澳洲棉 REVEILLE2 调控茉莉酸防御信号提高黄萎病抗性的分子机理

Verticillium wilt(VW) is a soil-borne fungal disease caused by Verticillium dahliae Kleb.(Vd). This disease is very difficult to control in infected plants owing to the long viability of resting structure, microsclerotia, and invasion of pathogen into xylem vessels. Up to now, there are no effective measure available to control VW once it occurs. Development and application of resistant varieties is the most economic and effective strategy to control VW. Therefore, based on insights into the molecular mechanisms of plant-Vd interaction, improving plant disease resistance via genetic engineering is an effective and sustainable strategy to control VW. Gossypium australe (2n=G2G2= 26), a wild relative of cotton, is a diploid wild species with resistant to both Fusarium wilt and Verticillium wilt, and it also has broad-spectrum resistance to various strains of Vd, which is an important gene resource for cotton resistance breeding. To narrow the mining range of disease resistance genes in G. australe, we first identified a G. hirsutum-G.australe translocation line TA01-7G, which was resistant to VW. Before and after Vd inoculation, the transcriptome of TA01-7G roots were andyzed, and 12 candidate genes from G. australe were screened. The resistance gene RVE2 was silenced in TA01-7G by using virus-induced gene silencing(VIGS) and transgenic overexpression technique in Arabidopsis and the resistance mechanism of RVE2 was further studied. First, RVE2 silencing reduced the resistance to Vd in TA01-7G. To further investigate RVE2 resistance function, we overexpressed RVE2 in Arabidopsis. The pathogenicity assays showed that overexpression of RVE2 increased the resistance to Vd in Arabidopsis. To elucidate the molecular mechanism of RVE2 resistance, possible interacting proteins of RVE2 were screened by yeast two-hybridassay(Y2H). Theresultrevealed that RVE2 physically interacted with TPL/TPRs and disturbed JAZ (Jasmonate ZIM-domain) proteins to recruit TPL and TPR1 in NINJA-dependent manner, which regulated Jasmonicacid(JA)response by relieving inhibited-MYC2 activity. ChIP-qPCR, electrophoretic mobility shift assay(EMSA)and Dual luciferase(Dual-LUC) experiments showed that, MYC2 bound to RVE2 promoter for activating its transcription, forming feedback loop. Interestingly, RVE2 gene sequence analysis in natural population showed that a unique truncated RVE2(GhRVE2D)widely existing in D-subgenome of upland cotton, which represses the ability of the MYC2 to activate GhRVE2A promoter but not GausRVE2 or GbRVE2. Subsequently, transient expression assays of different RVE2 genes in tobacco leaves indicated that the integrity of the RVE2 gene was of extremely importance. The result could partially explain why G. barbadense showed highervw resistance than G. hirsutum. Furthermore, disturbing the JA-signalling pathway using VIGS assays resulted in the loss of RVE2-mediated disease-resistance. RVE2 overexpression significantly enhanced the resistance to VW. RVE2 also physically interacted with MPK3 and MPK6, functioned in the downstream of BAK1 and SOBIR1 mediated innate immune response. Thus, we conclude that RVE2, as a new regulatory factor, plays a pivotal role in fine-tuning JA-signaling, which improves our understanding of the mechanisms underlying the resistance to cotton VW and provides a new gene for cotton breeding for resistance to VW.