The Sporisorium reilianum Effector Vag2 Promotes Head Smut Disease via Suppression of Plant Defense Responses
"> Figure 1
<p>Map of divergence region 19-1 of <span class="html-italic">Sporisorium reilianum</span> including delineation of subcluster deletions and comparison to the gene cluster 19A of <span class="html-italic">Ustilago maydis</span>. Arrows indicate open reading frames. Genes predicted to encode secreted proteins (SignalP 6.0) are colored in red. Gene numbers from Genbank are indicated on top of each gene. Regions deleted in subcluster deletion strains are indicated by brackets. The name of the deletion is indicated below the bracket. Region 19-1 of <span class="html-italic">S. reilianum</span> (<span class="html-italic">Sr</span>, top) is compared to cluster 19A of <span class="html-italic">U. maydis</span> (<span class="html-italic">Um</span>, bottom [<a href="#B44-jof-08-00498" class="html-bibr">44</a>]). Genes identified as virulence genes in <span class="html-italic">U. maydis</span> are named, and the homology of these genes to <span class="html-italic">S. reilianum</span> is indicated by grey shading.</p> "> Figure 2
<p>Dissection of virulence genes within divergence region 19-1 of <span class="html-italic">S. reilianum</span>. Virulence phenotype of subcluster gene deletion strains of <span class="html-italic">S. reilianum</span> on <span class="html-italic">Zea mays</span> cv. ‘Gaspe Flint’. Comparison of disease incidence and severity indexes (<b>left</b>), disease incidence distribution (<b>middle</b>), and disease severity distribution (<b>right</b>) are shown. Disease incidence gives the number of plants with the strongest displayed symptom (N, total number of evaluated plants), whereas disease severity gives the number of inflorescences displaying the strongest symptom (N, total number of evaluated inflorescences). N is indicated above the bar graph columns. The disease index indicates the average weighted strength of the displayed disease symptoms per total number of plants (incidence) or per total number of inflorescences (severity) relative to the respective values induced by wildtype (WT) infections, that were set to 10. Data are listed in <a href="#app1-jof-08-00498" class="html-app">Tables S3 and S4</a>. Student’s <span class="html-italic">t</span>-test was used for statistical analysis. * <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01. (<b>B</b>–<b>D</b>) represent cumulative data of three independent biological replicates testing about 20 plants for each replicate. Error bars indicate SEM. Strains used are listed in <a href="#app1-jof-08-00498" class="html-app">Table S1</a>. Mock indicates that plants were inoculated with water. (<b>A</b>) Virulence of subcluster A3 gene deletion strains in comparison to subcluster A1 deletion strains; (<b>B</b>) Virulence of subcluster A4 and A5 gene deletion strains; (<b>C</b>) Virulence of subcluster A8 and A9 gene deletion strains; (<b>D</b>) Virulence of region A8 gene deletion strains. Strains are lacking all three genes of region A8 (ΔA8), only the first gene <span class="html-italic">sr10050/vag2</span> (Δ50), only the second gene <span class="html-italic">sr10051</span> (Δ51), or only the third gene <span class="html-italic">sr10052.2</span> (Δ52). ΔA8 complementation strains contain either the gene <span class="html-italic">sr10050/vag2</span> (ΔA8 + 50) or <span class="html-italic">sr10051</span> (ΔA8 + 51) in the ΔA8 region.</p> "> Figure 3
