Molecular Mechanism Of Cell Death Induced By The RepA Protein Of Mulberry Mosaic Dwarf-associated Virus In Tobacco Plants

Geminiviruses are single-stranded circular DNA viruses that are encapsidated in germinate particles,causing devastating damage to tomato,cassava,cotton and other herbal plants in more than 50 countries and regions around the world.In recent years,with the development of high-throughput sequencing technology,new geminiviruses have been identified from many perennial woody plants,including citrus,grapevine,mulberry and apple trees.With a relatively small genome and limited coding capacities,geminiviruses rely on complex interactions with plants to complete their life cycles,such as replication,transcription and movement.Therefore,studying the interaction between plants and geminiviruses are crucial in understanding the mechanism of plant resistance to virus infection and the pathogenesis of viruses.Mulberry mosaic dwarf-associated virus(MMDaV)is a novel monopartite geminivirus that has been isolated from mulberry trees showing mosaic and dwarf symptoms.Because of its unknown biological properties and pathogenicity,MMDaV was classified as a tentative species of the family Geminiviridae by the International Committee on Virus Classification.In this research,we took advantages of Nicotiana benthamiana,a model plant for studying the interaction between plants and viruses,to study the interaction between MMDaV and plants as follows:Firstly,the infectious clone of MMDaV was constructed and inoculated into N.benthamiana plants.It was found that MMDaV infection caused hypersensitive response-like cell death in inoculated N.benthamiana leaves at three days post inoculation.Furthermore,V1,V2,V3,V4,V5 encoded on the virion strand,and C1(RepA)and C2(Rep)encoded on the complementary strand of MMDaV,were then constructed into the GFP-tagged plant binary expression vector.Agrobacterium-mediated transient infiltration of the constructs into N.benthamiana leaves showed that the expression of RepA induced cell death in N.benthamiana leaves.In order to identify the functional domains that are responsible for RepA-induced cell death,bioinformatics analysis was carried out to analyze the sequences and structure of RepA,and eight deletion mutants of RepA were constructed.We found that the RCA motif of RepA was required for RepA to induce cell death in N.benthamiana plants.Subcellular localization analysis of RepA and its mutants using confocal laser microscopy showed that RepA was located in the nucleus of the epidermal cells of N.benthamiana leaves,and the nuclear localization of RepA might be related to the cell death induced by RepA.In plants,several signaling pathways,such as MAPK cascade and jasmonic acid,are involved in plant resistance to diverse pathogens.In order to screen the host factors that are involved in RepA-induced cell death,virus-induced gene silencing was used to silence NDR1,RAR1,COI1,CTR1,NTF6,WRKY1,NPR1 and MEK2 in N.benthamiana plants,respectively.It was found that the silencing of the seven genes,including NDR1,RAR1,COI1,CTR1,NTF6,NPR1 and MEK2,did not affect RepA-induced cell death in N.benthamiana plants,whereas the silencing of WRKY1 inhibited RepA-induced cell death.Infiltration of the infectious clone of MMDaV into WRKY1-silenced N.benthamiana plants showed that silencing of WRKY1 also inhibited cell death caused by MMDaV infection.Real-time quantitative PCR was then used to analyze the expression level of WRKY1 after inoculation of RepA and MMDaV.It was found that either the expression of RepA or the infection of MMDaV significantly increased the expression of WRKY1.We speculated that the expression of RepA could induce cell death by activating the expression of WRKY1.