Molecular Mechanism Research Of MdATG8i In Regulating Resistance To Valsa Mali In Apple

Autophagy pathway is an evolutionary conservative material turnover process in eukaryotes.Autophagy function in cells is mainly reflected in the degradation of toxic substances,inactivated proteins and damaged organelles,promoting the recycling or repositioning of intracellular components to maintain cell homeostasis and cope with the stress response of cells.Remarkably,autophagy has emerged as a key defense mechanism against invading pathogens in a variety of ways.In turn,adapted pathogens have evolved to interfere with autophagic machinery by secreting host cell-entering effector proteins.In this study,based on the apple-Valsa mali interaction system,we analyzed the function of autophagy related protein Md ATG8 i in regulating the resistance to Valsa mali by targeting elongation factor Md EF-Tu,and revealed the molecular mechanism of the effector protein Vm1G-1794 involved in the above process during the Valsa mali infection.The main results are as follows:1.Md ATG8 i targets and degrades the susceptible factor Md EF-Tu to improve the resistance to V.mali.The overexpressing Md ATG8 i transgenic apples accumulated more autophagosomes,and showed higher autophagic activity to improve resistance after inoculation with V.mali.Md ATG8 i interact with Md EF-Tu,Md EF-Tu formed protein aggregates and was ubiquitinated and degraded during the V.mali infection.Further studies showed that the degradation of Md EF-Tu protein was accomplished through autophagy.The resistance of Md EF-Tu overexpressed transgenic apples to V.mali was reduced.In addition,the interaction between Md ATG8 i and Md EF-Tu results in a change of the localization of Md EF-Tu proteins.2.Valsa mali effector protein Vm1G-1794 interferes with the interaction between Md ATG8 i and Md EF-Tu.The effector Vm1G-1794,which could interact with Md ATG8 i,was screened by yeast two-hybrid.Experiments showed that Vm1G-1794 contained secretory signal peptides,which was highly expressed during the V.mali infection,and transient expression of Vm1G-1794 could inhibit the ROS production and callose deposition in N.benthamiana leaves.Further protein interaction experiments showed that both Vm1G-1794 and Md EF-Tu interact with Md ATG8 i through AIM(ATG8 interaction domain),and Vm1G-1794 competitively binds Md ATG8 i to release Md EF-Tu from MdATG8i-Md EF-Tu complex.3.Valsa mali effector protein Vm1G-1794 targets Md ATG8 i to inhibit autophagy activity in apple.Transient expression of Vm1G-1794 and Md ATG8 i in N.benthamiana leaves,we found that the interaction between effector protein Vm1G-1794 and Md ATG8 i could inhibit the autophagy activity by detecting the autophagy flux of MdATG8i-labeled fluorescent protein.The overexpression of Vm1G-1794 in apple calli accumulated fewer autophagosomes and showed a lower level of autophagy after inoculation with V.mali.Thus,the scavenging ability of autophagy on Md EF-Tu protein aggregates was reduced to promote the V.mali infection.The knockout mutant of Vm1G-1794 in V.mali induced ROS burst and callose deposition were reduced,triggering stronger autophagy activity during the infection.The results showed that Vm1G-1794 mutant significantly reduced the pathogenicity of V.mali.