The Screening Of Defensive Genes In Tobacco Specifically Induced By Bemisia Tabaci MEAM1 And The Role In Defense Against Myzus Persicae

Bemisia tabaci Middle East-Asia Minor 1(MEAM1)is a worldwide invasive pest.It has rapidly spread and cause great loss in agricultural production after invades in China.Our previous study showed that B.tabaci nymphs infestation decreases Myzus persicae performance on systemic but not local leaves of tobacco plants and feeding of Trialeurodes vaporariorum could not induce this aphid resistance.In the present study,transcriptomes of tobacco infested with B.tabaci and T.vaporariorum nymphs were sequenced using Illumina sequencing technology and the specific genes in systemic leaves induced by B.tabaci nymphs were screened.We investigated the role of selected genes in aphid resistance using tobacco curly shoot virus mediated virus-induced gene silencing technique.Also,we explored the mechanism of the spatial difference in aphid resistance by determining and analyzing the tobacco cell redox states and NPR1 subcellular localization after B.tabaci nymph infestation.This research had important academic meaning for showing competitive replacement mechanism of B.tabaci to other native insects and provide theoretical basis for pest resistance breeding and integrated pest management.Major research results as follows:1.Using Illumina sequencing technique to to monitor the tobacco transcriptome,a total of48377 transcripts were generated after an assembly in leaves of B.tabaci infested tobacco plants.The Blastn of the non-redundant(nr)tobacco database resulted in the identification of37462 transcripts,among which 24827 transcripts were mapped to 1690 GO terms and 5643 transcripts belong to 254 KEGG pathways.Most identified genes(766)involved in the metabolic process pathway,while there were 197,190 and 167 genes mapped to Plant hormone signal transduction,Phenylpropanoid biosynthesis and Plant-pathogen interaction pathway,respectively.25807 genes were up-regulated by B.tabaci nymph infestation,among which 11574 genes have GO annotation and 8260 transcripts mapped to different KEGG pathways.2.A total of 2204,7005 and 4951 transcripts were differentially expressed in systemic and local leaves of B.tabaci infested tobacco plants and systemic leaves of T.vaporariorum infested tobacco plants,respectively,among which 1547,3794 and 2809 transcripts were found to be up-regulated.Further analysis suggests 164 transcripts specifically induced by B.tabaci were screened.143 of which have NR annotation,104 genes mapped to 31 GOs.Therewere 34 genes mapped to 21 different KEGG pathways,and 9 genes belong to Phenylpropanoid biosynthesis Plant hormone signal transduction and Plant-pathogen interaction pathway.3.Based on the transcripts functional annotation,five differently expressed genes Nt WRKY41,Nt WRKY70,Nt EIG-E76,Nt Chi4,Nt Chi2 were screened and verified.Two of these genes,Nt WRKY70 and Nt EIG-E76,were shown to be involved in the aphid resistance induced by B.tabaci.Real-time quantitative pcr results showed that higher relative expression levels of Nt WRKY41,Nt WRKY70,Nt EIG-E76,Chi4 were observed in systemic than in local leaves of B.tabaci infested tobacco.This was consistent with the trends of RNA sequencing datas.Using gene-silencing technology,we found aphid survival rates in systemic leaves of WRKY70 or Nt EIG-E76 silenced plants were significant higher than that of wild-type plants after B.tabaci infestation.Nt WRKY41,Nt WRKY70 are key regulators of plant signal pathways,Nt EIG-E76 and chi have close relationship with glucanase and chitinase encoding.4.Using gene-silencing technology,we found that aphid resistance induced by B.tabaci was NPR1-dependent.And B.tabaci feeding induced variation of NPR1 subcellular localization contributed to the spatial difference in aphid resistance.Aphids survival in systemic leaves of NPR1-slienced tobacco plants had no difference with that of the wild-type plants after B.tabaci infestation.Real-time quantitative pcr results showed that,the expression level of NPR1 were increased in both local and systemic leaves after B.tabaci infestation.While subcellular localization assay demonstrated that localization of NPR1 in local and systemic leaves was different.In local leaves,B.tabaci infestation could inhibit nucleo-cytoplasmic transportantion,and most NPR1 proteins distributed in cytoplasm.On the contrary,NPR1 proteins were gathered in nucleus in systemic leaves.Then the nuclear localization of NPR1 was critical to active the aphid resistace.5.Variation of tobacco cell redox status induced by B.tabaci nymphs was close related with the aphid resistance and may affect the NPR1 subcellular localization.Levels of enzymatic antioxidants(CAT,APX)and non-enzymatic antioxidants(As A,GSH)were increased in systemic leaves while decreased in local leaves compared with respective controls after B.tabaci nymph infestation.Further exploration found that CAT was involved in B.tabaci induced aphid resistance.B.tabaci induced CAT activity decreased when the Cat1 expression was silenced.And Cat1 silencing made B.tabaci infested plants a more suitable host for aphids than infested control plants.While application of catalase from bovine liver could accelerate this aphid resistance.Moreover,silencing of Cat1 led to thesuppression of the B.tabaci mediated PR-1 and PR-2 expression.