| The plant diseases caused by Phytophthora cactorum are devastating ones worldwide, endangering more than 200 kinds of plants and causing heavy losses to the production of a variety of important crops including strawberries, apples, pears, tomatoes and flowers. In recent years, facility agriculture based on fruit, vegetable, flower production has been developed rapidly in China. The plant diseases caused by P. cactorum are becoming serious threat to facility agriculture. The knowledge learnt from the studies on the molecular mechanism of Phytophthora pathogenicity during their interactions with plants will help to develop new fungicide targets and provide important guidance to design control strategy for the plant diseases caused by Phytophthora.Based on the previous sequencing results of P. cactorum transcriptome, using semi-quantitative RT-PCR technique this study analyzed the transcriptional profiles of 32 selected pathogenicity-related genes during 4 developmental stages (mycelium, sporangium, zoospore and germinating cyst) and infection stages (1.5,3,6,12,24,48,96 hour post-inoculation) on Nicotiana benthamiana. The results showed that of them up-regulated during the developmental stages and infection stages were 13 genes, including 8 PcF/SCR genes.The 13 effector genes were cloned into a Potato virus X (PVX) vector pGR107 through high fidelity amplification and their ability to elicit plant cell death (PCD) was tested in solanaceous plants using Agrobacterium-mediated transient expression system. The results showed that 4 PcF/SCR effectors (U866, U14169, U16448 and U18031) can induce PCD in tomato(Solanum lycopersicum) and two of them (U866 and U16448) can trigger PCD in N. benthamiana. In addition, one elicitin (U7626) and one NLP effector (U2101) can trigger PCD in N. benthamiana. U7626 can also trigger PCD in Nicotiana tabacum var. Samsun NN.To further determine the roles of PcF/SCR effector in the pathogenesis of P. cactorum, in this study the protoplast transformation method of P. cactorum mediated by CaCl2-Polyethylene glycol (PEG) for Phytophthora gene silencing has been established. The resistance screening concentration of Geneticin (G418) in P. cactorum was first determined in wild type isolate and then the transformation method was verified by introducing the exogenous green fluorescent protein (GFP) gene into the pathogen. Green fluorescence in the mycelia of Gfp-transgenic transformants and their single-zoospore isolates can been observed under fluorescence microscope, indicating the success of establishing the stable transformation system of P. cactorum. Then, one PcF/SCR effector gene U866 was silenced in P. cactorum using this PEG-mediated protoplast transformation system. The pathogenicity of U866-silenced transformants was significantly decreased on N. benthamiana, in contrast with the wild type isolate and transformation controls. Additionally, the sensitivity of U866-silenced transformants to hydrogen peroxide was significantly increased, compared to the wild type isolate and transformation controls. The results indicated that the PcF/SCR effectors may play an important role during the interactions between P. cactorum and its host plants.In this study,8 PcF/SCR effectors of P. cactorum differentially expressed during the developmental and infection stages were obtained. Their full-length sequences were cloned and determined. Four PcF/SCR effectors were found to trigger PCD in N. benthamiana and S. lycopersicum by gene transient expression method. The expression of PcF/SCR genes was disturbed using the protoplast transformation system and it was found that one of them, U866, is important for the pathogenicity of P. cactorum. These results determined for the first time the important roles of PcF/SCR effectors in the pathogenicity of P. cactorum. They provide important experimental evidence for further understanding the molecular mechanisms of the pathogenicity of P. cactorum, and theoretical guidance for the design of the control strategies of plant diseases caused by the pathogen. |
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