Functional Analysis Of Tomato WRKY Transcription Factor SIDRW1and NAC Transcription Factor SISRN1in The Regulation Of Defense Response

Complex signaling networks that involve transcription factors play a central role in plant responses to surrounding crisises. The members of several transcription factor families, such as ethylene-responsive-element-binding factors (ERF)、basic-domain leucine-zipper (bZIP)、MYB、NAC and WRKY proteins have proven to involve in the plant defense response against abiotic and biotic stresses. Among them, NAC and WRKY are two of the biggest and most intensely researched transcription factor families in plants. However, most of the studies had been focused on rice and Arabidopsis, researches in these fields on tomato was rare. In this paper,12NAC and6WRKY transcription factors were cloned, and their VIGS vectors were constructed. Taken the VIGS as main tool, we identified a WRKY transcription factors gene, SlDRW1(defense-related WRKY1) and a NAC transcription factors gene SlSRN1(stress-related NAC1), respectively, which involved in tomato-disease interactions.The WRKY transcription factor gene SlDRW1is1731bp in size with a predicted open reading frame of1083bp. SlDRW1encodes a protein of360amino acids and the deduced protein contains all conserved domains characteristics of the WRKY group Ⅱ members. And the amino acids BLAST results demonstrated that SlDRW1has a99%and91%similarity in sequence with tomato other two WRKY transcription factors SlWRKY1and SlWRKY2respectively. Further alignment analysis of amino acid sequences for the ORF of SlDRW1and SlWRKY1, showed they are identical. Transactivation analysis demonstrated that SlDRW1can not activate reporter gene expression in yeast, indicating that SlDRW1maybe a transcriptional repressor. Subcellular localization analysis using GFP fusion protein showed that the SlDRW1protein is localized in the nucleus of Nicotiana benthamiana and tomato epidermal cells.The expression of SlDRW1gene was up-regulated dramatically24hours post infection (DPI) with Botrytis cinerea and peaked at48DPI. It also increased slightly by treatment with Pseudomonas syringae pv. tomato (Pst) DC3000, salicylic acid (SA) and jasmonate (JA), but not by1-aminocyclopropane-1-carboxylic acid (ACC), suggesting that SlDRW1may be involved in regulation of defense response in tomato.Then VIGS was adopted as vehicle to reveal the roles of SlDRW1in tomato-disease interaction. Quantitative real-time PCR analysis demonstrated that the transcripts of SlDRW1could be reduced by50-80%,even more than90%,around4weeks post VIGS treatment. After inoculation with Botrytis cinerea, the SlDRW1VIGS silenced tomatoes showed more susceptible phenotype than the control plants with bigger disease size. So we drew a conclusion that SlDRW1confers resistance to tomato against Botrytis cinerea. And the transient over-expression study confirmed the result. To grope for the mechanism underlying the phenomenon, the expression of active oxygen and pathogenesis-related proteins (PR) were tested. SlDRW1may modulate tomato defense response against Botrytis cinerea by regulation of the PR genes and active oxygen species expression.Nevertheless, SlDRW1has no effect on the response of tomato against Pst DC3000, drought and oxidative stress.The NAC transcription factor SlSRN1which contains a1470bp ORF and encodes a protein of490AA was also cloned and identified. The NAC gene endow resistance to tomato against necrotrophic fungus Botrytis cinerea and bacteria Pst DC3000, however, it act as a negative regulator in tomato drought responses. The expression of SlSRN1increased upon Botrytis cinerea and Pst DC3000infection, and the exogenous application of JA,SA, but not ACC. SlSRN1is a transcriptional activator since the transactivation analysis showed that SlSRN1activated reporter gene expression in yeast. Subcellular localization analysis showed that the SlSRN1protein is localized in the nucleus of cells. The mRNA products of SlSRN1in tomatoes can be reduced by an average of50%with VIGS technique. When the VIGS silenced tomatoes inoculated with B. cinerea, they showed more susceptible phenotypes and higher pathogen growth rate, indicating SlSRN1confers resistance to tomatoes against this pest. As to Pst DC3000, the silenced plants displayed similar symptoms, however, the bacteria grow much faster on SlSRN1silenced tomatoes, which made us draw a conclusion that SlSRN1acts as a positive regulator in tomato-Pst DC3000interaction. After withholding water for5days, SlSRN1silenced tomatoes showed less susceptible symptoms, indicating SlSRN1is a negative regulator in tomato response to drought. Results also hint that SlSRN1may be involved in regulating the expression of PR genes, via which to give rise to resistance of tomatoes against pathogens.