Studies On The Biological Functions Of The ESCRT In The Rice Blast Fungus

Magnaporthe oryzae is the initiator of the global rice disease that causes rice blast,and China is also a high incidence area of rice blast.Rice blast diseases are seriously threatening the output and quality of rice and affecting our country’s food security.The infection process and pathogenic mechanism of M.oryzae are very typical among filamentous pathogenic fungi and M.oryzae has become a model organism for studying molecular biology and pathogenic mechanism of filamentous fungi.The late endosomes containing multiple intralumenal vesicles(ILVs),known as the multivesicular body(MVB),is first observed in the nervous system in the 1950 s.In the early 21 st century,the identification of endosomal sorting complexes required for transport(ESCRT)leads to the discovery of MVB at the molecular level.After the formation of MVB,it will fuse with lysosome to degrade the cargo in ILVs,and this process is the down-regulation pathway of various membrane binding receptors.Therefore,ESCRT plays an extremely important role in maintaining intracellular protein levels,signal transduction,metabolism,growth and development.ESCRT is composed of four sub-complexes,ESCRT-0,Ⅰ,Ⅱ and Ⅲ,and key components and regulatory factors,such as Vps4 and Vta1.In this paper,ESCRT-0(Mo Hse1 and Mo Vps27)and the cofactor Mo Ist1 of ESCRT-III are systematically analyzed,and their regulatory mechanism in the pathogenesis of rice blast fungus is clarified.The deletion of Mo Hse1,Mo Vps27 and Mo Ist1 lead to the slow growth of rice blast fungus,serious damage to sporulation and significant decrease of pathogenicity.High pressure freezing microscope observation shows that ΔMohse1,ΔMovps27 and ΔMoist1 are basically unable to form MVB and ILVs,resulting in the degradation of many proteins,notably the tyrosine kinase receptor EGFR,to be blocked.Excessive accumulation of EGFR causes excessive activation of downstream mitogen activated protein kinase(MAPK),and Western blot analysis shows that the phosphorylation levels of Pmk1,Mps1 and Osm1 are significantly up-regulated.Transmission electron microscope observation founds ΔMohse1,ΔMovps27 andΔMoist1 vacuoles show fragmentation.Yeast two-hybrid and Pull-down test analysis show that Mo Vps27 interacts with autophagosome formation related protein Mo Atg5,and autophagosome and vacuole fusion associated protein Mo Vps18.Meanwhile,Western blot analysis shows that autophagosomes are excessive accumulation inΔMohse1,ΔMovps27 and ΔMoist1,and autophagy is abnormal.In addition,the plate stress test founds that ΔMohse1,ΔMovps27 and ΔMoist1 is sensitive to the endoplasmic reticulum(ER)stress inducer dithiothreitol(DTT).Western blot detection founds that,the degradation of the endoplasmic reticulum autophagy(ER-phagy)marker protein GFP-Mo Sec62 in ΔMohse1,ΔMovps27 and ΔMoist1 is reduced.Yeast two hybrid and Pull-down experiments have found that ESCRT-0 interacts with COPII(the subunit Lst1 of COPII has been shown to target ER degradation together with the ER-phagy receptor Atg40),indicating that the deletion of Mo Hse1,Mo Vps27,and Mo Ist1 affects ER-phagy.To sum up,the research shows that ESCRT complex participates in many processes such as MVB generation,MAPK pathway,autophagy,ER-phagy,and plays an important role in the pathogenesis of rice blast fungus,providing new insights into the pathogenesis and molecular mechanism of autophagy of M.oryzae.