Mechanistic Characterization Of Sumoylation And GPI-Anchor Modification During The Infection Of The Rice Blast Fungus

Protein post translational modifications(PTMs)play crucial roles in altering proteinprotein interactions,changing protein intracellular localization,or directing changes in the activities of the protein to the target protein.Recently,many studies have reported how plant fungal and bacterial pathogens use these PTMs to facilitate development,stress response,and host infection.However,the mechanisms of how PTMs regulate protein functions needs more in-depth research.In this study,we systematically analyze the function and mechanism of glycosylphosphatidylinositol(GPI)anchoring modification and small ubiquitin like modification(SUMO)during appressorium-mediated infection as well as stress responses in the rice blast fungus.Here are the main results:(1)The mechanism of GPI anchoring modification regulating appressorium penetration and immune escape of Magnaporthe oryzae was revealed.GPI anchoring modification is a main type of glycosylation.The target protein is fixed on the cell surface to take functions or transported to extracellular by GPI anchor.however,how this PTM participates in the regulation of pathogenicity of plant pathogens has not been well studied yet.In this research,we revealed that GPI anchored proteins can take a part in the integrity of cell wall structure and protect M.oryzae’s PAMPs from host immunity for the first time,thus attack the host successfully.First,we observed that GPI anchored proteins were located on the cell wall at different infection stags of M.oryzae,indicating that GPI anchored proteins take functions in maintenance of cell wall.Then we knocked out a key ethanolamine phosphate transferase GPI7 in the process of GPI anchor synthesis,causing GPI anchor modification was blocked in Δgpi7 deletions.The biological phenotype determination found that deletion of GPI7 resulted in defects in vegetative growth,cell division cycle,conidiation,host penetration and pathogenicity,indicating that GPI anchor modification can affect the appressorium mediated pathogenic process of M.oryzae.TEM observation showed the cell wall integrity of the conidia and appressorium in Δgpi7deletions were impaired.Further studies revealed Δgpi7 deletions were more sensitive to cell wall inhibitors compared to WT and were easier to hydrolyzed into protoplasts by lysing enzyme,which indicated that GPI anchored proteins were very important in the integrity of cell wall.In order to figure out how GPI anchored protein involved in pathogenicity regulation,we treated with HF pyridine to remove the GPI anchored protein on the surface of cell wall of WT strain,resulting in chitin and β-1,3-glucan were exposed to the host immune system and triggered the accumulation of reactive oxygen species in infected host cells.Interestingly,we found chitin and β-1,3-glucan were exposed to the host immune system in the Δgpi7 deletions too,indicating that GPI anchored proteins on the cell surface were indeed involved in host immune escape.Proteomic prediction shows that most GPI anchored proteins are secretory proteins and functional proteins on cell wall,including Gel family.We found that GPI anchoring modification can regulate the subcellular localization of Gel proteins to the cell wall,thus promote the penetration of appressorium.Interestingly,Gel3-GFP was discovered to locate at actin ring in appressorium.After GPI anchoring was blocked,Gel3-GFP could not form a ring,so its ability to penetrate the host decreased.Finally,it was found that GPI anchoring modification affected the secretion of effectors through BIC structure,but did not affect the secretion of effectors through traditional vesicles.In a word,a new mechanism was illustrated that GPI anchoring modification could promote the invasion of M.oryzae into host cells by affecting cell wall integrity and evade host immune recognition in this study.(2)The mechanism of SUMOylation regulating the pathogenicity of Magnaporthe oryzae,especially the formation of Septin ring at appressorium stage and the secretion of effectors,was systematically analyzed.Small ubiquitin like modification(SUMO)is another type of PTMs and shares similarity with the ubiquitination modification.In M.oryzae,only one SUMO protein called SMT3 is identified,and their conjugation to substrates occurs through a related enzymatic cascade involving the sequential action of an E1 activating enzyme coded by AOS1 and UBA2,an E2 conjugating enzyme coded by UBC9 and an E3 protein ligase(SIZ1).In this study,all genes involved in sumoylation were knocked out,and biological phenotypes of SUMO pathway gene deletions showed slow vegetative growth,weakened sporulation ability,abnormal conidia morphology,abnormal cell cycle during appressorium maturation,slow infection process and serious decline of pathogenicity,which indicated that sumoylation played an important role in these physiological processes.Besides,we found sumoylation have functions in regulating diverse stress responses.Specifically,SUMO pathway gene deletions were extremely sensitive to various environmental stresses,and a higher level of sumoylation was detected in the total protein of WT,as well as up-regulated expression levels of SUMO genes under different environmental stresses.Affinity purification from SMT3 interacting proteins identified 940 putative SUMO substrates,many of which were reported to be involved in pathogenicity.Bioinformatics analysis revealed that sumoylation is involved in regulating a variety of signal transduction pathways and cell metabolism,including glucose / lipid metabolism,amino acid synthesis,stress response,cell cycle regulation,autophagy,transcription regulation and other biochemical processes.These data show that there is a complex regulatory network between sumoylation and a variety of signal pathways,thus leading to the defects of various biological phenotypes in SUMO pathway gene deletions.More interestingly,we found four Septins involved in the penetration of appressorium to host are sumoylated proteins.Mutation of consensus sumoylation sites in each Septin protein resulted in reduced virulence to the host and dislocation of Septins in appressorium.Moreover,sumoylation is also involved in extracellular secretion of different effectors.InΔsmt3 mutant,the effectors AVR-Pia and AVR-Pizt secreted through BIC structure and the effector Slp1 secreted by traditional vesicles could not be secreted normally.All in all,this study systematically explained the important functions of SUMOylation in the vegetative growth,virulence development,signal transduction,stress response and host infection in the rice blast fungus.(3)The regulation mechanism of sumoylation to Pmk1 MAPK signal pathway was clarified.Pmk1 MAPK signaling pathway regulates appressorium formation,host penetration and the growth of late infection hyphae of Magnaporthe oryzae.This study suggested that K347 site of PMK1 protein is involved in the regulation of pathogenicity in M.oryzae in dependent of sumoylation.SMT3 protein interacted with PMK1 protein directly,indicating that PMK1 was indeed the target protein of sumoylation.Proteomics predicted that K347 of PMK1 protein was the sumoylation site,when the lysine was mutated to arginine without sumoylation,the level of PMK1 sumoylation decreased.Phenotypic analysis showed that the mutation of K347 of PMK1 led to the obstruction of appressorium mediated penetration and the expansion of late infection hyphae,resulting in the reduction of pathogenicity,which indicated that K347 sumoylation site of PMK1 was very important for its regulation of pathogenicity.In addition,PMK1-GFP gradually gathered in the nucleus during appressorium maturation,but the K347 mutation of PMK1couldn’t located in nucleus in its mutation strains and slightly expressed after sumoylation was blocked,indicating that K347 of PMK1 is very important for its stability and functional localization.Furthermore,the phosphorylation of PMK1 increased along with K347 of PMK1 mutated.In general,SUMOylation of PMK1 is of great significance to maintain its own stability,subcellular localization,and pathogenicity regulation.In conclusion,GPI anchoring modification in M.oryzae is involved in maintaining cell wall integrity,protecting PAMPs from host immune recognition.Sumoylation regulated the stress response,growth differentiation and pathogenicity of M.oryzae by coordinating many signal pathways and biochemical processes.