| Rice blast caused by Magnaporthe oryzae is one of the most devastating diseases in rice production,it causes substantial yield losses enough to feed 6000 million people every year,and poses a serious threat to global food security.Thus,it is urgent to elucidate the pathogenicity mechanism to formulate new strategies to control rice blast.The cell wall is a physical support to maintain the shape of fungal cells and also a barrier to resist environmental stress.As the first contact point,cell wall is also crucial in the interaction between fungus and plant.The cell wall structure of M.oryzae was remodeled under the synergistic action of hydrolases and synthases during the infection of rice.In this study,the cell wall of M.oryzae was selected as a target to elucidate the relationship between the proteins involved in cell wall remodeling（especially β-1,6-glucan component）and the development and pathogenicity of M.oryzae and its mechanism,so as to provide specific targets for screening new antifungal drugs.The results obtained in this paper are as follows:（1）Loss of the protein MoKre5 involved in β-1,6-glucan synthesis led to the cell wall remodeling of M.oryzae,thereby breaking homeostasis in the endoplasmic reticulum,resulting in the accumulation of reactive oxygen species and the decrease in virulence of the strain △Mokre5. β-1,6-glucan,as the crital component of cell wall,crosslinks mannoprotein, β-1,3-glucan and chitin in cell wall.Although β-1,6-glucan is crucial in cell wall,its synthesis remains unclear.In this study,six homologous genes were identified in M.oryzae by sequence alignment with the KRE genes in Saccharomyces cerevisiae,considering the effect of gene deletion on the content of β-1,6-glucan in S.cerevisiae and the different localization of genes in cells,four homologous genes were selected and studied;among which,MoKRE5 was the most important in the synthesis of β-1,6-glucan.Deletion of MoKRE5 fromM.oryzae resulted in cell wall remodeling,in the knockout strain △Mokre5,the content of β-1,6-glucan decreased by 60%,and the content of chitin increased by 26%,and the distribution of chitin in cell wall changed from apical concentrated distribution to diffuse distribution.The synthesis of melanin in cell wall was lost which produced albino hyphae.Furthermore,cell wall stress factor SDS and osmotic pressure stress factors inhibited the growth rate of △Mokre5;cell wall stress factor CFW affected the growth state of mycelia,and high concentration of CFW led to humidification occurred in the center of mycelia.MoKAR2 and MoSIL1 are two target genes in the endoplasmic reticulum unfolded protein pathway（UPR）.The deletion of MoKRE5 increased their expression levels by2.89 fold and 5.09 fold,respectively;the addition of ER inducer DTT increased their expression levels by 8.91 and 39.11 times in the wild-type strain Guy11,and 10.39 and73.04 times in the knockout strain,respectively,indicating that deletion of MoKRE5 activates the UPR pathway in the endoplasmic reticulum,and the addition of DTT further enhances the response of UPR pathway.After△Mokre5 was incubated with H2DCFDA,green fluorescence appeared in mycelia;after incubation with NBT,mycelia turned brown;however,mycelia of Guy11 did not change significantly,suggesting the loss of MoKRE5 induced the accumulation of intracellular reactive oxygen species.After incubation of △Mokre5 and propidium iodide（PI）,no red fluorescence appeared in mycelia,suggesting that cell wall permeability of △Mokre5 did not change.After 48 h culture,the content of malondialdehyde（MDA）in △Mokre5increased by 35%,indicating that reactive oxygen species producted led to peroxidation of the lipid in cell membrane.In addition,mitochondrial membrane potential fluorescent probe（JC-1）existed as a green fluorescent monomer in △Mokre5 cultured for 36 h,while a similar phenomenon appeared in Guy11 cultured for 48 h,suggesting that intracellular reactive oxygen species producted made the strain △Mokre5 entered the early stage of cell apoptosis in advance.At the same incubation time（12 h,24 h,36h and 48 