overlooked how elevated O3 and N addition affect tree immunity in face of pathogen infection, as well as of the important roles of phyllosphere microbiome community in host–pathogen–environment interplay. Here, we examined the effects of elevated O3 and soil N addition on poplar leaf rust [Melampsora larici-populina] severity of two susceptible hybrid poplars [clone ‘107’: Populus euramericana cv. ‘74/76’; clone ‘546’: P. deltoides Í P. cathayana] in Free-Air-Controlled-Environment plots, in addition, the link between Mlp-susceptibility and changes in microbial community was determined using Miseq amplicon sequencing. Rust severity of clone ‘107’ significantly increased under elevated O3 or N addition only; however, the negative impact of elevated O3 could be significantly mitigated when accompanied by N addition, likewise, this trade-off was reflected in its phyllosphere microbial α-diversity responding to elevated O3 and N addition. However, rust severity of clone ‘546’ did not differ significantly in the cases of elevated O3 and N addition. Mlp infection altered microbial community composition and increased its sensitivity to elevated O3, as determined by the markedly different abundance of taxa. Elevated O3 and N addition reduced the complexity of microbial community, which may explain the increased severity of poplar rust. These findings suggest that poplars require a changing phyllosphere microbial associations to optimize plant immunity in response to environmental changes. Full article
(This article belongs to the Topic Infectious Diseases)
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<p>Rust severity of poplar foliar rust in four ambient ozone concentration FACE plots (A-O<sub>3</sub>) and four elevated ozone concentration FACE plots (E-O<sub>3</sub>) under two N treatments (N0 = no addition of nitrogen, N60 = addition of 60 kg/ha nitrogen every month). The significant differences between treatments at the 0.05 probability were indicated as the asterisk (*) according to the two-tailed Wilcoxon test; ns: not significant.</p> Full article ">Figure 2
<p>Taxonomic structures of phyllosphere bacterial microbiota at the class level (<b>a</b>) and fungal microbiota at the genus level (<b>b</b>). Only the 12 families with the largest mean relative abundance are shown.</p> Full article ">Figure 3
<p>Shannon indices of phyllosphere communities of non-infected leaves and <span class="html-italic">Mlp</span>-infected leaves of the clone ‘107’ (<b>a</b>,<b>b</b>) and the clone ‘546’ clone (<b>c</b>,<b>d</b>) from ambient ozone concentration plots (A-O<sub>3</sub>) and elevated ozone concentration plots (E-O<sub>3</sub>) with nitrogen addition (N60) and without nitrogen addition (N0). Box plots showed the range of estimated values between 25% and 75%, the median, the minimum, and the maximum observed values within each dataset.</p> Full article ">Figure 4
<p>PCoA of bacterial and fungal communities using the weighted_unifrac distance for bacteria (<b>a</b>) and unweighted unifrac distance for fungi (<b>b</b>). Samples are sorted for ozone concentration (A-O<sub>3</sub> vs. E-O<sub>3</sub>) and nitrogen treatment (N0 vs. N60).</p> Full article ">Figure 5
<p>The Mental tests and Spearman’s correlation coefficients between α-diversity indices of <span class="html-italic">Melampsora larici-populina</span>-infected poplar phyllosphere fungal and bacterial communities with nitrogen addition, ozone concentration (Ozone), leaf dry weight (DW), leaf nitrogen and carbon content, and rust severity.</p> Full article ">Figure 6
<p>The co-occurrence networks (<b>a</b>) and trends of microbial network association indices (<b>b</b>) of phyllosphere microbiome for two hybrid poplars (‘107’ and ‘546’) in five conditions. No (<b>a</b>) biotic stresses (control), N addition (N), elevated O<sub>3</sub> (O<sub>3</sub>), N addition with elevated O<sub>3</sub> (N + O<sub>3</sub>), N addition, elevated O<sub>3</sub> with <span class="html-italic">Melampsora-larici populina</span> infection (N + O<sub>3</sub> + <span class="html-italic">Mlp</span>).</p> Full article ">
21 pages, 2999 KiB  
Article
Heme Oxygenase-1 (HMX1) Loss of Function Increases the In-Host Fitness of the Saccharomyces ‘boulardii’ Probiotic Yeast in a Mouse Fungemia Model
by Alexandra Imre, Renátó Kovács, Zoltán Tóth, László Majoros, Zsigmond Benkő, Walter P. Pfliegler and István Pócsi
J. Fungi 2022, 8(5), 522; https://doi.org/10.3390/jof8050522 - 18 May 2022
Viewed by 3253
Abstract
The use of yeast-containing probiotics is on the rise; however, these products occasionally cause fungal infections and possibly even fungemia among susceptible probiotic-treated patients. The incidence of such cases is probably underestimated, which is why it is important to delve deeper into the [...] Read more.
