Thermo-regulation of genes mediating motility and plant interactions in Pseudomonas syringae

Abstract

Pseudomonas syringae is an important phyllosphere colonist that utilizes flagellum-mediated motility both as a means to explore leaf surfaces, as well as to invade into leaf interiors, where it survives as a pathogen. We found that multiple forms of flagellum-mediated motility are thermo-suppressed, including swarming and swimming motility. Suppression of swarming motility occurs between 28° and 30 °C, which coincides with the optimal growth temperature of P. syringae. Both fliC (encoding flagellin) and syfA (encoding a non-ribosomal peptide synthetase involved in syringafactin biosynthesis) were suppressed with increasing temperature. RNA-seq revealed 1440 genes of the P. syringae genome are temperature sensitive in expression. Genes involved in polysaccharide synthesis and regulation, phage and IS elements, type VI secretion, chemosensing and chemotaxis, translation, flagellar synthesis and motility, and phytotoxin synthesis and transport were generally repressed at 30 °C, while genes involved in transcriptional regulation, quaternary ammonium compound metabolism and transport, chaperone/heat shock proteins, and hypothetical genes were generally induced at 30 °C. Deletion of flgM, a key regulator in the transition from class III to class IV gene expression, led to elevated and constitutive expression of fliC regardless of temperature, but did not affect thermo-regulation of syfA. This work highlights the importance of temperature in the biology of P. syringae, as many genes encoding traits important for plant-microbe interactions were thermo-regulated.

Figure 1. Thermo-regulation of motility in P. syringae.

Swarming of Pseudomonas syringae B728a at 20°C (gray bar, top photo of inset) and 30°C (white bar, bottom photo of inset) after 24 hours (A). Area of swimming colonies of Pseudomonas syringae B728a after 24 hours growth at various temperatures (B). Biosurfactant area on plates following 24 hours of incubation at 20°C (gray bar) or 30°C (white bar) (C). The vertical bars represent the standard deviation of the mean.

Figure 2. Thermo-regulation of fliC and syfA expression.

Cell normalized GFP fluorescence of cells grown for 48 hours at various temperatures that harbored a fusion with promoters of fliC (square), syfA (diamond), or nptII (triangle). The vertical bars represent the standard deviation of the mean.

Figure 3. Lack of thermo-regulation of fliC in a ΔflgM mutant.

Relative expression of either fliC (black bars) or syfA (white bars) in wild type Pseudomonas syringae B728a or a ΔflgM mutant at either 20°C or 30°C. Expression normalized to WT, 20°C. Error bars represent the 95% confidence interval.

Figure 4. Reduction of fliC expression in ΔflgM by complementation.

Cell-normalized GFP fluorescence of either wild type Pseudomonas syringae B728a (black squares) or a ΔflgM mutant (open diamonds) harboring a gfp reporter gene fusion with a promoter of fliC (A). Cell normalized GFP fluorescence of wild type P. syringae or a ΔflgM mutant harboring a gfp reporter gene fusion with the promoter of fliC as well as either a flgM complementing vector (p519Mcomp) (black and dark gray bar, respectively) or vector control (p519empty) (white and light gray bar, respectively) when grown at either 20°C or 30°C (B). The vertical bars represent the standard deviation of the mean.

Figure 5. salA positively regulates syfA.

Cell-normalized GFP fluorescence exhibited by wild type and various mutants of Pseudomonas syringae B728a harboring a gfp reporter gene fused either to syfA (A) or syfR (B) following 24 hours of incubation at 20°C. Vertical bars represent the standard deviation of the mean.

Figure 6. Temperature-dependent epiphytic survival.

Epiphytic population size of wild type Pseudomonas syringae B728a (A) or a ΔflgK mutant (B) incubated at 20°C (open diamond) or 30°C (black square) for 6.5 hours at 100% RH prior to being exposed to desiccation at 26–28°C and 60–65% RH. Log-transformed population size decline was measured during the desiccation period that occurred between 6.5 and 12 hours after inoculation (C). This experiment was repeated several times with similar results. Treatments noted with the same letter do not differ significantly at a p-value ≤0.05 as determined by least significant difference test (LSD). The vertical bars represent the standard deviation of the mean. Error bars are present in panel B, but in some cases are small and obscured by the treatment symbol.