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  • Bacterial strains, plasmids and growth conditions: Bacterial strains and plasmids used in this study are described in Table 1, and primers used in this study are listed in S1 Table. A spontaneous rifampicin-resistant strain of P. chlororaphis 30–84 and its derivatives were grown at 28°C in Luria-Bertani medium (LB) (5 g of NaCl per liter), AB minimal media (AB), AB amended with 2% casamino acids (AB-C) (Difco, Franklin Lakes, NJ), or pigment production medium-D (PPM-D) [16, 17, 40, 44]. Where applicable, antibiotics were used in the following concentrations: gentamicin (Gn; 50 μg/ml), kanamycin (Km; 50 μg/ml), rifampicin (Rif; 100 μg/ml), tetracycline (Tc; 50 μg/ml) and piperacillin (Pip; 50 μg/ml) for P. chlororaphis 30–84, and ampicillin Ap (100 μg/ml), Gn (15 μg/ml), Tc (25 μg/ml) for E. coli. DNA manipulations, sequence analysis, and PCR: Standard methods were utilized for plasmid DNA isolation, restriction enzyme digestion, ligation, transformation, and agarose gel electrophoresis [63]. Plasmids were introduced into P. chlororaphis 30–84 and its derivatives using either triparental matings or electroporation using methods described previously [1, 40]. Standard polymerase chain reaction (PCR) were carried out using FideliTaq DNA polymerase (Affymetrix, Santa Clara, CA) as described previously [45]. DNA sequencing was performed by the Laboratory for Genome Technology within Institute for Plant Genomics and Biotechnology, Texas A&M University using an ABI 3130xl Genetic Analyzer. To obtain the nucleotide sequence upstream of phenazine biosynthetic operon, the 250 bp nucleotide sequence upstream of the translation start site of phzX were blasted against the Pseudomonas Genome Database (www.pseudomonas.com) and National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/). A total of 27 phenazine producing strains were retrieved and analyzed for the types of a phenazine modifying enzyme. The nucleotide sequences (250 bp flanking sequences from the translation start site of each strain’s phenazine biosynthetic operon) were aligned and edited using MUSCLE (MEGA7). MEGA7 was used to build a, Maximum Likelihood (ML) trees based on the Tamura-Nei Model [64] [65]. ML bootstrapping was performed with 1,000 replicates to assess the relative stability of the branches. Construction of phzX transcriptional fusion derivatives: To determine the function of the 5’-UTR of phenazine biosynthetic operon in phenazine gene expression, a 1.5-Kb DNA fragment contains a deletion of 90-bp in the 5’-UTR of phenazine biosynthetic operon was synthesized (GeneScript, Piscataway, NJ) and obtained as pUC57-Enh (Table 1). This fragment lacks 90-bp of sequence (including both SspI sites and the second direct repeat) starting at the +8 bp (from the TSS) to the RBS of phzX, but maintaining the endogenous RBS and start codon of PhzX. PCR fragments of 271 bp and 181 bp containing sequence from -124 to +147 of phzX were amplified from genomic DNA of P. chlororaphis 30–84 and pUC57-Enh using the primers, phzXF1/phzXR1 (S1 Table) and cloned into the promoter trap vector pGT2-lacZ resulting plasmids pJMYX1 and pJMYX2, respectively (Fig 2 and Table 1). To determine the function of upstream sequence of phenazine biosynthetic promoter region, PCR fragments of 354bp (from -124 to +230), 310bp (from -80 to +230), and 274bp (from -44 to +230) containing the promoter of phenazine biosynthetic operon, 5’-UTR and 115bp of phzX coding sequence were amplified from P. chlororaphis 30–84 genomic DNA using the following primer sets, phzXF1/phzXR2, phzXF2/phzXR2 and phzXF3/phzXR2, respectively (S1 Table) These fragments were cloned into the promoter trap vectors pKT2-lacZ, creating the plasmids pJMYX3, pJMYX4 and pJMYX5, respectively (Table 1 and Fig 2). The plasmids pKT2-lacZ and pGT2-lacZ contain a promoterless lacZ gene with its own ribosome binding site (RBS) located downstream