Screening, Cloning And Functional Analysis Of Nicotiana Benthamiana Gene Involved In Hypersensitive Cell Death Induced By Three Elicitors

To avoid the invasion of pathogen, plant has evolved an immune mechanism to limit the pathogen growth, by which the plant recognizes the pathogen and makes effective immune reaction in time. There are two kinds of innate immunity in plant:PTI (PAMP-triggered immunity) and ETI (Effector-triggered immunity). PAMPs are a kind of conservative microbial substance, which can activate the plant signaling and induce plant immunity. In our research, virus-induced gene silencing (VIGS) method was used to identify genes which involved in hypersensitive cell death (HCD) induced by three elicitors (Nep1, harpin and INF1). Moreover, we started a further study to elucidate the molecular mechanism of a gene, NbMADsl, involved in HCD caused by harpin.In our research, we constructed the cDNA of Nicotiana benthamiana to PVX vector to form a cDNA library. Virus-induced gene silencing (VIGS) method was used to identify genes from the cDNAs library involved in hypersensitive cell death induced by three elicitors. Agrobacterium tumefeciens (Gv3101) transfermed with PVX::cDNA was used to silence the Nicotiana benthamiana. As a result,16genes were identified to suppress the formation of a necrotic lesion around the inoculation site and3were identitied to suppress the plant growth from2500clones, which were individually toothpick-inoculated or injection-inoculated onto the leaflets by using VIGS. Among them,12were found the homologous sequence by blast in the gene database. We find that Nb34-22and Nb34-3encode ATP-dependent protease (CD4A) and UDP-D-apiose/UDP-D-xylose synthase respectively, and silencing both of genes suppresses the plant growth and makes the plant a drawlf. Nbl6-5encodes ADP-ribosylation factor1(ARF1) in Nicotiana benthamiana, and silencing of Nb16-5makes the Nicotiana benthamiana dead. Our results suggest that it is a feasible strategy to identify and clone cDNAs of Nicotiana benthamiana that regulate hypersensitive cell death by Virus-induced gene silencing. We identified and cloned the genes involved in elicitors-induced HCD, and our work will help to further elucidate molecular mechanism of hypersensitive cell death and the elicitor-induced signal pathway in plants. In this study, we find that harpin-induced cell death, NO and AOS stimulation could be inhibited by the virus-induced gene silencing of NbMADsl in N. benthamiana but not in INF1or Nepl treatment, which reveals that the molecular mechanism in stomatal closure, NO and H2O2accumulation activated by the3elicitors is different. A number of studies have shown that the MADs-box family is involved in floral organ differentiation and regulation of the downstream genes expression level in plants, but there is no report about whether MADs-box is involved in plant defense triggered by elicitors. So it shows great significance of the study for the molecular mechanism of how MADs-box family involved in the regulation of plant defense responses induced by harpin. We also find that the NbMADs1-silenced plants impaire harpin-triggered stomatal closure, HCD, and defense-related genes expression. Further more, stomatal closure in NbMADs1-silenced plants and PVX Nb is observed after the application of H2O2and sodium nitroprusside (NO donor), and both harpin and H2O2stimulated guard cell NO synthesis. Taken together, these results demonstrate that the NbMADsl-H2O2-NO pathway mediates multiple harpin-triggered responses, including stomatal closure, HCD and defense-related gene expression.