Plant pathogenic fungi deploy secreted effectors to suppress plant immunity responses. These effectors operate either in the apoplast or within host cells, so they are putatively glycosylated, but the posttranslational regulation of their activities has not been explored. In this study, the ASPARAGINE-LINKED GLYCOSYLATION3 (ALG3)-mediated N-glycosylation of the effector, Secreted LysM Protein1 (Slp1), was found to be essential for its activity in the rice blast fungus Magnaporthe oryzae. ALG3 encodes an a-1,3-mannosyltransferase for protein N-glycosylation. Deletion of ALG3 resulted in the arrest of secondary infection hyphae and a significant reduction in virulence. We observed that Dalg3 mutants induced massive production of reactive oxygen species in host cells, in a similar manner to Dslp1 mutants, which is a key factor responsible for arresting infection hyphae of the mutants. Slp1 sequesters chitin oligosaccharides to avoid their recognition by the rice (Oryza sativa) chitin elicitor binding protein CEBiP and the induction of innate immune responses, including reactive oxygen species production. We demonstrate that Slp1 has three N-glycosylation sites and that simultaneous Alg3-mediated N-glycosylation of each site is required to maintain protein stability and the chitin binding activity of Slp1, which are essential for its effector function. These results indicate that Alg3-mediated N-glycosylation of Slp1 is required to evade host innate immunity.

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Glutamate homeostasis plays a vital role in central nitrogen metabolism and coordinates several key metabolic functions. However, its function in fungal pathogenesis and development has not been investigated in detail. In this study, we identified and characterized a glutamate synthase gene MoGLT1 in the rice blast fungus Magnaporthe oryzae that was important to glutamate homeostasis. MoGLT1 was constitutively expressed, but showed the highest expression level in appressoria. Deletion of MoGLT1 resulted in a significant reduction in conidiation and virulence. The ΔMoglt1 mutants were defective in appressorial penetration and the differentiation and spread of invasive hyphae in penetrated plant cells. The addition of exogenous glutamic acid partially rescued the defects of the ΔMoglt1 mutants in conidiation and plant infection. Assays for MoAtg8 expression and localization showed that the ΔMoglt1 mutants were defective in autophagy. The ΔMoglt1 mutants were delayed in the mobilization of glycogens and lipid bodies from conidia to developing appressoria. Taken together, our results show that glutamate synthase MoGlt1-mediated glutamate homeostasis is important for pathogenesis and development in the rice blast fungus, possibly via the regulation of autophagy.

Glycine-rich proteins (GRPs) have diverse amino acid sequences and are involved in a variety of biological processes. The role of GRPs in plant pathogenic fungi has not been reported. In this study, we identified and functionally characterized a novel gene named MoGRP1 in Magnaporthe oryzae, which encodes a protein that has an N-terminal RNA recognition motif (RRM) and a C-terminal glycine-rich domain with four Arg-Gly-Gly (RGG) repeats. Deletion of MoGRP1 resulted in dramatic reductions in fungal virulence, mycelial growth, and conidiation. The ΔMogrp1 mutants were also defective in cell wall integrity and in their responses to different stresses. MoGrp1 was localized to the nucleus and was co-immunoprecipitated with several components of the spliceosome, including subunits of the U1 snRNP and U2 snRNP complexes. Moreover, MoGrp1 exhibited binding affinity for poly(U). Importantly, MoGrp1 was responsible for the normal splicing of genes involved in infection-related morphogenesis. Domain deletion assays showed that both the RRM domain and its two adjacent RGG repeats were essential to the full function of MoGrp1. Notably, the nine amino acids between the first and the second RGG repeats were indispensable for nuclear localization and for the biological functions of MoGrp1. Taken together, our data suggest that MoGrp1 functions as a novel splicing factor with poly(U) binding activity to regulate fungal virulence, development, and stress responses in the rice blast fungus.