Abstract
Rhizoctonia solani is an important necrotrophic fungal pathogen which causes disease on diverse plant species. It has been classified into 14 genetically distinct anastomosis groups (AGs), however, very little is known about their genomic diversity. AG1-IA causes sheath blight disease in rice and controlling this disease remains a challenge for sustainable rice cultivation. Recently the draft genome sequences of AG1-IA (rice isolate) and AG1-IB (lettuce isolate) had become publicly available. In this study, using comparative genomics, we report identification of 3,942 R. solani genes that are uniquely present in AG1-IA. Many of these genes encode important biological, molecular functions and exhibit dynamic expression during in-planta growth of the pathogen in rice. Based upon sequence similarity with genes that are required for plant and human/zoonotic diseases, we identified several putative virulence/pathogenicity determinants amongst AG1-IA specific genes. While studying the expression of 19 randomly selected genes, we identified three genes highly up-regulated during in-planta growth. The detailed in silico characterization of these genes and extent of their up-regulation in different rice genotypes, having variable degree of disease susceptibility, suggests their importance in rice–Rhizoctonia interactions. In summary, the present study reports identification, functional characterization of AG1-IA specific genes and predicts important virulence determinants that might enable the pathogen to grow inside hostile plant environment. Further characterization of these genes would shed useful insights about the pathogenicity mechanism of AG1-IA on rice.
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Acknowledgments
SG was supported by SPM fellowship from Council of Scientific and Industrial Research (Govt. of India) and SKG was supported by Post-Doctoral Research fellowship from Department of Biotechnology (DBT, Govt of India). We acknowledge Praveen Raj S, CromDx Solutions Pvt. Ltd for assistance in computational analysis and central instrumentation facility at NIPGR for sequencing and qRT-PCR analysis. We also thank Dr G.S. Laha, DRR, Hyderabad for providing R. solani strains and AiPing Zheng, SICAU, China and Daniel Wibberg, CeBiTec, Bielefield University, Germany for making draft genome sequences of AG1-IA and AG1-B, R. solani strains publicly available. This work was supported by core research grant from the National Institute of Plant Genome Research, India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Communicated by S. Hohmann.
S. Ghosh and S. K. Gupta contributed equally to this study.
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294_2014_438_MOESM2_ESM.xlsx
Online Resource 2: Blast analysis against AG1-IB genomic contigs and Inparanoid database based ortholog prediction (XLSX 22 kb)
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Online Resource 7: The relative proportion of AG1-IA specific GO terms compared to that present in entire AG1-IA genome. The relative fraction of GO terms was computed using custom script and top 20 terms in Biological Process (A) and Molecular Function (B) are displayed. (PDF 667 kb)
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Online Resource 8: KOG and KEGG classification of AG1-IA specific genes. KOG and KEGG classification of AG1-IA specific genes were performed using eggNOG application and KAAS annotation server, respectively. The analysis classified 613 AG1-IA specific proteins into 23 KOG groups (A) and 187 genes across KEGG pathways. (PDF 727 kb)
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Online Resource 10: KEGG annotation of AG1-IA specific proteins that demonstrate homology in PHIDIAS database. (PDF 589 kb)
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Online Resource 11: Microscopic analysis of trypan blue stained mycelia of different Rhizoctonia strains. The analysis was performed using Nikon Eclipse E100 microscope at 400X magnification. (TIFF 846 kb)
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Online Resource 13: PCR analysis of selected AG1-IA specific genes across geographically diverse strains of R. solani. The 19 selected genes were PCR amplified from different R. solani strains (A. BRS-1, B. BRS-2, C. BRS-6, D. BRS-7, E. BRS-4) analysed on 1.5 % agarose gel. The presence of these genes across different strains is summarized in F. Dark dot reflects confirmative presence of these genes, while cases having ambiguity are reflected as empty dots. The lane L2 and M2 represent the same gene, however the primer binding sites are different. M represents molecular weight marker. (PDF 1310 kb)
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Online Resource 14: PCR analysis to confirm the presence of some ambiguous AG1-IA specific genes. The genes RS_L1, RS_L5, RS_L6, and RS_M5 which showed ambiguous results in PCR (Online Resource 11) were individually PCR amplified in different AG1-1A isolates. M represents molecular weight marker. (PDF 420 kb)
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Online Resource 16: Differential expression profile of AG1-IA specific genes across different time intervals (XLSX 1131 kb)
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Online Resource 17: Expression dynamics of selected AG1-IA specific genes. Semi-quantitative PCR based expressions of 19 selected candidate AG1-IA specific genes were analysed. The expression at 3 and 6 DPI of BRS-1 growth in susceptible rice IR64 is depicted in A and D, respectively. The expression at 3 and 6 DPI of BRS-1 growth in partially resistant rice cultivar Tetep is depicted in B and E, respectively while the expression during its growth in potato dextrose broth (PDB) at 3 and 6 DPI is reflected in C and F, respectively. The expression pattern of these genes at different stages is summarized in G. Dark dot reflects confirmative expression of these genes, while ambiguous cases are reflected as empty dots. R. solani specific 18S rDNA primers were used as a normalization control. The data is a representative of at least two biological and three technical replicates. The lane L2 and M2 represent the same gene, however the primer binding sites were different. M represents molecular weight marker. (PDF 2540 kb)
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Online Resource 18: Domain architecture of in-planta induced AG1-IA specific genes. The domain architecture of RS_P1 (A), RS_P3 (B), RS_P4 (C) proteins as revealed by Prosite and Pfam analysis. a: represents AG1-IA specific sequence while b: represents AG3 sequence, the nearest ortholog as per BLASTP analysis. Red flag in A.a represents PkC phosphorylation site and CAMP phosphorylation site is denoted by grey flag. In B.a Violet flag represents a PkC phosphorylation site while red and grey flag denotes N myristylation and Asn Glycosylation sites, respectively. Red flags in C.a represent N- Myristylation sites at N-terminal. (PDF 416 kb)
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Ghosh, S., Gupta, S.K. & Jha, G. Identification and functional analysis of AG1-IA specific genes of Rhizoctonia solani . Curr Genet 60, 327–341 (2014). https://doi.org/10.1007/s00294-014-0438-x
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DOI: https://doi.org/10.1007/s00294-014-0438-x