Potatoes, superior and resistance cultivar CT206-10 (Kim-Lee et al., 2005), were propagated in sterile containers containing Murashige and Skoog, 1962 (MS) medium supplemented with 3% sucrose and 0.8% agar. The pH of the medium was adjusted to 5.8 prior to autoclaving. All cultures were maintained at 20 ± 1°C under 16 h light/8 h dark photoperiod. Seedlings were transferred to greenhouse after 4-5 weeks and allowed to grow in natural light condition. Rs strain KACC10722 (race 3, biovar 2) was grown on CPG medium (0.1% casamino acid, 1% peptone, 0.5% glucose, pH 6.5), cultured at 28°C for 24-48 h. The bacterial concentration was diluted to 10⁷ cfu/ml prior to the plant infection.

For infiltration of pathogen Rs, the bacterial solutions at 10⁷ cfu/ml in an approximately 1 ml volume, measured according to indicator syringes no needle, were infiltrated into potato leaves. Rs infection to roots was carried out as described earlier (Deslandes et al., 1998) with slight modifications. Approximately 2 cm of roots from the bottom of five to six weeks old potato seedling were cut and infected with Rs for 1 hr. The infected potato plants were then transferred to Jippy pots and bacterial wilt disease caused by Rs was observed for 14 days. Whole potato tissues from ten plants, each of superior and resistance cultivar CT206-10 were harvested after 7, 14 and 21 days of post-infection and stored at −80°C until used.

Trypan-blue staining was performed as described earlier (Peterhansel et al., 1997). In brief, potato leaves were boiled in trypan-blue solution [0.033% (w/v) trypan-blue, 8% (v/v) lactate, 8% (v/v) glycerol, 8% (v/v) phenol, 8% (v/v) water, and 67% (v/v) ethanol] until the green color disappeared. The leaves were then washed with water and transferred to the saturated chloral hydrate solution (2.5 mg/ml) to remove non-specific staining. The leaves were then immersed in the 50% (v/v) glycerol solution until analysis.

Total protein from ten grams of potato root was extracted using Mg/NP-40 extraction buffer [0.5 M Tris-HCl, pH 8.3; 2% v/v NP-40; 20 mM MgCl₂; 2% v/v β-mercaptoethanol] followed by phenol extraction method as described earlier (Kim et al., 2001). Approximately, 600 μg protein was dissolved in the rehydration buffer [7 M (w/v) urea, 2 thiourea, 4% (w/v) CHAPS, 0.002% (w/v) bromophenol blue, 20 mM DTT, 0.5% (v/v) IPG buffer (pH 4-7)] and loaded on 24 cm IPG strips, pH 4-7 by rehydration loading overnight. First dimensional separation was carried out using following protocol: 50 V 4 h, 100 V 1 h, 500 V 1 h, 1,000 V 1 h, 2,000 V 1 h, 4,000 V 2 h, 8,000 V 5 h, and 8,000 V 9 h by IPGPhore2 platform (GE healthcare). Focused IPG strips were equilibrated using equilibration buffer containing 1% DTT as first step which was replaced by 2.5% iodoacetamide in the second step. Second dimensional separation was carried out using 12% SDS-PAGE after which the gels were stained with colloidal Coommassie brilliant blue G-250 (CBB).

Differential protein spots were excised from the 2D gels and were subjected to in-gel digestion as described previously (Kim et al., 2013). Prepared samples of tryptic peptides were subjected to MALDI-TOF/TOF MS using ABI 4800 Plus TOF-TOF Mass Spectrometer (Applied Biosystems, Framingham, MA, USA). The ten most and least intense ions per MALDI spot with signal/noise ratios >25 were selected for subsequent MS/MS analysis in 1 kV mode using 800-1,000 consecutive laser shots. Data were subjected to a Mass Standard Kit for the 4700 Proteomics Analyzer (calibration Mixture 1). MS/MS spectra were searched against the Uniprot/Swiss-Prot (14926175 sequences; 5299740401 residues) by Protein Pilot v.3.0 software (AB Sciex, Framingham, MA, USA) using MASCOT as search engine (ver. 2.3.0, Matrix Science, London, UK). The search parameters were as follows: fixed modifications-carbamidomethylation of cysteines, variable modification-methionine oxidation, peptide and fragment ion mass tolerances- 50 ppm, maximum trypsin missed cleavage- 1 and instrument type- MALDI-TOF/TOF. Only significant hits, as identified by the MASCOT probability analysis (p<0.05) were accepted.

First-strand cDNA was synthesized from 1 μg of total RNA from the roots of CT206-10 potato cultivar infected with Rs in 20 μl of reaction mixture using Superscript III reverse transcriptase (Life Technologies, NY, USA). The semi-quantitative RT-PCR was performed using Actin and Tubulin genes as internal control. The PCR reaction conditions were initial denaturation at 94°C for 5 min, 22-35 cycles of 94°C for 30 s, 52-58°C for 30 s, 72°C for 30 s and then final extension at 72°C for 5 min. The PCR amplified products were electrophoresed on 1.5% agarose gel and transcript levels of genes were estimated.