The use of yeast-containing probiotics is on the rise; however, these products occasionally cause fungal infections and possibly even fungemia among susceptible probiotic-treated patients. The incidence of such cases is probably underestimated, which is why it is important to delve deeper into the pathomechanism and the adaptive features of S. ‘boulardii’. Here in this study, the potential role of the gene heme oxygenase-1 (HMX1) in probiotic yeast bloodstream-derived infections was studied by generating marker-free HMX1 deletion mutants with CRISPR/Cas9 technology from both commercial and clinical S. ‘boulardii’ isolates. The six commercial and clinical yeasts used here represented closely related but different genetic backgrounds as revealed by comparative genomic analysis. We compared the wild-type isolates against deletion mutants for their tolerance of iron starvation, hemolytic activity, as well as kidney burden in immunosuppressed BALB/c mice after lateral tail vein injection. Our results reveal that the lack of HMX1 in S. ‘boulardii’ significantly (p < 0.0001) increases the kidney burden of the mice in most genetic backgrounds, while at the same time causes decreased growth in iron-deprived media in vitro. These findings indicate that even a single-gene loss-of-function mutation can, surprisingly, cause elevated fitness in the host during an opportunistic systemic infection. Our findings indicate that the safety assessment of S. ‘boulardii’ strains should not only take strain-to-strain variation into account, but also avoid extrapolating in vitro results to in vivo virulence factor determination. Full article
(This article belongs to the Topic Infectious Diseases)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Schematic depiction of free heme and hemoglobin-heme acquisition and utilization in <span class="html-italic">C. albicans</span> and <span class="html-italic">S. cerevisiae</span>: (<b>a</b>) <span class="html-italic">C. albicans</span> have heme-binding, GPI anchored CFEM cell surface proteins, with which uptake of external heme is possible through endocytosis. CaHmx1 is able to break down heme; however, several steps of heme-iron utilization have not yet been fully understood (indicated by question marks); (<b>b</b>) <span class="html-italic">S. cerevisiae</span> does not have heme-binding cell surface molecules, and thus it is unable to utilize external free heme or hemoglobin-heme. In this yeast, Hmx1 is an ER-bound enzyme that utilizes intracellular heme as an iron source.</p> Full article ">Figure 2
<p>Genomic comparison of the yeast isolates used for the deletion of <span class="html-italic">HMX1</span>. (<b>a</b>) Heterozygous variant density (per 10 kb) across chromosomes for the isolate PY0001 (top) and changes in allele ratios, i.e., gains and losses of heterozygous regions in the other five isolates (bottom). (<b>b</b>) Gene copy number variations in the six sequenced isolates. (<b>c</b>) The distribution of protein-coding genes affected by high- and medium-effect mutations. The total number of affected genes for combinations of isolates are shown at the bottom, each genome is represented by a circle colored according to isolate type (commercial, non-mycosis, mycosis).</p> Full article ">Figure 3
<p>Relative yeast growth under iron starvation: (<b>a</b>) bars show the mean results for the wild type <span class="html-italic">S. ‘boulardii’</span> isolate triplicates (full grey bars) and for the <span class="html-italic">ΔΔHMX1</span> mutant strain triplicates (striped bars) (for the second spot with 5000 plated cells). Individual data points are shown in blue (40 µM BPS), orange (80 µM BPS), and red (120 µM BPS), and whiskers show the standard deviation of the data. Comparisons with significant differences are marked (*: <span class="html-italic">p</span> &lt; 0.05; **: <span class="html-italic">p</span> &lt; 0.01; ***: <span class="html-italic">p</span> &lt; 0.001; ****: <span class="html-italic">p</span> &lt; 0.0001). (<b>b</b>) Photos of iron deprivation spot plate assay. Columns represent the wild type isolate vs. <span class="html-italic">ΔΔHMX1</span> mutant strain pairs by BPS concentration. Raw photographs, including control plates without BPS, are uploaded to FigShare.</p> Full article ">Figure 4