h）,ATP content in △Mokre5 decreased about 45%compared with Guy11,suggesting that reactive oxygen species producted results in impaired part mitochondrial function.Compared with Guy11,△Mokre5 showed weak extracellular laccase activity,and the expression levels of eight genes related to laccase synthesis were down-regulated.Expression level of bilirubin oxidase gene（MGG＿08046）in △Mokre5 was up-regulated by 4.5 times after treatment with 1 mM H2O2,however,it increased 35 times in Guy11,indicating that deletion of MoKRE5 inhibited the expression of genes related to laccase synthesis,leading to a decrease in exocrine production.Compared with Guy11,△Mokre5 showed no catalase activity,and 10 out of 14 genes related to catalase synthesis were not expressed,and 4 genes had low expression levels.Treatment by 1 mM H2O2 made the expression level of peroxidase gene（MGG＿07871）in △Mokre5 increase by 3.9 times,made the expression of ligninase gene（MGG＿07790）in Guy11 increase by 15.8 times,indicating that deletion of MoKRE5 also inhibited the expression of genes related to catalase synthesis,leading to the decrease of the exocrine amount of catalase.Infection of △Mokre5 increased the expression of genes related to resistance in rice.Compared with rice leaves infected with Guy11,after 48 h of infection by△Mokre5,the expression level of AOS2 increased by 1.7-times,the expression level of PAD4 increased by 22%;after 72 h of infection,the expression level of PBZ1 and CHT1 increased by 1.1 times and 89%,respectively,the expression level of LOX increased by37%;after 96 h of infection,the expression level of PR1a increased by 42%.In addition,infection of △Mokre5 also increased the activity of defense enzymes related to disease-resistance.After 48 h of infection by△Mokre5,activity of phenylalnine ammonialyase（PAL）increased by 37%;after 72 h of infection,activity of superoxide dismutase（SOD）and peroxidase（POD）increased by 48%and 56%,respectively.These results suggested that the cell wall structure of △Mokre5 may be more easily recognized by rice,thus enhancing the defense of rice against M.oryzae.（2） β-1,6-glucanase MoGlu16 is involved in cell wall remodeling,and the dynamic regulation of hydrolysis and synthesis during cell wall remodeling is crucial for maintaining normal cell growth.In this study,the β-1,6-glucanase MoGlu16 from the GH30 family was identified from the genome of M.oryzae.Compared with the mycelia cultured in liquid culture for 1 day,expression level of MoGlu16 was increased 9.8 times and 10.8 times in liquid culture for 2 and 6 days,respectively;compared with the conidia,it was upregulated3.3 times after 48 h of infection,suggesting that MoGlu16 may be involved in remodeling of cell wall in mycelia.Compared to those of Guy11,growth rate of the knockout strain △Moglu16 decreased by 17%on CM medium,the ability of the infected mycelia to form branches in rice cells was weakened,the disease index of rice leaves infected by△Moglu16 decreased by 83.6%,the gene copy number of the strain in rice cells decreased by 97.8%;these results suggested that MoGlu16 was involved in the growth of invasive mycelia.Deletion of MoGLU16 resulted in a 13.7%decrease in the content of β-1,6-glucan and a 12.2%increase in the content of chitin in the alkali-insoluble cell wall;moreover,△Moglu16 showed the decreased tolerance to cell wall stressors and osmotic stressors,increased tolerance to cell wall degrading enzymes,indicating that deletion of the gene MoGLU16 led to cell wall remodeling.To further determine the function of MoGlu16,in this study,MoGlu16 was expressed heterologously by Pichia pastoris GS115.At p H 5.0 and 50℃,the purified MoGlu16exhibited the highest hydrolytic activity against pustulan,reaching 219 U/mg,and only0.002 U/mg against cell wall of M.oryzaae.In addition,incubation of high concentration MoGlu16（0.030μg/μl）with conidia significantly inhibited the formation of bud tubes and appressoria,the treatment of mycelia by MoGlu16 induced the accumulation of intracellular reactive oxygen species,and then stimulated the up-regulation of expression level of genes associated with cell wall polysaccharides synthesis.Among which,the expression levels of chitin synthase genes MoCHS5,MoCHS6 and MoCHS7 increased by 9.3,8.6 and 8.8 times,respectively,and the expression levels ofα-1,3-glucan synthase gene MoAGS2 increased by 4.2 times.These results suggest that the addition of MoGlu16 in vitro disrupts the dynamic balance between cell wall synthesis and hydrolysis,and affects the normal cell growth of strain.