Oxygen Availability Influences Expression of Dickeya solani Genes Associated With Virulence in Potato (Solanum tuberosum L.) and Chicory (Cichorium intybus L.)

Abstract

Dickeya solani is a Gram-negative necrotrophic, plant pathogenic bacterium able to cause symptoms in a variety of plant species worldwide. As a facultative anaerobe, D. solani is able to infect hosts under a broad range of oxygen concentrations found in plant environments. However, little is known about oxygen-dependent gene expression in Dickeya spp. that might contribute to its success as a pathogen. Using a Tn5 transposon, harboring a promoterless gusA reporter gene, 146 mutants of D. solani IPO2222 were identified that exhibited oxygen-regulated expression of the gene into which the insertion had occurred. Of these mutants 114 exhibited higher expression under normal oxygen conditions than hypoxic conditions while 32 were more highly expressed under hypoxic conditions. The plant host colonization potential and pathogenicity as well as phenotypes likely to contribute to the ecological fitness of D. solani, including growth rate, carbon and nitrogen source utilization, production of pectinolytic enzymes, proteases, cellulases and siderophores, swimming and swarming motility and the ability to form biofilm were assessed for 37 strains exhibiting the greatest oxygen-dependent change in gene expression. Eight mutants expressed decreased ability to cause disease symptoms when inoculated into potato tubers or chicory leaves and three of these also exhibited delayed colonization of potato plants and exhibited tissue specific differences in gene expression in these various host tissues. The genes interrupted in these eight mutants encoded proteins involved in fundamental bacterial metabolism, virulence, bacteriocin and proline transport, while three encoded hypothetical or unknown proteins. The implications of environmental oxygen concentration on the ability of D. solani to cause disease symptoms in potato are discussed.

Keywords: Tn5 transposon mutagenesis; abiotic stress; anaerobic conditions; colonization; hypoxia; virulence.

FIGURE 1

Morphology of Dickeya solani IPO2222 Tn5 transposon mutants B7 and B18 colonies grown on M9 agar with glucose for 24 h at 28°C visualized with stereomicroscopy with low magnification (10–40×); size bar: black – 150 mm, white – 5 mm (A) and individual bacterial cells visualized by transmission electron microscopy (TEM); size bar: 1000 nm. TEM analyses were conducted on wild type and Tn5 mutant cells of D. solani IPO2222 grown overnight in Tryptone Soya Broth (TSB) with shaking (200 rpm) at 28°C. Photos were taken directly after bacteria collection from liquid cultures. For this, bacteria were adsorbed onto carbon-coated grids (Sigma) stained with 1.5% uranyl acetate and directly examined with electron microscope (Tecnai Spirit BioTWIN, FEI) (B). The figure shows representative colonies and cells.

FIGURE 2

Ability of four Dickeya solani IPO2222 Tn5 mutants to cause maceration (rotting) of potato tuber tissue. Quantitative determination of average weight of rotting tuber tissue (in grams) collected after 72 h incubation at 28°C in a humid box. Per mutant, ten individual potato tubers were inoculated in two independent experiments (n = 20). Results were considered to be significant at p = 0.05 and pair-wise differences were obtained using the t-test. Error bars represent standard deviation (SD) (A) and visual estimation of symptom expression in potato tubers inoculated with individual IPO2222 Tn5 mutants or wild type (IPO2222, control) in Ringer’s buffer (Merck) after 72 h incubation in a humid box (B). Dotted line in (B) shows extent of tissue maceration – softening of plant tissue by the wild type IPO2222 strain.

FIGURE 3

Ability of seven Dickeya solani IPO22222 Tn5 mutants to cause maceration of chicory leaves. Quantitative determination of the average area of rotting (in cm²) after 48 h incubation at 28°C in a humid box. Per mutant, five individual chicory leaves were inoculated in two independent experiments (n = 10). Results were considered to be significant at p = 0.05 and pair-wise differences were obtained using the t-test. Error bars represent standard deviation (SD) (A) and visual estimation of symptom expression in chicory leaves inoculated with individual IPO2222 Tn5 mutants or wild type (IPO2222, control) in Ringer’s buffer (Merck) after 48 h incubation in a humid box (B).

FIGURE 4

Ability of the three Dickeya solani IPO2222 Tn5 mutants to colonize in vitro grown potato plants cv. Kondor measured as a percentage of symptomatic plants 6 and 16 days post inoculation (DPI). Two week old plants were inoculated with 10 μl of 10⁸ cfu mL^-1 (final:10⁶ cfu per plant) suspension of individual D. solani Tn5 mutant (treatment) or wild type D. solani IPO2222 (control) by applying bacterial suspension on the interspace between the stem base and MS medium of each individual plant. As a negative control 10 μl of sterile 1/4 Ringer’s buffer was used. Each treatment consisted of 10 potato plants grown in individual culture tubes and the entire experiment was repeated independently one time with the same setup (n = 20 per treatment). Inoculated plants were visually inspected after 6 and 16 dpi for wilting, typical blackleg symptoms, stem desiccation and/or plant death.