Pangenome Analysis of the Soilborne Fungal Phytopathogen Rhizoctonia solani and Development of a Comprehensive Web Resource: RsolaniDB

Abstract

Rhizoctonia solani is a collective group of genetically and pathologically diverse basidiomycetous fungi that damage economically important crops. Its isolates are classified into 13 Anastomosis Groups (AGs) and subgroups having distinctive morphology and host ranges. The genetic factors driving the unique features of R. solani pathology are not well characterized due to the limited availability of its annotated genomes. Therefore, we performed genome sequencing, assembly, annotation and functional analysis of 12 R. solani isolates covering 7 AGs and select subgroups (AG1-IA; AG1-IB; AG1-IC; AG2-2IIIB; AG3-PT, isolates Rhs 1AP and the hypovirulent Rhs1A1; AG3-TB; AG4-HG-I, isolates Rs23 and R118-11; AG5; AG6; and AG8), in which six genomes are reported for the first time. Using a pangenome comparative analysis of 12 R. solani isolates and 15 other Basidiomycetes, we defined the unique and shared secretomes, CAZymes, and effectors across the AGs. We have also elucidated the R. solani-derived factors potentially involved in determining AG-specific host preference, and the attributes distinguishing them from other Basidiomycetes. Finally, we present the largest repertoire of R. solani genomes and their annotated components as a comprehensive database, viz. RsolaniDB, with tools for large-scale data mining, functional enrichment and sequence analysis not available with other state-of-the-art platforms.

Keywords: NGS—next generation sequencing; Rhizoctonia solani; basidiomycetous fungi; genome database; genomics; pangenome analyses; pathogenicity genes; soilborne plant pathogen.

FIGURE 1

(A) Circos plot. The Circos plot represents the syntenic relationship between genomes of different AGs of Rhizoctonia solani Kühn. Each line represents the region of genomic similarity predicted with Synima. Only the regions with coverage > 40,000 bases were enumerated and shown. (B) The plot highlights the number of high-similarity syntenic regions (coverage > 40,000 bp) shared between each pair of genomes, including T. calospora. The red connection represents corresponding isolates sharing a comparatively large number of syntenic relationships relative to other pairs of isolates. Here, self-hits were removed or not shown. (C) ITS2 phylogeny. ITS2 sequences of the tester strain were obtained from the NCBI database and were clustered with ITS2 sequences from assembled R. solani genomes (highlighted with blue color and *), along with ITS2 sequences from previously published R. solani genome assemblies (marked with **). The phylogenetic tree was constructed using megax software with 10,000 bootstrapping steps (see section “Materials and Methods”), after which the resulting tree and corresponding alignment were visualized together using Phylogeny.IO.

FIGURE 2

OrthoMCL clustering of the predicted proteomes in R. solani AGs. (A) Heatmap showing protein conservation across all sequenced R. solani AGs and T. calospora. Each row represents one orthoMCL cluster, and color is proportional to the number of protein members shared within a given cluster from the given species (black: no member protein present; red: large number of protein members present). The hierarchical clustering (hclust; method: complete) analysis enumerates the similarities between different fungal isolates based on proteins shared by them across all orthoMCL clusters. (B) Cluster frequency. The line plot represents the number of orthoMCL clusters shared by different fungal isolates used in this study. Example, > 1,400 orthoMCL clusters are shared by 14 different fungal isolates (including positive and negative controls) used in this study. The bimodal nature of the plot represents high similarities across independent proteomes as large numbers of clusters share protein members from 13 fungal isolates. The red line represents the smoothed curves after averaging out the number of clusters. (C) Protein classification based on the orthoMCL clusters. The “core” proteins represent the sub-set of proteomes (from each R. solani AG and T. calospsora) with a conserved profile across all the isolates. Similarly, the “unique” sets represent the isolate-specific protein subset. The rest of the protein subsets make up the “Auxiliary” proteome which are conserved in a limited number of isolates. (D) Shared orthoMCL clusters. The number of orthoMCL clusters shared between any two isolates. A shared cluster means, a given orthoMCL cluster contains proteins from both the isolates.

FIGURE 3

InterPro domain analysis of the unique proteome. In the unique proteome of each fungal isolate, InterPro protein domain families were predicted using InterProScan (Version 5.45–80.0). Only the top five most enriched protein families are shown. The number marks the corresponding annotation of InterPro family domain in the circular bar plot.

FIGURE 4

The secreted proteins. (A) Number of predicted proteins in the secretome of each fungal isolate (highlighted in yellow). The secreted proteins predicted in the unique proteome of each isolate is highlighted in red. (B) Comparative analysis of the top six highly enriched InterPro domains in the secretome. (C) Comparative analysis of the total number of secreted proteins predicted in R. solani isolates as compared to other basidiomycetes used in this study. P-value is computed using the unpaired Wilcoxon-rank sum test. (D) Heatmap showing the pairwise comparison of the InterPro terms commonly shared by the secretome of R. solani isolates as well as other basidiomycetes. The strong hierarchical clustering of R. solani isolates highlights their functionally unique and distinct secretome profile as compared to other basidiomycetes.

FIGURE 5

Effector proteins. (A) The number of cysteine rich effector proteins predicted in the predicted secretome of each fungal isolate. (B) The proportion of cysteine observed across all the effectors predicted in each isolate. (C) Topmost enriched InterPro domains in effector proteins of Rhizoctonia species (not T. calospora) and other Basidiomycetes (including T. calospora). (D) The comparative analysis of the distribution of number of effector proteins predicted in R. solani AGs as compared to other Basidiomycetes. The p-value is computed using the unpaired Wilcoxon-rank sum test.

FIGURE 6

CAZymes. (A) The number of carbohydrate-metabolizing enzymes (CAZymes) predicted in the proteome of each fungal isolate. (B) Heatmap showing the CAZyme conservation across all the R. solani AGs and T. calospora. Each row represents one CAZyme family of proteins, and color is proportional to the number of protein members shared within a given family from the given species (black: no member protein present; red: large number of protein members present). The hierarchical clustering (hclust; method: complete) enumerates the similarities between different fungal isolates based on proteins shared by them across all CAZyme families. For simplicity only the CAZyme families enriched in more than 50 enzymes across all proteomes are shown.