The Role Of Geminivirus Betasatellite In Begomovirus-Plant-Whitefly Tripartite Interactions

Geminivirus is a group of plant single-stranded circular DNA viruses that occur widely around the world.Among them,Begomovirus is the largest genus and cause serious damages.Begomoviruses are transmitted by whitefly Bemisia tabaci,and they can be classified into monopartite and bipartite virus according to their genome components.Bipartite begomoviruses contain two single-stranded circular DNA molecules,DNA-A and DNA-B.Monopartite begomoviruses contain only a single stranded circular DNA molecule,some of which are associated with betasatellites.Plant mediated begomovirus-whitefly interactions play significant roles in plant viral epidemics and whitefly invasions.?C1 protein encoded by betasatellite,the key factor of tripartite interactions,can inhibit plant defense response to whtifly through protein-protein interaction.Geminivirus betasatellites mainly cluster into two clades according to phylogenetic relationship,those isolated from plant species belonging to the family Malvaceae and those associated with other families.At present,most studies about betasatellite in tripartite interactions is focused on Tomato yellow leaf curl China betasatellite(TYLCCNB)associated with non-Malvaceae plants.However,there are few studies on betasatellite associated with Malvaceae plants.Cotton leaf curl Multan virus(CLCuMuV),associated by Cotton leaf curl Multan betasatellite(CLCuMuB),is one of the most important pathogens causing cotton leaf curl disease.CLCuMuB and TYLCCNB belong to different evolutionary clades.Here,we established a model containing begomovirus,tobacco and whitefly,for the analysis about the diverse functions and molecular mechanisms of CLCuMuB and TYLCCNB in tripartite interactions.The main results of study were as follows:(1)Difference of infection characteristics and amino acid sequence between CLCuMuB and TYLCCNBCLCuMuV/TYLCCNB or TYLCCNV/CLCuMuB could not induce the symptoms of begomovirus in Nicotiana benthamiana.TYLCCNB and CLCuMuB belong to different clades according to the phylogenetic tree analysis,and their amino acid X sequences were totally different.(2)CLCuMuB ?C1 was the key factor in plant mediated interaction between CLCuMuV/CLCuMuB and whitefly Asia ? 1CLCuMuV/CLCuMuB is mainly transmitted by B.tabaci Asia ? 1.Biological assays showed that the survival rate and fecundity of B.tabaci Asia ? 1 were both enhanced on CLCuMuV/CLCuMuB infected N.tabacum and CLCuMuB ?C1transgenic plants than controls.However,survival rates had no difference between nonviruliferous and viruliferous whiteflies fed by artificial diet.Olfactory selectivity test showed that whiteflies tended to CLCuMuV/CLCuMuB infected N.tabacum plants and CLCuMuB ?C1 transgenic plants.Co-infection of CLCuMuV/CLCuMuB and B.tabaci Asia ? 1 could significantly increase the content of salicylic acid(SA)in N.tabacum.The survival rate of whitefly was increased by the treatment of Me SA on tobacco,while decreased by the treatment of MeJA.(3)CLCuMuB ?C1 inhibited plant defense against B.tabaci by interacting with NtSBT1.7N.tabacum subtilisin-like protease 1.7(NtSBT1.7)was identified as putative CLCuMuB ?C1 binding protein via yeast two-hybrid system.The full-length interaction between CLCuMuB ?C1 and NtSBT1.7 was confirmed by bimolecular fluorescent complimentary(BiFC),pull-down and yeast two hybrid assay.NtSBT1.7belong to serine proteases,localized to plant cell membrane and mainly expressed in vascular bundle tissues.The expression of GUS driven by NtSBT1.7 promoter was affected by whitefly infestation,mechanical damage and MeJA treatment.B.tabaci Asia ? 1 laid more eggs on NtSBT1.7-silenced tobacco plants,and NtSBT1.7 positively regulated the transcript level of JA response gene PDF1.2.N.tabacum prepro Hydroxyproline-rich Systemin B(NtpreproHypSysB)is a precursor protein of tobacco systemin.The transcript level of NtpreproHypSysB could be induced by whitefly infestation.B.tabaci Asia ? 1 performed better on NtpreproHypSysB-silenced tobacco plants,and NtpreproHypSysB also positively regulated the transcript level of PDF1.2.Then,the interaction between NtSBT1.7 and NtpreproHypSysB was confirmed by BiFC and subcellular co-localization assay.CLCuMuB ?C1 could interfered the interaction between these two proteins.(4)TYLCCNB ?C1 inhibited plant defense against B.tabaci by interacting with Nt SKP1The interaction between TYLCCNB ?C1 and N.tabacum S-phase kinase associated protein 1(Nt SKP1)was confirmed again via GST pull-down assay based on the previous study in our lab.B.tabaci MEAM1 performed better on Nt SKP1-silenced tobacco plants,and Nt SKP1 positively regulated the transcript level of JA-related genes MYC2 b and PDF1.2.In addition,Nt SKP1 positively regulated the degradation of JAZ1 protein,an inhibitor of JA signaling pathway,thus initiating JA signal transduction.And TYLCCNB ?C1 could interfere the degradation of JAZ1 protein.In conclusion,our study confirmed the plant-mediated mutualism between CLCuMuV/CLCuMuB and B.tabaci Asia ? 1 and verified the key role of CLCuMuB?C1 protein in this tripartite interaction.At the same time,our results revealed the molecular mechanism about how CLCuMuB ?C1 and TYLCCNB ?C1 inhibited plant defense against whitefly through protein-protein interaction.Our results enriched the tripartite interactions about CLCuMuV/CLCuMuB and provided important ecological insights for the viral epidemics and insect invasions.In addition,our results supplemented the potential molecular mechanism network of betasatellite in tripartite interactions,which provided valuable reference for the study on molecular breeding of resistance crop varieties.