The Study On TMV-resistance Mechanism Of Nicotiana Rustica-derivated CNGene

TMV(tobacco mosaic virus) belongs to Togaviridae and it is a single-stranded RNA(ssRNA) virus which can infect more than 350 kinds of plants, including Solanaceae plant. Tobacco is one of Solanaceae plants with special economic value, and TMV-infection usually causes 30-50% losse at least. It is statistically reported that the losses caused by TMV-infection around every year are more than $100 million all over the world. TMV-infection occurs in many areas where tobacco are widely planting in China, inculding Jilin, Shangdong, Henan, Yunan and Guizhou province. Part of these tobacco-growing areas suffer more severe damage resulted from TMV and the incidence rate of TMV in the field is nearly 5-20%. As one of the tobacco-planting provinces, many tobacco varieties are planted in Guizhou province, including Najing 3, Guiyan 4, Yunyan 87, K326 and Yunyan 97, etc. And among of them, tobacco K326 is wildly planted in Guizhou province because of its high leaf quality and the planting area in 2012 reached 12200 hectares and it was nearly 33.8% of the total planting area of tobacco in Guizhou. But the variety is susceptible to TMV, the tobacco losses cuased by TMV infection in Guizhou province was as high as 0.32 million RMB. The resistance breeding via genetic engineering is one of the most economic, safe and effective method for improving the TMV-resistance in susceptible tobacco. In this study, a N. rustica-derivated resistance gene CN as resistant source to construct three plant expression vectors, namely pSH-CN, pGM626-DL-SAF-UCn and pSH737-U4Cn-RcHak for genetic transformation of TMV-susceptible tobacco varity K326 and transgenic plants were artificially inoculated with TMV. The inoculation performance and the mechanism of CN-mediated disease resistance were disscussed as follows:1. The TMV-inoculation in transgenic plants caused the hypersensitivity(HR) occurrance at inoculated-leaf, the lipid peroxidation product malondialdehyde(MDA) content reducing and chlorophyll content decreasing. The introduction of CN gene delayed mosaic plaques formation caused by TMV proliferation, indicating the CN could induce HR and form necrotic spots at the inoculated leaf which limited the spread of the virus and restrained the systemic infection in whole plants. Meanwhile, the content of MDA was significantly reduced in response to TMV infection in transgenic tobaccos, which protected better plant cells from damage of membrane peroxidation and improved the pathogen-defense capability of plant cells; In addition, the introduction of CN gene also caused the systemic hypersensitivity(SHR), which could induce the systemic resistance in transgenic plants to resist invading pathogens.2. The TMV-inoculation significantly increased the activities of antioxidant enzymes(AOEs) in transgenic plants, including superoxide dismutase(superoxide dismutase, SOD), peroxidase(peroxidase, POD) and peroxidase(catalase), which indicated the transgenic tobaccos could significantly improve AOEs activity to enhance the resistance to TMV infection. It is generally believed there is positive correlation between AOEs activity and the content of reactive oxygen species(ROS) and ROS content was rapidly increased in vivo during biotic and abiotic stress, high levels of ROS causing serious damage of cells. Therefore, the transgenic plants on the one hand protected plant cells from ROS-caused oxidative damage by increasing the activity of the AOEs, protecting cells integrity and improving pathogen-defense capability of plant tissue; on the another hand the higher AOEs activity played an important role in activating and regulating these genes expression meidiated virusresistance pathway.3. In TMV-inoculated transgenic plants, the content of hydrogen peroxide(H2O2) and salicylic acid(SA) significantly increased and both identified as signal molecule mediated the regulation and activation of disease resistance-related genes in signaling pathway downstream. H2O2 accumulation caused the enhancement of AOEs activity. The invading pathogens led to O·2- content increasing and O·2- could be catalyzed to H2O2 by SOD; thereby reducing oxidative damage caused by excessive accumulation of O·2- content. The H2O2 as substrate could be further metabolized by POD and CAT, and the former mainly oxidized variety of substrates with H2O2, such as phenols, ethanol