The Interaction Between Malvastrum Yellow Vein Virus And Tobacco Curly Shoot Virus Associated With Their Betasatellites

Previous studies have found that betasatellite associated with Tomato yellow leaf curl China virus(TYLCCNV) and Tobacco curly shoot virus(TbCSV) can be trans-replicated by non-cognate begomoviruses, symptoms induced by the two viruses were significantly different when associated with homologous and heterologous betasatellite, and the accumulation of the virus was not directly related to the severity of the symptoms. After inoculation with TYLCCNV and TbCSV associated with their cognate betasatellite, viruses and their cognate betasatellite are detectable during the whole infection process in Nicotiana benthamiana, TYLCCNV and its betasatellite are detectable just in the early stage after inoculation in N. glutinosa. Malvastrum yellow vein virus(MaYVV) and TYLCCNV are of the same pathogenic type, their cognate betasatellite is necessary for typical symptoms induction; TbCSVcan infect plants and induce typical symptoms without betasatellite. In order to clearify the interaction relations between the two kinds of pathogenic type, study was carried out with MaYVV Y47 isolate(Y47A), TbCSV Y35 isolate(Y35A), the betasatellite associated with MaYVV(MaYVB) Y47(Y47β) and betasatellite associated with TbCSV(TbCSB) Y35(Y35β) as the study object, N.benthamiana as the host plant.Real-time quantitative PCR(qPCR) detection system was established using specific primers designed based on the conserved sequence of Ma YVV, TbCSV and their betasatellite, DNA extracted from sample leaf and optimized, then the sensitivity and reproducibility of this system was tested. The standard curve was established based on the templates of seven concentration gradient(102-108copies/μL). The standard curve cycle threshold and template concentration exhibited good linear relationship and amplification efficiency was between 90%-110%. In qPCR, the correlation coefficient of the standard curve was 0.998-1.000 within the amplification efficiency, itshowed that the system has better detection performance. The minimum detection limit of this system is 10 copies/μL of the templet, which is 100 times the sensitivity compared to that of PCR.After infectious clone of Y47 A, Y47A+Y47β and Y47A+Y35β infecting N.benthamiana, no symptom was induced by Y47 A infection. The plants inoculated with Y47A+Y47β performed downward leaf rolling, leaf shrinking and yellow vein symptoms, whereas the plants inoculated with Y47A+Y35β performed serious stem distortion and dwarf, and no symptom of yellow vein or leaf shrinking was observed. The infection rate of Y47A+Y47β in N. benthamiana was 100% while Y47A+Y35β was 72.7%, and the time that symptoms displayed on N. benthamiana inoculated with Y47A+Y47β(4d) was earlier than those inoculated with Y47A+Y35β. The qPCR results indicated the accumulation of Y47 A in N. benthamiana inoculated with Y47A+Y47β and Y47A+Y35β was higher than that inoculated with Y47 A alone. These results showed that betasatellite helped to increase the Y47 A accumulation. When Y35β and Y47 A were co-inoculated N. benthamiana, Y35β replicated stably, but its replication level is lower than that of Y47β in the N. benthamiana inoculated with Y47A+Y47β. These results showed a lower replication level of betasatellite when associated with non-cognate helper virus.The infectious clone combination of Y47A+Y47β+Y35A+Y35β was used to inoculate N. benthamiana. We observed the symptoms were serious in the early stage as shrinking, downward leaf rolling, yellow vein, distorted stem, dwarfing and enation, but yellow vein symptom disappeared in the late stage. Specific PCR detection showed that Y35 A and Y35β increasingly accumulated during the infection with a peak on the 90 th day post inoculation(dpi). However, Y47 A and Y47β decreasingly accumulated during the process, Y47 A was undetectable in 180 dpi and Y47β was undetectable in 120 dpi. It indicates that the disease complex of Y47A/Y47β and Y35A/Y35β are competing for the resources of the host replication system. The accumulation of Y47 A rank from high to low in different inoculated N. benthamiana at 60 dpi are as follows: Y47A+Y47β, Y47A+Y47β, Y35A+Y35β, Y47A+Y35β and Y47 A, which further confirmed the betasatellite has a stronger specific interaction with its cognate begomovirus than that with a non-cognate begomovirus, and begomovirus preferentially transreplicate its cognate betasatellite.