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.

Involvement of rppH in Thermoregulation in Pseudomonas syringae

“…rppH and acyl-coenzyme A (CoA) dehydrogenase (ACDH) deletion mutants were constructed in a fashion similar to that previously described for flgM (24). Briefly, approximately one kilobase of upstream and downstream genomic DNA flanking either gene was amplified using Phusion DNA polymerase (Thermo Scientific [previously Finnzymes], Lafayette, CO) with the knockout (KO) primers listed in Table S2 in the supplemental material.…”

“…RNA isolation and quantitative reverse transcription-PCR (qRT-PCR) were performed as described in other studies (24). Briefly, cultures were harvested with RNAlater (Life Technologies, Carlsbad, CA) and stored for no longer than 1 week at 4°C prior to RNA isolation.…”

“…All protocols and analyses (including statistical tests for differentially expressed transcripts and assessment of gene functional enrichment) related to mRNA sequencing were performed exactly as described previously (24). Briefly, total RNA was isolated as described above, with 16S and 23S rRNA removed using a Ribo-Zero rRNA removal kit (Gram-negative bacteria) (Epicentre, Madison, WI) according to the manufacturer's protocol.…”

“…Previously, we demonstrated that the production of both the flagellum and the lipopeptide surfactant syringafactin were thermoregulated in P. syringae and that this regulation was due, at least in part, to reduced transcription of fliC and syfA at 30°C compared to their transcription at cooler incubation temperatures (24). While we found that flgM was necessary for the thermoregulation of fliC, it did not play any role in the thermoregulation of syfA, suggesting that additional means of transcriptional regulation must be functioning in P. syringae.…”

Comparative genomics of Pseudomonas syringae pathovar tomato reveals novel chemotaxis pathways associated with motility and plant pathogenicity

“…323 Surface but not swimming motility is temperature-dependent in Pto 324 Production of surfactants, flagella components and other products required for motility by P. 325 syringae pv. syringae are thermo-regulated with expression reduced at temperatures greater than 326 25 o C and completely repressed at 30 o C (Hockett et al 2013). We therefore tested both swimming 327 and surface motility of all chemotaxis and motility mutant strains at both 21 o C and 28 o C to 328 ascertain whether our previously observed motility phenotypes were affected by the temperature 329 they were originally performed (28 o C).…”