and formaldehyde and formed non-toxic H2 O, while the latter could directly catalyze the reaction of H2O2 to H2 O. At early stage after TMV inoculation, CN-contained tobaccos could significantly increase AOEs activity; And the trans-CN plants significantly reduced CAT activity at late stage after TMV inoculation which indicated the increasing H2O2 content. The H2O2 mediated HR occurance and produced the systemic acquired resistance(SAR) in in disease-resistance plants. Additionally, TMV-inoculation also caused a significant increasement SA content in transgenic plants, which is usually considered as endogenous signal molecule to regulate pathogen-resistance response. The study had showed that SA could bind as-l-like regulatory region of PR-1a promoter to activite its expression and improved plant defenses against pathogens;4. The CN-contianed tobacco plans significantly induced the expression of protein components, inculding CN(N-like protein), RAR1(required for MLa 12 resistance), STG1(suppressor of G2 allele of skp1), MEK1(mitogen-activated protein kinase kinase), NTF6(mitogen-activated protein kinase), PR-1a(pathogenesis-related proteins 1a) and transcription factor WRKY1 after TMV-inoculation. It is generally believed that Avr protein encoded by pathogen avirulence combine with corresponding R protein encoded by plants and regulate downstream transcriptional activation of disease-related genes via ?R-Avr? way. After transgenic plants inoculated with TMV, this ?CN-TMV-Avr? caused a significant upregulation of CN gene expression, indicating that TMV invasion induces the formation of TMV-Avr- CN complex protein and after that the expression levels of RAR1 and STG1 components were also significantly increased, indicating the Avr-CN complex protein could further combine with RAR1 and STG1 to form an original complex called ?pathogen-host? to regulate initiation of resistance-defending pathway downstream. After STG1 and RAR1 were upregulated, the expression of other protein components NTF6, MEK1 and WRKY1 were also enhanced, which indicated that the complex functioned by phosphorylation via mitogen-activated protein kinase cascade(MAPK cascades) pathway signaling pathway and regulated the expression of the downstream PRs, enabling the capacity of TMV-resistance in transgenic plants;5. In this study, the plant expression vector pGM626-DL-SAF-UCn was used to genetically tranform TMV-susceptible tobacco varity K326 and the foreign genes, including the gene screening reporter gene Bar::GUS, recombinase gene FLP and resistance genes CN was used to analyze the exogenous genes removal. With the gradual mature and aging of transgenic tobacco leaves, the sensitivity to herbicide phosphinothricin(PPT) gradually increased and GUS staining activity decreased step by step, indicating that ?Gene-deletor system? in transgenic tobacco plants implemented exogenous genes(Bar::GUS, FLP and CN) removal. The applications of ?Gene-deletor? technology facilitated the creation of the new disease-resistant germplasm without selectable marker, which provided a theoretical basis for the new tobacco breeding;6. The tobacco K326 genetically transformed with pSH737-U4Cn-RcHak not only improved the transgenic tobacco plants resistance to TMV by CN-mediated response mechanism, but it also enhanced the leaf thickness and reduced sizes of necrosis spot at inoculated leaves by synergistic effect of overexpression of tobacco high-affinity potassium transporter protein(HAK). The result showed that the synergistic expression of CN and HAK on the one hand improved the transgenic plants to TMV-resistance via internal defense mechanism(CN-mediated disease regulation); on the other hand enhanced the plants defense against TMV by the external defense(leaf thickening). Meanwhile, the salt-tolerance in HAK-overexpression tobacco plants was also improved. The study showed that the plants under abiotic stress were liable to be infected by pathogens. The HAK-overexpression in transgenic tobaccos not only imporved the K+-enriching ability and enhanced photosynthesis, but also strengthened the salt-stress tolerance of tobacco plants. These studies showed the application of co-transfromation method mediated by two engineering Agrobacterium tumefaciens with two plasmids respectively could create selectable marker-free, higher potassium-enriching and salt-tolerant and stronger TMV-resistant tobacco germplasm successfully.