Cloning of the egl gene of Pseudomonas solanacearum and analysis of its role in phytopathogenicity

Abstract

The egl gene of Pseudomonas solanacearum was cloned on a cosmid and expressed in Escherichia coli. Restriction endonuclease mapping, transposon mutagenesis, and subclone analysis showed that the egl gene was located on a 2.7-kilobase XhoI-SalI P. solanacearum DNA fragment. Immunoabsorption experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that the egl gene encodes the 43-kilodalton endoglucanase that is the major excreted endoglucanase of P. solanacearum. In E. coli, the egl gene appeared to be expressed from its own promoter, but its product was restricted to the cytoplasm. The cloned egl gene was mutagenized with Tn5 and used to specifically mutate the chromosomal egl gene of P. solanacearum by site-directed mutagenesis. The resultant mutant was identical to the wild-type strain in production of extracellular polysaccharide and extracellular polygalacturonase as well as several other excreted proteins but produced at least 200-fold less endoglucanase. This mutant strain was significantly less virulent on tomato than the wild-type strain in plant bioassay experiments. Virulence of the endoglucanase-deficient strain was restored to near wild-type levels by complementation in trans with the cloned egl gene, indicating that the egl gene is important but not absolutely required for pathogenesis.

Deconstructing the Cell Wall

“…Plant pathologists have generally felt that cellulases are not particularly important in pathogenesis, since extensive cellulose degradation typically occurs only late in infection, if at a11 (Cooper, 1984). However, when the major endoglucanase genes of the phytopathogenic bacteria Pseudomonas solanacearum and Xanthomonas campestris pv campestris were disrupted, virulence decreased (Gough et al, 1988;Roberts et al, 1988). …”

Root-mediated signal transmission of systemic acquired resistance against above-ground and below-ground pathogens

“…A 10 mL suspension of Pta (OD 600 ¼ 1) was sprayed on the leaves of N. benthamiana plants at 1 week after leaf infiltration with the chemical BTH (Figs 1 and 3). The severity of R. solanacearum symptoms was scored from 0 to 5 as follows: 0, no leaves wilted; 1, 1-20 % of leaves wilted; 2, 21-40 % of leaves wilted; 3, 41-60 % of leaves wilted; 4, 61-80 % of leaves wilted; and 5, 81-100 % of leaves wilted (Roberts et al, 1988). The severity of Pta symptoms was scored from 0 to 5 as follows: 0, no symptoms; 1, yellowish colour; 2, chlorosis only; 3, partial necrosis and chlorosis; 4, necrosis of the inoculated area and expanded chlorosis; and 5, complete necrosis of the inoculated area.…”

“…Ralstonia solanacearum, Pta and Psy were grown on solid Casamino acid-Peptone-Glucose (CPG) and King's B medium respectively containing 100 lg mL -1 rifampicin for selection at 30 C for 2 d, scraped off the plates and re-suspended in 10 mM MgCl 2 (Roberts et al, 1988;Song et al, 2015). A 50 mL aliquot of bacterial suspension of R. solanacearum at OD 600 ¼ 1 was freshly prepared and drenched into the root system of every N. benthamiana seedling (Chandrasekaran et al, 2016).…”

The Virulence Factors of the Bacterial Wilt Pathogen Ralstonia solanacearum

“…solanacearum secretes several CWDEs, including three polygalacturonases (PehA , PehB and PehC) [27,28], an endoglucanase (Egl) [29], a pectin methylesterase (Pme) [30], and a cellobiohydrolase (CbhA) [31]. Gene disruption analysis revealed that Egl, PehA, PehB and CbhA, each contribute to the pathogen's ability to cause wilt [31][32][33].…”