Horizontal gene transfer through natural competence and recombination in the generalist plant pathogen Xylella fastidiosa by

Homologous recombination is one of many forces contributing to the diversity, adaptation, and emergence of pathogens. For naturally competent bacteria, transformation is one possible route for the acquisition of novel genetic material. This study demonstrates that Xylella fastidiosa, a generalist bacterial plant pathogen responsible for many emerging plant diseases, is naturally competent and able to homologously recombine exogenous DNA into its genome. Several factors were identified that affect transformation and recombination efficiencies, such as nutrient availability, cell growth, and methylation of transforming DNA. Recombination was observed in at least one out of every 10 cells when exogenous plasmid DNA was supplied and one out of every 10 cells when different strains were grown together in vitro. Based on previous genomic studies and experimental data presented here, there is mounting evidence that recombination can occur at relatively high rates and could play a large role in shaping the genetic diversity of X. fastidiosa. INTRODUCTION Naturally competent bacteria, which are able to uptake DNA under natural growth conditions, are found in a wide range of phyla, suggesting that this trait is functionally important and could confer a fitness benefit (42). For these bacteria, DNA acquired through natural transformation could recombine into the genome, providing a source of genetic diversity and potentially a means of horizontal gene transfer, although it is possible that natural transformation evolved as a nutrient uptake system (37). Many factors affect the onset of competence in different bacteria. Bacteria can become competent in response to environmental signals or cues, such as antibiotics or alkaline conditions (10). Nutritional factors can also play a role. For example, the presence of chitin induces competence in Vibrio cholerae (32), while starvation conditions induce competence in Haemophilus influenzae (28). Growth stage can also be a regulating factor (10). A notable outlier, however, is Neisseria gonorrhoeae, which is not known to regulate its competence, and is able to acquire DNA during all phases of growth (21). Recently, interest has risen in the consequences of horizontally transferred DNA on the evolution of microbial pathogens (2). Populations of plant pathogenic bacteria, especially those colonizing crops, are faced with unique environmental pressures that impact the genetic diversity observed in populations. Because host plants tend to be genetically similar, pathogens may undergo periodic selective sweeps where allelic diversity is reduced or eliminated by the emergence of a genotype with increased fitness (18). In practice, such a process mimics the effects of bottlenecks on genetic diversity, as clonal individuals with a shared common ancestor dominate the population. Hostspecialized plant pathogens may undergo selective sweeps, eventually embarking on a coevolutionary arms race with host plants (11). However, a different scenario is plausible for generalist plant pathogens. For these organisms, homologous recombination may lead to the generation and maintenance of allelic diversity in populations (22), which would potentially permit them to explore a wider variety of host plants. While natural competence has been observed in the generalist plant pathogen Ralstonia solanacearum (4), it has not been documented in other bacterial plant pathogens. However, there is mounting evidence that recombination of homologous DNA acquired through natural transformation and other