（3） β-1,3-glucanase MoGluB had high hydrolytic activity on insoluble glucan,and high concentration of MoGluB inhibited conidial germination and appressorium formation.Besides β-1,6-glucan, β-glucan in cell wall includes β-1,3-glucan. β-1,3-glucanases,the important hydrolases during fungal cell wall remodeling,can hydrolyze β-1,3-glucan to oligosaccharide.In this study,the β-1,3-glucanase MoGluB was identified from the genome of M.oryzae,sequence alignment revealed that MoGluB was a new member of the GH64-GluB-like subfamily of the GH64 family.Compared with the mycelia cultured in liquid culture for 1 day,expression level of MoGluB was increased 30.8 times and 9.5 times in liquid culture for 2 and 6 days,respectively;compared with the conidia,it was upregulated 6.1 times after 48 h of infection,suggesting that MoGluB was involved in the growth of mycelia.MoGluB showed high hydrolytic activity for insoluble glucan like yeast glucan and pachymaran,which was 8.18 U/mg and 2.16 U/mg,respectively.However,the hydrolytic activity of MoGluB against cell wall of M.oryzae was only 0.005 U/mg.High concentration of MoGluB can significantly inhibit conidial germination and appressorium formation,the minimum inhibitory concentration of MoGluB on the germ tube and appressorium was0.1μg/μl,and when the concentration reached 0.2μg/μl,appressorium formation was completely inhibited.The function of MoGluB was compared with that of β-1,3-glucanases from other sources（Gns6 from rice and Ac Glu A from the biocontrol bacterium Archangium sp.Ac19）.Compared with MoGluB,Gns6 and Ac Glu A belong to the GH16 and GH55 families,respectively,suggesting different evolutionary pathways of β-1,3-glucanases from different sources.The optimal substrate for Gns6and Ac Glu A was laminarin,and their hydrolysis activities were 1.20 and 112.34 U/mg,respectively,indicating that they were β-1,3;1,6-glucanases.Similar to MoGluB,Gns6also inhibited conidial germination and appressorium formation,while Ac Glu A did not;Gns6 protein with catalytic sites mutation lost both hydrolysis activity and inhibition;indicating that β-1,3-glucanases showed a nonlinear correlation between its antibacterial activity and hydrolytic activity.Hydrolysates of MoGluB showed immunostimulating activity.MoGluB hydrolyzed pachymaran into oligosaccharides mainly composed of laminaripentaose,and pretreatment of the hydrolysates induced the expression of resistance-related genes in rice plants.After pretreatment with 1600 mg/l hydrolysates for 12 h,expression level of PBZ1 and AOS2 increased by 62.23 times and 26.70 times,respectively;after pretreatment for 24 h,expression levels of PR1a and CHT1 increased 18.82 and 6.96 times,respectively;LOX increased 3.4 times,PAD4 and PAL increased 3.99 and 16.39 times,respectively.In addition,after pretreatment with 1600 mg/l hydrolysates for 24h,the contents of jasmonic acid and salicylic acid in rice increased by 25.5%and 26.7%,respectively.The rice pretreated with high concentration hydrolysates for 24 h exhibited control efficiency of 68.42%to M.oryzae.In conclusion,the decrease in content of β-1,6-glucan from cell wall,imbalance of hydrolysis and synthesis in cell wall remodeling and activation of rice immune activity inducted by cell wall decomposition products would lead to the reduction of pathogenicity of M.oryzae.In this paper,the relationship between proteins associated with cell wall and pathogenicity of M.oryzae and their mechanism were preliminarily clarified,laying the theoretical foundation for the development of novel fungicides. |
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