<p>Comparison of growth curves of the isolates and <span class="html-italic">HMX1</span> deletion strains in medium of 50% RPMI-1640 and 50% human serum. Individual data points for wild-type strains are indicated with black, while data for the knockout mutants are indicated with red. Data points of each replicate are connected by a line. Comparisons with significant differences are marked (*: <span class="html-italic">p</span> &lt; 0.05; **: <span class="html-italic">p</span> &lt; 0.01; ***: <span class="html-italic">p</span> &lt; 0.001; ****: <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 5
<p>Hemolytic index. Bars show the mean results for the wild type <span class="html-italic">S. ‘boulardii’</span> isolates (full grey) and for the <span class="html-italic">ΔΔHMX1</span> mutant strains (striped). Individual data points are shown in green (α-hemolysis) and black (β-hemolysis), and whiskers show the standard deviation of the data. Comparisons with significant differences are marked (*: <span class="html-italic">p</span> &lt; 0.05; **: <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 6
<p>Kidney burden of mice injected with yeast probiotic isolates and <span class="html-italic">HMX1</span> deletion mutant strains. Individual data points represent the colony forming units per kidney weight (g) for every mouse. Data from mice that died during or were killed at the end of the experiment are both plotted. Horizontal black lines show the median of the datapoints within a dataset. Comparisons with significant differences are marked (*: <span class="html-italic">p</span> &lt; 0.05; **: <span class="html-italic">p</span> &lt; 0.01; ***: <span class="html-italic">p</span> &lt; 0.001; ****: <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">
21 pages, 7872 KiB  
Article
Encephalartos villosus Lem. Displays a Strong In Vivo and In Vitro Antifungal Potential against Candida glabrata Clinical Isolates
by Moneerah J. Alqahtani, Engy Elekhnawy, Walaa A. Negm, Sebaey Mahgoub and Ismail A. Hussein
J. Fungi 2022, 8(5), 521; https://doi.org/10.3390/jof8050521 - 18 May 2022
Cited by 15 | Viewed by 2358
Abstract
Recently, Candida glabrata has been recognized as one of the most common fungal species that is highly associated with invasive candidiasis. Its spread could be attributed to its increasing resistance to antifungal drugs. Thus, there is a high need for safer and more [...] Read more.
Recently, Candida glabrata has been recognized as one of the most common fungal species that is highly associated with invasive candidiasis. Its spread could be attributed to its increasing resistance to antifungal drugs. Thus, there is a high need for safer and more efficient therapeutic alternatives such as plant extracts. Here, we investigated the antifungal potential of Encephalartos villosus leaves methanol extract (EVME) against C. glabrata clinical isolates. Tentative phytochemical identification of 51 metabolites was conducted in EVME using LC–MS/MS. EVME demonstrated antifungal activity with minimum inhibitory concentrations that ranged from 32 to 256 µg/mL. The mechanism of the antifungal action was studied by investigating the impact of EVME on nucleotide leakage. Additionally, a sorbitol bioassay was performed, and we found that EVME affected the fungal cell wall. In addition, the effect of EVME was elucidated on the efflux activity of C. glabrata isolates using acridine orange assay and quantitative real-time PCR. EVME resulted in downregulation of the expression of the efflux pump genes CDR1, CDR2, and ERG11 in the tested isolates with percentages of 33.33%, 41.67%, and 33.33%, respectively. Moreover, we investigated the in vivo antifungal activity of EVME using a murine model with systemic infection. The fungal burden was determined in the kidney tissues. Histological and immunohistochemical studies were carried out to investigate the effect of EVME. We noticed that EVME reduced the congestion of the glomeruli and tubules of the kidney tissues of the rats infected with C. glabrata. Furthermore, it decreased both the proinflammatory cytokine tumor necrosis factor-alpha and the abnormal collagen fibers. Our results reveal, for the first time, the potential in vitro (by inhibition of the efflux activity) and in vivo (by decreasing the congestion and inflammation of the kidney tissues) antifungal activity of EVME against C. glabrata isolates. Full article
(This article belongs to the Section Fungal Pathogenesis and Disease Control)
