Screening Of Tobacco Mosaic Virus Mild Mutants And Evaluation Of Their Cross Protection Efficiency

Tobacco is an important cash crop in China,however,its production is affected by tobacco mosaic virus（TMV）.TMV has a wide host range and mainly infects solanaceous plants including tobacco.TMV causes mosaic,chlorosis,blister spot,and dwarfing symptoms,and seriously affects the yield and quality of infected crops.Currently,the main tobacco cultivars are susceptible to TMV,and there are no specific chemical agents available to control tobacco mosaic viral diseases.Using cross-protection strategy to control viral diseases is relatively safe and environmentally friendly,but there are few attenuated mutants available.In this study,we used TMV full-length infectious clone as the template to produce mutants by site-directed mutagenesis and then screened the attenuated mutants.The cross-protective effeciency and genetic stability of the attenuated double-mutant TMV-E614G-S643F was assessed and its potential for field application was studied.The main results are as follows:1.Four attenuated mutants were constructed by introducing mutation to the non-conserved site of TMV p126 replicase.Four TMV mutants were created by site-directed mutagenesis using the full-length infectious clone of TMV as the template.After inoculating Nicotiana benthamiana and Nicotiana tabacum cv.Zhongyan100 plants,there were no obvious symptoms on the plants,and their virulence were significantly reduced.Semi-quantitative RT-PCR（SqRT-PCR）results showed that the RNA accumulations of the four mutants in Nicotiana benthamiana and Nicotiana tabacum cv.Zhongyan100 were less than that of the wild-type TMV.Compared to the wild-type TMV,the accumulation levels of the four mutants in Nicotiana benthamiana systemic leaves decreased by 93%,67%,50%,and 93%,respectively.However,only TMV-E614G and TMV-S643F were detected in Nicotiana tabacum cv.Zhongyan100 and their relative coat protein（CP）accumulations decreased by 85%and 63%,respectively,compared to the wild-type TMV.2.TMV-E614G-S643F,a TMV attenuated with double mutations and lower concentration,were obtained.Six attenuated TMV double-mutants consisting of a combination of the initial four mutants were constructed,and they all showed no obvious symptoms on Nicotiana tabacum cv.Zhongyan100 plants.SqRT-PCR and Western blot results showed that only TMV-E614G-S643F double-mutant was virulent and the accumulation of the p126 gene and CP was less than that of the wild-type TMV.Its relative CP accumulation in inoculated and non-inoculated leaves decreased by 90%and 89%,respectively,compared to the wild-type TMV.3.The genetic stability of TMV attenuated double-mutant TMV-E614G-S643F was determined.It was observed proved that when transferred every fortnight,the mutant TMV-E614G-S643F was genetically stable for 6 generations.Also,it remained genetically stable after 90 days of continuous culture in Nicotiana tabacum cv.Zhongyan100 plants.4.The TMV attenuated double-mutant TMV-E614G-S643F had a good cross-protective effect on Nicotiana tabacum cv.Zhongyan100 and Solanum lycopersicum plants.Plants of Nicotiana tabacum cv.Zhongyan100 and three tomato cultivars were pre-inoculated with TMV-E614G-S643F.Plants of Nicotiana tabacum cv.Zhongyan100 was mechanically challenged with the wild-type TMV at 15 and 20 dpi,and Solanum lycopersicum plants was mechanically challenged with the wild-type TMV at 5and 10 dpi.The results showed a relative protection efficiency of 68.7%and 77.9%,respectively,at 15 and 20 days of protection interval respectively on the Nicotiana tabacum cv.Zhongyan100.Furthermore,SqRT-PCR detection results showed that TMV-E614G-S643F double-mutant pre-protective inoculation of Nicotiana tabacum cv.Zhongyan100 could enhance the plant’s immunity against the wild-type TMV.The disease severity of all tested plants was investigated at 21 days post-challenged inoculation.We found that when the protection interval was 5 days,the relative protection efficiency of TMV-E614G-S643F double-mutant was 85.4%,61.5%,and 72.5%,respectively,on the tomato cultivars Huangying,Danpo,and Jinboli.At 10 days of protection interval,the relative protection efficiency was 92.0%,81.6%,and 84.9%,respectively.5.The efficiency of indoor spray inoculation of TMV attenuated double-mutant TMV-E614G-S643F was determined,which laid a foundation for field application.We selected two commonly grown tobacco cultivars Nicotiana tabacum cv.Zhongyan100 and Nicotiana tabacum cv.NC55 for spray inoculation.The RT-PCR results showed that,after15 days of spray inoculation,the spray inoculation efficiency on Nicotiana tabacum cv.Zhongyan100 for culture with OD₆₀₀ of 0.1,0.3,and 0.5 was 86.7%,93.3%,and 93.3%,respectively,and the spray inoculation efficiency on Nicotiana tabacum cv.NC55 was66.7%,73.3%,and 93.3%,respectively.