DRAMATICALLY REDUCED VIRULENCE OF MUTANTS OF PSEUDOMONAS-SOLANACEARUMDEFECTIVE IN EXPORT OF EXTRACELLULAR PROTEINS ACROSS THE OUTER-MEMBRANE

Pseudomonas solanacearum is an important wilt-inducing pathogen that i nfects a wide variety of important crop plants throughout the world. S tudies using artificial inoculation methods suggest that some of its e xtracellular proteins play a significant, but auxiliary role in produc tion of wilt disease. We isolated mutants of race 1 and race 3 strains of P. solanacearum with Tn5 insertions at a single locus (eep) whose culture supernatants lack all of its known extracellular enzymes and m ost other detectable extracellular proteins (EXPs). Analysis of subcel lular fractions of eep::Tn5 mutants showed that they still synthesized many of these EXPs but accumulated them inside the cell. Experiments with PhoA fusion proteins showed that export of proteins across the in ner membrane was not affected by the eep mutation, suggesting that eep functions only in protein export across the outer membrane. Productio n of extracellular polysaccharide was not obviously affected by the ee p mutation. Analysis of eep mutants in stem-inoculated tomato plants s howed that they had lost the ability to cause wilt symptoms or kill th e plant, possibly because they colonized stems much more slowly than w ild types. Plants grown in soil inoculated with the eep mutants did no t develop any visible disease symptoms over a 20-day period, and their stems contained fewer than 10(3) P. solanacearum cells, whereas wild types killed plants in 14 days, and more than 10(10) cells were found in their stems. These results suggest that an individual or group of e xtracellular proteins of P. solanacearum is required for infection via the roots, as well as wilting and killing of host plants.