<p>Quantification of <span class="html-italic">vag2</span> mRNA and fungal DNA in inoculated maize seedlings. (<b>A</b>) Relative expression of <span class="html-italic">vag2</span> (<span class="html-italic">sr10050</span>) in <span class="html-italic">S. reilianum</span>-inoculated <span class="html-italic">Z. mays</span> cv. ‘Gaspe Flint’ tissues. Samples were harvested from a mating mixture of sporidia grown in liquid culture (0 dpi), from inoculated maize leaves at 2, 4, 6, and 9 dpi, or from ears of inoculated plants at 31 dpi. Total RNA isolated from collected samples was used for quantitative RT-PCR. The expression of <span class="html-italic">vag2</span> in all samples was normalized to expression values of the <span class="html-italic">S. reilianum</span> glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene (<span class="html-italic">sr10940.2</span>) and is represented relative to the expression value of axenic culture. Error bars represent the SEM of three biological replicates. Each biological replicate is a pool of at least 10 tissue samples of independent plants. (<b>B</b>) Proliferation density of <span class="html-italic">Δ</span><span class="html-italic">vag2</span> deletion strains (Δvag2) in leaves and nodes of colonized <span class="html-italic">Z. mays</span> cv. ‘Gaspe Flint’ relative to wildtype (WT) strains. Total DNA was isolated from 3-cm pieces of inoculated leaves at 3 dpi and of nodes at the base of inoculated leaves at 14 dpi. The fungal GAPDH and maize actin genes were used to quantify relative fungal proliferation by quantitative PCR. The ratio of the fungal proliferation of <span class="html-italic">Δ</span><span class="html-italic">vag2</span> deletion strains relative to wildtype strains was calculated as a measure of proliferation density. Error bars represent the SEM of three biological replicates. Each biological replicate is a pool of 10 samples from different plants. Data were analyzed with Student’s <span class="html-italic">t</span>-test (* <span class="html-italic">p</span> < 0.05).</p> "> Figure 4
<p>Vag2 contains a secretion signal peptide, but a Vag2-GFP Fusion is not functional. (<b>A</b>) Yeast secretion trap assay confirms the predicted secretion signal peptide of Vag2. <span class="html-italic">Saccharomyces cerevisiae</span> SEY6210 was used for transformation with the empty vector containing the <span class="html-italic">SUC2</span> gene and lacking the N-terminal sequences encoding the secretion signal peptide (pYST-1), or with the pYST-1 vector containing the Vag2 open reading frame in fusion to the <span class="html-italic">SUC2</span> gene of pYST-1 (pYST-vag2). Serial dilutions of different transformants were plated on SD minimal medium lacking leucine but containing glucose (SD-Leu + Glc) as growth control, and on SD minimal medium lacking leucine but containing sucrose (SD-Leu + Suc) as a test. Only strains containing <span class="html-italic">vag2</span> were able to use sucrose as a carbon source. (<b>B</b>) A Vag2-GFP fusion does not complement the lack of Vag2 during maize colonization by <span class="html-italic">S. reilianum</span>. Seedlings of <span class="html-italic">Z. mays</span> cv. ‘Gaspe Flint’ was used for inoculation with combinations of mating-compatible wild-type strains (WT), strains lacking <span class="html-italic">vag2</span> (Δvag2), or strains lacking <span class="html-italic">vag2</span> and containing a <span class="html-italic">vag2-GFP</span> fusion gene at the native locus of <span class="html-italic">vag2</span> (Δvag2+Vag2GFP). Three different combinations of strains were used (<a href="#app1-jof-08-00498" class="html-app">Table S1</a>). Comparison of disease incidence and severity indexes (<b>left</b>), disease incidence distribution (<b>middle</b>), and disease severity distribution (<b>right</b>) are shown, see legend in <a href="#jof-08-00498-f002" class="html-fig">Figure 2</a> for more explanations. (<b>C</b>) Fluorescence microscopic analysis of fungal hyphae expressing Vag2-GFP instead of Vag2 (Δvag2+Vag2GFP). Pictures represent merged bight field and GFP images of hyphae of <span class="html-italic">S. reilianum</span> colonizing leaves at 3 dpi (<b>top</b>) or ears at 31 dpi (<b>bottom</b>). Size bars: 10 µm.</p> "> Figure 5
