Highly virulent strains of Pseudomonas solanacearum that are defective in extracellular-polysaccharide production

Abstract

Extracellular polysaccharide (EPS) has long been regarded as one of the most important factors involved in wilting of plants by Pseudomonas solanacearum. By means of transposon Tn5 mutagenesis, we have isolated a class of mutants that have an afluidal colony morphology but retain the ability to cause severe wilting and death of tobacco plants. One such mutant, KD700, was studied in detail. By marker exchange mutagenesis, the altered colony morphology was shown to be the result of a single Tn5 insertion in a 14.3-kilobase EcoRI fragment. This defect could be corrected by introducing a homologous clone from a cosmid library of the wild-type, parental strain K60. The Tn5-containing fragment was introduced into other P. solanacearum wild-type strains by marker exchange, and these altered strains had the same afluidal phenotype as KD700. N-Acetylgalactosamine (GalNac), the major constituent of EPS of all wild-type strains of P. solanacearum, was not detected by gas chromatography-mass spectrometry analysis of vascular fluids from wilting plants infected by KD700. In contrast, GalNac was readily detected in similar fluids of plants infected by K60. Polysaccharides extracted from culture filtrates of KD700 contained approximately one-fifth of the GalNac present in polysaccharides from K60. No differences in growth rates in culture or in planta between the mutant and the parental strains were observed. Since strains that are deficient in EPS production can remain highly virulent to tobacco, we conclude that EPS, or at least its GalNac-containing component, may not be required for disease development by P. solanacearum.

Genetic and biochemical characterization of a Pseudomonas solanacearum gene cluster required for extracellular polysaccharide production and for virulence

“…Five complementation units were identified. These appear to encode functions that are distinct from those affected in the EPS-virulent TnS mutant KD700, based on comparisons of restriction map data (36). They are probably also distinct from the function(s) affected in the EPS-nonvirulent TnS mutants KDS00 and KD600 (37), because Tn3 insertions that have no effect on colony morphology or virulence separate epsA-epsD and rvrA from the TnS insertions of KDS00 and KD600 (Fig.…”

“…Most of the known or presumed virulence factors in P. solanacearum are extracellular products, including the plant cell wall-degrading enzymes polygalacturonase (3,27) and endoglucanase (26) and the plant hormones auxin, cytokinin, and ethylene (9). Although this array of extracellular products would seem to constitute a formidable arsenal for virulence, the most intensively studied virulence factor from P. solanacearum is a relatively inert product, extracellular polysaccharide (EPS) (1,11,13,19,31,36,38). This high-molecular-weight polymer is thought to be responsible for induction of the wilt symptoms that characterize the disease caused by P. solanacearum.…”

Extracellular polysaccharide is required for wild-type virulence of Pseudomonas solanacearum

“…Only one mutant, KD700, was characterized biochemically (18). This mutant produced no detectable amount of EPS in planta and only 20% of the wild-type amount of EPS in culture.…”

“…Several P. solanacearum EPS mutants which, quite unexpectedly, remained highly virulent when assayed by stem inoculation of tobacco plants were isolated (18,19). Only one mutant, KD700, was characterized biochemically (18).…”

“…Since this mutant appears to contradict the dogma that EPS is required for virulence in P. solanacearum, Xu et al cloned out the TnS and flanking DNA from the KD700 genome and used the flanking DNA as a hybridization probe to identify the corresponding wild-type DNA in a cosmid library (18). The cosmid named pL700A hybridized to the flanking DNA and was able to complement KD700 to the wild-type level of EPS production and virulence (18).…”