Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease of tomato and pepper. The disease process is interactive and very intricate and involves a plethora of genes in the pathogen and in the host. In the pathogen, different genes are activated in response to the changing environment to enable it to survive, adapt, evade host defenses, propagate, and damage the host. To understand the disease process, it is imperative to broaden our understanding of the gene machinery that participates in it, and the most reliable way is to identify these genes in vivo. Here, we have adapted a recombinase-based in vivo expression technology (RIVET) to study the genes activated in X. campestris pv. vesicatoria during its interaction with one of its hosts, tomato. This is the first study that demonstrates the feasibility of this approach for identifying in vivo induced genes in a plant pathogen. RIVET revealed 61 unique X. campestris pv. vesicatoria genes or operons that delineate a picture of the different processes involved in the pathogen-host interaction. To further explore the role of some of these genes, we generated knockout mutants for 13 genes and characterized their ability to grow in planta and to cause disease symptoms. This analysis revealed several genes that may be important for the interaction of the pathogen with its host, including a citH homologue gene, encoding a citrate transporter, which was shown to be required for wild-type levels of virulence.

A recombinase-based in vivo expression technology (RIVET) approach with Xanthomonas campestris pv. vesicatoria (Xcv) revealed that lipA, annotated as putative secreted lipase, is expressed during the interaction between this pathogen and tomato. Here, the tnpR and uidA reporter genes were used to show that lipA is strongly induced in XVM2 minimal medium and during the early stages of tomato infection by Xcv. A mutant strain impaired in lipA was generated by insertional mutagenesis. This mutant grew in a similar manner to the wild-type in rich medium, but its growth was significantly compromised in a medium containing olive oil as a single carbon source. The lipolytic activity of the extracellular fraction of the lipA mutant was reduced significantly relative to that of the wild-type strain, thus confirming that lipA indeed encodes a functional secreted enzyme with lipolytic activity. A plasmid carrying a wild-type copy of lipA complemented the lipA mutant for extracellular lipolytic activity. Dip inoculation experiments with tomato lines Hawaii 7998 (H7998) and Micro Tom showed that the lipA mutant grew to a lesser extent than the wild-type in tomato leaves. Following leaf syringe infiltrations, the mutant strain induced disease symptoms that were less severe than those induced by the wild-type strain, supporting a significant role of lipA in the pathogenicity of Xcv.