of multiple cloning locus, which enables the study of transcriptional activities. All transcriptional fusion plasmids were separately introduced into 30-84Ice via triparental mating and the transcriptional activity was determined by β-galactosidase activity [66] in 30-84Ice after 24 h growth in LB with rapid agitation. Strain 30-84Ice was used for the transcriptional fusion assays because it contains a phzB::inaZ insertion (Table 1) and as a consequence does not produce phenazine, which interferes with the β-galactosidase assay. Generation of a phenazine enhanced mutant: In order to generate the phenazine enhanced mutant strain, the 1.5 Kb sequence from pUC57-Enh was cloned into the vector, pLAFR3, generating pLAF-phzEnh (Table 1). This 1.5 Kb fragment contains flanking regions upstream from the EcoRV site in the first half of phzR and downstream to the BamHI site at the 3’-end of phzY (the second gene of phz operon) to facilitate homologous recombination. The pLAF-phzEnh plasmid was introduced into P. chlororaphis 30–84 strains containing pUCP18-RedS via triparental mating, and the chromosomal 90-bp deletion of 5’-UTR of phenazine biosynthetic operon was obtained with the support of λ phage recombinases [40, 48]. A dark orange colony (for 30-84Enh) from the PPMD plate or a dark blue colony (for 30-84ZN-Enh) from PPMD plate supplemented with 2% X-gal were chosen, and pUCP18-RedS plasmid (containing sacB) was cured by counter selection on LB plates supplemented with 5% sucrose. A Tcs, Pips, and SucroseR colony was chosen and mutation was verified by PCR and sequencing. Construction of an rsmE deletion mutant: To inactivate rsmE gene in P. chlororaphis 30–84, fragments of upstream (611 bp) and downstream (706 bp) of the rsmE open reading frame (ORF) were amplified from P. chlororaphis 30–84 genomic DNA using primers RsmEUPF/RsmEUPR and RsmEDWF/RsmEDWR (S1 Table). These fragments were designed to carry a KpnI site that permitted the insertion of a kanamycin resistance cassette at the 3’ end of upstream fragment and 5’ end of downstream fragment with 183 bp deletion of rsmE ORF. Each fragment was simultaneously cloned into the pEX18Ap (Table 1). A 961 bp fragment containing kanamycin resistance cassette was PCR amplified from pUC4K (Table 1) using the primers, KmKpnF/KmKpnR, and inserted into KpnI site between upstream and downstream fragments in pEX18Ap. The resulting plasmid, pEX18Ap-rsmEKO (Table 1), was electroporated into P. chlororaphis 30–84, and mutant was selected for by amending LB plates with the appropriate antibiotics. A KmR, Pips, and SucroseR colony was chosen and the rsmE mutation was verified by PCR and sequencing. Construction of translational fusion vector with 90-bp deletion of the 5’-UTR of phenazine biosynthetic operon: To determine the function of the 5’-UTR on translation of the phenazine biosynthesis genes, translation fusion vectors were created containing the sequence from the phenazine biosynthetic operon promoter to the 20th codon of PhzX with or without 90-bp sequence of 5’-UTR of phenazine biosynthetic operon (Fig 4B). The fragments with the 90-bp sequence (299 bp) and without the 90-bp sequence (209 bp) were PCR amplified from genomic DNA of P. chlororaphis 30–84 and pUC57-Enh, respectively, using primer set phzXF1/phzXR3 (S1 Table). The products were cloned, in frame, with the 8th codon of lacZ in the translational fusion vector, pME6015 [34, 45], resulting pJMYX7 and pJMYX8 respectively (Fig 4B and Table 1). These plasmids were introduced in the wild-type, 30-84W and 30-84RsmE via electroporation, and translational activities were determined by β-galactosidase activity. RNA preparation for quantitative PCR: To isolate RNA from the wild-type and 30-84Enh, single colonies of each strain were grown with rapid agitation at 28°C in 3 ml of AB-C broth. When cell density reached OD620 = 1.8, 1 ml aliquots of each sample were mixed with 2 ml of Qiagen RNA Protect