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<p>The total ion chromatogram of EVME presented the major identified metabolites (D-(−)-quinic acid, luteolin-7-<span class="html-italic">O</span>-glucoside, apigenin-7-<span class="html-italic">O</span>-glucoside, naringenin, apigenin, and hesperetin according to the retention time) via LC–ESI–MS/MS in negative ion mode.</p> Full article ">Figure 2
<p>Time–kill curve of representative <span class="html-italic">C. glabrata</span> isolates showing a decrease in the count of CFU/mL by three log units after treatment with EVME with concentrations of (<b>A</b>) 1× minimum inhibitory concentration (MIC) for two hours and (<b>B</b>) 4× MIC for one hour.</p> Full article ">Figure 3
<p>A representative example of the significant decrease (<span class="html-italic">p</span> &lt; 0.05) in the membrane integrity by EVME by increasing the leakage of nucleic acids.</p> Full article ">Figure 4
<p>Minimum inhibitory concentration (MIC) values of EVME of six <span class="html-italic">C. glabrata</span> isolates showed a substantial increase (<span class="html-italic">p</span> &lt; 0.05) in the presence of sorbitol.</p> Full article ">Figure 5
<p>Downregulation of the gene expression of (<b>A</b>) <span class="html-italic">CDR1</span>, (<b>B</b>) <span class="html-italic">CDR2</span>, and (<b>C</b>) <span class="html-italic">ERG11</span> after treatment with EVME in <span class="html-italic">C. glabrata</span> isolates C2, C5, C6, C8, C10.</p> Full article ">Figure 6
<p>The number of colony-forming units per gram (CFU/g) of fungal cells in kidneys of the three groups. The symbol (*) represents a substantial decrease (<span class="html-italic">p</span> &lt; 0.05). There was a non-significant difference between group II and group III.</p> Full article ">Figure 7
<p>Survival curve of rats of the different groups via Kaplan–Meier survival analysis.</p> Full article ">Figure 8
<p>H&amp;E-stained kidney sections of (<b>A</b>) normal kidney tissues (negative control) showing average-sized glomeruli (red arrows) surrounded by average-sized tubules lined with columnar cells (blue arrows) (×100); (<b>B</b>) group I showing vascular congestion (red arrow) surrounded by atrophic glomeruli (blue arrows) and focal degenerated tubules (green arrow) (×100); (<b>C</b>) group II showing focal vascular congestion (red arrow) surrounded by normal-sized glomeruli (blue arrows) and some atrophic glomeruli (green arrows) surrounded by average-sized tubules (×100); (<b>D</b>) group III shows average-sized glomeruli (blue arrows) and one atrophic glomerulus (green arrow) surrounded by average-sized tubules (×100).</p> Full article ">Figure 9
<p>Masson’s trichrome staining of the kidney sections of (<b>A</b>) normal kidney tissues (negative control) showing slight amounts of blue-stained collagen fibers in glomeruli (blue arrows) (×100); (<b>B</b>) group I showing a marked increase in the abnormal collagen deposition in the walls of tubules (blue arrows) (×100); (<b>C</b>) group II showing vascular congestion with focal blue-stained collagen fibers deposition around the vessels (blue arrows), no collagen deposition in the tubules (×100); (<b>D</b>) group III showing a marked reduction in the abnormal collagen fibers deposition and slight amounts in the glomeruli (blue arrows), with no collagen deposition in the tubules or the blood vessels (×100).</p> Full article ">Figure 10
<p>Tumor necrosis factor-alpha (TNF-α) immunohistochemical staining of the kidney sections of (<b>A</b>) normal kidney tissues (negative control) showing negative TNF-α staining, with score (0) (×100); (<b>B</b>) group I showing strong TNF-α staining with score (3) (×100); (<b>C</b>) group II showing moderate TNF-α staining with score (2) (×100); (<b>D</b>) group III showing weak TNF-α staining with score (1) (×100).</p> Full article ">
20 pages, 12051 KiB  
Article
Taxonomy, Phylogenetic and Ancestral Area Reconstruction in Phyllachora, with Four Novel Species from Northwestern China
by Jin-Chen Li, Hai-Xia Wu, Yuying Li, Xin-Hao Li, Jia-Yu Song, Nakarin Suwannarach and Nalin N. Wijayawardene
J. Fungi 2022, 8(5), 520; https://doi.org/10.3390/jof8050520 - 18 May 2022
Cited by 4 | Viewed by 2942
Abstract
The members of Phyllachora are biotrophic, obligate plant parasitic fungi featuring a high degree of host specificity. This genus also features a high degree of species richness and worldwide distribution. In this study, four species occurring on leaf and stem of two different [...] Read more.
