Creation Of Tobacco AOC And COI1 Gene Silencing Materials And Their Laboratory Evaluation Against Scab And Black Shank

Brown spot and black shank diseases are two major diseases of tobacco,caused by necrotrophic pathogens Alternaria alternata and Phytophthora nicotianae,respectively.Previous results showed that both wild tobacco（Nicotiana attenuata）and cultivated tobacco（Nicotiana tabacum）increased plant hormone jasmonate（JA）after inoculation of Alternaria alternata or Phytophthora nicotianae.However,whether JA plays a role in host resistance to these two pathogens in cultivated tobacco is still unknown.By using cultivated tobacco K326 as the host,we generated transgenic plants silenced with genes for JA biosynthesis or perception through RNAi technology,and evaluated their resistance to both diseases.The main results are as follows:1)Laser confocal microscope study showed that some A.alternata hyphae penetrated leaf of tobacco cultivar‘K326’ through stomata at 24 h post inoculation.2)The levels of JA increased significantly after K326 was infected with A.alternata and P.nicotianae,and the results of qPCR analysis showed that the expression of genes related to JA signaling pathway was up-regulated.3)Through RNAi technique we successfully generated transgenic K326 plants silenced with allene oxide cyclase（AOC）,the key gene for JA biosynthesis,and COI1,the receptor gene,respectively.Homozygous single-insertion lines of T2 generation of AOC-RNAi and COI1-RNAi were obtained and used for experiments.4)The results of qPCR and HPLC-MS/MS showed that the AOC gene expression and JA level were significantly decreased in AOC-RNAi lines.5)COI1-RNAi lines not only showed that the COI1 gene expression was sucessfully inhibited,but also the insensitivity of root growth to MeJA treatment,which proved that the COI1 gene was successfully silenced;6)In COI1-RNAi plants,not only the expression of the JA biosynthesis gene AOC induced by A.alternata was significantly reduced,but also the expression of the transcription factor genes NAC90,bHLH92,MYB4,WRKY50 and the phytoalexin scopoletin synthase gene F6’H1 is also significantly reduced.The experimental results indicate that the JA signal may regulate the resistance of plants to A.alternata through the expression of these genes.7)AOC-RNAi and COI1-RNAi lines were more susceptible to brown spot disease and black shank disease,which proved that JA played an important role in the host resistance to these two diseases.Taken all together,RNAi techniques were used to generate transgenic tobacco plants silenced with genes for JA biosynthesis and reception,and through which we demonstrated the important role of JA in host resistance to both brown spot and black shank diseases,and conservation of using JA signaling pathway against A.alternata in both cultivated tobacco and wild tobacco Nicotiana attenuata.