reagent (Qiagen, Hilden, Germany) to stabilize bacterial RNA, and cells were harvested by centrifugation for 10 min at 2400 x g. Total RNA was extracted using a Qiagen RNeasy Mini Kit (Qiagen) according to the manufacturer’s recommended protocol. The genomic DNA was removed using on-column DNase-I digestion (Qiagen). Five micrograms of total RNA were reverse-transcribed using random primers (Invitrogen Life Technologies, Carlsbad, CA) and Superscript III (Invitrogen) at 50°C for 1 h and inactivated at 75°C for 15 min. For the negative control, the same reaction was performed using sterilized water instead of reverse transcriptase. SYBR Green reactions were performed using the ABI 7900 HT Fast System (Applied Biosystems, Foster City, CA) in 384 well optical reaction plates. Quantitative PCR (qPCR) assays were performed to measure the expression levels of the target genes as previously described [45]. Briefly, aliquots (1 μl) of cDNA (2 ng/reaction) or negative controls were used as template for qPCR reactions with Fast SYBR Green PCR Master Mix (Applied Biosystems) and primers (500 nM final concentration). qPCR amplifications were carried out at 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min, and a final dissociation curve analysis step from 65°C to 95°C. Technical replicate experiments were performed for each of the biological samples, in triplicate. Amplification specificity for each reaction was confirmed by the dissociation curve analysis. The Ct values were used for further ΔΔCt analysis. The 16S rDNA was used as a reference gene to normalize samples. A relative quantification value was calculated for each gene with the control group as a reference [27, 40, 45]. Quantification of phenazine and AHL production: P. chlororaphis 30–84 strains were grown at 28°C in AB minimal, LB and PPMD broth with rapid agitation. Phenazines were extracted from cell-free supernatants as described previously [1, 16, 45]. Phenazines concentration was calculated via serial dilution of the extract at absorbance of 367 nm. Total AHLs were extracted as described previously [39, 45] from 5 ml cultures, which were grown at 28°C with shaking in AB minimal, LB and PPMD broth. AHL production was quantified by inoculating the extracted AHLs with the AHL-specific reporter strain 30-84I/Z (phzI-, phzB::lacZ). 30-84I/Z is deficient in AHL production due to mutation of AHL synthase gene phzI, but responds to AHL by expressing the reporter gene lacZ. β-galactosidase activity was determined on cultures grown at 28°C for 24 h with rapid agitation. Construction of phzR transcriptional reporters: In order to determine whether the 90-bp of the 5’-UTR of phenazine biosynthetic operon also negatively influence phzR expression, phzR transcriptional fusions were constructed. PCR fragments containing a sequence from -359 bp to +155 of phzR was amplified from genomic DNA of the wild-type and 30-84Enh using the primers, phzRF-phzRR (S1 Table). The 514 bp and 424 bp PCR amplicons containing phzR promoter region with or without the 90-bp of the 5’-UTR of phzX, respectively, were ligated into promoter trap vector pGT2-lacZ to make the phzR transcriptional fusion vectors, pJMYR1 and pJMYR2 (Table 1 and S4 Fig). These reporters were separately introduced into 30-84Ice by triparental mating, and transcriptional activities were determined by β-galactosidase activity. Microtiter plate biofilm assay and eDNA quantification: To measure the ability of strains 30–84 wild-type and 30-84Enh to form a biofilm, static biofilm assays were conducted in 24-well polystyrene microtiter plates. Briefly, overnight cultures grown in 3 different media (AB-C, LB and PPMD) were adjusted to an OD620 of 0.8 with fresh medium. The adjusted cultures were diluted 1:100 into the appropriate media, and 1.5 ml of the dilution inoculate was transferred into 24-well plates. Plates were incubated at 28°C without shaking. After 48 h, the adherent cell population