The Stringent Response Mediated by (p)ppGpp Is Required for Virulence ofPseudomonas syringaepv.tomatoand Its Survival on Tomato

Abstract

The hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor in Pseudomonas syringae pv. tomato DC3000 (DC3000). In this study, the role of the second messenger (p)ppGpp on virulence and survival of DC3000 was investigated. Results have demonstrated that (p)ppGpp-deficient mutant (ppGpp(0)) of DC3000 exhibited lower levels of expression of the T3SS and genes of other virulence traits, such as coronatine toxin. The ppGpp(0) mutant of DC3000 was greatly impaired in causing disease and in growth in planta. Furthermore, (p)ppGpp was required for swarming motility, pyoverdine production, the oxidative stress response, as well as γ-amino butyric acid utilization. Screening of amino acids, major signals in activation of ppGpp biosynthesis, revealed that promoter activities of the avrPto gene could be either activated or suppressed by various amino acids in a ppGpp-dependent or -independent manner. Moreover, the ppGpp(0) mutant exhibited increased cell size and decreased survival on plant surfaces. Altogether, these findings indicate that ppGpp acts as an internal signal that regulates the T3SS as well as other virulence factors in pseudomonads and suggest that bacterial pathogens utilize intracellular messengers to sense environmental and nutritional signals for rapid, precise, and reversible control of their pathogenesis and survival.

Mutations in the β-Subunit of the RNA Polymerase Impair the Surface-Associated Motility and Virulence of Acinetobacter baumannii

“…Substitutions at position Ser531 of the ␤-subunit of the E. coli RNA polymerase (which corresponds to Ser540 of the A. baumannii ␤-subunit reported here) specifically alter the interaction of E. coli RpoB with certain stringent promoters (31). In N. meningitidis, mutations in rpoB cause global transcriptional changes that functionally mimic the stringent response (32); in Pseudomonas spp., a phylogenetic neighbor of Acinetobacter spp., stringent RNA polymerase is involved in twitching motility (33,34). Thus, by isolating mutants arising from point mutations in the A. baumannii rpoB gene, we were also able to identify genes involved in the surface-associated motility of the bacterium (Table 1).…”

Homologues of the RNA binding protein RsmA in Pseudomonas syringae pv. tomato DC3000 exhibit distinct binding affinities with non‐coding small RNAs and have distinct roles in virulence

“…Deletion mutations of rsmA1 , rsmA2 , rsmA3 , rsmA4 , rsmA2/rsmA3 , rsmA2/rsmA4 , rsmA3/rsmA4 , rsmA2/rsmA3/rsmA4 and rsmA1 / rsmA2/rsmA3/rsmA4 were generated using splice overlap extension mutagenesis as described previously (Chatnaparat et al , ). Briefly, in a first round of PCRs, about 1 kb upstream and downstream fragments of rsmA1 , rsmA2 , rsmA3 and rsmA4 in Pst DC3000 were amplified using primers unique to these regions (Table ).…”

“…Pyoverdine product was detected in mannitol–glutamate (MG) medium as previously described (Ambrosi et al , ; Chatnaparat et al , ; Imperi et al , ; Park et al , ). Bacterial cells of overnight cultures in KB were washed in PBS, resuspended to an OD 600 of 0.05 in MG medium and incubated with shaking at 28 °C for 24 h. Pyoverdine was quantified by measuring the absorbance at 405 nm of culture supernatants diluted 2:1 in 100 mM Tris‐HCl (pH 8.0) and normalized with OD 600 .…”

Validation of RT-qPCR approaches to monitorPseudomonas syringaegene expression during infection and exposure to pattern-triggered immunity

“…We identified nine RG genes to test based on historical utility or new global expression data that suggest they might be ideal candidates for use as reference genes in these types of studies. Previous studies of P. syringae have used gyrA, gap-1, hemD, recA and rpoD and 16S rRNA as individual RGs (Ferreira, Myers et al 2006, Records and Gross 2010, González-Lamothe, El Oirdi et al 2012, Vargas, Farias et al 2013, Ishiga and Ichinose 2015, Lee, Ryu et al 2015) (Tegli, Gori et al 2011, Freeman, Chen et al 2013, Chatnaparat, Li et al 2015). gap-1 and gyrA showed equal expression under in vitro experimental conditions as measured by microarray (Ferreira, Myers et al 2006).…”

“…We developed P. syringae-specific primers for the 16S ribosomal small subunit RNA (16S rRNA) to measure the concentrations of bacterial RNAs within mixed RNA samples. We tested the transcriptional stability of six published DC3000 reference genes (16S rRNA, gap-1, gyrA, hemD, recA, and rpoD) (Ferreira, Myers et al 2006, Records and Gross 2010, Tegli, Gori et al 2011, González-Lamothe, El Oirdi et al 2012, Freeman, Chen et al 2013, Vargas, Farias et al 2013, Chatnaparat, Li et al 2015, Ishiga and Ichinose 2015, Lee, Ryu et al 2015) as well as three new reference gene candidates selected based on a preliminary in situ RNAseq experiment, ultimately validating four for use in our approach (recA, rpoD, leuD, and oprF) . Using our protocol for RT-qPCR with multiple validated RGs, we examined the relative expression of three GOIs (hrpA, cfl and fliC) relevant to DC3000 host interactions of DC3000 in planta , and from in vitro media.…”