Positively charged membrane

To eliminate false-positive sequences that escaped the subtraction process, southern hybridization was used for primary verification. Cloned inserts obtained from protozoa-exposed-specific cDNA libraries were amplified by PCR with SCOTS01 primers. PCR amplicons of positive SCOTS clones were transferred to a positively charged membrane (Roche, Mannheim, Germany). Samples of gDNA and cDNA mixtures generated from protozoa-exposed strain NJ-35 and protozoa-unexposed strain NJ-35 were used as probes, followed by labeling with DIG-dUTP (Roche). Dot blot hybridization analysis using DIG Easy Hyb (Roche) was performed according to the manufacturer's instructions. The clones that hybridized positively with the protozoa-exposed probes but negatively with the protozoa-unexposed probes were termed SCOTS clones. Then, the inserts of positive cDNA clones were sequenced by GENEWIZ, Inc., and the nucleotide sequences were queried using BLASTn implemented in BLAST+ (version 2.2.29; ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/) against the genome of A. hydrophila NJ-35. To classify the functions of the preferentially expressed genes, BLASTp implemented in BLAST+ (version 2.2.29) was used to align the amino acid sequences against the COGs database (updated 2014), and some genes related to bacterial virulence were classified according to a previous study (Pang et al., 2015 (link)).

Telomere length assays were performed using the teloTAGGG kit (Roche Diagnostics Ltd., West Sussex, UK). One microgram of genomic DNA was digested with HinfI/RsaI. Digests were separated by gel electrophoresis and blotted onto positively charged membrane (Roche Diagnostics Ltd., West Sussex, UK). Membranes were UV cross-linked, baked at 120 °C and washed in 2×SSC solution. Hybridization of DIG-labeled telomeric probe was performed using buffers and probe provided. Membranes were washed, probed with alkaline phosphatase-conjugated anti-DIG and exposed to CDP-star. Experiments were performed twice.

Southern blot analysis was performed as per the method described by Tripathi et al. [23 (link)]. Briefly, 10 μg of genomic DNA from each sample was restricted with BamH1 for 12 h. The DNA samples, including plasmid pBIN19g-35S::AtEFR and genomic DNA sample from a control non-transgenic plant, were run for a 0.8% agarose gel at 50 V. The gel stained with GelRed® was viewed under ultraviolet light to confirm the digestion. The restricted DNA was denatured, then blotted onto a positively charged membrane (Roche Diagnostics, West Sussex, UK) and fixed using ultraviolet cross-linking. The blots were then hybridized with a digoxigenin (DIG) PCR-labeled 635-bp AtEFR-specific probe. Hybridization and probe detection was performed using a DIG Luminescent Detection Kit for Nucleic Acids (Roche Diagnostics, UK) as per the manufacturer’s protocol.

Total cellular RNA was obtained from yeast cultures (2.0×10⁷ cells) using a TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. Equal amounts (10 µg) of total RNA were separated by electrophoresis through 1.5% formaldehyde denaturing agarose gel, transferred onto a positively charged membrane (Roche Applied Science) and hybridized with a DIG-labeled HXK2-specific antisense RNA probe. The blot was then stripped with stripping buffer (50% formamide, 50 mM Tris/HCl, pH7.5, 5% SDS) and hybridized using PFK1 and PYK1 probes, respectively. Finally, the blot was hybridized with a nuclear encoded ACT1 probe as an internal control [38] (link). The probes were synthesized from the corresponding plasmid linearized by restriction enzymes using a DIG RNA labeling kit (Roche Applied Science). The plasmids used for preparing the probes were constructed by PCR-amplifying fragments of HXK2 (positions 24094-24972), PFK1 (positions 971431-970785), PYK1 (positions 71983-73069), and ACT1 (positions 54696-54187), and then cloning the fragments into the pCRII-TOPO vector carrying the SP6 and T7 promoters (Invitrogen).

Total RNA was isolated using the SV Total RNA Isolation System (Promega) as described [52 (link)]. 5’ RACE was performed using the 5’/3’ RACE Kit (Roche). For Northern blotting 10–20 μg total RNA were separated using 7 M urea/10% acrylamide/0.6 x TBE gels. The RNA was transferred onto positively charged membranes (Roche) by semi dry blotting in 1 x TBE and UV crosslinked. For radioactive labeling 20 μCi γ-ATP³² (Hartmann Analytic) were mixed with 10 U T4 Polynucleotide kinase (Fermentas) and 4 pmol oligonucleotide and incubated at 37°C for 1 h. The reaction was stopped by addition of STE buffer (100 mM NaCl, 10 mM Tris, pH 8, 1 mM EDTA) and the labeled oligonucleotides were purified using MicroSpin G-25 columns (GE Healthcare). The probes were mixed with 500 μg yeast tRNA (Invitrogen) and 250 μg salmon sperm DNA (Invitrogen) to reduce unspecific probe binding. Denaturation was carried out for 10 min at 95°C. Hybridization was performed in hybridization buffer (0.5 M Na₂HPO₄, pH 7.2, 1 mM EDTA pH 7.5, 7% [w/v] SDS) over night at 68°C. The blots were washed in washing buffer (40 mM Na₂HPO₄, pH 7.2, 1 mM EDTA, pH 7.5, 1% [w/v] SDS) and exposed to a Phosphorimaging screen and analyzed with a Typhoon FLA-9000 (GE Healthcare).

Overnight cultures were grown to stationary phase (OD₆₀₀ of 3). 2.5 ml culture were withdrawn, mixed with 0.2 volume of stop solution (5% water-saturated phenol, 95% ethanol) and snap-frozen in liquid nitrogen. After thawing on ice, bacteria were pelleted by centrifugation (2 min, 14.000 rpm, 4°C), and RNA was isolated using the SV total RNA purification kit (Promega) as described by the manufacturer. RNA concentration and quality were determined by measurement of A₂₆₀ and A₂₈₀. Total cellular RNA (10 μg) was mixed with loading buffer (0.03% bromophenol blue, 4 mM EDTA, 0.1 mg/ml EtBr, 2.7% formaldehyde, 31% formamide, 20% glycerol in 4 × MOPS buffer) and was separated on agarose gels (1.2%), transferred overnight onto positively charged membranes (Roche) in 20 × SSC and UV cross-linked. Prehybridization, hybridization to DIG-labeled DNA probes and membrane washing were conducted using the DIG luminescent Detection kit (Roche) according to the manufacturer’s instructions. The csrC and csrB transcripts were detected with a DIG-labeled PCR fragment (DIG-PCR nucleotide mix, Roche) with primer pair 23/24 and 25/26 (Supplementary Table 2), respectively.