Ubiquitin-specific protease (UBP) family is the largest group of deubiquitinases, which plays important roles in eukaryotic organisms. Comprehensive analysis of UBP genes has not been conducted in the plant pathogenic fungi. In this study, 11 putative UBP genes were identified and characterized in the rice blast fungus Magnaporthe oryzae. Expression profile analysis showed that UBP3, UBP6, UBP12 and UBP14 were highly expressed in different tissues of M. oryzae. In all ubp mutants, especially Δubp3, Δubp12 and previously reported Δubp14, the ubiquitination levels were evidently elevated, which is consistent with their molecular roles in de-ubiquitination. The Δubp1, Δubp3, Δubp4, Δubp8 and Δubp14 mutants were reduced in colony growth. Most of the ubp mutants were severely reduced in conidia production capacity, indicating important roles of the UBPs in conidia formation. Except for Δubp2 and Δubp16, all of the other mutants were decreased in virulence to host plants and defective in invasive growth. These ubp mutants also induced massive ROS accumulation in host cells. We also found that the UBPs may function as both positive and negative regulators in stress response and nutrient utilization of M. oryzae. Collectively, UBPs are important for development, stress response, nutrient utilization and infection of M. oryzae.

Summary Glycosylphosphatidylinositol (GPI) anchoring plays key roles in many biological processes by targeting proteins to the cell wall; however, its roles are largely unknown in plant pathogenic fungi. Here, we reveal the roles of the GPI anchoring in Magnaporthe oryzae during plant infection. The GPI‐anchored proteins were found to highly accumulate in appressoria and invasive hyphae. Disruption of GPI7, a GPI anchor‐pathway gene, led to a significant reduction in virulence. The Δgpi7 mutant showed significant defects in penetration and invasive growth. This mutant also displayed defects of the cell wall architecture, suggesting GPI7 is required for cell wall biogenesis. Removal of GPI‐anchored proteins in the wild‐type strain by hydrofluoric acid (HF) pyridine treatment exposed both the chitin and β‐1,3‐glucans to the host immune system. Exposure of the chitin and β‐1,3‐glucans was also observed in the Δgpi7 mutant, indicating GPI‐anchored proteins are required for immune evasion. The GPI anchoring can regulate subcellular localization of the Gel proteins in the cell wall for appressorial penetration and abundance of which for invasive growth. Our results indicate the GPI anchoring facilitates the penetration of M. oryzae into host cells by affecting the cell wall integrity and the evasion of host immune recognition.

In eukaryotes, N 6 -methyladenosine (m 6 A) is abundant on mRNA, and plays key roles in the regulation of RNA function. However, the roles and regulatory mechanisms of m 6 A in phytopathogenic fungi are still largely unknown.Combined with biochemical analysis, MeRIP-seq and RNA-seq methods, as well as biological analysis, we showed that Magnaporthe oryzae MTA1 gene is an orthologue of human METTL4, which is involved in m 6 A modification and plays a critical role in autophagy for fungal infection.The Dmta1 mutant showed reduced virulence due to blockage of appressorial penetration and invasive growth. Moreover, the autophagy process was severely disordered in the mutant. MeRIP-seq identified 659 hypomethylated m 6 A peaks covering 595 mRNAs in Dmta1 appressoria, 114 m 6 A peaks was negatively related to mRNA abundance, including several ATG gene transcripts. Typically, the mRNA abundance of MoATG8 was also increased in the single m 6 A site mutant Δatg8/MoATG8 A982C , leading to an autophagy disorder.Our findings reveal the functional importance of the m 6 A methylation in infection of M. oryzae and provide novel insight into the regulatory mechanisms of plant pathogenic fungi.

The ascomycete fungus Magnaporthe oryzae is the causal agent of rice blast, which has become a model organism for studying plantpathogen interactions (Dean et al., 2012;Ebbole, 2007). During infection, M. oryzae forms a melanized appressorium on the host surface, breaches the host cuticle with a penetration peg, and spreads across host cells through invasive hyphae (Ebbole, 2007;Wilson & Talbot, 2009). The formation and maturation of the appressorium is a key and complex process that is determined by spore storage components (such as glycogen and lipid) and is regulated by a variety of signalling pathways (Rho et al., 2009;Talbot, 2003).The cAMP signalling pathway is one of the most important signalling pathways in the formation of the appressorium in M. ory-