| Bemisia tabaci MEAM1（Middle East-Asia Minor 1）is a worldwide invasive pest.It has spread rapidly and has been the dominant species on a variety of plants since invasion,causing significant losses to agricultural production.As small molecule regulatory substances in plant,phytohormones play an important role in regulating plant growth and development and resisting external stress,and are an important means for plants to balance the growth-defense resource allocation.The regulation of phytohormone signaling by B.tabaci has been considered to be one of the main reasons for its rapid invasion and serious damage.Previous studies have found that infection of B.tabaci caused specific changes in host plant phenotypes and physiological.In addition,B.tabaci feeding on tobacco can activate the salicylic acid（SA）signaling pathway and induce defensive response,which has a significant adverse effect on subsequent insects.In this study,GC-MS/MS and ELISA were used to systematically compare the hormones contents changes of tobacco after B.tabaci and Trialeurodes vaporariorum feeding.And combined with tobacco growth and photosynthetic indicators,the specific inhibitory effect of B.tabaci feeding on tobacco growth was clarified,and the target hormone inhibited by B.tabaci was identified:gibberellin.By measuring gibberellin content and expression levels of important regulatory genes after feeding of B.tabaci,the effects of B.tabaci on gibberellin synthesis and signal transduction were clarified.Further,gene silencing/overexpression and yeast two-hybrid technology were used to analyze the interaction between salicylic acid and gibberellin,and elucidate the inhibition mechanism of B.tabaci feeding on tobacco gibberellin signaling pathway;combined with aphid performance assay,the role of gibberellin in resistance to aphid resistance induced by B.tabaci was explored.This study revealed the regulation mechanism of hormone signals by B.tabaci,and enrich the knowledge about the transduction of plant hormone signals and their interactions during feeding by phytophagous insects.The main findings are as follows:1.By systematically comparing changes in tobacco growth and photosynthetic indicators of MEAM1 and T.vaporariorum under different densities,it was found that B.tabaci can cause more significant growth inhibition in tobacco compared to T.vaporariorum.The growth index measurement results showed:50 whitefly/plant of B.tabaci feeding for 10 d resulted in a significant reduction in tobacco plant height and internode length,which were 18.21%and14.86%lower than that of the controls,respectively,and as the feeding density and the feeding time increased.However,200 whitefly/plant of T.vaporariorum feeding for 30 d only resulted in a 9.35%decrease in tobacco plant height and a 13.14%decrease in internode length.In addition,feeding of B.tabaci also specifically resulted in changes in leaf area,the leaf area decreased by 31.23%（lower leaf）and 23.00%（middle leaf）,respectively,compared with the control.And the dry weight per cm2 of the upper leaves was significantly reduced,which was26.77%lower than that of the control when feeding on 200 whitefly/plant of B.tabaci for 30 d.But,feeding of T.vaporariorum did not cause significant changes in upper leaf area and dry weight.The photosynthetic index measurement results show:50 whitefly/plant B.tabaci feeding also significantly reduced the chlorophyll content and inhibited the stomata opening of tobacco functional leaves,which led to a decrease in photosynthesis of tobacco.Feeding of T.vaporariorum did not significantly affect the chlorophyll content and photosynthesis of the upper leaves of tobacco.2.Analysis of the contents of six hormones in tobacco found that compared to T.vaporariorum,feeding of whitefly caused more significant changes in plant hormones,and that nymph feeding played the most important role.The results of the hormone content feeding of B.tabaci adults and nymphs showed that,feeding of B.tabaci resulted in significant changes in the six hormone contents of tobacco.The contents of gibberellin（GA3）,auxin（IAA）,and methyl jasmonate（Me JA）in the attacked tobacco plants were reduced by 36.79%,37.30%,and21.70%,respectively,compared