Requirement for two or more Erwinia carotovora subsp. carotovora pectolytic gene products for maceration of potato tuber tissue by Escherichia coli

Abstract

Several genes encoding enzymes capable of degrading plant cell wail components have been cloned from Erwinia carotovora subsp. carotovora EC14. Plasmids containing cloned EC14 DNA mediate the production of endo-pectate lyases, exo-pectate lyase, endo-polygalacturonase, and cellulase(s). Escherichua coil strains containing one of these plasmids or combinations of two plasmids were tested for their ability to macerate potato tuber slices. Only one E. coli strain, containing two plasnids that encode endo-pectate lyases, exo-pectate lyase, and endo-polygalacturonase, caused limited maceration. The pectolytic proteins associated with one of these plasmids, pDRI, have been described previously (D. P. Roberts, P. M. Berman, C. Allen, V. K. Stromberg, G. H. Lacy, and M. S. Mount, Can. J. Plant Pathol. 8:17-27, 1986) and include two secreted endo-pectate lyases. The second plasmid, pDR30, contains a 2.1-kiloabse EC14 DNA insert that mediates the production of an exo-pectate lyase and an endo-polygalacturonase. These enzymes are similar in physicochemical properties to those produced by EC14. Our results suggest that the concerted activities of endo-pectate lyases with endo-polygalacturonase or exo-pectate lyase or both cause maceration.Erwinia carotovora subsp. carotovora causes soft rot on many plants. Soft rot results from the activity of extracellular enzymes (11). Extracellular enzymes produced by E. carotovora subsp. carotovora capable of degrading plant cell wall and plant cell membrane components include the following: endo-pectate lyase, endo-polygalacturonase, cellulase, protease, phosphatidase C, and phosholipase A (2,8,9,18,20,22). In an effort to clarify the role of Erwinia pectic enzymes in pathogenesis, several laboratories have isolated genes encoding enzymatic pathogenic determinants from Erwinia spp. (1, 6, 10, 17a, 23).Pectolytic enzymes are key factors in soft rot pathogenesis (for a review, see reference 5); however, the individual roles of the six or more pectolytic enzymes produced by E. carotovora subsp. carotovora EC14 (17a,18) MATERIALS AND METHODS Bacterial strains and plasmids. The bacterial strains and plasmids constructed or used or both constructed and used in this research are listed in Table 1 and in Table 2, respectively. Strain names with the prefix L-indicate designations for strains in the culture collection of G. H. Lacy.Chromosomal DNA isolation. Chromosomal DNA was isolated from strain EC14 as previously reported (17a).Cloning and construction of recombinant strains. Strain EC14 chromosomal DNA was partially digested with BamHI; ligated into dephosphorylated, BamHI-cleaved pBR322 DNA; and transformed into E. coli HB101 by procedures described previously (17a). Transformants with chimeric plasmids had ampicillin-resistant, tetracyclinesensitive phenotypes when screened on YT agar medium (13) containing ampicillin (30 jig/ml) or ampicillin and tetracycline (10 ,ug/ml). Transformants producing pectolytic or cellulolytic enzymes were detected on ampicillin-amended PEC-YA medium (19) or...

Erwinia carotovora subsp. carotovora extracellular protease: characterization and nucleotide sequence of the gene

“…E. carotovora subsp. carotovora EC14 and cosmid pCA7 have been described previously (2,43). Escherichia coli DH5a (Bethesda Research Laboratories [BRL], Inc., Gaithersburg, Md.)…”

Differential Activation of Potato 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Genes by Wounding and Pathogen Challenge

“…For DNA isolation, young leaves were harvested from plants grown in the glasshouse. lhe potato soft rot pathogen, Ecc strain EC14, described in previous studies (Roberts et al, 1986;Yang et al, 1989), was cultured in a minimal medium as described by Yang et al (1989).…”

Analysis of the Pseudomonas solanacearum polygalacturonase encoded by pglA and its involvement in phytopathogenicity

“…Purified Erwinia pectic enzymes macerate plant tissue and cause plant cell death (2,23), and cloned Erwinia pectic enzyme genes confer the ability to macerate plant tissue on the non-plant pathogen E. coli (7,15,27,31). Biochemical and genetic data (5) indicate that the excreted Erwinia enzymes pectate lyase, polygalacturonase, and pectin methylesterase act in a concerted manner to degrade the pectic portion of plant cell walls and provide nutrition for the pathogen.…”