The 3-Hydroxy-2-Butanone Pathway Is Required for Pectobacterium carotovorum Pathogenesis

Pectobacterium species are necrotrophic bacterial pathogens that cause soft rot diseases in potatoes and several other crops worldwide. Gene expression data identified Pectobacterium carotovorum subsp. carotovorum budB, which encodes the α-acetolactate synthase enzyme in the 2,3-butanediol pathway, as more highly expressed in potato tubers than potato stems. This pathway is of interest because volatiles produced by the 2,3-butanediol pathway have been shown to act as plant growth promoting molecules, insect attractants, and, in other bacterial species, affect virulence and fitness. Disruption of the 2,3-butanediol pathway reduced virulence of P. c. subsp. carotovorum WPP14 on potato tubers and impaired alkalinization of growth medium and potato tubers under anaerobic conditions. Alkalinization of the milieu via this pathway may aid in plant cell maceration since Pectobacterium pectate lyases are most active at alkaline pH

Operon structure of <i>bud</i> genes in <i>Klebsiella</i>, <i>Pectobacterium</i>, <i>Dickeya</i>, and <i>Erwinia</i> species.

<p>Sequences were retrieved from ASAP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022974#pone.0022974-Glasner2" target="_blank">[66]</a>. Genes are indicated by arrows, with the direction of the arrowhead indicating the direction of the gene. Genes shaded with the same color are homologous and unlabeled genes have no known function. <i>budA</i> is an alpha-acetolactate decarboxylase, <i>budB</i> is an acetolactate synthase, <i>budC</i> is an acetoin reductase and <i>budR</i> is a LysR-family transcriptional regulator. In <i>Klebsiella</i> and <i>Enterobacter</i> (not shown), <i>budABC</i> are in one operon. The locus of the <i>budC</i> gene is found elsewhere in the chromosome in <i>Pectobacterium</i> and <i>Dickeya</i>. The <i>Erwinia</i> species lack <i>budC</i>.</p

WPP14Δ<i>budB</i> is not impaired in growth in potato tubers.

<p>Bacterial suspensions containing 10⁵ CFU were stab-inoculated into potato tubers (cv. “Superior”) (<i>n</i> = 5). Tubers were placed into plastic bags and incubated at 28°C. Bacterial population was determined every 24 hrs after inoculation. Error bars indicate standard error. The graph shows data from one of two experiments, neither of which showed significant difference in bacterial growth.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

WPP14 Δ<i>budB</i> is reduced in virulence in potato tubers.

<p>Potato tubers (n = 10) were inoculated with 10⁶ CFU, placed into plastic bags and incubated at 28°C for 3 days. After incubation, macerated tissue was scooped out and weighed. The graph shown is from one experiment. This experiment was repeated twice with similar results. Error bars indicate standard error.</p

The <i>P. c.</i> subsp. <i>carotovorum budAB</i> operon affects bacterial modification of pH in tubers slices that are incubated anaerobically.

<p>Potato slices were inoculated with 10⁶ CFU and incubated under aerobic (<b>A</b>) and anaerobic conditions (<b>B</b>) for 20 hours at room temperature (∼22°C). <b>PR</b>: pH indicator phenol red (yellow at pH below 6.4 and red violet at pH above 8.2), <b>BP</b>: pH indicator bromocresol purple (yellow at pH below 5.2 and purple at a pH above 6.8).</p

<i>P. c.</i> sp. <i>carotovorum budB</i> transcript is differentially expressed in potato tubers and stems.

<p>Relative expression of the <i>budB</i> transcript in potato stems and tubers was determined with four reference mRNA (<i>ffh</i>, <i>dspE</i>, <i>pelB</i>, and <i>hrcC</i> - <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022974#pone-0022974-t002" target="_blank">Table 2</a>). The mean relative expression ratio (RER) of the <i>budB</i> transcript in tubers was 8.5-fold elevated compared to stem tissue, which was used as the calibrator. The difference was found to be significant at a 95% confidence interval by <i>t</i>-test using Prism software (Mac version 5.0d, GraphPad Software, Inc.) with a <i>P</i>-value = 0.0425. Error bars represent standard error of the mean.</p

RT-qPCR primer sequences and efficiency.

A<p>: Primer efficiencies were calculated from dilution curve with the MyiQ Cycler software.</p