Heterologous Expression Of Effector Mi-ISC-1 From Meloidogyne Incognita And Its Functional Analysis

Salicylic acid(SA)is a kind of plant defense signaling molecules,and it has been proved to play an important role in resisitance of pathogen infection.Using isochorismatase to hydrolyze isochorismate which is the synthetic substrate of SA in plants,thereby to inhibit plant immunity,is an important pathogenic mechanism for Verticillium dahliae and Phytophthora sojae.The effector Mi-ISC-1 encoding isochorismatase from Meloidogyne incognita was focused on this study.The transgenic Nicotiana benthamiana lines overexpressing Mi-isc-1 gene were cultivated,and the root growth and nematode infection of the transgenic lines were detected.The isochorismatase activity of Mi-ISC-1 was measured in vitro and in vivo using the prokaryotic expression system and Agrobacteriummediated transient expression.The effects of transient expression of Mi-ISC-1 on SA accumulation in tobacco leaves were detected,and subcellular localization of Mi-ISC-1 on inhibiting plant immune were also determined.The main results are as follows:1.The overexpression of Mi-ISC-1 in N.benthamiana can promote the parasitism of M.incognitaThe transgenic N.benthamiana lines was cultivated through plant expression vector carried Mi-isc-1 gene using Agrobacterium-mediated transformation.A total of six transgenic lines were obtained through hygromycin screening.The transcriptions of Mi-isc-1 gene in transgenic lines were detected using the RT-PCR,and the result showed that a600 bp fragment of Mi-isc-1 gene was specifically amplified from 6 transgenic lines,not from the wild-type(WT)and the empty vector control(GL)lines,which indicated the Miisc-1 gene can be transcribed normally in these transgenic lines.The results of Western Blot revealed that the fusion protein of Mi-ISC-1 and GFP was detected in transgenic lines,indicating Mi-ISC-1 have been expressed normally in N.benthamiana.The transgenic lines of No.1,2,and 4 were selected,and the phenotypes of root growth were observed,showing the overexpression of Mi-ISC-1 did not significantly affect the growth and development of tobacco roots.The results of inoculation with M.incognita showed that the numbers of galls,females and egg masses produced on the transgenic lines were increased by 33.7%,39.1% and 38.1%,respectively,when compared with that on control lines.These results revealed that the overexpression of Mi-ISC-1 in N.benthamiana can promote the parasitism of M.incognita.2.Mi-ISC-1 has isochorismatase activity in vitro as well as in vivoUsing the prokaryotic expression system,the proteins Ent A,Ent B,and Ent C related to isochorismate metabolism were expressed in E.coli and purified subsequently.The proteins Mi-ISC-1 and GFP were transiently expressed in tobacco leaves by Agrobacterium-mediated transformation and purified.The hydrolysis isochorismate activity of Mi-ISC-1 were determined by enzymatic reactions in vitro.The dynamics of the absorbance value at 340 nm in Mi-ISC-1 reaction increased with the time,which is similar to that in Ent B reaction as positive control,while in GFP reaction as negative control,the absorbance value did not change with the time.These results indicated that the Mi-ISC-1has isochorismatase activity in vitro.The content of isochorismate hydrolysate DDHB in tobacco leaves transiently expressed Mi-ISC-1 was further determined,the significant increase was appeared when compared with that in the GFP control,indicating the Mi-ISC-1 also has isochorismate hydrolysis activity in plant cells.3.The key site for Mi-ISC-1 to inhibit plant SA accumulation and its mediated immunity is cytoplasmThe effector Mi-ISC-1 was transiently expressed in tobacco leaves through the Agrobacterium-mediated trasformation.After inoculation with Phytophthora capsici,the levels of free SA and salicylate glucoside in tobacco leaves transiently expressed Mi-ISC-1were significantly lower than that in GFP treatment.The resutls indicated that the Mi-ISC-1can reduce the accumulation of SA induced by the infection of pathogens.After the expression site of Mi-ISC-1 was changed by nuclear localization and nuclear export signal,the resultes of inoculation with P.capsici revealed that the Mi-ISC-1 expressed in fusion with nuclear export signals can promote the infection of P.capsici and reduce the SA accumulation.However,the Mi-ISC-1 with nuclear localization signal has no significant effects on the P.capsici infection and the SA accumulation.The q RT-PCR detection revealed that the expression of PR1 gene was significantly inhibited in tobacco leaves with transient expression of Mi-ISC-1 localized in cytoplasm,while was not inhibited in that localized in nuclear.These results confirmed that the cytoplasm is the key site where the effector Mi-ISC-1 inhibiting plant immunity.In summery,the effector Mi-ISC-1 of M.incognita has isochorismatase activity,which inhibiting the SA synthesis in plant cytoplasm,thereby affecting the immune response mediated by the SA and promoting the nematode parasitism.