Mechanism Of Potato Auxin Repressed Protein Gene StARP1 On Late Blight

Potato（Solanum tuberosum）is an important grain and vegetable crop in China.It is often threatened by various pathogens during its growth.The late blight caused by Phytophthora infestans（P.infestans）is the most destructive,causing serious economic losses to potato production in China.Investigating the mechanisms of disease resistance and finding resistance genes is particularly important for the development of new disease resistant potato varieties.Our previous study found that potato ubiquitin ligase gene St ATL80 had a negative regulatory effect on late blight caused by P.infestans.In order to further explore the regulatory mechanism of St ATL80 on late blight.In this study,we screened the interaction protein of St ATL80 as a starting point,and studied the function and molecular mechanism of its interaction protein auxin repressed protein（ARP1）regulating P.infestans.The main results and conclusions are as follows:（1）Auxin repressed protein StARP1 is an interaction protein of ubiquitin ligase St ATL80.Using String database combined with yeast two-hybrid screening experiment,we found that there were four suspected St ATL80 interacting proteins in potato,including CYP19-4,CAS,SGT1 and ARP1.It was further proved by Y2 H,LCI,Bi FC and Pull-down that auxin repressed protein StARP1 was the interaction protein of St ATL80.（2）Biological characteristics of StARP1.The structure of StARP1 protein was predicted by SMART,and the auxin-repressed domain was found,indicating that StARP1 belongs to the Auxin repressed protein family.Plant Care was used to analyze the promoter cis-acting elements of StARP1,and found that the promoter region contains hormone,salt stress,light,drought stress,low temperature,damage and other related response elements,indicating that StARP1 is involved in the regulation of plant growth and development and stress resistance processes.Fluorescence quantitative PCR showed that StARP1 gene was distributed in roots,stems and leaves of potato,with the highest expression in leaves.StARP1 gene could respond to the infection of P.infestans,and was up-regulated by salicylic acid,auxin and jasmonic acid.The 35S::StARP1-GFP fusion expression vector was constructed and instantaneously infected tobacco.Laser confocal microscopy showed that StARP1 protein was localized in cytoplasm and nucleus.（3）Plants overexpressing StARP1 enhanced their resistance to P.infestans.The transient overexpression of StARP1 in tobacco leaves was inoculated with P.infestans.Compared with the control,the lesion area of transient overexpression of StARP1 was relatively small,q RT-PCR showed that the silk expression level of P.infestans was also lower.The leaves of Arabidopsis and potato transgenic lines with stable expression of StARP1 were inoculated with P.infestans.It was found that the resistance of leaves of Arabidopsis and potato transgenic lines with overexpression of StARP1 to P.infestans was significantly enhanced compared with the leaves of wild-type lines respectively.These results indicated that overexpression of StARP1 gene enhanced plant resistance to P.infestans.（4）Down-regulated expression of Nb ARP1 and At ARP1 homologous genes of StARP1 in tobacco and Arabidopsis reduced the resistance of plants to P.infestans.After transient down-regulation of Nb ARP1 in tobacco leaves,P.infestans was inoculated.Compared with the control,the lesion area of transient down-regulation of Nb ARP1 was relatively larger,and q RT-PCR showed that the silk expression level of P.infestans was also higher.Inoculation with P.infestans on Arabidopsis leaves stably down-regulated At ARP1 showed that the resistance of Arabidopsis leaves to P.infestans was lower than that of wild type.These results suggested that down-regulated expression of ARP1 gene reduced the resistance of plants to P.infestans.（5）StARP1 enhances resistance to P.infestans by affecting multiple immune-related pathways in potato.DAB and NBT staining showed that the accumulation of reactive oxygen species in StARP1 transgenic potato leaf cells after P.infestans was less than that in WT.The detection results of hydrogen peroxide and superoxide anion content were consistent with DAB and NBT staining.Meanwhile detection of ascorbic acid peroxidase（APX）,superoxide dismutase（SOD）and catalase（CAT）activities,and it was found that the antioxidant enzyme activities of StARP1 overexpressed plants were increased.The genes related to auxin signaling pathway in StARP1 overexpression potato lines were detected,and it was found that the expression levels of St GH3,St IAA24 and St SAUR3 were up-regulated,while the expression levels of St SAUR1,St IAA6 and St ARF1 were down-regulated.Transcriptome sequencing analysis of transgenic potato lines revealed that five resistance-related genes were up-regulated among the differentially expressed genes in StARP1 overexpressed plants.These results suggest that StARP1 may enhance resistance to P.infestans in potato by affecting the clearance of reactive oxygen species,auxin signaling transduction and multiple disease resistance-related pathways.In summary,we found that StARP1 is the interaction protein of St ATL80.As a member of the auxin repressed protein family.StARP1 responds to the induction of P.infestans and various hormones,and may participate in the regulation of potato late blight through reactive oxygen species scavenging,auxin signal transduction and various disease-resistance related pathways.The results provided theoretical basis and practical significance for understanding the infection mechanism of P.infestans and breeding resistant potato varieties.