The members of Phyllachora are biotrophic, obligate plant parasitic fungi featuring a high degree of host specificity. This genus also features a high degree of species richness and worldwide distribution. In this study, four species occurring on leaf and stem of two different species of grass were collected from Shanxi and Shaanxi Provinces, China. Based on morphological analysis, multigene (combined data set of LSU, SSU, and ITS) phylogenetic analyses (maximum likelihood and Bayesian analysis), and host relationship, we introduce herein four new taxa of Phyllachora. Ancestral area reconstruction analysis showed that the ancestral area of Phyllachora occurred in Latin America about 194 Mya. Novel taxa are compared with the related Phyllachora species. Detailed descriptions, illustrations, and notes are provided for each species. Full article
(This article belongs to the Special Issue Women in Mycology)
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Figure 1
<p>Phylogenetic tree of maximum likelihood showing the relationships of Phyllachoraceae based on combined LSU, SSU, and ITS data set analysis. Bootstrap values of maximum likelihood higher than 50% are shown on the left, while values of Bayesian posterior probabilities above 0.5 are shown on the right. New species are given in bold, followed by the host of the species behind its strain number.</p> Full article ">Figure 2
<p>Ancestral character state reconstruction based on the Bayesian tree. Each event is represented with a number at the nodes. Bayesian posterior probabilities are presented (≥0.5). The colored circle near the number at the nodes indicate that blue represents Dispersal, green represents Vicariance, orange represents Extinction. New species are given in bold.</p> Full article ">Figure 3
<p><span class="html-italic">Phyllachora flaccidudis</span> (IFRD9445, holotype). (<b>a</b>) Black spots on <span class="html-italic">Cenchrus flaccidus</span> (Poaceae); (<b>b,c</b>) Stromata; (<b>d</b>) Vertical section of ascomata in cotton blue; (<b>e</b>) Paraphyses; (<b>f</b>–<b>i</b>) Asci; (<b>j</b>–<b>m</b>) Ascospores. Scale bars, (<b>b</b>) 1 mm; (<b>c</b>)0.5 mm; (<b>d</b>) 200 μm; (<b>e</b>) 50 μm; (<b>f</b>–<b>i</b>) 10 μm; (<b>j</b>–<b>m</b>) 5 μm. Microscopic techniques: DIC.</p> Full article ">Figure 4
<p>(<b>a</b>) <span class="html-italic">P. flaccidudis</span> ascospore in DIW (Deionized Water); (<b>b</b>,<b>c</b>) <span class="html-italic">P. flaccidudis</span> ascospores with gelatinous sheath in ink; (<b>e</b>) <span class="html-italic">P. sandiensis</span> ascospore in DIW; (<b>f</b>–<b>h</b>) <span class="html-italic">P. sandiensis</span> ascospores with gelatinous sheath in ink; (<b>i</b>) <span class="html-italic">P. virgatae</span> ascospore in DIW; (<b>j</b>,<b>k</b>) <span class="html-italic">P. virgatae</span> ascospores with gelatinous sheath in ink; (<b>m</b>) <span class="html-italic">P. jiaensis</span> ascospore in DIW; (<b>n</b>–<b>p</b>) <span class="html-italic">P. jiaensis</span> with gelatinous sheath in ink. Scale bars, (<b>a</b>–<b>p</b>) 5 μm. Microscopic techniques: DIC.</p> Full article ">Figure 5
<p><span class="html-italic">Phyllachora sandiensis</span> (IFRD9446, holotype). (<b>a</b>) Black spots on <span class="html-italic">Cenchrus flaccidus</span> (Poaceae); (<b>b</b>,<b>c</b>) Stromata; (<b>d</b>) Vertical section of ascomata; (<b>e</b>) Paraphyses; (<b>f</b>) Ascus in cotton blue; (<b>g</b>–<b>i</b>) Asci; (<b>j</b>) Ascospore in cotton blue; (<b>k</b>–<b>m</b>) Ascospores. Scale bars, (<b>c</b>) 0.5 mm; (<b>d</b>) 100 μm; (<b>e</b>) 50 μm; (<b>f</b>–<b>i</b>) 10 μm; (<b>j</b>–<b>m</b>) 5 μm. Microscopic techniques: DIC.</p> Full article ">Figure 6
<p><span class="html-italic">Phyllachora virgatae</span> (IFRD9447, holotype). (<b>a</b>) Black spots on <span class="html-italic">Chloris virgata</span> (Poaceae); (<b>b</b>,<b>c</b>) Stromata; (<b>d</b>) Vertical section of ascomata; (<b>e</b>) Paraphyses; (<b>f</b>) Ascus in cotton blue; (<b>g</b>–<b>i</b>) Asci; (<b>j</b>–<b>m</b>) Ascospores. Scale bars, (<b>b</b>) 1 mm, (<b>c</b>) 0.5 mm; (<b>d</b>) 100 μm; (<b>e</b>) 20 μm; (<b>f</b>–<b>i</b>) 10 μm; (<b>j</b>–<b>m</b>) 5 μm. Microscopic techniques: DIC.</p> Full article ">Figure 7
<p><span class="html-italic">Phyllachora jiaensis</span> (IFRD9448, holotype). (<b>a</b>) Black spots on <span class="html-italic">Chloris virgata</span> (Poaceae); (<b>b</b>,<b>c</b>) Stromata; (<b>d</b>) Vertical section of ascomata; (<b>e</b>) Paraphyses; (<b>f</b>) Ascus in cotton blue; (<b>g</b>–<b>i</b>) Asci; (<b>j</b>–<b>m</b>) Ascospores. Scale bars, (<b>c</b>) 0.5 mm; (<b>d</b>) 100 μm; (<b>e</b>) 20 μm; (<b>f</b>–<b>i</b>) 10 μm; (<b>j</b>–<b>m</b>) 5 μm. Microscopic techniques: DIC.</p> Full article ">
17 pages, 1369 KiB  
Article
Metabolomics Analysis and Antioxidant Potential of Endophytic Diaporthe fraxini ED2 Grown in Different Culture Media
by Wen-Nee Tan, Kashvintha Nagarajan, Vuanghao Lim, Juzaili Azizi, Kooi-Yeong Khaw, Woei-Yenn Tong, Chean-Ring Leong and Nelson Jeng-Yeou Chear
J. Fungi 2022, 8(5), 519; https://doi.org/10.3390/jof8050519 - 18 May 2022
Cited by 12 | Viewed by 2490
Abstract
Endophytic fungi are a promising source of bioactive metabolites with a wide range of pharmacological activities. In the present study, MS-based metabolomics was conducted to study the metabolomes variations of endophytic Diaporthe fraxini ED2 grown in different culture media. Total phenolic content (TPC), [...] Read more.
