| Verticillium wilt caused by Verticillium dahliae is one of the major threatens in cotton production.V.dahliae population presents typical differentiation in pathogenicity,and the pathogen V.dahliae can be divided into two groups – defoliating and nondefoliating according to the symptoms caused on cotton plant.Defoliating V.dahliae can lead to significant defoliation on cotton plant,which is much more harmful to cotton plant and has been the major limiting factor in cotton production.Up to now,molecular mechanism of the defoliation on cotton plant caused by V.dahliae remains unknow.In this study,defoliating V.dahliae strain XJ592 and nondefoliating V.dahliae strain XJ511 were used to sequence and compare to find differential genes between strain XJ592 and XJ511.The aim is to reveal molecular mechanism of cotton plant defoliation caused by V.dahliae strain XJ592 through studying on the role of these differential genes during the defoliation process.Main results are as following:1.During the process of XJ592 induced defoliation on cotton plant,auxin(AUX)signal pathway,jasmonic acid(JA)signal pathway,and cytokinin(CTK)signal pathway are negatively correlated with the defoliation;while ethylene(ET)signal pathway and salicylic acid(SA)signal pathway are positively correlated with the defoliation.Additionally,abscisic acid(ABA)may play a more indirect role on the process of the defoliation on cotton plant caused by XJ592.The concentration of ABA and SA significantly increased in cotton plant infected by XJ592 and XJ511 compared with the control.The concentration of ABA and SA in XJ592 infected cotton plant was 6.81 times and 11.12 times as that of XJ511 infected cotton plant at 35 days post inoculation(dpi),respectively.The concentration of ET precursor ACC showed no significant changes between XJ511 infected cotton plant and the control,which only significantly increased under XJ592 treatment during the defoliation process.The concentration of ACC in XJ592 infected cotton plant was 2.17 times as that of XJ511 infected cotton plant at 35 dpi.Results showed that the concentration of ACC only specifically increased during the defoliation process in XJ592 infected cotton plant.We further studied relative gene expression patterns under different hormone signal pathways,results revealed that AUX pathway,ET pathway,SA pathway,JA pathway,and CTK pathway all participate in the process of the defoliation caused by XJ592 on cotton plant.Meanwhile,the relative expression level of SA pathway relative gene Gh NPR1,ET pathway relative gene Gh EIN4,and CTK pathway relative gene Gh CKX3(negative responsive gene)in XJ592 infected cotton plant was 1.75,1.92,and 5.24 times as that of XJ511 infected cotton plant at 35 dpi,respectively;the relative expression level of AUX pathway relative gene Gh ASA1 and JA pathway relative gene Gh MYC2 in XJ592 infected cotton plant was 0.54 and 0.65 times as that of XJ511 infected cotton plant at 35 dpi,respectively;the expression level of ABA pathway relative gene Ghb HLH1 showed no significant difference between XJ592 and XJ511 infected cotton plants.2.The genomes of V.dahliae defoliating strain XJ592 and nondefoliating strain XJ511 were sequenced,assembled,annotated,and compared,forty-seven secretory protein coding genes in XJ592 were found different compared to XJ511.The final size of assembled XJ592 genome and XJ511 genome was 35.67 M and 34.90 M,respectively.XJ592 genome was predicted to contain 10305 function genes,XJ511 genome was predicted to contain 10462 function genes.Among these function genes,forty-seven secretory protein coding genes in XJ592 were found showing different sequence compared to XJ511.Further analysis revealed that extensive chromosome rearrangement exists in XJ592 genome compared to XJ511 genome,comparative genomic analysis showed that there are many unique sequences in XJ592 genome including 47 XJ592 unique sequences with length more than 500 bp.Eleven among these 47 sequences are only homologous with sequences from V.alfalfae or V.nonalfalfae,indicating that horizontal gene transfer happens in genomes between V.dahliae and V.alfalfae or V.nonalfalfae.3.Secretory protein coding genes including Vd SSP,Vd CUE,VdSAP,Vd NCD,Vd CR,Vd NAD,and Vd Xyn3 are significantly involved in the defoliation process and the pathogenic process of V.dahliae on cotton plant.Forty-seven secretory protein coding genes in XJ592 with sequence difference compared to XJ511 were annotated,after testing protein function to trigger plant cell death and considering protein annotation information,seven among them named Vd SSP,Vd CUE,VdSAP,Vd NCD,Vd CR,Vd NAD,and Vd Xyn3 were selected to be studied as candidate effectors.All these seven candidate effectors contain functional signal peptides.Further study indicated that these seven genes significantly participated in the defoliation process and the pathogenic process caused by XJ592 on cotton plant.4.V.dahliae VdSAP regulates the defense response pathway,IDA(Inflorescence Deficient in Abscission)induced abscission signal pathway and ET signal pathway in cotton plant to successfully infect and cause the defoliation on cotton plant through targeting to Gh8322X1 protein.A protein named Gh8322X1 from cotton plant was found as candidate interacting protein with VdSAP,and further yeast two-hybrid assay,bimolecular fluorescence complementation assay,MBP pulldown assay,and Co-IP assay all confirmed the interaction between VdSAP and Gh8322X1 protein.VIGS assay was conducted to evaluate the function of Gh8322X1 on the interaction between cotton plant and V.dahliae strain XJ592.Results showed that Gh8322X1 silenced cotton plant exhibited average Disease Index(DI)of 89.44 and average defoliation rate of 82.2%,which was increased by 20.1% and 24.4% compared to TRV:00 treated cotton plant,respectively.Transient expression of Gh8322X1 protein in Nicotiana benthamiana would lead to significantly higher expression level of defense genes relative with SA,JA,ET,and PTI,and lead to significantly lower expression level of genes relative with IDA induced abscission progress,these results indicate that Gh8322X1 plays a molecular bonding role in plant defense responses and plant development.Further,in vitro detecting results of Gh8322X1 protein level incubated with VdSAP and in vivo detecting results of Gh8322X1 protein level coexpressed with VdSAP revealed that VdSAP negatively regulates protein level of Gh8322X1 in cotton plant.By q RT-PCR assay on Gh8322X1 silenced cotton plant,the down-regulating of Gh8322X1 would enhance plant ET signal.Hence,ET signal functions as key role during the infection process and the defoliation process caused by V.dahliae defoliating strain XJ592 on cotton plant.Defoliating V.dahliae strain XJ592 can secret VdSAP and target to Gh8322X1 of cotton plant,the interacting between these two proteins will lead to the decrease of protein level of Gh8322X1 in cotton plant,and will significantly enhance the expression of plant ET signal and IDA induced abscission signal,finally results in the acceleration of disease process and the defoliation on cotton plant. |
