Study On The Regulation Mechanism Of Phosphodiesterase MoPdeH And Its Related Proteins In CAMP Signaling Pathway And Pathogenicity Of Magnaporthe Oryzae

Rice is the main crop in the world,has been deeply affected by rice diseases leading to large-scale production reduction.Rice blast is one of the most destructive diseases in rice production,which can cause a great decrease,even no harvest at all.Now,due to the rapid mutation of the strain from field,the resistant varieties will lose resistance in a few years.In recent years,with the development of molecular biology,it is helpful for us to better explore the molecular regulatory mechanism of Magnaporthe oryzae and develop new target sites of fungicides,so as to achieve the effect of persistent disease control.In eukaryotes,cAMP signaling pathway regulates signal exchange and perceives morphogenesis.Studies have shown that cAMP signaling pathway and its components in blast fungus play an important role in the growth,sexual/asexual reproduction,appressorium formation,cell wall integrity and plant infection.Intracellular cAMP is mainly balanced by the biosynthesis catalyzed by MoMacl and the hydrolysis catalyzed by MoPdeH and MoPdeL.Previous studies have indicated that the high-affinity MoPdeH plays an important role in the process of cAMP hydrolysis,participating in many important biological processes such as the conidia morphogenesis,appressorium formation,surface recognition,cell wall integrity and plant infection.However,the low-affinity MoPdeL had a weak ability to hydrolyze cAMP,and only participated in the conidia morphogenesis,having no effect on pathogenicity of M.oryzae.The purpose of this study was to investigate the characterization of intracellular cAMP hydrolase high affinity MoPdeH and related proteins in cAMP signaling pathway and pathogenicity of M.oryzae.In order to further understand the pathogenicity of MoPdeH,we analysed the roles of domains of MoPdeH and MoPdeL separately or in chimeras.The results indicated that the HD and EAL domains of MoPdeH are indispensable for its phosphodiesterase activity and function.Replacement of the MoPdeH HD domain with the L1 and L2 domains of MoPdeL,either singly or together,resulted in decreased cAMP hydrolysis activity of MoPdeH.All of the transformants exhibited phenotypes similar to that of the AMopdeH mutant,but also revealedthat EAL and L1 play additional roles in conidiation,and that L1 is involved in infectious growth.We further found that the intracellular cAMP level is important for surface signal recognition and hyphal autolysis.The intracellular cAMP level negatively regulates Mpsl-MAPK and positively regulates Pmkl-MAPK in the rice blast fungus.Our results provide new information to better understand the cAMP signalling pathway in the development,differentiation and plant infection of the fungus.To further explore the molecular regulatory mechanism of MoPdeH,we screened cDNA library using yeast two-hybrid system,obtained a conservative rate-limit enzyme inosine-5'-monophosphate dehydrogenase MoImd4 of de novo purine metabolic pathway.In our previous study,the deletion mutant of MoIMD4 was obtained,and it was found that the deletion of MoIMD4 blocked the de novo purine metabolism,and affected the growth,conidia and pathogenicity of rice blast.Further,we dectected the inteaction mechanism between MoPdeH and MoImd4.We found that MoImd4 and MoPdeH mutally promoted enzyme activity.The prediction of the reaction sites of MoImd4 and the inactivation mutation showed that all these sites could be used as the enzyme activity sites of MoImd4,but the mechanism of action were different:some sites only participated in the de novo purine metabolism;In addition,other sites also influenced the interaction between MoImd4 and MoPdeH,which in turn affected the enzymatic activity of MoPdeH.These results showed that MoImd4 and MoPdeH combined de novo purine metabolism with intracellular cAMP signaling pathway to jointly regulate the development and pathogenicity of M.oryzae.Besides that,the inhibitor MPA can specifically target the MoImd4 of M.oryzae,but the damage to the host rice homogenous gene IMPDH is small.Therefore,MPA can be used as the fungicide for the field disease control in the future.MoPdeH was used as the decoy using Co-IP test in vivo.Results of mass spectrometry obtained many potential proteins interacting with MoPdeH,including many mitochondrial proteins.To explain the relationship between MoPdeH in the cAMP signaling pathway and the mitochondrial proteins,we analyzed the function of mitochondrial outer membrane translocase MoTom70 and mitochondrial phosphate carrier protein MoPic in M.oryzae.Results showed that MoTom70 and MoPic were involved in development and pathogenicity of M.oryzae.MoTom70 input the MoPic from the cytoplasm to the mitochondria,instantaneously.Deletion of MoTOM70 causes continuous mitochondrial division and the state was irreversible.Whatsmore,MoTom70 and MoPic were involved in cAMP signaling pathway.Therefore,the input of mitochondrial protein may be related to the cAMP signaling pathway mediated by MoPdeH,which jointly regulate the development and pathogenicity of M oryzae.In conclusion,studying the function of MoPdeH and its related proteins may further elucidate the molecular regulatory mechanism of M.oryzae in development and pathogenicity,providing new drug targets for the prevention and treatment of rice blast.