Physiological And Molecular Response Mechanism Of Potato Tubers To Fusarium Sulphureum And The Functional Study Of StPALs

Potato dry rot(PDR),induced by Fusarium sulphureum(Fs),is one of the most serious storage diseases threatening the potato(Solanum tuberosum L.)industry.The systematic understanding of the disease resistance mechanism can provide reference for disease resistance breeding and integrated prevention and control,but related studies still need to be further developed.Based on this,in this study,the changes in potato antioxidant enzyme system,related metabolites and hormone content during Fs infestation were studied using two potato cultivars(Longshu No.7 and Atlantic,LS and Atl)as materials.Meanwhile,the gene expression pattern of potato tubers was revealed with the help of RNA-Seq sequencing technology.What’s more,phenylalanine ammonia-lyase gene StPAL4 was cloned and identified,and its function was studied.The main findings of this research were as follows:1.The lesion diameters of LS and Atl varieties began to expand from 2 days post inoculation(dpi)after Fs infection,and increased rapidly at 4 dpi.The contents of cellular inclusions and disease-resistance related defensive enzymes in Atl and LS varieties were regulated to varying degrees,and showed a trend of increasing first and then decreasing,reaching their maximum at 2 dpi and 4 dpi.These results suggested that the increased activity of antioxidant enzymes to remove reactive oxygen accumulation was an adaptive response of potato to Fs invasion.2.With the extension of Fs infection,PAL activity in LS and Atl tubers reached the maximums at 0.5 dpi and 2 dpi,respectively.Meanwhile,metabolites in phenylpropanoid and its downstream related pathways,such as total phenols,flavonoid,lignin,and eight phenolic acids,were induced to increase,which indicated that PAL-mediated phenylpropanoid metabolic pathways were involved in potato resistance to Fs,and the accumulation of phenolic metabolites and lignin were the important immune responses induced by PDR pathogens after infection.In addition,the characteristics of hormone changes were determined based on HPLC techniques,and it was found that the contents of abscisic acid,gibberellin and jasmonic acid showed a continuous increase in both varieties,while zeatin and IAA also showed specific response patterns,which revealed that the synthesis of plant hormones was also an important aspect of potato tuber response to Fs infection.3.To eliminate the effect of genetic background differences between varieties,two potato varieties(LS and Atl)were selected and the gene expression patterns of potato tubers in response to Fs were analyzed by RNA-Seq.The results showed that the number of common up-regulated differentially expressed genes(DEGs)at the same infestation time points(1,3,7 dpi)in both varieties,were 1025,1334 and 1394,and the common down-regulated DEGs were 645,2155 and 2091,respectively.DEGs were significantly clustered in profiles 0,1,2,7,17 and 19(p < 0.05)in both varieties,basing on gene expression pattern using STEM software.The profiles 17 and 19 displayed a continuous increasing trend in gene expression over time in response to Fs infection.Further KEGG enrichment analysis in these two profiles revealed that“Phenylalanine metabolism”(ko00360),“Flavonoid biosynthesis”(ko00941),“Phenylpropanoid biosynthesis”(ko00940)and MAPK signaling pathway–plant(ko04016)were significantly enriched in both varieties.The related family member genes of PAL,4CL,HCT,COMT,CCOAOMT,CAD and POD were significantly induced by Fs.4.Nine StPALs genes were identified in the potato genome and their family members were bioinformatically analyzed.q RT-PCR analysis indicated that StPALs were induced to be expressed after Fs infestation.In potato tubers,transient expression of StPAL4 gene attenuated the rate of disease spot development,increased lignin content,and induced up-regulated expression of PAL,4CL,CAD,and POD genes.5.Ten Atlantic positive StPAL4-transformed potato plants were obtained through an Agrobacterium tumefaciens mediated potato genetic transformation system.The lesion diametes of the overexpressed StPAL4 transgenic tubers were significantly lower(p<0.05)than the wild type,while their PAL activity,total phenols,flavonoids and lignin contents were significantly higher(p<0.05)than the wild type.These indicated that StPAL4 positively regulated potato resistance to dry rot.