The cysteine protease MoAtg4 interacts with MoAtg8 and is required for differentiation and pathogenesis in Magnaporthe oryzae

Atg4 is a unique cysteine protease responsible for the cleavage of the carboxyl terminus of Atg8 during the formation of autophagosomes in yeast. Here we report that MoAtg4, an Atg4 homologue in Magnaporthe oryzae, controls cell differentiation and pathogenicity by interacting with MoAtg8, an autophagic protein essential for autophagic cell death and pathogenicity. Yeast complementation assay revealed that MoATG4 can functionally complement the defects of the yeast ATG4 deletion mutant. The direct interaction between MoAtg4 and MoAtg8 was detected in both yeast two hybrid and bimolecular fluorescence complementation (BiFC) assays. We also specify a cysteine residue, Cys206, as the active residue within MoAtg4 for the cleavage of MoAtg8 in vitro. Expression pattern analysis revealed that MoATG4 gene is expressed throughout growth and development by M. oryzae and can be induced by starvation and MoAtg4 protein localized in the cytoplasm of M. oryzae. Deletion of MoATG4 in M. oryzae caused significant reduction of aerial hyphae, conidiation, perithecia formation and delay of conidial germination and appressorium formation. Furthermore, as a result of lower turgor pressure of the appressorium, the Delta Moatg4 mutant lost its ability to penetrate rice and barley. The developmental and pathogenic phenotypes were recovered by reintroduction of an intact copy of MoATG4 into the mutant, suggesting that MoATG4 is indispensable in the development of M. oryzae and essential to pathogenicity of this fungus.