Functional Analysis Of Wheat Adaptor Protein Gene TaAP2σ In The Regulation Of Fusarium Head Blight

Fusarium head blight is an important disease of wheat which causes serious threat to grain yield,quality and food safety.Research on the pathogenesis of Fusarium head blight,reveal the mechanism of wheat Fusarium head blight resistance,dig deeper into the resistance resources,and cultivate Fusarium head blight resistant varieties to control wheat Fusarium head blight has become an urgent problem with important practical value and social significance.We found that TaCLC1,a clathrin light chain protein gene of wheat,has a positive regulatory effect on Fusarium graminearum induced Fusarium head blight.In order to further investigate the regulatory mechanism of TaCLC1 on Fusarium graminearum,this study was conducted to investigate the function and molecular mechanism of TaCLC1 protein’s interacting proteins(Adaptor proteins,TaAP2σ)in regulating Fusarium graminearum.The main results are as follows:(1)The wheat genetic transformation vector PC186 of TaCLC1 with Ubi promoter was constructed using the Gateway system,and transgenic wheat overexpressing TaCLC1 was obtained.By sequencing its transcriptome,the analysis revealed that the expression of the articulated protein gene TaAP2σ in the endocytic pathway was up-regulated,and after verification by yeast two-hybrid,LCI(firefly luciferase complementation imaging)and Bi FC,(Bimolecular fluorescence complementation)it was found that the articulated protein TaAP2σinteracts with TaCLC1.(2)Biological characterization of TaAP2σ.Visualization of the structural domain of TaAP2σ using TBtools revealed that it contains a small chain structure of a clathrin protein complex.Analysis of the promoter cis-acting elements of the TaAP2σ gene using the Plant Care website revealed the presence of hormone-responsive elements such as salicylic acid and jasmonic acid in the promoter region,indicating that TaAP2σ may be regulated in expression by hormones.Fluorescence quantification experiments showed that TaAP2σ gene’s were differentially expressed in roots,stems and leaves of wheat,with higher levels in stems and leaves,and were able to respond to Fusarium graminearum infestation and induced expression by hormones such as salicylic acid and jasmonic acid.A fusion expression vector of TaAP2σ gene with green fluorescent protein GFP tag was constructed to transiently infiltrate Nicotiana tabacum,and laser confocal microscopy revealed that TaAP2σ was distributed in both Golgi Apparatus and Endoplasmic Reticulum.(3)Arabidopsis thaliana overexpressing TaAP2σ gene improved resistance to erysipelas.Stably inherited transgenic Arabidopsis thaliana was obtained by inflorescence infestation,and inoculation with Fusarium graminearum revealed that the leaf rot area of Arabidopsis thaliana transformed with TaAP2σ gene was smaller and the expression of Fusarium graminearum mycelium was lower compared with wild-type Arabidopsis leaves.The results suggest that overexpression of the TaAP2σ gene in Arabidopsis improves resistance to Fusarium graminearum.The expression levels of disease process-related protein(PR)genes in Arabidopsis transformed with TaAP2σ gene were detected,and the expression levels of PR3 and PR5 were found to be higher in Arabidopsis transformed with TaAP2σ gene than wild type.Spraying of endocytosis pathway inhibitor chlorpromazine followed by Fusarium graminearum inoculation revealed that Arabidopsis leaves not sprayed with chlorpromazine had a smaller area of putrefaction and lower expression of Fusarium graminearum mycelium compared with Arabidopsis leaves sprayed with chlorpromazine.The results suggest that spraying chlorpromazine attenuated the resistance of Arabidopsis thaliana to Fusarium graminearum by the TaAP2σ gene.These results suggest that the TaAP2σ gene may protect against Fusarium graminearum in Arabidopsis by promoting the expression of disease-process-related protein genes and regulating the endocytosis pathway.(4)Using barely stripe mosaic virus mediated gene silencing technology,we inoculated wheat leaves with Fusarium graminearum with the GFP label after transient silencing of the TaAP2σ gene.We found that the lesion area of BSMV::TaAP2σ wheat leaves was significantly larger than that of the control,and the fluorescence area of the lesion was also larger.At the same time,the content of Fusarium graminearum was also higher than that of the control,indicating that the growth of Fusarium graminearum was accelerated after the silenced TaAP2σ gene.The expression of TaPAL and TaOPR,key genes of salicylic acid and jasmonic acid synthesis pathways,were further examined in each wheat strain,and it was found that the expression of TaPAL and TaOPR were less up-regulated in the silenced strain than the wild type compared with the control.It showed that the expression of these two genes showed a positive correlation with the expression of TaAP2σ,indicating that TaAP2σ may regulate the resistance to Fusarium head blight through SA and JA.In conclusion,TaAP2σ,as an articulated protein,is an important component of the endocytic pathway,and TaCLC1 has a reciprocal relationship with TaAP2σ,responding to the induction of Fusarium graminearum and various hormones,and is involved in regulating plant resistance to Fusarium head blight,possibly by collaborating with TaCLC1 to regulate the endocytic pathway,as well as the expression of disease process-related proteins to resist the pathogen.