Study On Immune Inducing Function Of Phytophthora Effector RxLR116 And RxLR2932

Effectors are a class of biologically active substances secreted by pathogenic bacteria to disarm and manipulate the hosts.If recognized by the host resistant proteins,it can stimulate immune response of hosts.RxLR effectors are oomycetes cytoplasmic effectors with a highly conserved N-terminal RxLR-d EER motif responsible for transporting the effectors into host cells and highly divergent sequences of C-terminal responsible for exerting active functions.Phytophthora capsici is a kind of plant pathogenic oomycete that has the characteristics of rapid spread,extreme destructiveness,wide host range and difficulty in control.It causes huge economic losses to the vegetable production industry every year.As a new type of green biological control agents,the plant immune inducer can regulate metabolism of the plant by activating its immune system,thereby it can enhance ability of the plant to resist diseases and stress,and is in line with the national strategic demand of “medicine and fertilizer reduction”.Currently,the research and development of new plant immune inducers are booming,and the immune inducing effect of bacterial effector Harpin is remarkable.Therefore,effectors as protein immune inducers have broad development prospects in the prevention and control of plant diseases.In this study,phytophthora nicotianae effector RxLR116 and phytophthora sojae effector RxLR2932 were used as the research objects to study and analyze their immune-inducing functions.After using agrobacterium-mediated transient expression technology to clarify their immune decoy activity,a large number of high-purity protein solutions were prepared through the prokaryotic expression system.The main research contents and results of effector RxLR116 and effector RxLR2932 in this paper are as follows:(1)Transient expression of RxLR116 and RxLR2932 on leaves of nicotiana benthamiana showed that they were unable to cause plant cells necrosis.(2)The zoospore infection test of phytophthora capsici proved that RxLR116 and RxLR2932 can significantly inhibit the infection of phytophthora capsici.(3)In order to clarify the key functional domains that can trigger hosts immune resistance,we constructed truncated bodies RxLR116-25 and RxLR116-44,RxLR2932-15 and RxLR2932-58.Through the zoospore infection test of phytophthora capsici,the key functional domain of the effector RxLR116 was identified to exist in amino acids 116 to 159,and the key functional domain of the effector RxLR2932 existed in amino acids 131 to 188.(4)In order to explore the specific pathways by which effectors RxLR116 and RxLR2932 cause plant immunity,we conducted DAB staining and resistance gene detection experiments.The results showed that RxLR116 and RxLR2932 caused the burst of reactive oxygen species during the infection of P.capsici,and the up-regulated expression of four positively immune-related resistance genes including PR1 were detected by q RT-PCR.(5)The subcellular localization test was carried out to clarify the position of the effectors functioning,and the results showed that RxLR116 was mainly present in the nucleus and RxLR2932 was distributed in the nucleus and cytoplasm.(6)After clarifying that the effectors have immune inducing function,we carried out the purification of the protein preparations in next step.RxLR-p ET21 b prokaryotic expression vectors were constructed to fuse the target proteins with the his-tag of the vector.After transformation and induction,they were abundantly expressed in Escherichia coli.Through affinity chromatography,ion exchange chromatography and gel filtration chromatography,we obtained high-purity and homogeneous protein solutions of RxLR116 and RxLR2932 and their truncations.(7)Through the thermal stability test,it was verified that the protein solutions of the four truncated bodies had good stability.In this paper,we determined the optimal purification conditions for RxLR116 and RxLR2932,and clarified their characteristics to stimulate plant immunity,and preliminarily explained the mechanism of action,as well as providing novel candidate proteins for the development of plant immune inducer.