Cloning And Functional Analysis Of Nicotiana Benthamiana Gene Coding Hypersensitive Cell Death Induced By Three Pamps: Fungal Nep1, Bacterial Harpin, And Oomycete INF1

Elicitors can trigger the plant immune system, leading to rapid programmed cell death (hypersensitive cell death, HCD) and stomatal closure. Here, we cloned and functional analysed Nicotiana benthamiana gene coding HCD induced by three PAMPs:fungal Nep1, bacterial harpin, and oomycete INF1 via virus-induced gene silencing. From these genes, we investigated the role of NbALY916.The cDNAs from Nicotiana benthamiana were cloned into a binary potato virus X(PVX)-based expression vector and transfermed into Agrobacterium tumefeciens(Gv3101).6000 clones were individually toothpick-inoculated or injection-inoculated onto leaflets of Nicotiana benthamiana.35 cDNAs were identified to suppress or attenuate formation of a necrotic lesion around the inoculation site,14 of that have different sequence. Nb14-4, one of the clones, encodes a specific ALY protein. Clone Nb 14-56 encodes a protein highly homologous to PhotosystemⅡlight-harvesting chlorophyll a/b-binding protein in Nicotiana tabacum; Nb38(2)-59 encodes a Ribonucleoprotein RNP-1; Nb3-9 and Nb4-26 encode Malate dehydrogenase and Fructose-bisphosphate aldolase respectively. Nb4-28 encodes 14-3-3 d-2 protein in Nicotiana tabacum and so on. No homologous genes of another 21 clones were found in the public database, suggesting that the cloned fragments were too small or no homologous EST sequence appeared in the database. Based on the result, it firmly confirms that this functional screening method is a feasible strategy to identify cDNAs of Nicotiana benthamiana that regulate hypersensitive cell death. And this work will help to further elucidate molecular mechanism of hypersensitive cell death in plants.Functioning as a regulator of HCD, a gene coding for NbALY916 was obtained from the library of Nicotiana benthamiana using the well-established Potato virus X (PVX)-based virus-induced gene silencing (VIGS). We used VIGS method to produce single-silenced (NbALY916, NbALY615, NbALY617 and NbALY1693) N. benthamiana plants. NbALY916-silenced plants attenuated Nepl or harpin-induced HCD and H2O2 production, but developed a normal HCD after INF1 treatment. However, other ALYs-silenced plants developed a normal HCD after Nepl, harpin or INF1 treatment suggesting that NbALY916 may be specifically involvd in regulation of elicitor induced HCD and regulation of HCD by NbALY916 is elicitor dependent. Surprisingly, NbALY916-silenced plants showed significantly impaired elicitor-induced stomatal closure and elicitor-promoted nitric oxide (NO) production in guard cells, but not elicitor-triggered AOS production in guard cells. These results suggest elicitor-induced stomatal closure, NO accumulation and H2O2 accumulation may be uncoupled and NbALY916 function specifically in regulation of elicitor-induced stomatal closure and elicitor-promoted nitric oxide (NO). We also found HCD caused by overexpression of MAPKKKa was suppressed in NbALY916-silenced plants; the transcript accumulation of NbALY916 was downregulated in MAPKKKa, MEK2 and WIPK-silenced plant while NbALY916 was required for WRKY2 transcripts accumulation. In addition, NbALY916 gene silencing was able to suppress expression of PR genes induced by Nepl and impacted plant disease resistance. Overall, these results indicate that, NbALY916 may be located at downstream of MAPK cascade composed by MAPKKKa, MEK2 and WIPK to regulate Nepl-induced cell death and disease resistance, while other signaling pathways may be involved in harpin or INF1-induced HCD.