Comparative secretome analysis of Rhizoctonia solani isolates with different host ranges reveals unique secretomes and cell death inducing effectors

Abstract

Rhizoctonia solani is a fungal pathogen causing substantial damage to many of the worlds' largest food crops including wheat, rice, maize and soybean. Despite impacting global food security, little is known about the pathogenicity mechanisms employed by R. solani. To enable prediction of effectors possessing either broad efficacy or host specificity, a combined secretome was constructed from a monocot specific isolate, a dicot specific isolate and broad host range isolate infecting both monocot and dicot hosts. Secretome analysis suggested R. solani employs largely different virulence mechanisms to well-studied pathogens, despite in many instances infecting the same host plants. Furthermore, the secretome of the broad host range AG8 isolate may be shaped by maintaining functions for saprophytic life stages while minimising opportunities for host plant recognition. Analysis of possible co-evolution with host plants and in-planta up-regulation in particular, aided identification of effectors including xylanase and inhibitor I9 domain containing proteins able to induce cell death in-planta. The inhibitor I9 domain was more abundant in the secretomes of a wide range of necrotising fungi relative to biotrophs. These findings provide novel targets for further dissection of the virulence mechanisms and potential avenues to control this under-characterised but important pathogen.

Figure 1

Similarity between AG8, AG1-IA and AG3. Proteins from the entire proteomes (a) or secretomes (b) from AG8, AG1-IA and AG3 were grouped into orthology groups (OGs) using Orthofinder and shared and isolate-specific OGs plotted in Venn diagrams. The number in each sector represents the number of OGs with members from those isolates and the percentage is the percent of all OGs. (c) The percent of genes to which at least one AG8 genomic DNA Illumina read mapped, indicating the potential conservation of the gene even though the complete gene may not have been assembled in the draft genome.

Figure 2

Hierarchical clustering of protein tribes reveals tribes with shared effector-like characteristics. (a–o) are heatmaps representing tribe scores for the following characteristics; (a) up-regulation of the AG8 member of the tribe during infection of wheat roots at 2 days after infection; (b) proteins predicted to be effectors by the EffectorP algorithm; (c) proteins containing site-specific diversifying selection; (d) proteins exhibiting whole protein diversifying selection; (e) annotation with a Pfam domain; (f) small cysteine rich proteins; (g) containing an effector motif-like sequence; (h) containing internal repeats; (i) containing nucleotide localisation sequences; (j) having a flanking intergenic region greater than 1 Kb; (k) conserved among AG8, AG1-IA and AG3; (l) only present in one isolate; (m) having orthologs in plant pathogenic fungi; (n) having orthologs in animal pathogenic fungi; (o) not having homologs in non-pathogenic fungi. Further details of the proteins and characteristics associated with each tribe are presented in Supplementary Data S3.

Figure 3

A R. solani AG8 Inhibitor I9 domain containing protein induces cell death when transiently expressed in N. benthamiana leaves. (a) Images acquired 5 days after agroinfiltration. (b) Lesion diameter on transiently transformed leaves without R. solani inoculation (light grey), transiently transformed leaves three days following R. solani AG8 inoculation (dark grey). Asterisks indicate values significantly different to the corresponding mock or R. solani inoculated GFP sample according to Dunnett’s test. Average values from three biological replicates is shown with standard error. The experiment was repeated three times with similar results. (c) Number of secreted proteins containing the Inhibitor I9 domain in fungi with different lifestyles. Abbreviations for fungal species names described in Supplementary Data S3b.

Figure 4

An AG8 xylanase gene induces cell death and chlorosis phenotype in N. benthamiana. (a) Images acquired 5 days after agroinfiltration. (b) Lesions on leaves 3 days after inoculation with R. solani AG8. (c) Closer view of a region expressing RSAG8_07159. (d) Light microscopy of cell death regions resulting from expression of RSAG8_07159. (e) Fluorescence image corresponding to (d) showing expression of GFP from a separate open reading frame on the same construct as RSAG8_07159. (e) UV light image corresponding to (d). Red colour indicates auto-fluorescence from chlorophyll, blue colour indicates fluorescence from necrotic cells. (g) Lesion diameter at 5 days after inoculation with R. solani AG8. Dark grey bars without R. solani inoculation, light grey bars with R. solani inoculation. The average and standard error of four replicates are presented. Asterisks indicate values significantly different to the corresponding mock or R. solani inoculated GFP sample according to Dunnett’s test. Scale bars 10 mm in (a) and (b) 5 mm in (c) and 1 mm in (d to f). The experiment was repeated three times with similar results.