Molecular Mechanisms Of ?b Protein In Suppressing Autophagy And ROS Burst To Facilitate Barley Stripe Mosaic Virus Infection

Autophagy is a eukaryotic mechanism that removes damaged or unwanted cellular materials,and is a conserved defense strategy against virus infection.Many animal viruses are known to evade or hijack the autophagy pathway to promote their infection or transmission,however,counter-defense strategies of plant RNA viruses to interfere with autophagy have not been reported.Reactive oxygen species(ROS)is one of the earliest cellular responses against pathogen infection.Although evidence suggests that ROS bursts are correlated with successful plant virus infection and disease development,the mechanism whereby ROS affects virus infection have not been carefully investigated.Barley stripe mosaic virus(BSMV)is a ss(+)RNA virus,the multifunctional yb protein encoded by RNA? plays multi-roles during virus infection.In this study,we will focus on the interactions between yb and the ATG7 and GOX host factors,and molecular mechanisms of yb protein in suppressing autophagy and ROS burst to facilitate BSMV infection.The levels of mRNAs encoding autophagy-related genes and the abundance of autophagosomes both decreased,whereas the accumulation of NBRl increased substantially during BSMV infection.We found that BSMV inhibits the autophagy pathway via activities of the yb viral suppressor of RNA silencing(VSR)protein,however the results revealed that the autophagy-inhibition function of yb protein is independent of its VSR activity.The ?b protein interacted directly with AuTophGy-related gene 7(ATG7)protein in vivo andin vitro,and mutagenesis experiments demonstrated that yb tyrosine 29 is required for the yb-ATG7 complex interactions,as mutant ybY29A failed to interact with ATG7.Co-IP,competitive pull-down,and ATG8/ATG8-PE detection assays verified that the interaction between yb and ATG7 is much stronger than that of ATG7-ATG8,which is required for host autophagy.Thus,?b protein is posited to inhibit autophagy by disrupting the interactions between ATG7 and ATG8 to facilitate virus replication and movement during BSMV infection.Moreover,we found that BSMV RNA accumulation was greater and the symptoms were exacerbated in the ATG7-orATG5-silenced plants.Furthermore,BSMV infection was inhibited in the ATG7-overexpressing plants,and the BSMVY29A mutant virus infections were diminished in symptom severity and viral accumulation.These results collectively demonstrate that autophagy has an antiviral role during BSMV infection.Taken together,these findings reveal that BSMV yb protein subverts autophagy-mediated antiviral defenses by disrupting ATG7-ATG8 interactions to promote BSMV infection,and provide evidence that ATG7 is a newly discovered target of pathogen effectors that functions in the ongoing plant defense and viral counter-defense arm race.In addition,we demonstrated that BSMV infection suppressed ROS bursts in N.benthamiana by DAB-and NBT-staining assays.These studies revealed that yb,but not other proteins encoded by BSMV,is responsible for suppression of ROS bursts that affect pathogenesis.A series experiments including BiFC,Co-IP,GST pull-down,and gel filtration analyses showed that yb can interact directly with glycolate oxidase(GOX)in vivo and in vitro.Additional in vitro and in vivo biochemical studies indicated that the inhibitory role of ?b in ROS bursts affect GOX enzymatic activity,leading to redox state disruption of peroxisomes.Silencing of GOX mRNA expression impaired BSMV infection in N.benthamiana by disrupting ROS metabolites.We propose a model whereby BSMV manipulates host metabolic processes by interfering with ?b-GOX interactions,and that these activities contribute to reduced plant anti-viral responses and suppression of virus infection.These new discoveries demonstrate that ?b inhibits autophagy by disrupting interactions between ATG7 and ATG8,and reduction of peroxisomal ROS generation by binding with GOX and blocking its enzyme activities.The findings also contribute to our knowledge about strategies used for host defense and pathogen counter-defenses,and increase our understanding of the novel functions of the ?b protein.