The Molecular Mechanism Of Hypersensitive Cell Death And Stomatal Closure In Response To Three Pamps: Bacterial Harpin, Fungal Nep1, And Oomycete Boehmerin

Elicitors or pathogen-associated molecular patterns (PAMP) can trigger the plant immune system, leading to rapid programmed cell death (hypersensitive response, HR) and stomatal closure. Here, we describe the role of plant respiratory burst oxidase homologs (RBOH), vacuolar processing enzyme (VPE), heterotrimeric G-proteins (G proteins), and mitogen-activated protein kinase (MAPK) from Nicotiana benthamiana in response to three PAMPs:bacterial harpin, fungal Nepl, and oomycete boehmerin, induced HR and stomatal closure via virus-induced gene silencing. RBOH, VPE, and G protein can function in elicitor-induced stomatal closure. MAPKKKa-MEK2-WIPK play a role in Nepl-triggered HR. Ga, Gβ2, and VPE la are involved in harpin-induced HR. These results indicate signaling component is conserved in different elicitor-triggered stomatal closure. Despite this conservation, the complexity of elicitor signaling also exists.RBOH play a prominent role in AOS production. NbrbohA was constitutively expressed at a low level, whereas NbrbohB was induced when protein elicitors exist (such as boehmerin, harpin, or INF1). We examined the HR cell death and pathogenesis-related (PR) gene expression of these single-silenced (NbrbohA or NbrbohB) and double-silenced (NbrbohA and NbrbohB) N. benthamiana plants, induced by various elicitors. The HR cell death and transcript accumulation of genes related to the defense response (PR1) were slightly affected, suggesting that RBOH are not essential for elicitor-induced HR and activation of these genes. Interestingly, gene-silenced plants impaired elicitor-induced stomatal closure and elicitor-promoted nitric oxide (NO) production, but not elicitor-induced cytosolic calcium ion accumulation and elicitor-triggered AOS production in guard cells. These results indicate that RBOH from N. benthamiana function in elicitor-induced stomatal closure, but not in elicitor-induced HR.VPE is a cysteine proteinase with the caspase-1-like activity. Although NbVPE silencing didn't affect H2O2 accumulation triggered by boehmerin, harpin, or Nep1, the HR was absent in NbVPE 1a-and NbVPEl a/1b-silenced plants treated with harpin alone. However, NbVPE-silenced plants developed a normal HR after boehmerin and Nepl treatment. These results suggest that harpin-triggered HR is VPE-dependent. Surprisingly, all gene-silenced plants showed significantly impaired elicitor-induced stomatal closure and elicitor-promoted nitric oxide (NO) production in guard cells. Dual-silenced plants showed elicitor-triggered AOS production in guard cells. The accumulation of transcripts associated with defense and cell redox was examined using semi-quantitive RT-PCR, and it was found that an Nbrboh gene and WKRY transcription factor are VPE-regulated. Overall, these results indicate that VPE from N. benthamiana functions not only in elicitor-induced HR, but also in elicitor-induced stomatal closure, suggesting that VPE may be involved in PAMP-triggered immunity (PTI).Signaling through G proteins is a conserved mechanism found in all eukaryotes. Here, Ga, Gβ1, Gβ2, and AC cDNA were isolated. The HR was absent in Gα-and Gβ2-silenced plants treated with harpin alone. However, all the gene silenced plants developed a normal HR after boehmerin and Nep1 treatment. These results suggest that harpin-triggered HR is Ga and G02 dependent. Surprisingly, Gα, Gβ1, and Gβ2-silenced plants showed significantly impaired elicitor-induced stomatal closure and elicitor-promoted nitric oxide (NO) production in guard cells. It suggests elicitors signaling mechanisms are different. We show that both Gβsubunits, each provides functional selectivity to the plant heterotrimeric G proteins, revealing a mechanism underlying the complexity of G protein-mediated signaling in plants.MAPK cascades are composed of three protein kinase modules:MAPKK kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs, which are linked in various ways to upstream receptors and downstream targets. Silencing of genes encoding a motigen-activated protein kinase (MAPK) WIPK, an MAPK kinase (MAPKK) MEK2, or an MAPK kinase kinase (MAPKKK) MAPKKKa attenuated Nepl-induced hypersensitive response (HR); two MAPK, SIPK and NTF6, an MAPKK, MEK1-silenced plants developed the normal HR after boehmerin, harpin or Nepl treatment. We also found the transcript accumulations of a WRKY2 transcription factor and a defense-related gene (PR2b) were down-regulated in all the gene-silenced plants after Nepl treatment. WRKY2-silencing also abolished Nepl-triggered HR, but not boehmerin and harpin. We also found MEK2-NTF6 participated in elicitor-induced H2O2 production and SIPK-silencing attenuated elicitor-induced NO production in guard cells.Above all, the identification of these genes that play a role in elicitor signaling advances our knowledge of elicitor signaling, and it provides further evidence for the convergence and divergence of downstream signaling of different elicitors.