At first, we compared the resistance of “CT206-10” with that of wild type “superior” against Rs infection (Kim-Lee et al., 2005). Leaf wilting is one of the early symptoms of Rs infection in plants therefore; we observed the leaf wilting on both the cultivars after infection. Leaves of both the cultivars showed varied degree of wilting after infection with Rs, suggesting successful colonization of Rs in both the cultivars (Fig. 1). Although both the cultivars showed wilting of leaves, wilting percentage was far less in leaves of CT206-10 cultivar as compared to superior, suggesting increased resistance of former cultivar to Rs than later (Fig. 1). To further cross-check this observation, leaves of both the plants were subjected to trypan blue staining. Trypan blue staining is a measure of cell viability as it stains selectively the dead cells (Peterhansel et al., 1997). Results of trypan blue staining showed stronger blue color in the green leaves of superior plants in comparison with the CT206-10 leaves, indicating that cell death was weakly induced in CT206-10 cultivar (Fig. 2). Taken together, the results of disease resistance assay and trypan blue staining indicate that CT206-10 cultivar is relatively more resistant to Rs in comparison with the superior cultivar.

Rs is a soil borne pathogen which invade susceptible plant through root. Therefore, proteins were extracted from the potato roots infected with Rs and used for 2-DE analysis. More than 500 reproducible spots were observed on the high-resolution 2-DE gels using Image Master 2D Platinum software (Fig. 3). Among these, 12 spots showed differential expression in Rs infected samples, of which 8 spots were identified by MALDI-TOF-MS (Table 1). The identified proteins were Actin 51 partial protein (Spot 2), Hypothetical protein of Oryza sativa Japonica group (Spot 4), Glycine-rich RNA binding protein (GRP; Spot 6), Tomato stress induced-1 protein (TSI-1; Spot 7), Hypothetical protein POPTR, Populus trichocarpa (Spot 8), Pathogenesis-related protein STH-2 (Spot 9), Putative pathogenesis protein (Spot 10) and Pentatricopeptide repeat containing protein (PPR; Spot 12).

In the present study, we observed that expression of glycine rich RNA binding protein in potato up-regulated upon Rs infection. Up-regulation of GRP in maize and carrot has also been reported in response to wounding and abscisic acid (ABA) (Gomez et al., 1988; Sturm, 1992). Homologs of glycine rich proteins were also found to be induced in response to cold in barley and Arabidopsis (Carpenter et al., 1994). The mutant plant of GRP7 (glycine rich protein 7) showed higher suceptibility for Pseudomonas syringae infection as compared to wild plant (Fu et al., 2007).

TSI-1 protein was found to up-regulated upon Rs infection. It belongs to intracellular pathogenesis related protein which is characterized in tomato, tobacco and pea during wounding, osmotic stress and pathogen colonization (Chiang and Hadwiger, 1990; Iturriaga et al., 1994; Vidya et al., 1999). TSI-1 protein also showed 71% homology with potato’s STH2 protein at nucleotide level (Vidya et al., 1999). This protein also has RNase activity against biotic stresses in some plants (Moiseyev et al., 1994).

Pathogenesis related protein (STH2, spot 9) was also found to be upregulated upon Rs infection. Similar result has also been reported earlier in potato in response to Phytophthora infestans infection (Constabel and Brisson, 1992). Homologs of STH2 gene have also been identified in different plants like pea, bean and parsley upon infection with different pathogens or elicitation (Chiang and Hadwiger, 1990; Somssich et al., 1988; Walter et al., 1990).

In contrast to the above mentioned proteins, the expression of PPR protein was down-regulated upon Rs infection. PPR protein is a RNA binding protein which control variety of post-transcriptional regulation like RNA editing, splicing, stability, polyadenylation and translation of organelle genes (Schmitz-Linneweber and Small, 2008; Tan et al., 2014). It was reported that WSL, a PPR protein targets to the chloroplast and wsl mutant plant showed defect in splicing of rpl2 transcript, resulted in transcript accumulation and reduction in its protein synthesis. The wsl mutant showed higher sensitivity to abiotic stresses like ABA, salinity and sugar (Tan et al., 2014). It has also been reported that in absence of PPR protein, Arabidopsis plant became susceptible to necrotrophic fungal pathogen and hypersensitive to abiotic stresses like ABA, glucose and salinity (Laluk et al., 2011).

Spot nos. 4 and 8 were identified as hypothetical proteins due to their unknown functions. Taken together, our results have shown up-regulation of both the proteins after Rs infection, suggesting their roles in plant defense. However, it needs further characterization of these identified proteins. Based on our observations and previous reports, it can be speculated that induction of these proteins under Rs infection may be involved in increasing Rs tolerance in potato.

MALDI-TOF-MS analysis identified eight differentially expressed proteins upon Rs infection, among them, five genes (StGRP, StTSI-1, StSTH-2, Hypothetical protein POPTR of Populus trichocarpa and StPPR) were selected to quantify their transcript levels by semi-quantitative RT-PCR. The present study identified up-regulation of all five genes after 7 days of Rs infection (Fig. 5). Interestingly, StTSI-1 was not expressed in control, whereas others were induced after Rs infection, suggesting it may play a role in defense response against Rs infection.

GRP protein expression increased 7 days after Rs infection and almost constant throughout all the investigated stages. The GRP transcript levels correspondingly upregulated after Rs infection. TSI-1, Hypothetical protein POPTR and STH-2 proteins were not detected in control plant but induced 7 days after Rs infection, and consistently decreased at later stages (7 to 21 days). Similar to the trend observed in TSI-1 protein, its transcripts were also not detected in control sample. However, the transcript levels of TSI-1 were increased consistently from 7 days to 21 days of post Rs-infection.

Overall, not 1:1 correlations were found in most of the examined transcripts level and their proteins level. This discrepancy can be due to various factors including post-transcriptional and post-translational modifications. Besides, functioning of some other regulatory mechanism cannot be neglected (Griffin et al., 2002; Gry et al., 2009; Velez-Bermudez and Schmidt, 2014).