The evolution and pathogenic mechanisms of the rice sheath blight pathogen

Abstract

Rhizoctonia solani is a major fungal pathogen of rice (Oryza sativa L.) that causes great yield losses in all rice-growing regions of the world. Here we report the draft genome sequence of the rice sheath blight disease pathogen, R. solani AG1 IA, assembled using next-generation Illumina Genome Analyser sequencing technologies. The genome encodes a large and diverse set of secreted proteins, enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, which probably reflect an exclusive necrotrophic lifestyle. We find few repetitive elements, a closer relationship to Agaricomycotina among Basidiomycetes, and expand protein domains and families. Among the 25 candidate pathogen effectors identified according to their functionality and evolution, we validate 3 that trigger crop defence responses; hence we reveal the exclusive expression patterns of the pathogenic determinants during host infection.

Figure 1. Genomic sequence and paired reads coverage.

(a) Distribution of genomic coverage (upper) and paired reads coverage (bottom). A major peak was detected at 121 × and 105 × coverage, and a second peak was detected at 66 × and 55 × that showed nearly half of the coverage of the main peak, which indicates highly heterozygous regions that were not merged in the assembly or hemizygous regions. (b) Frequency distribution of GC content. Reads of library with an insert size of 173 bp were used for analysing. Reads were aligned to the assembly using Burrows–Wheeler Aligner. (c) Comparison of the assembled genome scaffold 17 with a fosmid cdsdaxa sequence. The sequence depth was calculated by mapping the Illumina Genome Analyser short reads with an insert size of 173 bp for the genome assembly. The remaining unclosed gaps on the scaffolds are marked as white blocks. (d) The sequence depth and a coverage of the genome assembly bases for mismatches of fosmid and genome assembly bases.

Figure 2. Phylogenetic relationship of R. solani AG1 IA with other Basidiomycetes fungi and orthologs.

(a) Venn diagram showing orthologs between and among the four sequenced Basidiomycete fungi. The values explain the counts of ortholog groups and the counts of genes in parentheses. (b) Distribution of tree branch lengths of 608 single-copy orthologs. The tree branch length of each ortholog group was calculated using codeml with the JTT amino-acid substitution model from the PAML package, based on the CLUSTALW alignment. The branch lengths indicate different evolutionary rates of these ortholog groups. The distribution of the branch length values was determined by the Shapiro–Wilk test (P<0.01). A peak (111 ortholog groups, 18.26%) is distributed near the tree length of 3, and the tree length of 40 ortholog groups (6.58%) was estimated. (c) The phylogeny was constructed using TREEBEST with the neighbour-joining method from 608 single-copy orthologs (each fungus has only one gene in an ortholog group). Protein alignments were analysed using MUSCLE.

Figure 3. The expression pattern of genes coding for the degradative enzymes of R. solani AG1 IA.

There are totally 114 GH and 12 PL family members expressing in the six infection stages after inoculation (the 10-, 18-, 24-, 32-, 48- and 72-hr stage). The colour scale indicates transcript abundance relative to the mycelium before inoculation: red, increase in transcript abundance; blue, decrease in transcript abundance. The black bold genes are the enriched GH and polysaccharide lyase (PL) transcripts of R. solani AG1 IA, compared with the other sequenced pathogens. R. solani AG1 IA had conspicuously enriched GH and PL families: GH13, GH5, GH95, GH35, GH88, GH7, GH79, PL1 and PL4. Among them, GH13 member α-amylase catalyse the hydrolysis of starch and sucrose in crops. The twofold changes in transcript abundance are marked with circles at the different infection stages; blue, at the 24-h stage; green, at the 32-h stage; dark orange, at the 48-h stage. The enzyme families are defined according to the carbohydrate-active enzyme database. The substrate of CAZy families: CW, cell wall; ESR, energy storage and recovery; FCW, fungal cell wall; PCW, plant cell wall; PG, protein glycosylation; U, undetermined; a-gluc, a-glucans (including starch/glycogen); b-glyc, b-glycans; b-1,3-gluc, b-1,3-glucan; cell, cellulose; chit, chitin/chitosan; hemi, hemicelluloses; inul, inulin; N-glyc, N-glycans; N-/O-glyc, N-/O-glycans; pect, pectin; sucr, sucrose; and treh trehalose. Pearson correlation was used for distance calculations, and the average linkage method was chosen for clustering.

Figure 4. Candidate effectors cause necrotic phenotypes in rice, corn and soybean.

(a) Phenotypes observed on rice and maize leaves but not observed on soybean leaves. The effectors are encoded by the AG1IA_09161, AG1IA_07795 and AG1IA_05310 genes, respectively, and the cell death phenotypes were visible after inoculation with purified proteins after 48 h. CK represents the total expressed proteins of the E. coli strain harbouring the empty pEASY-TlTM E1 vector. Three replicates of three plants of each line were evaluated for toxin sensitivity. Scale bars (rice), 3 cm; scale bars (maize), 4 cm; scale bars (soybean), 3 cm. (b) Genes with signal peptides (red) and domain structures. CtaG-cox11 domain(blue), peptidase_S8 domain (green), inhibitor I9 domain (light purple) and glycosyltransferase GT family 2 domain (dark purple) are identified in the Pfam database. (c) Clustering of predicted effectors. The hierarchical clustering statistical method based on the Pearson correlation (correlation>0.98) and the average linkage was used.