Stemphylium lycopersici Nep1-like Protein (NLP) Is a Key Virulence Factor in Tomato Gray Leaf Spot Disease
<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> "> 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> < 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> < 0.01, Student’s <span class="html-italic">t</span> test); 8 denotes significant difference (<span class="html-italic">p</span> < 0.05); ns denotes no significant differences.</p> "> 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> < 0.05) by Duncan’s new multiple range test.</p> "> 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> < 0.05) by Duncan’s new multiple range test. ns denotes no significant differences.</p> "> 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> < 0.05) by Duncan’s new multiple range test.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Materials and Growth Conditions
2.2. Fungal Strain Isolation and Growth Conditions
2.3. Gene Knockout and Overexpression in S. lycopersici
2.4. Genetic Transformation of S. lycopersici
2.5. Bioinformatics Analysis and Phylogenetic Assay
2.6. Tomato Disease Assay
2.7. DNA Constructs and Plant Transformation
2.8. ROS Staining
2.9. RNA Extraction and RT-qPCR
2.10. Quantification and Statistical Analysis
3. Results
3.1. A Stemphylium lycopersici Type I Nep1-like Protein (NLP) Is Induced during Infection of Tomato
3.2. NLP Affects Conidiospore Production but Not Vegetative Growth in S. lycopersici
3.3. NLP Weakly Affects Osmotic and Oxidative Stress Adaptation in S. lycopersici
3.4. NLP Is Required for Full Virulence of S. lycopersici in Tomato
3.5. NLP Suppresses S. lycopersici Infection-Induced ROS Production in Tomato Leaves
3.6. Expression of S. lycopersici NLP in Tomato Leads to Constitutive Plant Immune Responses and Reduced Growth
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Lian, J.; Han, H.; Chen, X.; Chen, Q.; Zhao, J.; Li, C. Stemphylium lycopersici Nep1-like Protein (NLP) Is a Key Virulence Factor in Tomato Gray Leaf Spot Disease. J. Fungi 2022, 8, 518. https://doi.org/10.3390/jof8050518
Lian J, Han H, Chen X, Chen Q, Zhao J, Li C. Stemphylium lycopersici Nep1-like Protein (NLP) Is a Key Virulence Factor in Tomato Gray Leaf Spot Disease. Journal of Fungi. 2022; 8(5):518. https://doi.org/10.3390/jof8050518
Chicago/Turabian StyleLian, Jiajie, Hongyu Han, Xizhan Chen, Qian Chen, Jiuhai Zhao, and Chuanyou Li. 2022. "Stemphylium lycopersici Nep1-like Protein (NLP) Is a Key Virulence Factor in Tomato Gray Leaf Spot Disease" Journal of Fungi 8, no. 5: 518. https://doi.org/10.3390/jof8050518