| Phytophthora capsici Leonian, a ubiquitous soil facultative parasite, can cause pepper blight disease in the world-wide and which is one of the important pathogens in reducing pepper production. In pepper-growing areas around the world, the widespread occurrence of the pepper blight disease seriously affects the yield and quality of pepper, and causes enormous economic losses as well as environmental damage in natural ecosystems.For decades, pepper-producing countries have carried out a lot of researches about breeding and disease resistance, and have performed extensive work on physiological and biochemical mechanisms of disease resistance, genetic resistance, resistance gene mapping and cloning and disease resistance breeding but the progress is slow. Now, the prevention of P. capsici rely on chemical control, 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.The results show that in the plant pathogen and host interaction process, a diverse number of plant pathogenic microbes can manipulate biochemical, physiological, and morphological processes of their host plants through delivering a series of effector molecules that can either infect parasitic colonization or modulate plant defense responses. In 2003, Torto et al. discovered CRN1 and CRN2 from Pex (Phytophthora extracellular protein) cDNAs that triggered crinkling and necrosis of leaves (hence their moniker, crinklers or CRN proteins) when overexpressed in Nicotiana benthamiana transient expression assays used a vector derived from PVX (Potato virus X).Currently, the CRN protein, as a new member in effector family, is only found in Phytophthora and there is few research on this protein family. In the interaction process of host and pathogen, the mechanism of CRN protein, the mechanism of enter cellular and the host target(s) and so on have known little, so it become a research hot spot. In this study, the highly pathogenic strain of Phytophthora capsici SD33 saved in our lab was used as experimental material, and the work we did for the crinkling and necrosis genes was as follows:(1) CRN-like sequences were collected from JGI database according to the conserved motifs reported and then blasted in NCBI database.(2) Two CRN sequences, PcCRN1 and PcCRN2, were isolated from phytophthora capsici genome and analyzed using the NCBI online to confirm that both sequences were homologous to CRNs from nonredundant database.(3) To examine the pathogenicity of PcCRN1 and PcCRN2 in P. capsici and the function of conserved motifs WL and LQLFLAK, a series of mutant constructs were generated and agrobacterium-mediated transient expression were used to assay in vitro. The PcCRN1 and PcCRN2 transient expression in Agrobacterium can produce chlorotic symptoms, and the symptoms of conserved motif-mutations before and after were different. This suggested that PcCRN1 and PcCRN2 play a role in pathogenicity and the conserved motif also had an impact on its pathogenicity.(4) With pHam34 vector and PEG-mediated protoplast cotransformation techniques were implemented to produce transformants and RT-PCR and qRT-PCR technology were used to test and verify the CRN function in P. capsici.Through this study, we can see the function of CRN 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. |
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