Functional Analysis Of Unfolded Protein Response Associated Genes MoHAC1 And MoIRE1 In Magnaporthe Oryzae

Rice is the most food crop in China,the staple food of nearly 60 percent of thepopulation is rice,thus the stability and safety of rice production relate to the nationalwelfare and the people’s livelihood.M.oryzae is the causal agent of rice blast,the mostdestructive disease of cultivated rice worldwide.Each year rice blast causes losses ofbetween 10 and 30%of the rice harvest,severely threats to world food safety.Currently,control measures of the rice blast is mainly used by cropping resistant cultivars andsupplemented by chemical pesticides.But the resistant cultivars decrease the resistance toM.oryzae due to easy variation of the pathogen races.Meanwhile,application of a singlechemical pesticides lead to anti-fungicide population formation result in the loss of controleffect of the rice blast.Therefore,the control of the rice blast disease is still a momentousproblem on rice production,and the detail studies of the pathogenesis of M.oryzae providea new strategy for disease resistance breeding and a candidate target for the development ofnovel fungicides for blast control.In this paper,we focused on functional analysis ofunfolded protein response(UPR)associated genes MoHAC1 and MoIRE1 during itsdevelopment and pathogenicity in M.oryzae,and the main results are showed as follows:We have previously showed that bZIP transcription factor(TF)MoApl and MoAtflmediate the oxidative stress response and are required for pathogenicity.Here,we report theidentification and characterization of a bZIP TF MoHac1,a homologous protein of Hacl inSaccharomyces cerevisiae.The MoHac1 protein contains a conserved bZIP domain,weobtained MoHAC1null mutant by gene knockout.Compared with the wild type strainGuy 11,the △Mohacl mutant showed a reduced growth rate in vegetative growth,theasexual development and pathogenicity were seriously impaired,the pathogenicity of△Mohacl mutant was significantly reduced on rice and barley leaves.In addition,MoHac1contribute to the resistance to the cell wall and osmotic stress inducer,but not the peritheciaand asci development,suggesting that MoHac1 play an important role in growth,asexual development and pathogenicity in M.oryzae.Homeostasis of the endoplasmic reticulum(ER)is important for cellular development,this procession is mediated by UPR.In yeast,the UPR is activated by the accumulation ofunfolded or misfolded protein,result in the unconventional cytoplasmic splicing of HAC1mRNA.The translated Haclp then moved into the nucleus to regulate the UPR-targetedgenes.The second chapter of this paper found that the △Mohacl mutant was hypersensitiveto the ER stress inducer,indicateing that MoHac1 contribute to ER stress response in M.oryzae.Similar to the yeast HAC1,the MoHAC1 mRNA harbors a 23 bp unconventional intron which would be spliced under ER stress condition,and then the translated protein moved into the nuclear to regulate the UPR-targeted genes.Meanwhile,UPR is activated during the asexual development and pathogenesis of M.oryzae.A mutant carrying several site-directed mutations in the unconventional intron was generated in the △Mohac1 background,and showed similar defects on conidiation and pathogenicity.Although deletion of MoHAC1did not affect the appressorium developed by hyphal tips,the mutant could not penetrate the surface cells of rice,suggesting that a fully functional UPR is required for conidiation and invasive hyphae growth in M.oryzae.We have previously showed that the bZIP TF MoHac1 mediate the UPR and is required for growth,asexual development and pathogenicity,but the function of upstream ER-stress sensor Irelp is still unknown.The third chapter of this paper reported the identification and characterization of ER transmembrane protein MoIre1,a homologous protein of Ire1p in S.cerevisiae.We generated a MoIRE1 null mutant by gene knockout.Compared with the wild type strain Ku80,the △Moirel mutant showed a reduced growthrate in vegetative growth,the conidia formation and pathogenicity were seriously impaired,the pathogenicity of △Moirel mutant was significantly reduced on rice and barley leaves.In addition,MoIre1 contribute to the resistance to the cell wall stress,oxidative stress andER stress inducer,and is essential for hydrophobic of hyphae surface.Furthermore,loss of MoIre1 result in an unspliceable MoHAC1 mRNA in response to ER stress.The results suggest that MoIre1 play an important role in growth and asexual development and providea comprehensive analysis of UPR in M.oryzae.In summary,UPR is essential for the development of M.oryzae,the UPR associated genes,MoHac1 and MoIre1,control the UPR and have important roles in growth,asexual development and pathogenisis of M.oryzae.