Screening And Functional Analyses Of Pathogenicity-Related Genes In Pectobacterium Carotovorum Subsp.Carotovorum

Pectobacterium carotovorum subsp. carotovorum（Pcc） is an important plant pathogen causing serious bacterial soft rot on Zantedeschia spp. and other host plants. Extracelluar cell wall-degrading enzymes, such as pectate lyase（Pel）, cellulose（Cel）, and protease（Prt）, are considered to be the main factors responsible for Pcc pathogenicity. The soft rot disease caused by Pcc has brought serious economic loss in the agriculture around the world. Therefore, it has become increasingly important to study the pathogenic mechanism of pathogen.In our previous study, we found that both proteins of glucose-6-phosphate1-dehydrogenase（Zwf, PC1₁832） and aspartate ammonia-lyase（AspA, PC1₀506） showed upregulated expression when the PccS1interacted with Zantedeschia elliotiana. Through the pathogenicity analysis of single crossover mutant and the double crossover mutant of zwf and asp A genes in this study, we found that compared with the wild type strain, zwf or aspA single crossover mutant strains significantly reduced virulence, while the double crossover mutants of zwf or aspA consistent with the wild type.The gene2-keto-3-deoxy-6-phosphogluconate aldolase was found downstream of zwf in the same direction （eda, PC1₁833） by analyzing the complete sequence of PccSl, and Eda is one of two key enzymes in Entner-Doudoroff pathway（ED pathway）. Deletion mutant Δeda and complementary strain Δeda（edd） were successfully constructed by using homologous recombination method. The result of the mutant phenotype analysis showed that Δeda could not grow normally when sodium acetate used as a sole carbon source while the wild-type PccS1with a good growth, but the growth rate of Δeda in LB and MMX medium consistent with PccS1. The virulence of Δeda on host plants, Zantedeschia elliotiana’Yellow Queen’ and cabbage, decreased significantly, and the lesion area decreased by80.35%and97.51%respectively. The forming ability of pectinase decreased with a59.9%reduction in the area of pectinase tablet hydrolysis circle; At the transcriptional level, the expression of regulation genes kdgR, hexA and rsmA increased remarkablely, meanwhile, the transcriptional expression levels of pectinase genes pel and peh reduced significantly. Therefore, the virulence reduced in the single crossover mutant of zwf was probably through affecting eda gene expression, and eda play an important role in PccS1virulence in a certain extent by regulating negatively on kdgR, for kdgR upregulated expression after the deletion of eda, and pel, peh which are negatively regulated by KdgR according to the results of the reports previously.At the same time, by using the homologous recombination gene knockout technology, three deletion mutants of downstream genes of aspA were constructed, i. e. AdcuA, ΔcutA1and AcycZ. The phenotype of the mutants, such as movement, biofilm formation, extracellular enzyme activity and pathogenicity were measured. Compared with wild type, in addition to hydrogen peroxide tolerability and biofilm formation capacity of the mutants were improved, other phenotypes were similar to that of PccSl. The results showed that, the deletion mutation of dcuA, cutAl and cycZ genes did not affect the pathogenicity of the strain PccSl, and the pathogenic decline of single crossover mutant of asp A gene may have a smaller relationship with the downstream genes.