The Research On Pathogenic Genes Of Verticillium Dahliae Using Host-induced Gene Silencing (HIGS) Method

Verticillium Wilt of cotton, named “cotton cancer”, is a kind of soil-borne, systematic vasculardisease, which produces the heavy influence of cotton production and cotton fiber qualify. Thepathogenic fungus was Verticillium dahliae. The V. dahliae was easy variation to generate newphysiological strains, which brought enormous difficulties and nondeterminacy. The research of V.dahliae has being a hot topic for a long time. The study was used host-induced gene silencing (HIGS)technology and the highly virulent strain V991as experimental materials. A series of tobacco rattlevirus (TRV) RNA interference (RNAi) vectors were constructed aiming to fungal targeting genes. TheRNAi vectors were transformed into the seedlings of Nicotina benthamianaby agroinfiltration. Then theV. dahliae spore was inoculated into the plants. In the end, the disease index and reactive type wasrecorded and statistics. Meanwhile, the fungal biomass and transcriptional level of targeting level wasdetected by molecular biological way. The ultimate goal is to get the key developmentalandpathogenicgenes.The main results of this research were listed as follows:1. The statistics of the published the peotein sequence of V. dahliae in database. According to thebiological function, the sequences were classified into12groups according the biological function,including energetic metabolism, protein metabolism, DNA replication, RNA transcription, and so on.The5-10sequences from different groups were selected and cloned as targeting genes from the V991transcriptome. The92TRV RNAi vectors were constructed and transformed into Agrobacteriumtumefaciens strain GV3101.2.The host-induced gene silencing (HIGS) platform was built to select the targeting genes. Firstly, theconcentration of injected Agrobacterium and the inoculated time of V. dahliaewere optimized.Afteragroinfiltration of92TRV RNAi vectors and fungal inoculation, the disease index was statistics.Compared to the control groups, the disease index reduced in the plants injected with the Agrobacteriumcontaining targeting genes. Meanwhile, the resistant active type was improved. Among them, the20genes were significant resistance to V. dahliae, mainly including glycometabolism, energy metabolism,protein metabolism, and so on.3.The AK (adenylate kinase), AACP (ADP,ATP carrier protein) and OST STT3(oligosaccharyltransferase STT3subunit) was further studied. Compared to the negative groups, the fungal biomass ofthe RNAi groups significantly decreased in root, stem and leaves, up to50%. These results wereconsistent to the trypan blue staining. Meanwhile, the transcriptional level of targeting genes wasremarkably higher in negative groups,3.5fold (AK),3.7fold (AACP) and6.9fold (OST STT3)respectively.4. To get the transgenic plants, the Gateway RNAi vectors were constructed and transformed into N.benthamiana. The research was focused on obtaining the key developmental and virulent genes in virtue of HIGS.A new way to observe the plant-pathogen interaction directly and record the disease index was exploredand detected by the molecular analysis. This study provided the experimental dataand theoretical base toprevent the agricultural pest and disease.