Cloning And Functional Analysis Of Carnitine Acyl Transferase Genes From Phytophthora Capsici

Phytophthora capsici Leonian, a ubiquitous soil facultative parasite, can attack solanaceous and cucurbit hosts, which include pepper, tomato, eggplant, cucumber, pumpkin, melon and so on, causing pepper blight disease in the world-wide. In1922, Leonian described P. capsici after observing a blight of peppers at the New Mexico Station. The pepper blight disease seriously affects the yield and quality of pepper, causing enormous economic losses as well as environmental damage.Now, the prevention of P. capsici rely on chemical control, progress of studies on the anti-disease breeding is slow, pesticide residues and other problems still threaten the pepper production, human health and environmental safety. Therefore, the search to explore important virulence factor and pathogenic mechanisms of P. capsici become the focus of the study.Many plant pathogens can form a specific infection structure called appressorium, which can result in significant mechanical stress, promoting the infection hyphae invade the host tissues. In recent years, more and more studies about appressorium related genes are concentrated in the fungi field. Bastmeyer (2002) shown that oomycetes growth power is from tugor in the appressorium. The pth2gene, isolated from the rice blast fungus, encoding peroxidase type carnitine acyl transferase, is necessary for appressorium formation. The pth2gene deletion mutant strain completely lost pathogenicity, which is related with function lost appressorium and infection hyphae can not effectively extens on the host surface (Gurpreet et al.,2006). Therefore, the research on the appressorium-related genes is necessary.In this study, the highly pathogenic strain of. Phytophthora capsici SD33saved in our lab was used as experimental material, and the work we did for the carnitine acyl transferase genes was as follows:According to the CAT gene reported in the literature, we filter the CAT sequences from Phytophthora capsici genome by biological software, and then NCBI-BLAST online to confirm screening of genes belong to the CAT gene family; We successfully cloned and analyzed the two CAT genes, PcCAT1and PcCAT2, from the highly pathogenic P. capsici SD33; Using qRT-PCR technology to analyze expression pattern during the early interaction of the two gene, PcCAT1and PcCAT2, the results show that the two genes were efficiently expressed at4h after inoculation;To examine the function of PcCAT1and PcCAT2in P. capsici, agrobacterium-mediated transient expression was used to assay in vitro. The PcCATl and PcCAT2transient expression in Agrobacterium can produce chlorotic symptoms, dead spot phenomenon after inoculated pepper. This suggested that PcCAT1and PcCAT2play a role in pathogenicity as functional genes; With pHAM34vector and PEG-mediated protoplast cotransformation techniques were implemented to produce gene silenced transformants of PcCAT1and PcCAT2, RT-PCR and qRT-PCR technology were used to test and verify the CAT function in P. capsici. Aniline blue staining and immunofluorescent staining experiments confirmed the PcCAT1and PcCAT2genes can affect the formation of infection structure, germ tube and appressorium, in the early interaction.Through this study, we can see the function of CAT in the pathogenesis of P. capsici, and we proved that PEG-mediated protoplast co-transformation technology can be implemented in P. capsici and it was determined that stable transformation successfully made a molecular tool to exploit the pathogenic mechanism of P. capsici...