Isolation and Characterization of Exoenzyme Over-Producing GFP-Tagged Mutant of Soft Rot Pathogen, Pectobacterium versatile
Soft rot pathogens produce plant cell wall degrading enzymes (PCWDEs) as a major virulence factor. Mutants that over-produce the enzymes are hyper-virulent while enzyme-deficient mutants are non-virulent. The main objective of this research was to isolate and characterize pleiotropic mutants of Pectobacterium versatile and identify the mutated genes and their role in regulation of virulence. We constructed a stable, independent, and saturated GFP-tagged mutant library of Pectobacterium versatile by the process of mini-Tn5 transposon insertion mutagenesis with the conditional Pir-dependent R6K-based suicide vector. We screened for and isolated mutants that were over-producing extracellular protease (Prt). Quantitative, and semi-quantitative enzyme assays revealed the increased production of other extracellular enzymes such as- cellulase, polygalacturonase and pectate-lyase. The mutants and parental strains were inoculated on celery petioles, potato tubers and carrot root discs for pathogenicity test. The hyper virulent mutants macerated more host tissues than the parental strain. The mutant gene was identified using the modified protocol for direct Sanger sequencing of mutant genomes. We found that the mutants had transposon insertion in several previously known regulatory genes such as rsmA and rsmC, and unknown ones such as degQ. The cosmid clone of degQ obtained through the PCR screening of Ecc71 genomic library, complemented the mutant phenotype. An understanding of the role of these genes in the disease process can be used in designing effective management strategies for soft rot disease.
Plant sciences|Plant Pathology
"Isolation and Characterization of Exoenzyme Over-Producing GFP-Tagged Mutant of Soft Rot Pathogen, Pectobacterium versatile"
ETD Collection for Tennessee State University.