Relationship of Genes Conferring Epiphytic Fitness and Internal Multiplication in Plants in Erwinia herbicola

Most bacterial plant pathogens colonize the surface of healthy plants as epiphytes before colonizing internally and initiating disease. The epiphytic phase of these pathogens is thus an important aspect of their epidemiology and a stage at which chemical and biological control is aimed. However, little is known of the genes and phenotypes that contribute to the ability of bacteria to grow on leaves and survive the variable physical environment in this habitat. In addition, while genes such as hrp awr and others which confer pathogenicity and in planta growth ability have been described, their contribution to other aspects of bacterial epidemiology such as epiphytic fitness have not been addressed. We hypothesized that bacterial genes conferring virulence or pathogenicity to plants also contribute to the epiphytic fitness of these bacteria and that many of these genes are preferentially located on plasmids. We addressed these hypotheses by independently identifying genes that contribute to epiphytic fitness, in planta growth, virulence and pathogenicity in the phytopathogenic bacterium Erwinia herbicola pv gypsophilae which causes gall formation on gypsophila. This species is highly epiphytically fit and has acquired a plasmid (pPATH) that contains numerous pathogenicity and virulence determinants, which we have found to also contribute to epiphytic fitness. We performed saturation transposon mutagenesis on pPATH as well as of the chromosome of E.h. gypsophilae, and identified mutants with reduced ability to grow in plants and/or cause disease symptoms, and through a novel competition assay, identified mutants less able to grow or survive on leaves. The number and identity of plasmid-borne hrp genes required for virulence was determined from an analysis of pPATH mutants, and the functional role of these genes in virulence was demonstrated. Likewise, other pPATH-encoded genes involved in IAA and cytokinin biosynthesis were characterized and their pattern of transcriptional activity was determined in planta. In both cases these genes involved in virulence were found to be induced in plant apoplasts. About half of avirulent mutants in pPATH were also epiphytically unfit whereas only about 10% of chromosomal mutants that were avirulent also had reduced epiphytic fitness. About 18% of random mutants in pPATH were avirulent in contrast to only 2.5% of random chromosomal mutants. Importantly, as many as 28% of pPATH mutants had lower epiphytic fitness while only about 10% of random chromosomal mutants had lower epiphytic fitness. These results support both of our original hypotheses, and indicate that genes important in a variety of interactions with plant have been enriched on mobile plasmids such as pPATH. The results also suggest that the ability of bacteria to colonize the surface of plants and to initiate infections in the interior of plants involves many of the same traits. These traits also appear to be under strong regulatory control, being expressed in response to the plant environment in many cases. It may be possible to alter the pattern of expression of such genes by altering the chemical environment of plants either by genetic means or by additional or chemical antagonists of the plant signals. The many novel bacterial genes identified in this study that are involved in plant interactions should be useful in further understanding of bacterial plant interactions.