Establishment Of An Efficient Expression Of Resistance-Inducing Factor PsXEG1 And Detection Of Resistance Response In Soybean

Soybean root rot caused by Phytophthora sojae is a devastating disease in the world’s soybean production.The global soybean production causes losses of more than one billion dollars per year.The traditional fungicides against Phytophthora sojae and the effect is small,based on the special disease resistance of the disease-resistant varieties due to the rapid evolution of Avr genes,making resistant varieties difficult to exert long-lasting resistance,how to improve soybean resistance is an important issue in soybean production.Plant immunity inducers are a new class of biological agents,which are different from traditional agrochemicals such as insecticides or herbicides that kill and destroy pests and diseases through chemical compounds.They mainly enhance the plants by stimulating immune responses.The defense response allows the host to obtain disease resistance.However,research on plant immunity inducers for improving soybean resistance has progressed slowly,and there are no mature product preparations.This study is based on an elicitor PsXEG1 identified in the previous laboratory,which can be used by host and non-host plants to recognize the immune response of plants.The immune-stimulating function of PsXEG1 has the potential to be prepared as a bio-pesticide-plant immune inducer.However,it is difficult to carry out large-scale production by cultivating Phytophthora,and the protein expressed by the prokaryotic expression system has poor activity and extremely low expression level.How to obtain high-quality PsXEG1 protein is a problem to be solved by developing into an immune inducer.This study established a Pichia past oris expression system that efficiently expressed PsXEG1,which created the necessary conditions for its development as a plant immune inducer.High expression and resistance identification of PsXEG1 in Pichia pastoris:by using recombinant pastoris to express the heterologous protein PsXEG1,after purification,an average of 5 mg of PsXEGl protein can be obtained per 1 L of Pichia fermentation product,a single 2.5 L fermentation The purified product PsXEG1 protein 12 mg was obtained,which greatly increased the yield of PsXEG1.PsXEG1 induced soybean resistance was detected.The concentration of PsXEG1 was the best at 500 pM,and it inhibited the normal growth of soybean yellowing at more than 700 pM due to its toxic function.It will promote the infection of soybean with Phytophthora sojae,and in order to find the effective inhibitory concentration of PsXEG1,the EC50 value was determined to be 73 pM.Expression and resistance assay of PsXEG1 active mutant PsXEGlE136D:Due to the toxic function of high concentration of PsXEG1,it inhibits the growth of soybean and promotes the infection of pathogenic bacteria.The 136th glutamic acid in the domain of GH12 of PsXEG1 is its the key site of enzyme activity,so we constructed the enzyme activity mutant PsXEG1E136D expression vector,and heterologous expression of the enzyme mutant in Pichia pastoris,Ni-NAT afinity purification of the fermentation product by AKTA The purer protein was tested and its induced soybean resistance was tested.It still retains the function of stimulating soybean immune resistance,which not only can induce soybean resistance to Phytophthora sojae,but also enhance the layering slash.Resistance to bacteria,Fusarium graminearum,and stalk-like species rot,when compared to wild-type PsXEG1 at a concentration of 5 mol,does not inhibit the elongation of hypocotyls of soybean seedlings,and its toxic function is also lost.Provides a theoretical basis for the safety of biological agents.The simplified PsXEG1E136D fermentation broth can induce soybean disease resistance:Due to the high purification cost of PsXEG1,the purification process is more complicated,so we simplified the culture medium of Pichia pastori inducer.High expression and resistance identification of PsXEG1 in Pichia s.Compared with the original medium,the simplified medium removes yeast powder and peptone,and only the yeast nitrogen source YNB is retained.Biotin,phosphate buffer solution.The simplified Pichia pastoris fermentation product is removed by high-speed centrifugation,and then dried to a powder by a low-temperature freeze-drying apparatus,and then reconstituted with sterilized water or PBS to obtain a crude protein solution.The soybean resistance induced by the soybean is detected,which still retains the function of stimulating soybean resistance,and can still induce the soybean to enhance the resistance to Phytophthora sojae,and at the same time enhance the soybean to the fusarium and cereal.Resistance to bacteria,stalk-like species,and Rhizoctonia solani.By simplifying the culture medium,not only the fermentation product retains the function of inducing soybean disease resistance,but also simplifies the purification process and reduces the fermentation production cost.In summary,the biologically active PsXEG1 can be expressed in large quantities by Pichia pastoris,which simplifies the fermentation method of PsXEG1E136D,and lays a foundation for the future application of PsXEGl to improve crop disease resistance.