Mechanism Of Downregulation Of Light-harvesting Complex Ⅱ Inducing ROS-mediated Defense Against TuMV Infection In Nicotiana Benthamiana

Turnip mosaic virus（Tu MV）,as the classical member of genus potyvirus which is the largest genus of plant RNA viruses and causes huge economic losses,has an extremely broad host range that comprises mainly of Brassica species and approximately 320 species of dicotyledonous plants which belong to at least 156 genuses.Virus disease caused by Tu MV seriously affects the quality and yield of vegetables products,has represented a serious threat to the agricultural production.Identification the interaction between Tu MV and host plant,exploration the relative key host factors of Tu MV and potential resistance genes in the host,can deepen the understanding of interaction between virus and host,the mechanism of highly pathogenic of virus and resistance mechanism of host,and provide theoretical basis for managing of biotic threats as well as have important significance for disease control and safety of vegetable production.In our previous research,Nicotiana benthamiana plants which were infected with Tu MV were sequenced by transcriptome,the data was analyzed and a gene which belongs to light-harvesting chlorophyll a/b complex protein of photosystem II named Nb LHCB3 was screened and was significantly downregulated.Previous researches suggest that LHCB3 plays important roles in photosynthesis like modulating the rate of state transition,distributing the excitation energy and charge separation.However,the functions of LHCB3 and even whole LHCBs and LHCAs in plant immunity have not been well investigated.Basing on the hypothesis that whether LHCB3 involves in suppressing the infection of virus,we aimd at the effect of Nb LHCB3 on Tu MV infection,and attempted to explore the mechanism.In this study,we found that accumulation of both Nb LHCB3transcripts and Nb LHCB3 protein showed a significant decrease in the Tu MV-infected plants,which further proved the transcriptional data.And Tu MV NIa is responsible for downregulation of Nb LHCB3 in our further study.When Nb LHCB3 was systemically silenced by the technique named tobacco rattle virus-induced gene silencing,systemic infection of Tu MV was inhibited.Further research found that H₂O₂ was over-accumulated in Nb LHCB3-silenced plants.Genes which involved in ROS production encoding like AO and NADPH oxidase were upregulated.By contrast,genes participate in ROS scavenging like CAT,SOD and GR were all downregulated.All these results suggest that ROS have an effect on suppressing infection of Tu MV in Nb LHCB3-silenced plants.To explore the relation between ROS,Nb LHCB3 and Tu MV,we treated the Nb LHCB3-silenced plants with DPI（a chemical inhibitor which works in ROS production）and DMTU（an activating agent which works in ROS scavenging）and found that the treatment of chemical inhibitors promoted virus infection.We then co-silencing of Nb LHCB3with genes involved in ROS production and found that the plants showed less ROS and compromised the resistance of plants to Tu MV but plants which co-silencing of Nb LHCB3 with genes in the ROS scavenging pathway represented more ROS and increased resistance to the virus.To further determine the biological function of Nb LHCB3,the CDS of Nb LHCB3 was amplified and was linked to a vector which was drove by 35S promoter,and then stable transgenic lines of N.benthamiana overexpressing Nb LHCB3 was obtained by transgenic technology named leaf disk transformation which was mediated by Agrobacterium tumefaciens.The transgenic lines accumulated less ROS than wild type,and were proved to be susceptible to Tu MV infection.All these results illustrate that Nb LHCB3 mediates susceptibility to Tu MV by negatively regulated ROS production.Taken together,the results presented here indicate that downregulation of Nb LHCB3 is involved in defense against Tu MV by inducing ROS production.