was quantified by crystal violet staining as described previously [17, 67]. The concentration of eDNA was determined quantitatively using Qubit 2.0 Fulorometer (Invitrogen), as described previously with few modification [19]. Briefly, overnight cultures grown in AB-C broth at 28°C with agitation were adjusted to an OD620 of 0.8. The adjusted cultures were re-inoculated at a 1:100 dilution into 20 ml AB-C broth. Cultures were grown at 28°C with rapid agitation and sampled every 8–12 h. Cell-free supernatant by centrifugation and filtration were mixed with double-strand DNA fluorescent dyes (dsDNA BR) from Qubit (Invitrogen Life Technologies, Carlsbad, CA), and the concentration of eDNA was quantified using Qubit 2.0 Fluorometer (Invitrogen Life Technologies). The amount of eDNA was reported as μg/ml. To determine the ability of colonizing to the host plant, the wild-type and 30-84Enh were inoculated to wheat (cultivar TAM112) by two different methods. For the root-dip inoculation methods, bacterial cultures were grown in 10 ml KMB broth for 24 h, and inoculum was standardized to OD620 = 0.8 (ca. 2 x 109) in sterile 1X PBS. Seeds were surface sterilized as previously described [44], and surface sterilized seeds were pregerminated on sterilized germination paper for two days. Seedling roots were dipped into the bacterial solution for 10 min, and sown into 25 × 200 mm cone-tainers that contain a natural wheat rhizosphere (Uvalde, TX) soil mix (soil: sand, 2:1, v:v). Plants were grown for 30 days before the entire root system was processed for the CFU calculation, as described previously [44]. For the soil inoculation method, bacterial cultures were prepared as described above. Bacteria cultures were washed 3 times with sterilized water, and inoculum were thoroughly mixed with natural wheat rhizosphere soil mix (soil: sand, 2:1, v:v) for final bacterial population of 106 CFU/g of soil. Pregerminated wheat seedlings (two days) were sown into the bacterial amended soil mix and were grown for 24 days. Bacterial populations were collected from the entire root system and quantified by CFU. Total populations were determined by serial dilution on LB agar amended with rifampicin. Fungal inhibition and take-all suppression assay: To quantify the ability of strains 30–84 wild-type and 30-84Enh to inhibit the take-all causal agent Ggt strain ARS-A1, an in vitro dual culture assay was conducted as described previously [45]. After 7 days of co-culture on potato dextrose agar plates, the zone of inhibition was measured as the distance between edge of the bacterial colony and the fungal mycelium. Assays to determine the ability of strains 30–84 wild-type and 30-84Enh to suppress take-all disease on wheat seedlings were conducted as described previously [1, 51]. Briefly, bacteria-coated seeds or control seeds (coated with methyl cellulose) were sown in tubes (25 × 200 mm) filled with 5g of sterilized vermiculate layer overlaid with 20 g of a natural wheat rhizosphere soil mix (soil: sand, 2:1, v:v) amended with Ggt colonized oat kernels fragments (0.85%, w/w). For the control, sterilized oat kernels were ground and amended to the soil mix with same amount (0.85%, w/w). Seeds (cv. TAM112) were covered with 1cm of sterilized vermiculate and incubated for 3 days at room temperature to facilitate germination. Seedlings were arranged in a complete randomized block design, and transferred to a growth chamber (16°C, 12 h dark-light cycle). After 20 days, root disease was evaluated on a scale of 0–5, where 0 = no disease and 5 = nearly dead. as described previously [1]. All data presented are mean ± the standard error of the mean (STE) from at least two experiments. Data were analyzed using ANOVA and Fisher’s protected Least Significant Difference (LSD) test (P<0.05) or unpaired t-test. Data were processed with GraphPad Prism (GraphPad Software, San Diego, CA).
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