Functional Characterization Of ACTIN7 In Immune Response Of Pepper To Ralstonia Solanacearum Infection

Capsicum annuum（pepper）is the largest vegetable in China,during it growth and development,pepper frequently suffers from various soil borne diseases including bacterial wilt caused by R.solanacearum,which tends to be more serious under the conditions of high-temperature-high-humidity（HTHH）.The most effective way to solve the problems caused by the soil-borne pathogens is to develop and utilize cultivars with high levels of disease resistance,and a better understand of the mechanisms underlying disease resistance will benefit the development of pepper cultivars with high levels of disease resistance.Actin is one of the most abundant plant cellular proteins,which are highly conservative and mediate almost every form of cell movement.Previous studies have shown that besides that they might fulfill their functions in the cytoplasm,actins can also polymerize and depolymerize in the nucleus,and have implicated in direct regulation of gene transcription,DNA rearrangement and repair.For example,ACTIN7 in Arabidopsis thaliana has been found to be the only actin that responds to external stimuli and plant hormone cues,and its expression is induced by both auxin and light,but is inhibited by abscisic acid signaling.However,the role of ACTINs in plant immune defense response is still unclear.In this study,we functionally characterized ACTIN7 in defense response of pepper to R.solanacearum infection.The main results are as followings:（1）The results of Quantitative Real-time PCR showed that CaACTIN7in pepper plants infected by R.solanacearum under room temperature and HTHH conditions was significantly down-regulated compared with the control plants.Furthermore,exogenous application of salicylic acid（SA）or abscisic acid（ABA）also down-regulated CaACTIN7,indicating it relationship to defense response of pepper plant to R.solanacearum infection.In addition,the result of subcellular localization analysis carried out by transient overexpression in N.benthamiana leaf epidermal cells showed that CaACTIN7 distributes in the whole cell of N.benthamiana leaf epidermal cells.（2）Through the analysis of virus-induced gene silencing,it was found that CaACTIN7 silencing enhanced the resistance of pepper plants to inoculation with R.solanacearum,and promoted the up-regulated expression of SA signaling mediated marker genes CaNPR1 and CaPR1 as well as CaABR1 that is mediated by ABA signaling.（3）In order to confrim the result from virus-induced gene silencing,we carried out N.benthamiana genetic with 35S::CaACTIN7-GFP vector,and three strains T₀plants and their corresponding T₁and T₂lines were acquired.Using homozygous T₂plants,it was found that the overexpression of CaACTIN7 significantly enhanced the susceptibility of N.benthamiana to R.solanacearum infection.The results of the above loss-of-function and gain-of-function assay consistently suggest that CaACTIN7 is involved in the regulation of defense response of pepper plants to R.solanacearum and acts as a negative regulator.（4）By using the purified prokaryotic expressed CaACTIN7-His as bait,the possible interaction proteins of CaACTIN7 were found by LC-MS/MS analysis.It was found that CaACTIN7 might interact with the pathogen-associated molecular patterns（PAMP）EF-Tu of R.solanacearum.This interaction was further confirmed by bimolecular fluorescent complimentary（BiFC）and pull-down assay,and it was found that the interaction between CaACTIN7 and EF-Tu occurred in cell membrane,cytoplasm and nucleus.Through the subcellular localization analysis of transient overexpression in N.benthamiana leaves,the results showed that EF-Tu was located on the cell membrane,cytoplasm and nucleus,which was consistent with the result that interaction between CaACTIN7 and EF-Tu occurred in the whole cell.Moreover,we found that overexpression of EF-Tu in pepper leaves could activate the expression of SA and ABA induced disease resistance genes,indicating that EF-Tu could be recognized by pepper specific receptor as R.solanacearum PAMP and stimulate PTI.（5）Considering that CaACTIN7 plays a negative regulatory role in the defense response of pepper to Ralstonia solanacearum and it can interact with Ralstonia solanacearum EF-Tu,we further analyzed the possible interaction between EF-Tu and CaACTIN7 by combining virus-induced gene silencing and agrobacterium-induced overexpression.The results showed that the negative regulatory effect of CaACTIN7 on SA signal under R.solanacearum infection was not caused by EF-Tu.The above results collectively indicate that CaACTIN7 acts as a negative regulator in the defense response of pepper to R.solanacearum infection.R.solanacearum EF-Tu can activate the SA and ABA mediated immune response of pepper,and it can interact with CaACTIN7.However,the possible biological function of this interaction has not been found yet.