Functional Analysis Of PITG₁5718.2 In Transgenic Potato

Potato late blight caused by Phytophthora infestans is the most devastating disease in potato production,and it is the world's largest crop disaster.During the stage of co-evolution between pathogens and plant,the pathogens exhibit their pathogenic property by secreting effectors such as RxLR.Previously,we had identified a candidate avirulence gene,PITG₁5718.2,which triggered hypersensitive response?HR?in many collections of Solanum nigrum.Preliminary analysis was indicated that there are two different alleles among different strains of P.infestans for the effector PITG₁5718.2,PITG₁5718.2^HLJ and PITG₁5718.2⁸⁸⁰⁶⁹.The C-term of PITG₁5718.2^HLJ and PITG₁5718.2⁸⁸⁰⁶⁹ alone could stimulate HR,while the complete PITG₁5718.2⁸⁸⁰⁶⁹ could not stimulate HR on Solanum nigrum.It was revealed that the N-term of PITG₁5718.2⁸⁸⁰⁶⁹ could supperss the HR elicited by its C-terminus.In this study,further studies were conducted on the pathogenic function of the PITG₁5718.2.By using transient expression on N.benthamiana and stable expression in potato,we try to explore the function of the effector during the potato late blight infection.Meanwhile,the fuction of the N-term of PITG₁5718.2⁸⁸⁰⁶⁹ to suppress the PTI and ETI was further investigated on S.nigrum and N.benthamiana.And,the following results were obtained:?1?The N-term of PITG₁5718.2⁸⁸⁰⁶⁹ were co-infiltrated with any of following effectors on S.nigrum,including with PITG₁6245.2,PITG₁5556.2,PITG₂1190.2,PITG₀4089.1,PITG₂0301.2,Avh238,Avh240 and Avh241,which could stimulate HR responses.The results showed that the N-term of PITG₁5718.2⁸⁸⁰⁶⁹ could inhibit the HR elicited by other effector in addition to Avh241.The N-term of PITG₁5718.2⁸⁸⁰⁶⁹ were co-expressed in N.benthamiana with INF1,SFI2,NIP,Bax,Avr3a and R3a,Avr2 and R2respectively.The results showed that the N-term of PITG₁5718.2⁸⁸⁰⁶⁹ could not inhibit the PTI nor ETI triggered by these effectors.Therefore,it is concluded that the the N-term of PITG₁5718.2⁸80698069 specifically inhibits the HR response mediated by partial effectors on S.nigrum.?2?By using transient expression system,the N.benthamiana leaves were expressed the PITG₁5718.2^HLJ,then inoculated with the spore suspension of P.infestans HLJ.The results showed that PITG₁5718.2 could promote the infection and play a pathogenic role during the infection of late blight.?3?Total 13 positive transgenic plants with PITG₁5718.2^HLJLJ were obtained by genetic transformation of potato.Based on the detection of DNA,RNA and protein,it is verified that PITG₁5718.2^HLJ gene could be stably expressed in potato.The transgenic plants were inoculated with HLJ spore suspension to observe the area of lesions and their mycelial biomass.The results showed that the transgenic plants were more susceptible than the wild type.The growth parameters of Desiree and the transgenic plants were measured.The plant height of the transgenic plants was significantly lower than that of the wild type.It is indicated that the effector was implicated as a virulence factor involved in the inhibition of plant immune response and growth.?4?We also investigated the SA-,JA-,and ET-pathway-related genes in transgenic plants.We found that both SA-and ET-signaling marker genes were up-regulated and JA-related genes was down-regulated expressed in transgenic plants upon inoculation.After24 h post inoculation,comparing with wild type,the JA-related gene LOX was dramatically up-regulated expression,while SA-related gene PR1b was deduced expressed in transgenic plants.It is speculated that JA may promote the infection of late blight on potato.?5?By subcellular localization analysis,PITG₁5718.2 was localized in the cytoplasmic membrane.In summary,by studying the biological function of PITG₁5718.2^HLJ in potato,we further analyzed the role of this avirulence gene in the infection of potato late blight.And isolated the relevant non-host disease resistance genes will helps to understand the specific inhibition process in the future.