<p>Vag2 interacts with ZmCM2. (<b>A</b>) Vag2 lacking its signal peptide was cloned in fusion with the GAL4 binding domain (BD-Vag2ΔSP) and introduced into the Y2HGold strain. One of the retrieved plasmids containing maize chorismate mutase-derived sequences in fusion to the GAL4 activation domain (AD-ZmCM2) was introduced into Y187. Both strains were mated, and diploids were selected on SD minimal media lacking tryptophan and leucine (-TL). Reconstitution of the GAL4 transcription factor by the interaction of Vag2ΔSP with ZmCM2 was verified by checking the expression of the GAL4-promoter controlled genes ADE2 and HIS3 as well as a gene allowing resistance to the drug aureobasidine A on SD medium lacking tryptophan, leucine, adenine and histidine, and containing aureobasidine A (-TLAH + Au). As a positive control, strains expressing tumor suppressor p53 (AD-P53) and the strong interaction partner SV40 T-antigen (BD-T), were used. As negative controls, strains were expressing Vag2 (BD-Vag2ΔSP) or Lamin (AD-Lam) and T-antigen (BD-T). (<b>B</b>) Bimolecular fluorescence complementation confirms in-planta interaction between Vag2 and ZmCM2. The N-terminal part of YFP was expressed alone (N-YFP) or as a fusion with ZmCM2 (N-YFP-ZmCM2), whereas the C-terminal part of YFP was expressed alone (C-YFP) or as a fusion with Vag2 lacking its signal peptide (C-YFP-Vag2ΔSP) after infiltration of <span class="html-italic">Nicotiana benthamiana</span> leaves with <span class="html-italic">Agrobacterium tumefaciens</span> cells delivering the indicated constructs. At 3 days after infiltration, the leaves were analyzed by fluorescence microscopy. YFP fluorescence was only seen when both fusion proteins were co-expressed. Shown are representative cells under conditions enabling detection of GFP fluorescence (<b>left</b>), and a merge of the GFP fluorescence and brightfield pictures (<b>right</b>). Size bars: 50 µm.</p> "> Figure 6
<p>SA and SA-induced defense genes are upregulated in maize colonized by <span class="html-italic">S. reilianum</span> lacking <span class="html-italic">vag2</span>. (<b>A</b>,<b>B</b>) SA levels were quantified in maize tissues inoculated with <span class="html-italic">S. reilianum</span> wildtype (WT) and <span class="html-italic">Δ</span><span class="html-italic">vag2</span> deletion strains (Δvag2), or with water (Mock). Samples were collected from maize leaves below the inoculation site at 6 dpi (<b>A</b>), or of maize ears collected at 31 dpi (<b>B</b>). (<b>C</b>–<b>F</b>) Relative expression of the SA-induced defense genes <span class="html-italic">PR1</span> (AC205274.3_FG001) and <span class="html-italic">PR5</span> (GRMZM2G402631) was measured by real-time PCR. Samples were collected from inoculated leaves at 6 dpi (<b>C</b>,<b>E</b>) and from ears at 31 dpi (<b>D</b>,<b>F</b>) and used for extraction of total RNA. RNA samples were subjected to qRT-PCR with <span class="html-italic">PR1</span> and <span class="html-italic">PR5</span> gene-specific primers (<a href="#app1-jof-08-00498" class="html-app">Table S2</a>). Expression levels of <span class="html-italic">PR1</span> (<b>C</b>,<b>D</b>) and <span class="html-italic">PR5</span> (<b>E</b>,<b>F</b>) were normalized to the maize actin reference gene (GRMZM2G126010), and expression of WT was set to 1. For each biological replicate, leaves or ears of 10 different plants were collected. Error bars indicate SEM of three biological replicates, and asterisks indicate a significant difference (Student’s <span class="html-italic">t-</span>test, * <span class="html-italic">p</span> < 0.05) of mutant relative to wildtype-inoculations.</p> "> Figure 7
<p>Model of the possible function of Vag2. (<b>A</b>) Visualization of metabolite quantification based on GC-EI/TOF-MS analysis of total metabolome of control-inoculated maize seedlings (Mock) or maize seedlings inoculated with <span class="html-italic">S. reilianum</span> wildtype strains (WT) or strains lacking <span class="html-italic">vag2</span> (Δ50). Sections of inoculated leaves were collected 3 days post-inoculation and consisted of the colonized part (D) below the inoculation marks and the non-colonized part of the leaf above the inoculation wounds (U). Data show averages of seven to eight individual