Role of Extracellular Polysaccharide and Endoglucanase in Root Invasion and Colonization of Tomato Plants by Ralstonia solanacearum.

ABSTRACT Ralstonia solanacearum is a soilborne plant pathogen that normally invades hosts through their roots and then systemically colonizes aerial tissues. Previous research using wounded stem infection found that the major factor in causing wilt symptoms was the high-molecular-mass acidic extracellular polysaccharide (EPS I), but the beta-1,4-endoglucanase (EG) also contributes to virulence. We investigated the importance of EPS I and EG for invasion and colonization of tomato by infesting soil of 4-week-old potted plants with either a wild-type derivative or genetically well-defined mutants lacking EPS I, EG, or EPS I and EG. Bacteria of all strains were recovered from surface-disinfested roots and hypocotyls as soon as 4 h after inoculation; that bacteria were present internally was confirmed using immunofluorescence microscopy. However, the EPS-minus mutants did not colonize stems as rapidly as the wild type and the EG-minus mutant. Inoculations of wounded petioles also showed that, even though the mutants multiplied as well as the wild type in planta, EPS-minus strains did not spread as well throughout the plant stem. We conclude that poor colonization of stems by EPS-minus strains after petiole inoculation or soil infestation is due to reduced bacterial movement within plant stem tissues.

Quantitative immunofluorescence of regulated eps gene expression in single cells of Ralstonia solanacearum.

Ralstonia solanacearum, a phytopathogenic bacterium, uses an environmentally sensitive and complex regulatory network to control expression of multiple virulence genes. Part of this network is an unusual autoregulatory system that produces and senses 3-hydroxypalmitic acid methyl ester. In culture, this autoregulatory system ensures that expression of virulence genes, such as those of the eps operon encoding biosynthesis of the acidic extracellular polysaccharide, occurs only at high cell density (>10(7) cells/ml). To determine if regulation follows a similar pattern within tomato plants, we first developed a quantitative immunofluorescence (QIF) method that measures the relative amount of a target protein within individual bacterial cells. For R. solanacearum, QIF was used to determine the amount of beta-galactosidase protein within wild-type cells containing a stable eps-lacZ reporter allele. When cultured cells were examined to test the method, QIF accurately detected both low and high levels of eps gene expression. QIF analysis of R. solanacearum cells recovered from stems of infected tomato plants showed that expression of eps during pathogenesis was similar to that in culture. These results suggest that there are no special signals or conditions within plants that override or short-circuit the regulatory processes observed in R. solanacearum in culture. Because QIF is a robust, relatively simple procedure that uses generally accessible equipment, it should be useful in many situations where gene expression in single bacterial cells must be determined.