Exogenous Application Of Salicylic Acid Enhanced Plant Tolerance To Tomato Yellow Leaf Curl Virus Via Triggering Rnai And Mapk Cascade Signaling Pathway In Tomato

Tomato yellow leaf curl disease,caused by Tomato yellow leaf curl virus(TYLCV),is a serious threat for tomato production all of the world.Currently,two methods are used to control this virus,one is breeding by infiltrated resistant genes or markers from wild type tomatointo cultivated varieties,and the other is transgenic technology through RNA interference(RNAi)to suppress the replication of virus.The former is time-consuming and resistance is not stable.While the latter transgenic technology has not been widely accepted by consumers.However,plant induced resistance provides us a new way to enhance tolerance to TYLCV.Induced Resistance is a phenomenon that plants can quickly and strongly response the subsequent pathogen infection,if these plants had been under certain elicitor by biological or abiotic factors.Induced Resistance mainly includes induced systemic resistance(ISR)and systemic acquired resistance(SAR).ISR is induced by Plant Growth-Promoting Bacteria(PGPB),which relies on jasmonic acid(JA)and ethylene(ET)signaling pathways,while SAR is induced by pathogen infection or chemicals,such as SA or its derivatives benzothiadiazole(BTH).Therefore,we proposed a hypothesis that Induced Resistance could increase tolerance to TYLCV in tomato plants.Moreover,MAPK cascade signal participated in Induced Resistance,however,RNAi plays a key role in plant antiviral defense.What is the relationship between MAPK cascade signal and RNAi in SA-meidated defense?This study firstly demonstrated that exogenous foliar application of SA enhanced tomato tolerance to TYLCV.It was confirmed that a proper concentration of SA can enhance Induced Resistance in tomato plants.Then,the function of SlDCL2 and Sl DCL4,two key genes involved in plant RNAi signal,was studied in tomato plant against TYLCV.In addition,SlMAPK3,which is involved in Induced Resistance,was studied after artificial inoculation of TYLCV in SlMAPK3 transgentic plants.Further more,the relation betweenRNAi,SlMAPK3 and SA signaling pathway was investigated in Arabidopsis mutant.The mechanism of the SA-mediated induced resistance to TYLCV was studied deeply in this study,which providing a theoretical basis for Induced Resistance antiviral defense in plant.Main results are as follows:1The optimal concentration was 0.5 mM for SA-mediated resistance enhanced the tolerance to TYLCV in tomato plants of ‘TTI112B-2'.Tomato plants of ‘TTI112B-2' was treated with different concentrations of SA for two days,then were inoculated with TYLCV infection cloning.The disease incidence(%)and the disease index were calculated at 30 days after inoculation(dpi).Results demenstrated that the disease incidence(51.1%)and the disease index(29.3)in SA-treated plantswere significantly differentfrom those of control plants with a 68.8% of disease incidence,and 35.7 of disease index.Further,it was found that SA treatment induced most of RNAi-related genes,decreased TYLCV-infected cell membrane damage,increased the antioxidation ability,and enhanced thephotosynthetic ability.Lastly,it was confirmed that systemic acquiredresistance(SAR)had been induced in plant by SA pretreatment.2SlDCL2 and SlDCL4 were essential in tomato antiviral defense against TYLCV.25 genes involved in RNAi signal were detected in resistance('Y19')and sensitive('TTI112B-2')material after TYLCV infection by qRT-PCR.Resultsdemenstratedthatmost RNAi-related genes were induced in both materials,however,the expression of these genes were relatively higher in resistance materialas compared to sensitive material.Two key genes in RNAi pathway,SlDCL4 and SlDCL2,were silenced by Virus-Induced Gene Silencing(VIGS)technology in both materials.It was found that TYLCV content and disease index were increased,the disease symptoms appeared earlierand resistance was damaged in both the materials.3SA-mediated resistance needs the participation of DCL2 and DCL4.Arabidopsis wild type Col-and mutant dcl2,dcl4 and dcl2/4 were pretreated with 0.1 mM SA,then inoculated with TYLCV.The content of TYLCV were determined by qPCR and ELISA at 14 dpi.The resultsdemenstrated that SA pretreatment inhibited the replication of TYLCV in Col-,however,in mutant of dcl2,dcl4 and dcl2/4,TYLCV could not be inhibited by pretreatment of SA.In addition,H2O2 and O2–contents were found to be higher in all plants after TYLCV inoculation,especially for mutant plants of dcl2,dcl4 and dcl2/4.These results suggested that DCL2 and DCL4 play a key role in SA-induced tolerance to TYLCV.4 MAPK3 participated in regulating the tolerance to TYLCV in tomato plants.SlMAPK3 was induced in RNA and protein levels by TYLCV inoculation in ‘Y19'materialusing the method ofqPCR and ELISA technology,respectively.The function of SlMAPK3 were investigated by VIGS and transgenic techniques.It was found that SlMAPK3 participated in regulating of SA/JA-signaling genes(SlPR1,SlPR2/ SlLapA,SlPI-I,and SlPI-II).Overexpression of SlMAPK3 enhanced the tolerance to TYLCV in transgenic plants of OE4,OE6 and OE7 compared to wild type(WT)and had a lower disease severity,but had a higher antioxidant capcity.5 SA signal participated inthe pathway of MAPK3 activation induced by TYLCV.The wild type Arabidopsis(Col-),npr1(SA signal were blocked in npr1)and mapk3 mutants were pretreated with 0.1 mM SA for 24 h,then was inoculated with TYLCV.The activity of MAPK3 were determined by western blot.Results demenstrated that theTYLCV induced MAPK3 activity was enhanced by 0.1 mM SA pretreatment in Col-,but the increasing MAPK3 activityin npr1 was diminished.These result suggested that SA signaling participated inMAPK activity pathway induced by TYLCV pathogene.