Inlfuence Of The Stably Expressing PVX P25Protein On Virus Resistance And Phenotype Of Tobacco

RNA silencing is a kind of sequence-specific degradation mechanism commonly foundin eukaryotic organisms to resist the invasion of heterologous nucleic acid. RNA silencing isused against virus infection in plants, but viruses interfere RNA silencing pathway byencoding silencing suppressors. RNA Silencing suppressors affect on different phases of RNAsilencing pathway. Studies on the modes of action of suppressors would make usunderstanding RNA silencing mechanism and virus-host interaction deeply, and providetheoretical basis of antiviral crops breeding. P25, a25kD protein encoded by the Potato virusX, is a kind of weak RNA silencing suppressor and pathogenic factor. In this study, weacquired non-translated PVYNCP and P25double transgenic tobacco using conventional crossbreeding. Then PVYNresistance and growth abnormalities of P25stably expressing plants andthe effect of P25protein on the miRNA level were analysized. The main results are asfollows:(1) Acquiring and identification of P25-PVYNCP double transgenic tobacco. P25transgenic plants were screened by PCR and Western blot analysis; PVYNCP transgenic viralresistant plants were screened by PCR and PVYNresistance analysis. Transgenic tobaccoexpressing P25as the male parents were hybrided with PVYNCP-mediated virus resistanttobacco whose stamens were removed. The double transgenic hybrid progenies with PVX P25and PVYNCP genes were obtained successfully by PCR and Western blot analysis.(2) PVYNresistant analysis of double transgenic tobacco. Double transgenic plants wereinoculated with PVYNwhen they were4to5leaves. ELISA analysis suggested that high titerPVYNcould be detected in the all inoculated plants. Northern blot indicated that the level ofPVYNCP mRNA of double-transgenic plants was obviously higher than the female parentlines showing RNA silencing. The results above showed that heterologous expressing P25protein could play the role of RNA silencing suppressor, resulting in the loss of PVYNresistance in the double transgenic plants. Although P25is a weak silencing suppressor, itshows a strong silencing suppressor activity due to CaMV35S promoter. (3) Physiological indexes examination of P25transgenic tobacco related to the abnormalphenotype. Comparing with wild-type tobacco, the leaves of P25and double transgenic plantsturned yellow and wilt. Above phenotypes usually result from chlorophyll degradation, andchlorophyll degradation is the original process and the sign of leave senescence. So wepresumed that P25protein can boost leave senescence. We analysed the chlorophyll contentand two enzymes activity which were related to chlorophyll degradation, superoxidedismutase (SOD) and peroxidase (POD) in P25transgenic tobacco. The results showed thatthe chlorophyll content of P25transgenic tobacco decreased54%, and chlorophyll a/chlorophyll b of P25transgenic plants was less than wild type. SOD could eliminatesuperoxide anion which results in chlorophyll decomposition and decrease of ratio ofchlorophyll a/chlorophyll b, while POD participates in chlorophyll degradation of whichactivity has a high negative correlation with senescence. The results showed that in P25transgenic plants, SOD activity decreased25%and POD activity increasd2.5times,comparing with wild type plants. In addition, a series of physiological indexes related tophotosynthetic capacity were detected. The results showed that in P25transgenic tobacco, Netphotosynthesis rate(Pn), transpiration rate(EVAP), stomatal conductance(Gs) and intercellularconcentration(Ci) decreased45%,36%,50%and3%respectively comparing with wild typeplants. There is almost not change in Ci, Pn is usually determined by stomatal limitation onlywhen both Gs and Ci change in the same way. So we prosume that in P25transgenic tobacco,the decline of Pn is not caused by stomatal limitation.(4) Analyses of P25transgenic tobacco miRNA expression. MiRNA takes part in almostall the signaling pathways, such as the regulation of gene expression related to growth anddevelopment, cell differentiation, proliferation and apoptosis and involves in many pathologicand physiological processes. MiRNA pathway is also affected by viral RNA silencingsuppressors like siRNA silencing. MiRNA and siRNA share some similar proteins andpathways. Both of them need a helicase and the complex containing at least one Agonautefamily protein. The genes of DCL1and AGO1which play an important role in the miRNApathway, are under the negative regulation of miR162and miR168respectively. MiR162regulated DCL1expression is significantly accumulated in P25transgenic tobacco, speculatedthat P25could influence DCL1expression. qRT-PCR was used to analyze the expression level of several miRNAs. The expression levels of miR162and miR168in the P25transgenictobacco increased2.8and3.7times respectively comparing with the wild type plants. Theresults show that P25, as a silencing suppressor, interfered siRNA pathway and miRNApathway at the same time. Expression levels of miR156, miR165, miR167and miR171whichwere closely related to growth and flowering, development, hormone response andtranscription factors respectively in plant, increased2.0,3.2,4.7and1.8times respectively inthe P25transgenic plants. The results indicated that P25protein generally influenced theexpression levels of the miRNAs related to growth and development, causing abnormalphenotye such as senescence and flowering delaying. The results also revealed the mechanismby which P25protein worked as a virus pathogenic factor.