Endophytic fungi are a promising source of bioactive metabolites with a wide range of pharmacological activities. In the present study, MS-based metabolomics was conducted to study the metabolomes variations of endophytic Diaporthe fraxini ED2 grown in different culture media. Total phenolic content (TPC), total flavonoid content (TFC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and ferric reducing antioxidant power (FRAP) assays were conducted to assess the antioxidant potential of the fungal extracts. Multivariate data analysis (MVDA) was employed in data analysis and interpretation to elucidate the complex metabolite profile. The supplemented culture medium of D. fraxini fungal extract stimulated the production of metabolites not occurring in the normal culture medium. Antioxidant activity studies revealed the potential of supplemented cultured fungal extract of D. fraxini as a source of antioxidants. The present findings highlight that fungal culture medium supplementation is an effective approach to unravelling the hidden metabolome in plant-associated fungal diversity. Full article
(This article belongs to the Special Issue Fungi: What Have We Learned from Omics?)
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<p>Effects of culture medium supplementation on TPC and TFC in DFC and DFS. Values given are means ± SD, with <span class="html-italic">n</span> = 6. * Significantly different from DFC.</p> Full article ">Figure 2
<p>PCA pairwise score plot in unsupervised analysis of DFC and DFS. Different PCs illustrate variability in the spatial distribution of the sample groups.</p> Full article ">Figure 3
<p>PCA scores plot in unsupervised analysis. PC1 versus PC2 showing the discrimination of DFC and DFS growing in different culture media.</p> Full article ">Figure 4
<p>PCA loading plot in unsupervised analysis obtained from DFC and DFS. Shown data are the Var ID (peak number).</p> Full article ">Figure 5
<p>Heatmap overview showing the discriminations of DFC and DFS. The colour scale was set to default ranging from red (high) to blue (low).</p> Full article ">Figure 6
<p>HCA plot showed as dendrogram in unsupervised analysis for DFC and DFS corresponding to the PCA model.</p> Full article ">Figure 7
<p>PLS-DA scores plot in supervised analysis. Predictive components 1 versus 2 showing the supervised separation between the two sample groups (DFC and DFS) based upon the culture supplementation.</p> Full article ">Figure 8
<p>PLS-DA loading plot in supervised analysis obtained from DFC and DFS. Shown data are the Var ID (peak number).</p> Full article ">Figure 9
<p>VIP score plot of the most important discriminant metabolites by PLS-DA for DFC and DFS. The relative abundance of each important metabolite is indicated with a colour code scaled from blue (low) to red (high). A high VIP score indicates a high impact of the metabolite as a discriminant feature among the sample groups.</p> Full article ">Figure 10
<p>Chemical structures of discriminant putatively identified metabolites in DFC and DFS.</p> Full article ">
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Article
Stemphylium lycopersici Nep1-like Protein (NLP) Is a Key Virulence Factor in Tomato Gray Leaf Spot Disease
by Jiajie Lian, Hongyu Han, Xizhan Chen, Qian Chen, Jiuhai Zhao and Chuanyou Li
J. Fungi 2022, 8(5), 518; https://doi.org/10.3390/jof8050518 - 18 May 2022
Cited by 6 | Viewed by 2598
Abstract
The fungus Stemphylium lycopersici (S. lycopersici) is an economically important plant pathogen that causes grey leaf spot disease in tomato. However, functional genomic studies in S. lycopersici are lacking, and the factors influencing its pathogenicity remain largely unknown. Here, we present [...] Read more.
