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Article
Zengyan Zhang
Institute of Crop Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing, China
Corresponding Author
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Wheat (Triticum aestivum) is one of the most important staple crops. The necrotrophic binucleate fungus Rhizoctonia cerealis is the causal agent for the devastating disease wheat sharp eyespot and additional diseases of other agricultural crops and bioenergy plants. In this study, we present the first high-quality genome assembly of R. cerealis Rc207, a highly aggressive strain isolated from wheat. The genome encodes expand and diverse sets of virulence-related proteins, especially secreted effectors, carbohydrate-active enzymes (CAZymes), metalloproteases, Cytochrome P450 (CYP450), and secondary metabolite-associated enzymes. Many of these genes, in particular those encoding secretory proteins and CYP450, showed markedly up-regulation during infection in wheat. Of 831 candidate secretory effectors, ten up-regulated secretory proteins, such as CAZymes, metalloproteases and antigens, were functionally validated as virulence factors required for the fungal infection in wheat. Further intra-species and inter-species comparative genomics analyses showed that repeat sequences, accounting for 17.87% of the genome, are the major driving force for the genome evolution, and frequently intraspecific gene duplication contributes to expansion of pathogenicity-related gene families. This is the first genome-scale investigation elucidating the pathogenesis mechanisms and evolutionary landscape of R. cerealis. Our results provide essential tools for further development of effective disease control strategies.
Keywords
wheat, Rhizoctonia cerealis, wheat sharp eyespot, R. cerealis Rc207
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This is a list of supplementary files associated with this preprint. Click to download.
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Supplementary Figure 1 Scanning electron microscopy images of R. cerealis hyphae on the wheat leaf sheath during early infection stages. The red arrows represent the fungal hyphae that are infecting to wheat and signals of potential de-structuring by the fungal secreted enzymes.
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Supplementary Figure 2 The sharp eyespot symptom development in leaf sheaths during R. cerealis infection to the susceptible wheat cv. Wenmai 6. The red arrow represents sharp eyespot symptom.
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Supplementary Figure 3 The distribution of cellulose degrading enzymes (blue), hemi-cellulose degrading enzymes (orange) and pectin degrading enzymes (green) in the R. cerealis Rc207.
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Supplementary Figure 4 Heap map showing expression levels of CAZymes in R. cerealis Rc207 during wheat infection. A total of 234 genes were significantly up-regulated, and these genes were clustered. Color bar represents the log2 of (FPKM+1) value, ranging from blue (0) to red (14).
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Supplementary Figure 5 Significantly up-regulated genes of CAZymes during R. cerealis Rc207 infection in wheat. (A) cellulose degrading enzymes, (B) hemi-cellulose degrading enzymes, (C) pectin degrading enzymes, (D) xylan degrading enzymes.
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Supplementary Figure 6 qRT-PCR assay on transcript profiles of the CAZyme-encoding genes in R. cerealis Rc207 during the infection. The tested genes include 1 RcAA9 (Rc_00897.1), 1 RcCE5 (Rc_03129.1), 2 RcCBM1 (Rc_14069.1, Rc_02907.2), 3 RcGH5 (Rc_06583.1, Rc_08522.1, Rc_05200.2), 3 GH10 (Rc_02727.1, Rc_10772.1, Rc_06659.1), 1 GH51 (Rc_09878.1), 1 GH6 (Rc_00803.1) and 1 GH28 (Rc_03586.1). The R. cerealis Actin gene was used as an internal control to normalize the data. SE bars were calculated based on three replicates and Student’s t-tests (*, P < 0.05; **, P < 0.01).
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Supplementary Figure 7 The lesion areas/infection caused by R. cerealis liquid mycelia inoculation for three days on leaves infiltrated with heterologously-expressed RcGH6-1 (5 μM) and heterologously-expressed RcGH28 proteins (5 μM) for 6h.
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Supplementary Figure 8 (A) Statistic analysis of cysteine-rich candidate effectors in R. cerealis genome; and (B) A. tumefaciens-mediated transient expression in N. benthamiana showed that cell death-induced activity of RcRNase. Photographs were taken five days post agro-infiltration.
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Supplementary Figure 9 qRT-PCR analysis of the secretory effectors in R. cerealis Rc207 during wheat infection, including RcAA9 (Rc_05515.1), RcLP (Rc_08199.1), RcGH5 (Rc_08801.1), RcGH6-2 (Rc_07778.1), RcTP (Rc_02278.1), RcOV16-1 (Rc_05066.1), RcOV16-2 (Rc_11267.1), and RcRNase (Rc_06868.1). The R. cerealis Actin gene was used as an internal control to normalize the data. SE bars were calculated based on three replicates and Student’s t-tests (*, P < 0.05; **, P < 0.01).
