Study Of Bemisia Tabaci MEAM1 Meditated Nitric Oxide In Suppression Of Defense Responses In Tobacco

Bemisia tabaci Middle East-Asia Minor 1(MEAM1)is a worldwide invasive pest,it causes extensive crop damage and is hard to be controlled.It has rapidly spread and causing great loss in agricultural production after invades in China.Reseach data shows it can suppress effectual defense in plants by manipulating defense signaling,thereby enhancing their performance.Nitric oxide(NO)is an important multifunctional signaling molecule that acts in complex plant signaling networks.However,its role in plant defense mediated by B.tabaci infestation is largely unclear.In this study,we found that,NO contents,the activities of the NO-related biosynthetic enzyme nitric oxide synthase(NOS),and the transcript levels of the signaling component gene Nitric oxide-associated protein 1(NOA1)were significantly induced following B.tabaci infestation.In a bioassay,the exogenous application of NO donor SNP resulted in the tobacco plants being better preferred by the B.tabaci adults than the controls and also accelerated nymphal development.On the contrary,plants treated with NO scavenger c PTIO repelled B.tabaci adults,and nymphal development was prolonged in these plants comparison to the controls.However,neither the NO donor nor scavenger treatment significantly affected the survival of B.tabaci.Furthermore,silencing of NOA1 suppressed the NO level produced by B.tabaci infestation.The adult preference was decreased and nymphal development was slowed down in NOA1-selienced plants compared with uninfested controls.Importantly,silencing of NOA1 compromised the suppression of jasmonic acid(JA)levels and the downstream of JA-related defense genes mediated by B.tabaci.These results indicated that,NO signaling was triggered by B.tabaci infestation,and may play a key role in suppressing the JA-dependent basal response,thereby enhancing the performance of B.tabaci.These findings enhance our knowledge in B.tabaci-plant interactions,which are potentially important for developing new methods to control this pest.Major research results as follows:1.In tobacco plants,variation in NO contents,the activities of the NO biosynthetic enzymes NOS and NR,and the transcript levels of the NO biosynthetic NOA1,NIA-1 and NIA-2were measured following B.tabaci infestation.Feeding of B.tabaci caused a significant accumulation of NO,which increased by 53.41% of that of the control at day 15.Simultaneously,leaves infested with B.tabaci showed greater fluoresce intensity compared with the uninfested controls.Furthermore,NOS activity and the transcript levels of NOA1 increased,being 55% higher than that of the uninfested control at day 15.However,the NR activity and transcript levels of NIA-1 and NIA-2 did not show obvious alterations during the entire feeding period compared with the uninfested controls.These data indicate that B.tabaci infestation could trigger the NO signaling pathway in tobacco plants.2.To investigate the relationship between NO level and the performance of B.tabaci,we quantified the settle preference,development rate and survival of B.tabaci in plants with or without chemical application.Chemical application would alter the settle preference of B.tabaci adults.Compared with the water treated plants,71.09% of adults settled on the plants treated with SNP,whereas only 28.9% of adults settled on the plants applied with c PTIO.Furthermore,at 20 days postinfestation,the nymph development rate differed on the SNP-,c PTIO-,and water treated plants.The fourth instar proportion was remarkably higher on SNP-treated plants than on watered treated control plants.On the contrary,the fourth instar proportion in c PTIO treated plants was significantly lower than that of watered treated control plants.However,the survival rate of adult whiteflies did not differ on plants with different chemical applications.These data indicate that a positive correlation exists between the NO level and B.tabaci performance.To investigate the function of NO in the JA-mediated defense against B.tabaci,the NO synthesis related gene NOA1 was silenced using the virus-induced gene silencing technique.As shown in the results,the transcript levels of NOA1 and NO decreased by 0.634-fold and 51.9%,respectively,in NOA1-silienced plants after 15 d of B.tabaci infestation compared with the control.Similar to c PTIO application,NOA1 silencing decreased the performance of B.tabaci.Compared with the empty vector-injected plants,68.77% of adults settled on the NOA1-silenced plants,and the fourth instar proportion in the NOA1-silenced plants was significantly lower than that of the empty vector injected control plants.However,NOA1 silencing had no significant effect on the adult survival rate.3.We then analyzed the effect of silencing NOA1 on JA content and the expression levels of JA signaling-related genes.The JA content in B.tabaci nymph infested leaves did not differ significantly from that of the uninfested controls during the entire feeding period.However,B.tabaci nymph infestation obviously impacted JA accumulation in NOA1-silienced plants.The JA content was remarkably higher in the leaves of the B.tabaci-infested NOA1-slienced plants than in the leaves of uninfested empty-vector injected plants at 0-20 d after infestation.Furthermore,the JA content peaked at 15 d,at which point it was 83.07% higher than that of the uninfested control plants.In response to B.tabaci infestation,the variation in AOC,and LOX3 transcript levels did not differ between the empty-vector injected and the NOA1-silienced plants.However,the transcript levels of PI-Ⅱ and TPI were decreased by 0.408-and 0.591-fold,respectively,in the empty vector-injected plants,but were increased by 1.468-and 1.5-fold,respectively,in the NOA1-silienced plants following B.tabaci infestation.These results indicate that the silencing of NOA1 could partially alleviate the suppression the of JA-dependent defense response mediated by B.tabaci infestation.4.Through iodoTMT labeling and nitrosation enrichment technology and high-resolution liquid chromatography-mass spectrometry quantitative proteomics research strategy,the expression level and nitrosation of whole protein in tobacco before and after the damage to Bemisia tabaci MEAM1 levels were tested and differential sites of proteins related to nitrosation modification were identified.The results showed that a total of 6,733 proteins were identified after Bemisia tabaci ingested tobacco,of which 5,416 proteins contained quantitative information.Using 1.5 and 2 times as the threshold for differential expression,the significance analysis showed that 350 and 108 protein expressions occurred,respectively.Up-regulation,234 and 30 protein expressions were down-regulated;541 nitrosolation sites were identified on 374 protein,of which 463 of317 proteins contained quantitative information.Using the 1.5-fold change threshold as the standard,it was found that the modification level of 47 sites in the T / C comparison group was up-regulated,and the modification level of 5 sites was down-regulated.The protein containing quantitative information sites was systematically adjusted.Bioinformatics analysis,including protein annotation,functional classification,functional enrichment and cluster analysis based on functional enrichment,combined with proteomics data to screen out alternative target proteins such as AOC and TRX.