leaf samples. Error bars indicate standard error. The values depicted on the Y-axis are arbitrary abundance units used to determine metabolite amounts relative to a spike of <sup>13</sup>C<sub>6</sub>-sorbitol. The <span class="html-italic">cis</span>- and <span class="html-italic">trans</span>- forms of 4-hydroxy-cinnamic acid and of caffeic acid were measured individually and summed before the calculation of error bars. Statistical analysis indicated no significant differences between wildtype- and <span class="html-italic">Δvag2</span>-inoculated samples (Student’s <span class="html-italic">t</span>-test, <span class="html-italic">p</span> < 0.05). (<b>B</b>) Schematic depiction of the two main pathways of SA generation in plants and how Vag2 might influence metabolite flow in modulating the activity of the maize chorismate mutase ZmCM2. The main route of SA generation in plant-microbe interactions may be from shikimate via chorismate and isochorismate (pink arrows). The second possibility is from phenylalanine via cinnamic acid and benzoic acid (green arrows). The chorismate mutase (CM) catalyzes the reversible interconversion of chorismate and prephenate, a precursor of tyrosine, tyramine, and phenylalanine. Phenylalanine is a substrate of the phenylalanine-ammonia-lyase (PAL), the key enzyme for SA generation via the second route. Metabolite analysis depicted in (<b>A</b>) indicates that when maize seedlings were infected with <span class="html-italic">S. reilianum</span> wild type, the levels of tyrosine, tyramine, phenylalanine, and shikimate were elevated relative to that of control plants and relative to plants inoculated with <span class="html-italic">vag2</span> deletion strains, whereas the levels of benzoate, 4-hydroxy-cinnamate and caffeate were slightly lower. SA levels were shown to be increased in <span class="html-italic">Δvag2</span>-inoculated samples relative to wildtype-inoculated samples (<a href="#jof-08-00498-f006" class="html-fig">Figure 6</a>A,B). Vag2 interaction with ZmCM2 might make the ZmCM2-catalysed reaction unidirectional (red arrow), which would result in the redirection of the metabolite flow into the generation of tyrosine, tyramine, and phenylalanine, thus lowering the SA levels by lowering the SA precursor concentration.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains and Growth Conditions of S. reilianum and Maize, Symptom Scoring
2.2. Generation of S. reilianum Gene Deletion and Complementation Strains
2.3. Genomic DNA and RNA Isolation, and qRT-PCR Analysis
2.4. Bimolecular Fluorescence Complementation and Fluorescence Microscopy
2.5. Yeast Two-Hybrid Screening
2.6. Yeast Secretion Trap Assay
2.7. Metabolite Analysis
3. Results
3.1. Diversity Region 19-1 of S. reilianum Contains the Virulence Gene vag2
3.2. S. reilianum vag2 Is Transcriptionally Upregulated during Fungal Biotrophic Growth
3.3. vag2 Deletion Mutants Show Reduced Systemic Spread in Maize
3.4. Vag2 Has a Functional Secretion Peptide
3.5. Interaction Partners of Vag2 Are Involved in Various Plant Processes
3.6. Vag2 Interacts with the Maize Chorismate Mutase 2 (ZmCM2)
3.7. S. reilianum Δvag2 Deletion Strains Slightly Increase the SA Level in Colonized Tissue and Induce SA-Related Defense Gene Expression in Maize
3.8. Metabolite Flux Is Redirected from SA Generation to Aromatic Amino Acid Accumulation
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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Interaction Partner | Frequency | Gene ID | Description |
---|---|---|---|
Metabolism | |||
IP1 | 24 | GRMZM2G179454 | Zea mays chorismate mutase (ZmCm2) |
IP2 | 11 | GRMZM2G027663 | Putative ThiC superfamily protein |
IP3 | 10 | GRMZM2G006329 | Zea mays Enzyme: pleckstrin homology (PH) domain-containing protein |
IP4 | 6 | GRMZM2G135588 | Putative citrate synthase family protein |
IP5 | 4 | GRMZM2G081585 | Chloroplastic iron-superoxide dismutase (sodB) |
IP6 | 3 | AC208571.4_FG001 | Hypothetical protein containing a haloacid dehalogenase-like hydrolase family domain / NHL repeat domain |
