Conserved effectors with a ribonuclease domain are involved in virulence of phytopathogenic Colletotrichum fungi

Members of the genus Colletotrichum infect many commercially important crops. Colletotrichum fungi secrete small effector proteins, which are thought to be important for successful plant invasion. By comparing Colletotrichum genomes, we selected 37 highly conserved genes encoding small-secreted proteins that are expressed during infection in both Colletotrichum higginsianum and Colletotrichum orbiculare. Using the transient gene expression system in Nicotiana benthamiana, we expressed these candidates to investigate their cell death-inducing activity. We identified a cell death-inducing gene that encodes a protein with a ribonucleases domain, designated as secreted ribonuclease (SRN). Phylogenetic tree analysis revealed that SRN homologs are conserved in all tested Pezizomycotina species, but not in Taphrinomycotina and Saccharomycotina, implying that SRNs in fungi originated after the diversification of Pezizomycotina from these groups. In particular, SRN homologs are significantly expanded in Blumeria graminis (Pedersen et al., 2012, BMC Genomics). In C. orbiculare, there are four members of the SRN family, SRN1 to SRN4. To characterize these further, we established knockout mutants of SRN genes in C. orbiculare and found that they are required for full virulence of the pathogen. Quadruple mutants of SRNs, but not triple mutants, showed reduced virulence on plants, indicating that all SRNs have a redundant function. In N. benthamiana, transiently expressed SRN1, 2, and 4 induced cell death. A SRN2 variant that has a mutation in the ribonuclease catalytic residue did not induce cell death, indicating that the ribonuclease activity is required for cell death. Interestingly, B. graminis has 29 SRN homologs, however all of them lack the ribonuclease catalytic residue required for induction of cell death by C. orbiculare SRN2. In summary, SRNs encode virulent effectors in C. orbiculare but their function may be distinct from those in B. graminis.