with the control;the contents of cytokinin（CK）,abscisic acid（ABA）,and ethylene（ET）were increased by 62.29%,13.5%,and 15.45%.T.vaporariorum feeding resulted in changes in four hormones content except GA3 and CK.IAA was only 19.50%lower than the control,while Me JA,ABA and ET content was 26.80%,25.00%,and 27.24%higher than the control.When the whitefly adults and nymphs were feeding separately,we found that the changes in the hormone content（except ET）caused by the whiteflies nymphs alone was consistent with adults and nymphs feeding together.And feeding of B.tabaci adults only increased the content of Me JA and ET.Therefore,changes in hormones content of tobacco caused by B.tabaci are mainly caused by nymphs.In addition,gibberellin content was significantly inhibited after B.tabaci damage,which is consistent with the inhibition of tobacco growth phenotype.3.Bemisia tabaci inhibited tobacco GA signaling pathway by activating the GA inhibitor gene RGL and inhibiting the GA synthesis gene GA20ox expression.GC-MS/MS results showed that,the GA3 content began to decrease after B.tabaci feeding for 10 d,and the decrease was more significant with B.tabaci feeding,which were 21.21%（10 d）,37.40%（20 d）,and42.40%（30 d）lower than the control,respectively.q RT-PCR results showed that,Bemisia tabaci feeding caused a significant decrease in the expression level of GA20ox,which was 33.66%（5 d）,71.70%（10 d）,and 82.08%（20 d）of the control.The expression of the RGL gene was increased by 21.75-fold compared with the control when the B.tabaci feeding for 20 d.The results of exogenous spraying of GA3 also proved that sprayed GA3（1μM and 2μM）could partially alleviate the reduction of tobacco plant height and internode length caused by B.tabaci,which increased by 77.57%and 93.82%compared with control tobacco,respectively.4.It was found that B.tabaci feeding on tobacco inhibits gibberellin synthesis and signal transduction through the salicylic acid signaling pathway and its important regulator WRKY70.Transplantation of Nah G gene to block salicylic acid would weaken the inhibitory effect of B.tabaci on gibberellin signaling pathway and tobacco growth.B.tabaci feeding Nah G tobacco only resulted in a slight decrease in GA3 content of 14.80%compared to the control,a decrease in GA synthesis gene GA20ox expression by 44.15%,and RGL expression increased by 2.12-fold,which was significantly lower than the change level of wild-type tobacco.In addition,Silencing WRKY70 did not cause significant changes in the tobacco GA signaling pathway,and B.tabaci feeding only resulted in a decrease in GA20ox expression by 29.00%compared to the control,while RGL expression increased by only 3.63-fold compared to the control.The inhibition of the tobacco GA signaling pathway was significantly alleviated by B.tabaci feeding.Overexpression WRKY70 exacerbated the inhibition of GA signaling pathway causing by B.tabaci,and the expression of RGL gene was increased by 24.22-fold compared with the control.WRKY70 and RGL are key regulators of the salicylic acid and gibberellin signaling pathways,respectively.Yeast two-hybrid experiments have shown that WRKY70 and RGL can interact with each other to regulate the transduction of salicylic acid and gibberellin signaling pathways.5.It was found that gibberellin played a negative regulatory role in the resistance of B.tabaci to tobacco aphids through its antagonistic effect with salicylic acid.Exogenous spraying of chlormequat（CCC,GA synthesis inhibitor）can significantly reduce the survival rate and fertility of M.persicae on tobacco,the survival rate and fertility of M.persicae were reduced by 18.94%and 16.52%,respectively,compared with sprayed water.Survival rate of M.persicae on GA20ox-silenced tobacco was significantly reduced,while overexpression-GA20ox did not affect the survival rate of M.persicae.In addition,GA20ox-overexpressing tobacco caused inhibition of SA signaling pathway induced by B tabaci,the expression levels of SA signaling pathway important defense genes WRKY70,NPR1 and PR1-a were only 2.71-,1.25-,and 1.23-fold higher than those of the uninjured control,which was significantly lower than the increase of wild-type tobacco. |
