Homeostasis Of Cell Wall Integrity Pathway Phosphorylation Regulates The Growth And Pathogenicity Of Rice Blast Fungus Magnaporthe Oryzae

The rice blast is the destructive fungal disease that caused by Magnaporthe oryzae,which leads to a loss of more than 3 billion kg of rice in China every year,and poses a huge threat to the food security of the world.The current control measures including cultivate disease-resistant varieties and application of fungicide.Given to the rapid variation in the field,it is easy to cause resistant to disease-resistant varieties or the drugs.Therefore,to generate such smart plants an in-depth knowledge of the molecular requirements of pathogens to cause disease is essential.The cell wall is the first barrier between fungi and the outside world,which plays an important role in recognizing external signals,maintaining cell morphology and ensuring its internal environmental homeostasis.When exposed to external and host cell wall stress,fungi rapidly activate the cell wall integrity(CWI)signaling pathway,which upregulates the transcriptional quantity of downstream genes.Previous research in our lab found that The CWI signaling pathway are composed of MoMckl,MoMkk1 and MoMps1,which can transmit extracellular signals into the cell through cascade phosphorylation to control pathogenicity and respond to cell wall stress of M.oryzae.Although protein kinases in the CWI signaling pathway of this pathogen have been well studied.However,the mechanism by which that regulates the signaling pathway turn off remains unknown.Here,we identified several proteins that interacts with MoMokk1,including members of type 2C protein phosphatases(PP2C)MoPtc1/2 and a histone chaperone MoNap1.The further study elucidated the molecular mechanism that the balance of phosphorylation level regulated by these interacting proteins which involved in CWI signaling pathway regulate the pathogenicity of M.oryzae.The main results are as follows:Protein phosphatases MoPtc1 and MoPtc2 regulating the growth,development and pathogenicity of M.oryzae.In this chapter,five protein members of PP2C family(MoPtc1,MoPtc2,MoPtc5,MoPtc6 and MoPtc7)were identified.PP2C deletion mutants were obtained by homologous recombination technique.Among these mutants,only ΔMoptcl andΔMoptc2 caused defects on the pathogenicity of M.oryzae.Phylogenetic tree construction and yeast covering analysis showed that MoPtc1 and MoPtc2 were highly similar to PP2C domains of yeast Ptc1 and Ptc2/Ptc3.The transcriptional levels of MoPTC1 and MoPTC2 in different growth and development stages of M.oryzae were detected by qRT-PCR,and the results showed that MoPTC1 expression did not change significantly in the infection stage,while MoPTC2 expression significantly decreased during infection,suggesting that these PP2C may play a distinct role during the interaction between M.oryzae and rice.In order to further analyze the biological functions of MoPtc1 and MoPtc2,double mutant strains ΔMoptclΔMoptc2 was first obtained.The mycelia growth and conidiation ofΔMoptcl and ΔMoptc2 were analyzed.These mutants showed a significant reduction in vegetative growth than Guy11,and which produced approximately 70%fewer conidia than Guy11,while the ΔMoptcl ΔMoptc2 double mutant failed to produce any conidia,indicating that MoPtc1 and MoPtc2 are important for the vegetative growth and conidiation of M.oryzae.In order to further elucidate the mechanism of their regulation of pathogenicity,intracellular turgor pressure and the infection in rice leaf sheath cells were detected respectively in these mutants.And the results suggested that MoPtc1 and MoPtc2 play an important function in turgor generation and IH growth.MoAtg8 is a marker protein of autophagy,by which autophagy capacity can be determined by analyzing its degradation rate.As autophagy is an important process during infection,we also evaluated the autophagy of the