| Plants have evolved a sophisticated innate immunity during their long history coevolution with various pathogens,which include the two layers of interconnected branches termed PTI(PAMP triggered immunity)and ETI(effector triggered immunity).Although the Zigzag model of PTI and ETI are wildly accepted,but the conceptual discrimination between PTI and ETI has been challenged by their shared downstream signaling components,and there is a continuum between PTI and ETI.Dissection of the continuum contributes better understanding of the defence signaling.Elicitins,conserved in Phytophthora and trigger HR like cell death in plants,appear to be elicitors in the continuum between PTI and ETI.In the present study,the full length cDNA of two members of elicitins of Phytophthora capsici were isolated,and were functionally identified by a method of gain of function and loss of function,and their corresponding interactors were also isolated and functionally identified.The results are as followings.By cDNA library screening,two full length cDNA termed CAP1A and CAP2A was acquired.CAP1A exhibits a high degree of sequence identity among elicitins from different species,while CAP2A shares low degree of similarity with other elicitins.With the full length of cDNA of the two elicitins from Phytophthora capsici,their functionally identification was performed by transient overexpression,the result showed both of them induce HR like cell death and HR and PR gene expression,while the immunity induced by CAP2A is more intensive than that induced by CAP1A,especially in blight resistant pepper variety CMV334.The CAP1A and CAP2A induced immunity was found to be conserved in pepper,tobacco and Arabidopsis.The potential role of CAP2A and CAP1A were further confirmed by overexpression in transgenic tobacco.These results suggested that CAP2A and CAP1A are in the continuum of typical PAMPs and effectors and CAP2A may be evolvedly younger than CAP1A.By yeast two-hybrid,the interacting protein of CAP1A and CAP2A,CaSRC2 and CaSGT1,were isolated respectively,which were further confirmed by BiFC and co-IP,the result of BiFC showed that CAP1A-CaSRC2 and CAP2A-CaSGT1 interaction occurred in plasma membrane.The immunity induction by CAP1A-CaSRC2 and CAP2A-CaSGT-1 were investigated by individually transient expression or coexpression,and the results showed that transient overexpression of CaSRC2 and CaSGT1 induce HR cell death to a low extent,while coexpressed with CAP1A or CAP2A,CaSRC2 and CaSGT1 induced intensive HR cell death,and the silence of CaSRC2 and CaSGT1 by VIGS significantly attenuated the HR cell induction by CAP1A and CAP2A,strongly suggesting that CaSRC2 and CaSGT1 act as receptors of CAP1A and CAP2A in plant immunity induction,respectively.In addition,it was also found that C2 domain in CaSRC2 is necessary for plasma membrane localization of CaSRC2 as well as the interaction between CaSRC2 and CAP1A.Moreover,the localization of CAP1A-CaSRC2 complex in plasma membrane is also necessary for its immunity induction.Combining the results that the sequence of CAP2A is less conserved in different species but trigger more intensive immunity than CAP1A,we anticipate that elicitin CAP2A may be evolvedly younger than CAP1A and they may be evolvedly correlated.To examine this possibility,we designed a novel experiment to comparatively analysis the effect of transient overexpression of CAP2A on CAP1A-CaSRC2 triggered immunity in CaSGT1 silenced and un-silenced pepper plants,the results showed overexpression of CAP2A significantly suppressed CAP1A-CaSRC2 induced HR cell death,strongly suggesting a Zigzag interaction among CAP1A,CAP2A,CaSRC2 and CaSGT1.Our results afford the first evidence for CaSRC2 and CaSGTl act as receptors of CAP1A and CAP2A,respectively,in plant immunity induction as well as the zigzag interaction among CAP1A,CAP2A,CaSRC2 and CaSGT1,which supply new insight on quantitive evolution within PTI mediated by elictins. |
PDF Full Text Request