IP7 | 3 | GRMZM2G300862 | Aspartate kinase |
IP8 | 3 | GRMZM2G014788 | Unknown protein containing Carboxypeptidase regulatory-like domain |
IP9 | 2 | GRMZM2G135588 | Aspartate kinase homoserine dehydrogenase 2 (akh2) |
IP10 | 2 | GRMZM2G049538 | Terpene synthase1 |
IP11 | 1 | GRMZM2G151934 | Zea mays protein DA1-related 2-like |
IP12 | 1 | GRMZM2G043198 | Pyruvate dehydrogenase 2 (pdh2) |
IP13 | 1 | GRMZM2G121612 | Starch synthase |
IP14 | 1 | GRMZM2G118806 | Uncharacterized protein with proteolysis and peptidase activity |
IP15 | 1 | GRMZM2G088689 | 2-Oxoisovalerate dehydrogenase (acylating) |
IP16 | 1 | GRMZM2G448142 | Putative NADH-quinone oxidoreductase subunit K |
Transcription/DNA binding | |||
IP17 | 7 | GRMZM2G100246 | Unknown protein containing NOT2,3,5 domain |
IP18 | 4 | GRMZM2G440943 | Helicase/SANT-associated, DNA binding protein |
IP19 | 2 | GRMZM2G351304 | Uncharacterized protein containing chromosome segregation protein SMC domain |
IP20 | 2 | AC225308.2_FG005 | Putative homeodomain-like transcription factor superfamily protein |
IP21 | 1 | GRMZM2G133016 | MYB DNA-binding domain superfamily protein |
IP22 | 1 | GRMZM5G876621 | Zea mays putative RING zinc finger domain superfamily protein |
IP23 | 1 | GRMZM2G377369 | Uncharacterized protein containing DNA binding site |
IP24 | 1 | AC226373.2 | Zink finger C-x8-C-x5-C-x3-H type family protein |
IP25 | 1 | GRMZM2G340749 | General negative regulator of transcription |
Protein processes | |||
IP26 | 5 | GRMZM2G027282 | Proteasome 26S subunit 6A (RPT5a) |
IP27 | 3 | GRMZM2G168119 | Putative HSP20-like chaperone domain family protein |
IP28 | 2 | GRMZM2G134980 | Putative dnaJ chaperone family protein |
IP29 | 2 | GRMZM2G006781 | Conserved oligomeric Golgi complex subunit 8 |
IP30 | 1 | GRMZM2G551402 | Unknown protein containing a ubiquitin carboxyl-terminal hydrolase domain |
IP31 | 1 | GRMZM2G012631 | HSP protein (HSP90-2) |
IP32 | 1 | GRMZM2G137495 | DnaJ domain or J-domain. DnaJ/Hsp40 (heat shock protein 40) |
IP33 | 1 | GRMZM2G162968 | Chaperone protein ClpB2 |
IP34 | 1 | GRMZM2G154312 | Co-chaperone protein SBA1 |
Signaling | |||
IP35 | 4 | GRMZM2G038982 | Uncharacterized protein containing a STKc_MAP3K-like domain |
IP36 | 3 | GRMZM2G126946 | Zea mays putative calcium-dependent lipid-binding (CaLB domain) family protein |
IP37 | 1 | GRMZM2G152877 | Uncharacterized protein containing F-box-like and Cupin-like domain domain |
IP38 | 1 | GRMZM2G326472 | Uncharacterized protein containing a STKc_MAP3K-like domain |
Nuclear processes | |||
IP39 | 2 | GRMZM2G159028 | RNA binding protein |
IP40 | 1 | GRMZM2G111014 | Unknown protein containing DNA gyrase subunit |
IP41 | 1 | GRMZM2G588223 | Hypothetical protein ZEAMMB73 containing double-stranded RNA binding motif |
IP42 | 1 | GRMZM2G030128 | DNA repair-recombination protein (rad50) |
IP43 | 1 | AC205703.4_FG010 | Hypothetical protein ZEAMMB73_142911/ATPase involved in DNA replication, recombination, and repair |
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Zhao, Y.; Agrawal, N.; Ghareeb, H.; Habib, M.T.; Dickmeis, S.; Schwachtje, J.; Iven, T.E.; Kopka, J.; Feussner, I.; Schirawski, J. The Sporisorium reilianum Effector Vag2 Promotes Head Smut Disease via Suppression of Plant Defense Responses. J. Fungi 2022, 8, 498. https://doi.org/10.3390/jof8050498
Zhao Y, Agrawal N, Ghareeb H, Habib MT, Dickmeis S, Schwachtje J, Iven TE, Kopka J, Feussner I, Schirawski J. The Sporisorium reilianum Effector Vag2 Promotes Head Smut Disease via Suppression of Plant Defense Responses. Journal of Fungi. 2022; 8(5):498. https://doi.org/10.3390/jof8050498
Chicago/Turabian StyleZhao, Yulei, Nisha Agrawal, Hassan Ghareeb, Mohammad Tanbir Habib, Sascha Dickmeis, Jens Schwachtje, Tim E. Iven, Joachim Kopka, Ivo Feussner, and Jan Schirawski. 2022. "The Sporisorium reilianum Effector Vag2 Promotes Head Smut Disease via Suppression of Plant Defense Responses" Journal of Fungi 8, no. 5: 498. https://doi.org/10.3390/jof8050498