The fungus Stemphylium lycopersici (S. lycopersici) is an economically important plant pathogen that causes grey leaf spot disease in tomato. However, functional genomic studies in S. lycopersici are lacking, and the factors influencing its pathogenicity remain largely unknown. Here, we present the first example of genetic transformation and targeted gene replacement in S. lycopersici. We functionally analyzed the NLP gene, which encodes a necrosis- and ethylene-inducing peptide 1 (Nep1)-like protein (NLP). We found that targeted disruption of the NLP gene in S. lycopersici significantly compromised its virulence on tomato. Moreover, our data suggest that NLP affects S. lycopersici conidiospore production and weakly affects its adaptation to osmotic and oxidative stress. Interestingly, we found that NLP suppressed the production of reactive oxygen species (ROS) in tomato leaves during S. lycopersici infection. Further, expressing the fungal NLP in tomato resulted in constitutive transcription of immune-responsive genes and inhibited plant growth. Through gene manipulation, we demonstrated the function of NLP in S. lycopersici virulence and development. Our work provides a paradigm for functional genomics studies in a non-model fungal pathogen system. Full article
(This article belongs to the Special Issue Emergent Fungal Models for Genetics and Cell Biology)
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Figure 1

Figure 1
<p>Characterization of a type I Nep1-like protein (NLP) from <span class="html-italic">S. lycopersici</span>. (<b>A</b>) Schematic diagram of the NLP protein of gray leaf spot. The full sequence of the NLP protein of gray leaf spot was used to predict and analyze the conserved structural domains. SP denotes signal peptide. NPP1 denotes Necrosis-Inducing Phytophthora Protein 1 characteristic domain. nlp20 represents the conserved pattern of 20 amino acids. (<b>B</b>) Phylogenetic tree built using three types of NLP proteins with maximum likelihood method. Red, black, and blue represent the type I, type II, and type III NLPs, respectively. The black box highlights the NLP of <span class="html-italic">S. lycopersici</span>. (<b>C</b>) Expression of the <span class="html-italic">NLP</span> gene during pathogen infection. The expression of the <span class="html-italic">NLP</span> gene was measured at 6, 12, 24, and 48 h post-inoculation (hpi) on tomato leaves and in CM liquid medium (negative control). The <span class="html-italic">S. lycopersici ACTIN</span> gene was used as an internal reference.</p> Full article ">Figure 2
<p>NLP affects the adaptation of <span class="html-italic">S. lycopersici</span> to ionic and oxidative stress as well as its asexual reproduction. (<b>A</b>) Schematic diagram showing targeted replacement of <span class="html-italic">NLP</span> enhanced by CRISPR/Cas9. (<b>B</b>) Schematic diagram showing the <span class="html-italic">S. lycopersici NLP</span> gene overexpression construct. The mCherry-tagged <span class="html-italic">NLP</span> gene was driven by the promoter and first intron of <span class="html-italic">ACTIN</span>. (<b>C</b>) Effect of NLP on conidia production. Strains were inoculated on sporulation medium. All spores were collected and suspended in liquid medium. The conidia production was assessed by measuring spore concentration. Lowercase of a and b denotes significant difference among multiple groups (<span class="html-italic">p</span> &lt; 0.05) by Duncan’s new multiple range test. (<b>D</b>) Effect of NLP on the adaption of <span class="html-italic">S. lycopersici</span> to ionic stress and the cell wall disturbing agents sodium dodecyl sulfate (SDS, 0.005%) and Congo Red (CR, 300 μg/mL). Conidia of the wild-type (WT), ∆<span class="html-italic">nlp-1</span>, ∆<span class="html-italic">nlp-2</span>, <span class="html-italic">pACTIN:NLP-1</span>, and <span class="html-italic">pACTIN:NLP-2</span> strains were inoculated on CM medium containing NaCl (1 M) or KCl (1 M) and incubated at 25 °C for 8 days. CM medium without stress agents was used as the negative control. (<b>E</b>) Mycelial growth quantification of strains under ionic stress. (<b>F</b>) Effect of NLP on the adaption of <span class="html-italic">S. lycopersici</span> to oxidative stress. Fungal cakes (5 mm in diameter) of WT, ∆<span class="html-italic">nlp-1</span>, ∆<span class="html-italic">nlp-2</span>, <span class="html-italic">pACTIN:NLP-1</span>, and <span class="html-italic">pACTIN:NLP-2</span> strains were inoculated on CM plates containing H<sub>2</sub>O<sub>2</sub> (20 mM) and incubated at 25 °C for 8 days. CM medium without H<sub>2</sub>O<sub>2</sub> was used as the negative control. (<b>G</b>) Mycelial growth quantification of strains growing under oxidative stress. ** denotes very significant difference (<span class="html-italic">p</span> &lt; 0.01, Student’s <span class="html-italic">t</span> test); 8 denotes significant difference (<span class="html-italic">p</span> &lt; 0.05); ns denotes no significant differences.</p> Full article ">Figure 3
