Steroidal alkaloids (SAs) are triterpene-derived specialized metabolites found in members of the Solanaceae family that provide plants with a chemical barrier against a broad range of pathogens. Their biosynthesis involves the action of glycosyltransferases to form steroidal glycoalkaloids (SGAs). To elucidate the metabolism of SGAs in the Solanaceae family, we examined the tomato (Solanum lycopersicum) GLYCOALKALOID METABOLISM1 (GAME1) gene. Our findings imply that GAME1 is a galactosyltransferase, largely performing glycosylation of the aglycone tomatidine, resulting in SGA production in green tissues. Downregulation of GAME1 resulted in an almost 50% reduction in a-tomatine levels (the major SGA in tomato) and a large increase in its precursors (i.e., tomatidenol and tomatidine). Surprisingly, GAME1-silenced plants displayed growth retardation and severe morphological phenotypes that we suggest occur as a result of altered membrane sterol levels caused by the accumulation of the aglycone tomatidine. Together, these findings highlight the role of GAME1 in the glycosylation of SAs and in reducing the toxicity of SA metabolites to the plant cell.

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.

Acidovorax citrulli is the causal agent of bacterial fruit blotch of cucurbits. We have shown that functional type IV pili (T4P) are required for full virulence of this bacterium. To identify A. citrulli genes required for T4P activity, we screened a library of about 10,000 transposon mutants of A. citrulli M6 for altered T4P-mediated twitching motility. This screen led to the identification of 50 mutants impaired in twitching ability due to transposon insertions into 20 different genes. Representative mutants with disruptions in these genes were further characterized. All mutants were compromised in their virulence in seed transmission and stem inoculation assays and had reduced biofilm formation ability relative to wild-type M6. When grown on nutrient agar, most mutants produced colonies with a translucent and fuzzy appearance, in contrast to the opaque and smooth appearance of wild-type colonies. The colony morphology of these mutants was identical to that of previously reported phenotypic variants of strain M6. The exceptions were M6 mutants disrupted in genes tonB, pilT, pilW, and pilX that exhibited typical wild-type colony morphology, although lacking twitching haloes surrounding the colony. Transmission electron microscopy revealed that most mutants lacked the ability to produce T4P. The exceptions were mutants with disruptions in tonB, pilT, pilW, and pilX genes that were shown to produce these appendages. These findings support the idea that colony phenotypic variation in A. citrulli is determined by the lack of ability to synthesize T4P but not by lack of T4P functionality.

Xanthomonas campestris pv. vesicatoria (Xcv) is the causal agent of bacterial spot disease of tomato and pepper. Previously, we have reported the adaptation of a recombinase- or resolvase-based in vivo expression technology (RIVET) approach to identify Xcv genes that are specifically induced during its interaction with tomato. Analysis of some of these genes revealed that a citH (citrate transporter) homologous gene contributes to Xcv virulence on tomato. Here, we demonstrate that the citH product indeed facilitates citrate uptake by showing the following: citH is specifically needed for Xcv growth in citrate, but not in other carbon sources; the citH promoter is specifically induced by citrate; and the concentration of citrate from tomato leaf apoplast is considerably reduced following growth of the wild-type and a citH-complemented strain, but not the citH mutant. We also show that, in the Xcv-tomato interaction, the promoter activity of the citH gene is induced as early as 2.5h after Xcv is syringe infiltrated into tomato leaves, and continues to be active for at least 96h after inoculation. We identified an operon containing a two-component regulatory system homologous to tctD/tctE influencing citH expression in Xcv, as well as its heterologous expression in Escherichia coli. The expression of hrp genes does not seem to be affected in the citH mutant, and this mutant cannot be complemented for growth in planta when co-inoculated with the wild-type strain, indicating that citrate uptake in the apoplast is important for the virulence of Xcv.

Acidovorax citrulli causes bacterial fruit blotch (BFB) of cucurbits, a disease that threatens the cucurbit industry worldwide. Despite the economic importance of BFB, little is known about pathogenicity and fitness strategies of the bacterium. We have observed the phenomenon of phenotypic variation in A. citrulli. Here we report the characterization of phenotypic variants (PVs) of two strains, M6 and 7a1, isolated from melon and watermelon, respectively. Phenotypic variation was observed following growth in rich medium, as well as upon isolation of bacteria from inoculated plants or exposure to several stresses, including heat, salt and acidic conditions. When grown on nutrient agar, all PV colonies possessed a translucent appearance, in contrast to parental strain colonies that were opaque. After 72 h, PV colonies were bigger than parental colonies, and had a fuzzy appearance relative to parental strain colonies that are relatively smooth. A. citrulli colonies are generally surrounded by haloes detectable by the naked eye. These haloes are formed by type IV pilus (T4P)-mediated twitching motility that occurs at the edge of the colony. No twitching haloes could be detected around colonies of both M6 and 7a1 PVs, and microscopy observations confirmed that indeed the PVs did not perform twitching motility. In agreement with these results, transmission electron microscopy revealed that M6 and 7a1 PVs do not produce T4P under tested conditions. PVs also differed from their parental strain in swimming motility and biofilm formation, and interestingly, all assessed variants were less virulent than their corresponding parental strains in seed transmission assays. Slight alterations could be detected in some DNA fingerprinting profiles of 7a1 variants relative to the parental strain, while no differences at all could be seen among M6 variants and parental strain, suggesting that, at least in the latter, phenotypic variation is mediated by slight genetic and/or epigenetic alterations.