ΔMoptcl,ΔMoptc2 and ΔMoptclΔMoptc2 mutants of M.oryzae.Autophagy increased in ΔMoptc1,ΔMoptc2 and ΔMoptc1ΔMoptc2 than Guy11,suggesting that MoPtc1 and MoPtc2 were involved in the non-selective autophagy pathway of M.oryzae.We identified that MoPtc1 and MoPtc2 interact with MoMkk1,a key component of CWI signaling pathway,by yeast-two-hybrid(Y2H)and co-immunoprecipitation(co-IP).In order to detect whether they are involved in MoMkk1 by dephosphorylation,phosphorylation relationship between MoPtc1,MoPtc2 and MoMkk1 was detected by Mn2+-Phos-Tag and fluorescence detection in tube(FDIT)analysis.The results showed that the levels of phosphorylated MoMkk1 were significantly increased in the ΔMoptcl and ΔMoptc2 mutants,suggesting that MoPtc1 and MoPtc2 function on the dephosphorylation of MoMkk1.Based on the finding that MoPtc1 and MoPtc2 dephosphorylate MoMkk1,we speculated thatΔMoptc1 and ΔMoptc2 mutants might exhibit continuous activation of MoMkk1 phosphorylation that consequently results in pathogenicity defect.Mycelial pellets of theΔMomkk1 MoMKK1DD mutant were inoculated on the wounded rice leaves for 4 days,limited lesions were observed in comparison to the wild type,suggesting that continued activation of MoMkkl phosphorylation causes defect on the pathogenicity of M.oryzae.Further,co-IP analysis and Microscale Thermophoresis(MST)showed that the affinity between MoPtc1 and MoMkk1 was increased but the affinity between MoPtc2 and MoMkk1 was decreased under CWI stress.This finding suggested that MoPtc1 and MoPtc2 regulated the dephosphorylation of MoMkkl by an individual pattern under CWI stress.Ser179 in MoMkkl was further identified as the phosphorylation site depending on MoPtc2 by phosphorylation mass spectrometry.Analysis of vegetative growth,conidiation and pathogenicity showed the mutant exhibited defects on vegetative growth,conidia formation and pathogenicity,indicating that the phosphorylation of Ser179 of MoMkkl is important in M.oryzae.We also identified the interaction between histone chaperone MoNap1 and MoMkkl by Y2H and co-IP analysis.As the previous work showed that deletion of MoNAP1 caused defects on pathogenicity,the phosphorylation level of MoMps1 in ΔMonap1 was detected.And the result showed that the phosphorylation of MoMps1 was decreased significantly in the mutant than Guy11.Phosphorylation of MoNap1 in ΔMonapl and ΔMomkk1 was further analyzed by Mn2+ Phos-Tag and the results indicated that the phosphorylation of MoNap1 was independent to MoMkk1.In order to further analyze the mechanism by which MoNap1 regulates the pathogenicity of M.oryzae,the nuclear export-signal(NES)deletion mutation of MoNap1 was obtained.The pathogenicity of NES deletion mutant strain showed defect on the pathogenicity of M.We further mutated the predicted phosphorylated site on tyrosine 328.Activated mutation(Y328D)led to the accumulation of MoNap1 in the nucleus and also a rescue in the pathogenicity of ΔMonap1,suggesting that phosphorylation of MoNap1 regulated the pathogenicity of M.oryzae.In the nucleus,MoNapl interacted and histone H3 and regulated the acetylation level of H3K9,these results indicated that MoNap1 positively regulate the CWI signaling pathway,significantly regulate the acetylation level of H3K9 and regulate the pathogenicity of M.oryzae.In summary,we identified multiple regulators of MoMkkl including MoPtc1,MoPtc2 and MoNap1.MoPtc1 and MoPtc2 negatively regulate the phosphorylation level MoMkk1 which is important for pathogenicity and response to CWI stress of M.oryzae.MoNap1 positively regulates the phosphorylation level of CWI and the acetylation level of histone H3K9,thus regulating the pathogenicity of M.oryzae.These results not only provided new evidence to reveal the response of M.oryzae to CWI stress from environment and host,but alsoprovided theoretical basis for screening the drug targets based on CWI pathway of M.oryzae.