Single Amino Acid Change Underpin Avr3b Effector Function And Effector Screen Revealing New Resistance In One Chinese Potato Cultivar

Phytophthora spp.is plant destructive pathogens that seriously threaten crop production.The most representative ones are Phytophthora infestans and Phytophthora sojae.Phytophthora infestans secrete large amount of specific effector proteins while infecting plants.One of these proteins has a conserved sequence of "arginine-any amino acid-leucine-arginine(RXLR)" at the N-terminus and contains more than 400 coding genes in the Phytophthora genome.These RXLR effector proteins can suppress the plant's immune system and make it susceptible to pathogens.In the co-evolution,plants have also evolved disease-resistant genes encoding receptor proteins to recognize pathogen RXLR effector proteins,thereby inducing plants to produce disease-resistant responses to prevent further infection by phytophthora infestation.Avr3b is an RXLR effector protein secreted by Phytophthora sojae.During infection,Avr3b interacts with the cyclophilin GmCYP1 in soybean and activates its own Nudix hydrolase activity.The glycine-proline(GP)motif in the Avr3b of soybean strain P6497 conferred its binding to the cyclophilin GmCYP1 as a substrate,and the substitution of proline(P132)in the GP motif causes the loss of Avr3b hydrolase activity,hence plant susceptibility reduces.More importantly,the mutant Avr3bP132A cannot be activated by GmCYP1,so this mutant is not recognized by the soybean disease resistance gene Rps3b.We found that the natural mutation of P132 in the GP motif of Phytophthora sojae strain P7076 was naturally mutated to A132,and it was speculated that the function of the GP motif was lost.We replaced the A132 motif here with P132,using particle bombardmentmediated transient gene expression methods.By transient expression of LUC and Avr3bA13P in soybean,the significant drop in LUC-positive spots was observed by the multi-chemiluminescence imaging system,indicating that the number of viable cells was significantly reduced,which is in line with our previous predictions.Because Avr3bA132P can be recognized by the disease resistance gene Rps3b in soybean thus,resulting in more cell death and less infection.The study further confirmed the key amino acid position 132 of the non-toxic effector protein Avr3b.Since it is the binding site of the substrate,cyclophilin GmCYP1,hence its function is crucial for whether Avr3b can be recognized by disease-resistant genes,and it also explains that Avr3bP132A is carried in the field thats why the soybean Phytophthora strains can adapt to disease-resistant soybeans.Phytophthora infestans secretes a large amount of RXLR effector proteins,some of which can induce plant resistance.According to the theory of genes for genes,it is speculated that it may be recognized by potato disease resistance genes.Potato variety C88 is a popularized variety in southwestern China and has good resistance to late blight,but its resistance background is not clear.In this study,60 effectors of Phytophthora infestans cloned in the laboratory were tested against potato varieties C88.The PVX virus transient expressionsystem was used to express and detect potato variety C88 effector-induced cell death phenotype.The results of the study found that 3 of the 60 effectors(PITG-04085,PITG-06308,and PITG-23092)tested could strongly induce plant cell death.In this study,the cell death inducing protein NLP1 was used as a control.In addition,we also tested the response of PITG-04085,PITG-06308,and PITG-23092 to Desiree potato variety and Nicotiana benthamiana but neither of the two plants showed a cell death response,confirming that these effectors are not PAMP effectors.The hypersensitive response induced by potato C88 cultivar is specific,which may mean that there are new disease resistance genes in this variety,which indicates the direction for the future mining of new disease resistance genes.