UW551ΔlecM and UW551ΔaidC had reduced survival in potato tubers at 4°C.

<p>Potato tubers were injected with different <i>R</i>. <i>solanacearum</i> strains, and bacterial cell numbers were counted by grinding and dilution plating tubers at different times after inoculation. The experiment was repeated three times, with three tubers per strain per time point. At 6, 9 and 12 weeks after inoculation, the population sizes of UW551ΔlecM and UW551ΔaidC in tubers were significantly lower than those of the wild type parent strain (<i>P</i><0.05, ANOVA).</p

Comparative Transcriptome Analysis Reveals Cool Virulence Factors of <i>Ralstonia solanacearum</i> Race 3 Biovar 2

<div><p>While most strains of the plant pathogenic bacterium <i>Ralstonia solanacearum</i> are tropical, the race 3 biovar 2 (R3bv2) subgroup attacks plants in cooler climates. To identify mechanisms underlying this trait, we compared the transcriptional profiles of <i>R</i>. <i>solanacearum</i> R3bv2 strain UW551 and tropical strain GMI1000 at 20°C and 28°C, both in culture and during tomato pathogenesis. 4.2% of the ORFs in the UW551 genome and 7.9% of the GMI1000 ORFs were differentially expressed by temperature <i>in planta</i>. The two strains had distinct transcriptional responses to temperature change. GMI1000 up-regulated several stress response genes at 20°C, apparently struggling to cope with plant defenses. At the cooler temperature, R3bv2 strain UW551 up-regulated a cluster encoding a mannose-fucose binding lectin, LecM; a quorum sensing-dependent protein, AidA; and a related hypothetical protein, AidC. The last two genes are absent from the GMI1000 genome. In UW551, all three genes were positively regulated by the adjacent SolI/R quorum sensing system. These temperature-responsive genes were required for full virulence in R3bv2. Mutants lacking <i>lecM</i>, <i>aidA</i>, or <i>aidC</i> were each significantly more reduced in virulence on tomato at 20°C than at 28°C in both a naturalistic soil soak inoculation assay and when they were inoculated directly into tomato stems. The <i>lecM</i> and <i>aidC</i> mutants also survived poorly in potato tubers at the seed tuber storage temperature of 4°C, and the <i>lecM</i> mutant was defective in biofilm formation <i>in vitro</i>. Together, these results suggest novel mechanisms, including a lectin, are involved in the unique temperate epidemiology of R3bv2.</p></div

Mutation of <i>R</i>. <i>solanacearum</i> strain UW551 <i>lecM</i>, <i>aidA</i>, or <i>aidC</i> but not <i>solI</i> differentially reduced bacterial virulence at 20°C.

<p>Virulence was measured on wilt-susceptible tomato plants at 20°C and 28°C via soil soak inoculation (A-E) or at 20°C via cut petiole inoculation (F). Each point represents the mean of three biological replicates, each containing 16 plants per strain per temperature. The area under disease progress curve (AUDPC) was measured for each strain in A-C and each mutant’s AUDPC relative to wild type is shown (E). Asterisks indicate that virulence of wild type and mutant strains were significantly different (* <i>P</i>< 0.05, ** <i>P</i>< 0.01, ANOVA). Each mutant was also significantly reduced in virulence (<i>P</i>< 0.01, repeated measures ANOVA) compared to the wild type following cut petiole inoculation (F).</p

Expression of lectin genes in <i>R</i>. <i>solanacearum</i> strains GMI1000 and UW551 under various conditions.

<p>^aValues shown are scaled log2 signal intensities from strain-specific whole genome microarrays hybridized to labeled cDNA extracted from <i>R</i>. <i>solanacearum</i> cells grown in rich culture medium (CPG) or <i>in planta</i> (IP). These absolute expression values were used to allow comparisons across strains.</p><p>^b<i>np</i>: gene not present in this strain.</p><p>Expression of lectin genes in <i>R</i>. <i>solanacearum</i> strains GMI1000 and UW551 under various conditions.</p

A cluster of genes adjacent to those encoding the SolI/R quorum sensing system were up-regulated in <i>R</i>. <i>solanacearum</i> strain UW551 at 20°C, in culture.

<p>Some were also upregulated <i>in planta</i>. Arrows represent open reading frames. The numbers above the arrows indicate the expression fold-change for each gene at 20°C compared to 28°C, in culture/<i>in planta</i>, determined by whole-genome microarray analysis as described in the text. The arrangement and expression levels of the corresponding genes in strain GMI1000 are also shown.</p