For the in vitro treatments, the HMM-grown cells were resuspended in HMM medium lacking L-glutamine, FeCl3, AHL and (NH4)SO4, designated HMM-FeN medium. The cells were washed in HMM-FeN and resuspended in HMM-FeN to a final density of 2.5x109 CFU/ml. Aliquots were transferred to 5 test tubes and diluted to 2.5x108 CFU/ml with either 1) HMM medium, 2) HMM medium with NaCl to a final concentration of 0.23 M, 3) HMM medium with H2O2 to a final concentration of 0.5 mM, 4) HMM medium lacking FeCl3 but with N,N’-di(2-hydroxybenzyl)ethylenediamin-N,N’-diacetic acid monohydrochloride hydrate (HBED) to a final concentration of 100 µM, or 5) HMM medium lacking L-glutamine, (NH4)SO4 and AHL; these were designated as the basal medium, osmotic stress, Apoplastic growth, iron limitation and nitrogen limitation treatments, respectively. At the end of the treatments, the cells were immediately diluted with an RNA stabilizing agent, harvested by centrifugation, and stored as cell pellets at -20°C. All treatments were performed at 25°C with aeration via shaking. Two of the in vitro samples are missing; these are the B728a ∆rpoS cells in the basal medium (replicate 2) and the B728a ∆retS cells exposed to low N (replicate 2). For the in planta treatments, the HMM-grown cells were suspended in water, which contained 0.01% of the biosurfactant Silwet L-77, to a density of 1x106 CFU/ml for B728a, ΔahlR, ΔaefR, ΔgacS, ΔsalA, and ΔretS, to a density of 1x107 CFU/ml for ΔrpoS and ΔalgU, to a density of 1x108 CFU/ml for ΔhrpL, and to a density of 1x109 CFU/ml for ΔrpoN. The higher inoculum densities were to compensate for the reduced growth of the mutants in planta and to ensure sufficient RNA recovery. One of the in planta treatment samples is missing: the B728a ∆rpoN cells recovered from the apoplast (replicate 2). To establish epiphytic populations, the HMM-grown cells were suspended in water containing 0.01% of the biosurfactant Silwet L-77 to a density of 1x106 CFU/ml for B728a, ΔahlR, ΔaefR, ΔgacS, ΔsalA, and ΔretS, to a density of 1x107 CFU/ml for ΔrpoS and ΔalgU, to a density of 1x108 CFU/ml for ΔhrpL, and to a density of 1x109 CFU/ml for ΔrpoN. The higher inoculum densities were to compensate for the reduced growth of the mutants in planta and to ensure sufficient RNA recovery. The bacteria were spray inoculated onto leaves. Plants were incubated for 24 h in a 95% RH mist chamber, at which time the chamber was partially opened until the water on the leaves had visibly evaporated and was then re-sealed to allow incubation without active humidification for another 48 h. The incubations were at 25°C with ambient but not supplemental lighting. A total of 400 to 600 primary leaves were collected, immediately submerged in an RNA stabilizing solution, sonicated for 10 min, and then physically removed from the solution. To establish apoplastic populations, the HMM-grown cells were suspended in water, which contained 0.01% of the biosurfactant Silwet L-77, to the same densities as for the epiphytic treatment, with the exception of the ΔrpoS mutant, for which 106 cells/ml were used. The bacteria were introduced by vacuum infiltration into leaves that were submerged in the inoculum. Plants were incubated for 48 h under plant growth lights with a 12-h photoperiod. A total of 40 to 80 leaves were collected and immediately submerged in an RNA stabilizing solution. The leaves were cut into squares (~ 3x3 mm2) while submerged, and the plant tissues and liquid were sonicated for 10 min. The solution was filtered through Whatman filter paper #1, with repeated filter changes. For both in planta treatments, the cells in the suspension (epiphytic cells) or filtrate (apoplastic cells) were collected by centrifugation, suspended in the residual supernatant, separated from plant debris by filtration through a 5-µm filter with repeated filter changes, again harvested by centrifugation, and stored as cell pellets at -20°C.
Growth protocol
Cells were grown on solid King’s B medium containing rifampin (50 μg/ml). Cells were transferred to liquid HMM and subcultured twice in HMM with aeration via shaking at 25°C. HMM medium contained 0.2% D-fructose, 0.2% D-mannitol, 0.2% succinate, 10 mM L-glutamine, 10 µM FeCl3, 10 µM N-(β-ketocaproyl)-L-homoserine lactone (AHL), 50 mM potassium phosphate buffer, 7.6 mM (NH4)2SO4, 1.7 mM MgCl2, and 1.7 mM NaCl. When the cells reached late-log phase (5x108 CFU/ml), they were collected by centrifugation at 5,000xg for 10 min.
Extracted molecule
total RNA
Extraction protocol
In the in vitro treatments, RNA was stabilized using RNAprotect Bacteria Reagent (Qiagen Inc., Valencia, CA). In the in planta treatments, RNA was stabilized using an acidic phenol RNA stabilizing solution due to the large volumes required. Total RNA was extracted from the cells using a Qiagen RNeasy mini kit and DNA was removed using the on-column DNase I digestion with subsequent DNase I removal. RNA integrity was evaluated using an Agilent 2100 bioanalyzer.
Label
Cy3
Label protocol
Labeling was performed by Roche NimbleGen Inc., Reykjavík, Iceland, following their standard operating protocol. See www.nimblegen.com.
Hybridization protocol
Hybridization was performed by Roche NimbleGen Inc., Reykjavík, Iceland, following their standard operating protocol. See www.nimblegen.com.
Scan protocol
Scanning was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
Description
Washed, late log-phase cells were incubated in HMM medium lacking FeCl3 but with the strong chelator N,N’-di(2-hydroxybenzyl)ethylenediamin-N,N’-diacetic acid monohydrochloride hydrate (HBED) at a final concentration of 100 µM and 2.5x109 CFU/ml for 2 h at 25°C.
Data processing
Expression values were generated from the raw fluorescent readings for each probe (.pair files) using quantile normalization (Bolstad et al., 2003, Bioinformatics, 19:185), and the Robust Multichip Average (RMA) algorithm (Irizarry et al., 2003, Nucleic Acids Res. 31: e15 and Irizarry et al., 2003, Biostatistics 4:249) by Roche NimbleGen Inc. A linear model analysis of the data was conducted for each feature (ORF or predicted sRNA), and this model analysis included data from nine additional B728a mutants subjected to each stress treatment (data for the mutants are not included here). Each linear model included fixed effects for replications, treatments, strains, and treatment-by-strain interactions, as well as a fixed intercept parameter and one random error effect for each observation. LIMMA analysis (Smyth, 2004, Stat Appl Genet Mol Biol 3:Art 3) was applied to share information across genes when estimating error variances. This was done separately for distinct groups of treatments that had similar absolute median residuals. The resulting variance estimates were used to calculate Welch t-statistics and corresponding P-values among all pairwise treatment comparisons of interest. For each comparison of interest, q-values were estimated from the corresponding distribution of P-values (Nettleton et al., 2006, J Agr Biol Envir St 11:337-356).
Global regulatory networks active in Pseudomonas syringae B728 during leaf colonization and in response to target environmental stresses: the GacS, SalA, RetS, AlgU, HrpL, RpoN, RpoS, AhlR and AefR regulons
Data table header descriptions
ID_REF
VALUE
RMA-normalized, averaged gene-level signal intensity
