| Potato is one of the four major food crops in the world.While potato late blight caused by Phytophthora infestans is one of the most devastating diseases in potato production.P.infestans regulates plant immune response by secreting a large number of effector into plants.The effector secreted by pathogen has virulent function and/or avirulent function.The virulent function is manifested as: the effector is secreted to the host plant and inhibits the plant’s immune response and promotes the infection of pathogen;the avirulent function is manifested as: the effector is recognized by the host plant disease-resistant protein and then causes the plant’s immune response.The previous research of our lab found that the RxLR effector Pi16275 has both avirulent and virulent functions.This study analyzed the virulent function of Pi16275,and initially revealed the role of Pi16275 in the process of infecting potatoes by P.infestans.The main findings are as follows:1.The transient overexpression of Pi16275 promotes the infection of P.Infestans In this study,the q RT-PCR technology was used to detect the expression of Pi16275 at different stages of infection of potato.The results showed that the expression level of Pi16275 was the highest at 48 h after the infection,which was83.0 % higher than that at 0 h of the infection.This indicates that Pi16275up-regulated during early infection stages,suggesting that Pi16275 plays an important role in the process of infection.Subsequently,the Nicotiana benthamiana transient expression system mediated by Agrobacterium was used to transiently overexpress Pi16275 in N.benthamiana leaf epidermal cells.The experimental results showed that after overexpression of Pi16275 and GFP(control)in N.benthamiana,the area of diseased spots on N.benthamiana leaves overexpressing Pi16275 was significantly larger than that of the control,after inoculation of P.infestans.This indicated that the overexpression of Pi16275 significantly promoted the infection of P.infestans,and Pi16275 played a virulent function in the process of infection.2.Pi16275 is located in plant nucleus,cytoplasm and cell membrane The Agrobacterium-mediated transient expression system was used to express Pi16275 and GFP fusion protein(p RI101-GFP-Pi16275)in N.benthamiana epidermal cells.Laser confocal microscope observation showed that there were green fluorescent signals in the nucleus,cytoplasm and cell membrane of N.benthamiana epidermal cells.This indicating that Pi16275 was localized in the nucleus,cytoplasm and cell membrane of plant cells.Western Blot was used to detect the expression of Pi16275-GFP fusion gene,and the results showed that a band with the same size as the expected Pi16275-GFP fusion protein could be detected,indicating that Pi16275-GFP can be stably expressed in N.benthamiana leaves.3.The identification of the target protein of Pi16275 in potato and the preliminary study of its function Through the yeast nuclear library screening technology,a total of 10 possible candidate target proteins were initially screened in this study.Using point-to-point yeast two-hybrid technology,it was finally determined that the three proteins St RPSS,St MUC2 and St VAEL interact with Pi16275.Subsequently,virus-mediated gene silencing technology was used to silence the homologous genes of St RPS5,St MUC2 and St VAEL in N.benthamiana.Then the leaves of N.benthamiana silenced plants were inoculated with P.infestans,and it was found that silencing the homologous gene of St RPS5 significantly reduced the resistance of N.benthamiana leaves to P.infestans,indicating that St RPS plays an important role in the process of plant resistance to P.infestans infection. |
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