Function Analysis Of Pathogenicity-related Signalling Mucin MoMsb2 And Transcriptional Regulator MoWor1 In Magnaporthe Oryzae

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases in cultivated rice. Rice-rice blast fungus system has been developed as the model host-fungal pathogen interaction system for research. Several signaling pathways essential for virulence have been identified, containing c AMP-PKA, Pmk1 and Mps1 signaling pathways. Over the past 20 years, these three signaling pathways have been well studied, and some essential components were identified and functionally characterized. The Pmk1 pathway plays essential roles in maturation of appressorium, penetration and invasive growth and the Mps1 pathway is involved in cell wall integrity. However, little is known about the upstream receptors and downstream transcriptional factors of these MAPKs signaling pathways. Recently, research on effectors became one of hottest fields and more and more potential effectors have been discovered. It was reported in 2013 that in rice blast fungus two distinct secretion systems were applied for delivering effectors. One is the conserved secretion system related to endoplasmic reticulum and Golgi apparatus, and the other secretion system involves exocyst complex and t-SNARE which delivers effectors to biotrophic interfacial complex firstly. However, the regulation of these effectors wasn’t well studied. In this study, we conducted deeply analyses of the activation of one upstream receptor Mo Msb2 in Pmk1 pathway and its relationship with the other mucin Cbp1. In addition, one pathogenicity-related transcriptional regulator Mo Wor1 was well characterized.Various surface signals are recognized by Magnaporthe oryzae to activate the Pmk1 MAP kinase that is essential for appressorium formation and invasive growth. One of upstream sensors of the Pmk1 pathway is the Mo Msb2 signaling mucin. However, the activation of Mo Msb2 and its relationship with other sensors is not clear. In this study, we showed that the cleavage and transmembrane domains are essential for Mo Msb2 functions. Cleavage of Mo Msb2 was further confirmed by Western Blot analysis and five putative cleavage sites were functionally characterized. Expression of the extracellular region alone partially rescued the defects of Momsb2 in appressorium formation and virulence. The cytoplasmic region of Mo Msb2, although dispensable for appressorium formation, was more important for penetration and invasive growth. Interestingly, the Momsb2 cbp1 double mutant deleted of both mucin genes was blocked in Pmk1 activation. It failed to form appressoria on artificial surfaces and was non-pathogenic. In addition, we showed that Mo Msb2 interacts with Ras2 but not with Mo Cdc42 in co-IP assays. Overall, results from this study indicated that the extracellular and cytoplasmic regions of Mo Msb2 have distinct functions in appressorium formation, penetration, and invasive growth, and Mo Msb2 has overlapping functions with Cbp1 in recognizing environmental signals for Pmk1 activation.Complex infection-related morphogenesis and production of various effectors are known to be important for successful colonization and disease development. In this study, we characterized the activation of the Mo WOR1 transcription factor and its role in infection-related morphogenesis and effector gene expression. The Mowor1 mutant was non-pathogenic although it was normal in appressorium formation and turgor generation. Close examination showed that Mowor1 was defective in penetration and growth of normal invasive hyphae. The expression of Mo Wor1 appeared to be controlled by the Mps1 but not Pmk1 MAP kinase, and the mps1 and Mowor1 mutants had similar phenotypes in plant infection and cell wall integrity defects. However, lack of MAPK phosphorylation sites and dispensability of the putative MAPK docking site suggested that Mo Wor1 is not a direct target of Mps1. Site specific mutagenesis analyses showed that the putative PKA phosphorylation site was not essential for localization of Mo Wor1 to the nucleus but important for its normal function. Although the CDK phosphorylation site of Mo Wor1 is dispensable during vegetative growth and appressorium formation, the S77 A mutation affected penetration and invasive growth. Localization of Mo Wor1S77A-GFP to the nucleus in late stages of appressorium formation and during invasive growth was not observed, indicating a stage-specific CDK phosphorylation of Mo Wor1. q RT-PCR and fluorescence microscopy examinations showed that deletion of Mo WOR1 affected the expression of the majority of effector genes during plant infection. Overall, our data indicate that Mps1 may indirectly regulate the expression of Mo Wor1 in maintaining cell wall integrity, conidiation, and plant infection. Mo Wor1 is likely a stage-specific target of CDK and plays a crucial role in effector gene expression and morphogenesis related to the development of penetration pegs and invasive hyphae.