EIN3EIL1 Negatively Regulate Innate Immunity Through Inhibiting SA Pathway In Arabidopsis

During the coevolution of host-microbe interactions, the evolution of the plant immune response has culminated in a highly effective defense system that is able to resist potential attack by microbial pathogens. At first,based on the recognition of PRRs from plants to PAMPs from microbes, the PAMPs-triggered immunity (PTI) will be initiated rapidly. PTI may be the plant's first active response to microbial perception, which usually halts infection before the microbe gains a hold in the plant. So PTI play a significant role in the plant disease defense against pathogen infection.The recognition on PAMPs can induce a serial of defense response in plants, which is associated with MAP kinase signaling, transcriptional induction of pathogen-responsive genes, production of reactive oxygen species, and deposition of callose to reinforce the cell wall at sites of infection. Though much work has been done cataloguing PAMPs, it is still little known about the molecular mechanism of PTI.It is never denied that plant hormones such as SA, JA and ET, play crucial roles in signalling networks involved in plant defence responses. Generally, SA-dependent signalling is believed to be important for plant defences elicited by biotrophs, whereas the JA/ET pathway has been associated with defences against necrotrophs. More and more evidence demonstrates that ET pathway mutants showed different phenotype against biotrophic or semi-biotrophic pathogens. But the mechanism underlying the role of ET in plant susceptibility is still unknown.Our experiments suggested that EIN3EIL1 negatively regulate PAMPs-triggered innate immunity in Arabidopsis, including callose deposition in the site of infection, the transcription expression of pathogen-responsive genes. Furthermore, we found the negative regulation exert through inhibiting SA pathway. Both biochemic and genetic experiments supported that EIN3EIL1 protein can bind with the specific sites of the promoter of SID2. Following is the key conclusion on our experiments: 1 EIN3EIL1 negatively regulate plant defence responses against virulent and nonpathogenic pathogens. ebf1ebf2 mutant supported more bacterial growth than WT Col-0. It implied that EIN3 protein may be involve in defence responses. Furthermore, bacterial growth showed that ein3eil1 reduced susceptibility to virulent DC3000 and enhanced the resistance to nonpathogenic DC3000 hrpL, compared to WT Col-0. These results suggested EIN3EIL1 negatively regulate plant defence responses.2 EIN3EIL1 repress PAMPs-triggered innate immunity.Callose deposition induced by DC3000 hrpL and flg22 enhanced in ein3eil1 mutant, and reduced in EIN3 overexpressing plants compared to that in Col-0. In addition, ein3eil1 supports higher-level expression of FRK1 and PR1, constitutively and induciblely. All of these experiments demonstrated that EIN3EIL1 repress PTI.3 EIN3EIL1 repress transcriptional expression of flg22-induced genes.Our microarray data and results of Real-time PCR showed that EIN3EIL1 repress expression of flg22-induced genes. Among these flg22-induced and EIN3EIL1-repressed genes, many genes are components of SA pathway, such as SID2, PR1, PR2.4 SID2 mutation block the repression function of EIN3EIL1 on PTI.Genetic analysis showed that ein3eil1sid2 lost the resistance against to virulent or nonpathogenic pathogen. Additionally, callose deposition induced by DC3000 hrpL and flg22 reduced greatly in ein3eil1sid2. Expression of EIN3EIL1-repressed defense genes is also blocked by SID2 mutation. All of these results suggested that SID2 is at the downstream of EIN3EIL1 during the PTI.5 EIN3EIL1 can specifically bind with the promoter sequence of SID2.Our ChIP results showed that the sequence of SID2 promoter can accumulate to a much higher level after immunopricipitation with EIN3 or EIL1 protein. In another words, EIN3EIL1 specifically bind with the promoter sequence of SID2.6 EIN3EIL1-SID2 maybe is a node of ET and SA pathway.The growth of DC3000 and the inducible expression of PR1 showed that etr1 enhances the susceptibility to DC3000 and reduced PR1 expression. Contrary to etr1, ein2 showed reduced susceptibility to DC3000 and constitutively PR1 expression. All of them are consistent with our above conclusions. It implies that ET pathway has crosstalk with SA pathway through EIN3EIL1-SID2.Based on our results, we put forward that EIN3EIL1 negatively regulate PAMPs-triggered innate immunity through inhibiting SA pathway in Arabidopsis.Interestingly, we screened a novel ein3 allele and named as ein3-5. ein3-5 display enhanced susceptibility to both virulent DC3000 and nonpathogenic DC3000 hrpL. In addition, ein3-5 also showed reduced callose deposition induced by DC3000 hrpL and flg22. Northern blot analysis suggested that the expression of PR1 induced by bacteria is reduced in ein3-5 compared to Col-0. All of these phenotypes about defence are different to others allele of EIN3.To discover the susceptibility mechanism of ein3-5, we excluded two hypotheses that ERF1 expression or the more stability of EIN3-5 protein contribute to the susceptibility in ein3-5, on the basis of our experiments. Based on experiments in AtERF3 loss of function mutant andoverexpression plants, we found that AtERF3 contributes to the reduced callose deposition in ein3-5. It is surprised that AtERF3 never affect on the bacterial growth in both erf3 mutant plants and AtERF3 overexpressing plants. So we speculated that AtERF3 is not the major cause resulting in susceptibility in ein3-5, though AtERF3 can negatively regulate callose deposition induced by flg22.The further experiments will be done to explain the susceptibility in ein3-5.