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Supplementary Figure 10 RcLP protein did not induce cell death in infiltrated leaves of the susceptible wheat cv. Wenmai 6. Each heterologously-expressed protein was infiltrated in 5 μM.
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Supplementary Figure 11 Comparison of protease genes between R. cerealis Rc207 and R. solani AG1 IA.
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Supplementary Figure 12 The count of Metalloproteases families in R. cerealis Rc207 genome.
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Supplementary Figure 13 Heap map showing expression levels of metalloproteases of R. cerealis Rc207 during wheat infection. The color bar represents the log2 of (FPKM+1) value, ranging from blue (0) to red (10).
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Supplementary Figure 14 The cell death-induced activity of RcFL1 in the protein infiltrated leaves of wheat and N. benthamiana. (A) Trypan blue staining of wheat leaves infiltrated with His-TF-RcFL1 and His-TF (each 5 µM). Dead wheat leaf cells were stained by trypan blue. (B) The necrosis of N. benthamiana leaves infiltrated with His-TF-RcFL1 (5 µM).
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Supplementary Figure 15 Phylogenetic tree showing the phylogenetic relationship of R. cerealis Rc207 to eleven other sequenced fungi. F. graminearum was used as an outgroup. The scale is the time unit of differentiation in million years. MYA, million years ago.
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Supplementary Table 1 R. cerealis Rc207 genome assembly quality assessment Supplementary Table 2 Summary of predicted repeat sequences in R. cerealis Rc207 genome Supplementary Table 3 Summary of R. cerealis Rc207 genome annotation Supplementary Table 4 Detailed functional annotation of the 14,433 R. cerealis Rc207 genes Supplementary Table 5 The 169 predicted secondary metabolite-associated clusters of R. cerealis Rc207 Supplementary Table 6 Statistics of differentially expressed genes during R. cerealis Rc207 infection to wheat Supplementary Table 7 R. cerealis Rc207 genes up-regulated during infection to wheat Supplementary Table 8 The OrthoMCL-annotated groups with overrepresented up-regulated genes in R. cerealis Rc207 Supplementary Table 9 Functional annotation of R. cerealis Rc207 CAZymes Supplementary Table 10 Comparison of the CAZyme classes among R. cerealis Rc207 and other fungal species Supplementary Table 11 Detailed comparison of the CAZyme families among R. cerealis Rc207 and other fungal species Supplementary Table 12 Expression of the CAZyme genes during R. cerealis Rc207 infection to wheat Supplementary Table 13 Functional annotation of the R. cerealis Rc207 secretome Supplementary Table 14 Candidate effectors of R. cerealis Rc207 Supplementary Table 15 Novel candidate effectors of R. cerealis Rc207 Supplementary Table 16 Comparison of the predicted proteases among three Rhizoctonia species Supplementary Table 17 Expression of the Metalloprotease genes in R. cerealis Rc207 during wheat infection Supplementary Table 18 R. cerealis strains Rc207 and Rc301 genome assemblies and annotation by Illumina HiSeq 2000 Supplementary Table 19 Syntenic gene clusters within R. cerealis Rc207 genome Supplementary Table 20 Characterization and comparison of the R. cerealis Rc207 genomic scaffolds Supplementary Table 21 Primers and their sequences in this study.
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This preprint is under consideration at a Nature Portfolio Journal. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Nature journals have partnered with Research Square to offer In Review, a journal-integrated preprint deposition service.
Article
Zengyan Zhang
Institute of Crop Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing, China
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 11 Mar, 2021
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Wheat (Triticum aestivum) is one of the most important staple crops. The necrotrophic binucleate fungus Rhizoctonia cerealis is the causal agent for the devastating disease wheat sharp eyespot and additional diseases of other agricultural crops and bioenergy plants. In this study, we present the first high-quality genome assembly of R. cerealis Rc207, a highly aggressive strain isolated from wheat. The genome encodes expand and diverse sets of virulence-related proteins, especially secreted effectors, carbohydrate-active enzymes (CAZymes), metalloproteases, Cytochrome P450 (CYP450), and secondary metabolite-associated enzymes. Many of these genes, in particular those encoding secretory proteins and CYP450, showed markedly up-regulation during infection in wheat. Of 831 candidate secretory effectors, ten up-regulated secretory proteins, such as CAZymes, metalloproteases and antigens, were functionally validated as virulence factors required for the fungal infection in wheat. Further intra-species and inter-species comparative genomics analyses showed that repeat sequences, accounting for 17.87% of the genome, are the major driving force for the genome evolution, and frequently intraspecific gene duplication contributes to expansion of pathogenicity-related gene families. This is the first genome-scale investigation elucidating the pathogenesis mechanisms and evolutionary landscape of R. cerealis. Our results provide essential tools for further development of effective disease control strategies.
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