MAPK-Mediated Cell Wall Integrity Coordinates With CAMP Pathway And Autophagy To Regulate Growth And Virulence Of Magnaporthe Oryzae

Rice is one of the most important crop in China which covers a quarter of the planting area over the whole country and it annual output contributes to half of the total grain output of the country.However,M oryzae is the causal agent of rice blast,and the outbreak of the rice blast are a threat to production worldwide,the annual loss can feed about 60 million people.The prevention of M.oryzae mainly depends on the cultivation of resistant varieties and chemical control.The M.oryzae is easy to mutate,which makes the original resistant varieties lose their resistance,and the use of single chemical cause fungus became resistant to chemical drugs.Therefore,an effective control of rice blast is a difficult problem in food security.The molecular level study of M.oryzae can provide a new strategy for disease resistant and development of novel fungicides.In this study,we focused on the coordination and interaction between different signal pathways to regulate the differentiation and pathogenicity in M.oryzae.Cell wall is the first barrier for filamentous fungi to resist external stress,and also involved in signal transduction and substance exchange between cells and the environment.Our previous studies identified a high-affinity cAMP phosphodiesterase MoPdeH is important not only for cAMP signaling and pathogenicity but also for cell wall integrity(CWI)maintenance through an unknown mechanism.Utilizing affinity purification,we found that MoPdeH interacts with MoMckl,one of the components of the mitogen-activated protein(MAP)kinase cascade that regulates CWI.Overexpression of MoMCKl suppressed defects in autolysis and pathogenicity of the ?MopdeH mutant although partially,suggesting that MoPdeH has a critical role in CWI maintenance mediated by the MAP kinase pathway.We found that MoMckl and two other MAP kinase cascade components,MoMkkl and MoMpsl,modulate intracellular cAMP levels by regulating the expression of MoPDEH through a feedback loop.In addition,disruption of MoMKK1 resulted in less aerial hyphal formation,defective asexual development,and attenuated pathogenicity.Moreover,MoMkk1 has a role in the response to the osmotic stress via regulation of MoOsml phosphorylation levels,whereas endoplasmic reticulum(ER)stress enhances MoMpsl phosphorylation and loss of the MAP kinase cascade component affects the unfolded protein response(UPR)pathway.Taken together,our findings demonstrate that MoPdeH functions upstream of the MoMckl-MoMkkl-MoMpsl MAP kinase pathway to regulate CWI and that MoPdeH also mediates crosstalks among the cAMP-signaling pathway,the osmotic sensing HOG pathway,and the DTT-induced UPR pathway in M oryzae.M.oryzae relies heavily on the secretory pathway,both for the delivery of cell wall components to the hyphal and the production and secretion of extracellular proteins into host to suppress the host-derived defense thereby infecting host.Increased demand on the secretory system exerts stress on the endoplasmic reticulum,which is countered by the activation of a coordinated stress response pathway termed the unfolded protein response.However,the relationship between cell wall integrity pathway and the unfolded protein response remains unclear.Our previous work showed that exogenous dithiothreitol(DTT,endoplasmic reticulum stress inducer)can rescue the defects in cell wall integrity(CWI)of the MoMCK1 mutant.In this study,we started here as a breakthrough point and found that DTT can induce the autophagy in M.oryzae;we found MoAtgl-kinase-complex as the key protein that response to DTT signals and linking autophagy to CWI through screening interaction patterns of MoMkk1 under DTT treatment.We also found that deletion of CWI could block autophagy and overexpression of MoAtgl-complex could completely restore the defect in AMomckl mutant,indicating that CWI crosstalk with autophagy.Moreover,we found that overexpression of MoAtgl-complex in the wild type could enhance the virulence of M.oryzae on K23 rice variety,suggesting that the synergistic action of CWI and autophagy is essential for infection and pathogenicity of M.oryzae.Our work aims to understand molecular mechanism on cross-talk between CWI signaling pathway and autophagy and how such cross-talk events link to differentiation and pathogenicity in this fungus.Further work will reveal how the endoplasmic reticulum stress can activate CWI thereby promoting autophagy in response to cell wall integrity stress or during infection.These results can provide new insights for revealing the mechanism of crosstalk among CWI,endoplasmic reticulum stress and autophagy and how such crosstalk events link to differentiation and pathogenicity in the rice blast fungus.It is also a valuable finding for characterization of efficient and low toxic disease strategy against M.oryzae.RGS proteins function as negative regulators of G-protein signaling to also govern growth,development,and/or pathogenicity.Our previous studies have characterized eight RGS and RGS-like proteins from M.oryzae and we provided evidence showing all have certain roles in conidiation,appressorium formation,cAMP levels,and pathogenicity.We found MoRgs7 with a unique RGS-seven transmembrane(7-TM)domain motif is localized to the highly dynamic tubule-vesicular compartments during early appressorium differentiation followed by gradually degradation.To explore whether this involves an active signal perception of MoRgs7,we identified a Gbeta-like/RACK1 protein homolog in M oryzae MoMipll that interacts with MoRgs7.Interestingly,MoMipll selectively interacted with several components of the cAMP regulatory pathway,including Ga MoMagA and the high-affinity phosphodiesterase MoPdeH.We further showed that MoMipll promotes MoMagA activation and suppresses MoPdeH activity thereby up-regulating intracellular cAMP levels.Moreover,MoMip11 is required for the response to multiple stresses,a role also shared by Gbeta-like/RACK1 adaptor proteins.In summary,we revealed a unique mechanism by which MoMip11 links MoRgs7 and G-proteins to reugulate cAMP signaling,stress responses,and pathogenicity of M oryzae.Our studies revealed the multitude of regulatory networks that govern growth,development,and pathogenicity in this important causal agent of rice blast.In summary,the MAPK-mediated cell wall integrity pathway in the rice blast fungus regulates the growth,differentiation and virulence of the rice blast fungus by coordinating the cAMP pathway,the unfolded protein response pathway,and autophagy.