Cell Wall Integrity Coordinates With Mitosis To Regulate Development And Virulence Of Magnaporthe Oryzae

Rice blast caused by M.oryzae is the most destructive fungal disease on rice.M.oryzae can successfully infect rice at almost all stages of its life cycle,which can reduce rice yield by 10-35%,and the resulting food loss can feed more than 60 million people.Therefore,effective control of rice blast is very important for food security.At present,the prevention of rice blast mainly depends on breeding resistant varieties,scientific cultivation and chemical protection.However,the complex pathogenic field flora of M.oryzae weakened the resistance of the resistant varieties,and there is a risk of long-term use of a single type of chemical agent.Therefore,exploring the pathogenic mechanism of M.oryzae can provide potential targets to development new fungicides.As the first barrier for fungi to contact with the outside world,cell wall plays an important role in recognizing external signals,maintaining cell morphology and ensuring the homeostasis of internal environment.The cell wall integrity（CWI）signaling pathway of M.oryzae consists of MoMck1,MoMkk1 and MoMps1.Our previous studies found that the integrity of mycelial cell wall was damaged in the mutant strains（35）Momck1,（35）Momkk1and（35）Momps1 resulting in the acceleration of the mycelium self-digestion rate,and seriously affect sporulation and pathogenicity.However,cells need to elongate and division during the process of vegetative growth and infection,accompanied by cell wall remodeling which result in cell wall stress.How to balance mitosis and cell wall integrity remains unclear.In this paper,we identifided the cell division related kinase MoSep1 interacted with MoMkk1,and elucidated the melocular mechanism of MoSep1-dependent MoMkk1phosphorylation to coordinate CWI and MEN.The results are as follows:Identification of CWI protein kinase Momkk1 interacting proteinsIn this study,we analyzed the phosphorylation level of MoMkk1 in vegetative growth stage and infection stage,results showed that the phosphorylation level of MoMkk1increased with the increase of nuclear density in infection stage.It was found that the distribution of primary chitin was disordered and the phosphorylation level of MoMkk1was significantly decreased after the normal cell cycle of M.oryzae was blocked by cell mitosis inhibitor Nocodazole,and the addition of Nocodazole to conidia suspension resulted in the decrease of pathogenicity.The results showed that the integrity of cell wall integrity was destroyed when the cell division process of M.oryzae was blocked.In addition,there were more than 2 nuclei in the three mutants（35）MoMck1,（35）MoMkk1 and（35）MoMps1,indicated that when the CWI pathway is blocked,the process of cell division will also be destroyed.These results demonstrated that there is a connection between cell division and cell wall integrity in M.oryzae.To further explore the relationship between them,we screened the interaction proteins of MoMkk1 by co-immunoprecipitation technique and yeast two hybrid method.A protein kinase MoSep1（MGG＿04100）involved in cell division was identified and the interaction between MoMkk1 and MoSep1 was confirmed by yeast two hybrid,GST-pull down and co-immunoprecipitation.We speculated that this may be the key connection to the coordination of cell division and cell wall integrity in regulating the growth and development of M.oryzae.Mitotic exit network regulated the development and pathogenicity of M.oryzaeIn Schizosaccharomyces pombe,the core component of mitotic exit network（MEN）consists of Cdc15 and Dbf2-Mob1.The GTPase Tem1 activates Cdc15 in late mitosis,and then Cdc15 phosphorylates Dbf2.The activated Dbf2-Mob1 phosphorylates Cdc14 to regulate the transcription level of downstream genes that ensures the exit from mitosis.We identified two other components of MEN pathway besides MoSep1,MoDbf2（MGG＿02757）and Mob1（MGG＿03151）in M.oryzae.When the three genes MoSEP1,MoDBF2 and MoMob1 were deleted separately,the mycelial growth rate slowed down and the aerial mycelia became thinner.The asexual reproduction ability was also seriously affected,not only the number of conidia decreased,but also the morphology of conidia was seriously distorted,with more than 75%conidia only have one septum.Compared with the wild-type strain,the germ tubes formed during conidial germination of the mutant strains（35）Mosep1,（35）Modbf2 and（35）Momob1 was significantly longer,and the appressorium formation was obviously delayed.The turgor pressure of appressorium was significantly lower than that in the wild type and the pathogenicity of three mutants was significantly reduced.These results indicate that MoSep1,MoDbf2 and MoMob1 play an important role in the growth,development and pathogenicity of M.oryzae.The nuclear division and septum distribution of the three mutants were different from wild type strains.More than 80%hyphae cell contain more than two nuclei in a cell,and the distance between the septum was different.During the process of appressorium formation,new septa were formed in the germ tube,and some nuclei remained the germ tube even after the appressorium was formed,and these were different from wild type.These results showed that the deletion of these three genes,separately,blocked the mitosis.Revealed that MoSep1,MoDbf2 and MoMob1 participate in the regulation of mitotic exit process of M.oryzae as in S.pombe.However,the deletion of MoSep1 did not affect the phosphorylation of MoDbf2,but decreased the phosphorylation level of MoMob1 which was different from S.pombe.We identified that the 43rd serine of MoMob1 was an important phosphorylation site dependent on MoSep1 through mass spectrometry analysis,and proved that MoSep1-dependent MoMob1 phosphorylation is important for vegetive growth,conidiation,and pathogenicity in M.oryzae.MoSep1-dependent MoMkk1 phosphorylation is vital for development and pathogenicity in M.oryzaePrevious results indicated that there is a relationship between MEN and CWI in M.oryzae,and MEN kinase MoSep1 interacted with CWI component MoMkk1.To further reveal the relationship between MEN and CWI,we tested the cell wall integrity of mutant strains（35）Mosep1,（35）Modbf2 and（35）Momob1 by some related experiments.The results showed that the protoplast release rate of these three mutants was significantly faster than the wild-type strain,and they were more sensitive to congo red and CFW.The distribution of primary chitin at the tip of mycelium was disordered.These results indicated that the deletion of MoSEP1,MoDBF2 and MoMOB1 genes damaged the cell wall integrity of M.oryzae.In order to further explore the relationship between MEN and CWI,we verified the interaction between CWI and MEN through yeast two hybrid method.Results showed only MoSep1 and MoMkk1 can interact with each other in these two pathways.Mn²⁺-Phos-tag gel detection and in vitro phosphorylation analysis confirmed that MoSep1 was involved in the regulation of phosphorylation of MoMkk1 in M.oryzae.There were 6 phosphorylation sites（19S,24T,125S,136S,139T,207T）of MoMkk1 that depended on MoSep1 were identified by mass spectrometry.The phosphorylation of these six amino acids is essential in the regulation of cell wall integrity and the exit from mitosis.The phosphorylation of MoMkk1 depended on MoSep1 plays an important role during the development of M.oryzae.In this paper,we found that there is a connection between MEN and CWI in M.oryzae which is mediated by MEN kinase MoSep1 phosphorylated CWI component MoMkk1.MoSep1-dependent MoMkk1 phosphorylation is vital for development and pathogenicity in M.oryzae.This work provides new evidence in responding to cell wall stress during the development and pathogenicity of M.oryzae.