<p>NLP is a key virulence factor of <span class="html-italic">S. lycopersici</span> during infection on tomato leaves. (<b>A</b>) Infected tomato leaves of the WT, ∆<span class="html-italic">nlp</span>, and overexpression strains at 5 days post inoculation (dpi). (<b>B</b>) Lesion area of leaves of tomato cultivar M82 resulting from infection by WT, ∆<span class="html-italic">nlp</span>, and overexpression strains. (<b>C</b>) Fungal biomass of WT, ∆<span class="html-italic">nlp,</span> and overexpression strains on infected tomato leaves. The relative fungal growth was measured by RNA-based RT-qPCR using the threshold cycle value (<span class="html-italic">C<sub>T</sub></span>) of <span class="html-italic">S. lycopersici ACTIN</span> gene (locus_tag:TW65_02246) against the <span class="html-italic">C<sub>T</sub></span> of tomato <span class="html-italic">ACTIN2</span> gene (Solyc11g005330). Conidia of different strains were inoculated on the surface of tomato leaves, and mycelium biomass was measured at 5 days post inoculation (dpi). The assay was performed on intact plants, of which the infected leaves were detached for imaging. Lowercase of a, b, and c denotes significant difference among multiple groups (<span class="html-italic">p</span> &lt; 0.05) by Duncan’s new multiple range test.</p> Full article ">Figure 4
<p>NLP inhibits ROS production in tomato induced by <span class="html-italic">S. lycopersici</span> infection. (<b>A</b>,<b>B</b>) Relative expression levels of tomato <span class="html-italic">RbohA</span> (<b>A</b>) and <span class="html-italic">RbohB</span> (<b>B</b>) gene after infection by the WT, ∆<span class="html-italic">nlp</span>, and overexpression <span class="html-italic">S. lycopersici</span> strains. (<b>C</b>) DAB staining of tomato leaves showing H<sub>2</sub>O<sub>2</sub> production induced by the WT, ∆<span class="html-italic">nlp</span>, and overexpression <span class="html-italic">S. lycopersici</span> strains. (<b>D</b>) Quantification of DAB staining. (<b>E</b>) NBT staining of tomato leaves showing O<sub>2</sub><sup>−</sup> production induced by the WT, ∆<span class="html-italic">nlp</span>, and overexpression strains. (<b>F</b>) Quantification of NBT staining. The RGB values of images were converted into 16-bit grayscale, which were quantified by ImageJ with the grayscale statistics method. a, b, c, and d designate statistically significant differences determined using the DPS software. Lowercase of a, b, and c denotes significant difference among multiple groups (<span class="html-italic">p</span> &lt; 0.05) by Duncan’s new multiple range test. ns denotes no significant differences.</p> Full article ">Figure 5
<p>Expression of <span class="html-italic">S. lycopersici NLP</span> gene in tomato leads to constitutive expression of immunity genes and plant growth reduction. (<b>A</b>) RT-qPCR results showing the <span class="html-italic">NLP</span> gene expression in the leaves of <span class="html-italic">NLP</span>-overexpressing transgenic tomato lines. GFP-tagged NLP was driven by the 35S promoter (<span class="html-italic">p35S:NLP</span>). (<b>B</b>,<b>C</b>) Expression of the immune-responsive genes <span class="html-italic">PR-STH2</span> (<b>B</b>) and <span class="html-italic">ERF.C3</span> (<b>C</b>) in the <span class="html-italic">p35S:NLP</span> tomato lines. Tomato plants were incubated under sterile conditions for 2 weeks prior to RNA extraction and RT-qPCR analysis. (<b>D</b>,<b>E</b>) Fresh weight (<b>D</b>) and root length (<b>E</b>) of <span class="html-italic">p35S:NLP</span> tomato plants. Two-week-old tomato plants grown in growth container under sterile conditions were weighed (g per plant) and their root lengths were measured. (<b>F</b>–<b>H</b>) Growth phenotype (<b>F</b>), height (<b>G</b>), and fresh weight (<b>H</b>) of <span class="html-italic">p35S:NLP</span> tomato plants. Plants were grown in the greenhouse for 5 weeks. The scale bar indicates 10 cm. Lowercase of a and b denotes significant difference among multiple groups (<span class="html-italic">p</span> &lt; 0.05) by Duncan’s new multiple range test.</p> Full article ">
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