Differential gene expression of two potato cultivars in response to infection with Ralstonia solanacearum

INTRODUCTION

In Egypt, Potato is economic vegetable crops for local consumption, processing, and export, with a production of approximately 5.08 million tons, produced from approximately 175,161 hectares. Egypt is the 15^th worldwide and largest producers of potato in Africa (FAO STAT, 2019).Potato can be affected by many pathogens including bacteria, fungi, viruses, and nematodes. Bacterial wilt (Brown rot) disease, caused by Ralstonia solanacearum (Smith) is a major limiting factor for potato production worldwide, especially in the tropical and subtropical regions and warm areas such as the Mediterranean region and it causes huge economic losses (Lemaga et al., 2005;Kabeil et al., 2008; Khairy et al., 2021).

R. solanacearum is soil borne, gram-negative bacterium that can wilt hundreds of different species including potato, tomato, pepper, tobacco, eggplant, geranium, and banana (Hayward, 2000; OEPP/EPPO, 2004; Denny, 2006). The loss in yield due to the disease in potato has been reported to be 33-90% (Elphinstone, 2005). Rs can enter the plant roots via wounds or during lateral root emergence. Thereafter it penetrates the xylem and multiply in the xylem vessels, obstructs water flow in the plant then plants quickly wilt and die (OEPP/EPPO, 2004; Tan et al., 2016).

Soil-borne bacteria including Rs are difficult to control and planting resistance/tolerant varieties is considered the most effective and cheapest strategy for the control of potato bacterial wilt (Muthoni et al.,2014, 2020). So far, there are no complete resistant commercial varieties against Rs has been identified but some degree of resistance/tolerance are available in the potato germplasm (Laferriere et al., 1999; Huet, 2014).

Plants have diverse natural mechanisms to protect themselves against infections by pathogens. Understanding the basic mechanisms of host resistance is necessary for breeding resistant varieties. For study the molecular mechanisms of potato-Rs interactions, Li et al. (2010) identified 302 differentially expressed genes using suppression subtractive hybridization (SSH) and microarray techniques. Among them, 81 genes were considered Rs resistance related genes, and these genes involved in pathogen recognition, transcription factor functioning, signal transduction, systemic acquired resistance (SAR), hypersensitive response (HR), and cell protection. On the other hand, more than 140 genes related to pathogenesis-related, lignin biosynthesis, and hormone signaling genes were induced in resistant tomato cultivar, but not in susceptible cultivar after inoculation with Rs (Ishihara et al., 2012). Also,expression of b-1,3-glucanase genes and glucanase protein were increased in the resistant cultivar suggested that the ethylene (ET) and jasmonic acid (JA) signaling pathways are involved in signal transduction of resistance to Rs (Ishihara et al., 2012). Narancio et al. (2013) suggested a role of both salicylic acid (SA) and ET in the early defense responses to Rs when they used S. tuberosum cDNA microarrays for determined the transcriptome of the highly-resistant S. commersonii accession F100. Eight differentially proteins were identified by the proteome analysis of resistance against Rs in potato cultivar CT206-10, includes tomato stress induced-1 (TSI-1) protein, glycine-rich RNA binding protein (GRP), pentatricopeptide repeat containing (PPR) protein and pathogenesis-related (STH-2) protein (Park et al., 2016). More recently, Li et al. (2021a) found an increase in the number of upregulated differentially expressed genes in tobacco. WRKY transcription factors (WRKY6 and WRKY11), ERF transcription factors (ERF5 and ERF15), and PR5 encoding genes were upregulated in the resistant cultivar in response to Rs.

In plants, defense-related enzymes consist of an important protective system against pathogen attack. Superoxide dismutase (SOD), phenylalanine ammonia-lyase (PAL), peroxidase (POD) and polyphenol oxidase (PPO), are necessary defense-related enzymes and deeply studied in the research of plant- pathogen interaction (Vanitha and Umesha, 2008; Fortunato et al., 2015; Xie et al., 2017). Antioxidant enzymes (SOD, POD, and PPO) are important for defense against oxidative stress and membrane lipid peroxidation during pathogen attack (Foyer and Noctor, 2003; Li et al., 2008). PPO participates in the oxidation of polyphenols into quinones and lignification of plant cells during pathogen invasion (Mohammadi and Kazemi, 2002). POD enzyme involved in oxidation of phenols, and lignification of host plant cells during defense against the pathogen (Chittoor et al., 1999).

The present study was conducted to evaluate the defense mechanisms of two potato cultivars differing in resistance to Rs, by analyzing the alteration in gene expression using differential display technique, quantification of the defences gene expression using real-time PCR, and measuring the activities of the peroxidase and polyphenol oxidase enzymes,

MATERIAL AND METHODS

Plant Material and Growth Conditions:

The Potato cultivars, Lady Bedford and Diamant, which are partially resistance and susceptible to R. solanacearum (Rs), respectively (Elgayar, 2010), were used in this study. Healthy potato tubers were surface sterilized with 1% sodium hypochlorite for five min, washed with sterile water and planted in sterilized 25 cm diameter plastic pots filled with sterile clay, sand and peat moss (2:1:1). The plants were grown in greenhouse (28-30 °C) under natural light condition. Fertilizer solution (N–P–K: 20-20-20) was weekly applied at 0.2 % (w/v) and plants were irrigated with water when necessary. Four-week-old plants were used for inoculations.

Pathogen Inoculation and Disease Evaluation

A virulent isolate of Rs, obtained from Plant Pathology Department, Faculty of Agriculture, Damanhour university, was maintained ontetrazolium chloride (TZC) agar medium (Kelman, 1954) for 48 h at 28 ^oC. The bacterial cells were harvested in sterile distilled water and bacterial concentration was spectrophotometrically adjusted to 1 x 10⁸ cfu ml^-1.

Plants were inoculated by stem-puncture technique (Prior and Steva, 1990) by forcing sterilized dissecting needle into the stem through a drop of bacterial suspension. Similarly, plants inoculated with sterile distilled water were used as control. The experiment was arranged in a randomized block design with five replicates per treatment and five plants in each replicate.

Disease severity (wilt severity) was recorded 25 days post inoculation (dpi) for individual plants on the scale of 0 to 4 (Kempe and Sequeira, 1983) as follow: 0, no wilting; 1, 1 to 25% of leaves wilted; 2, 26 to 50% of leaves wilted; 3, 51 to 75% of leaves wilted; and 4, 76 to 100% of leaves wilted or dead. The percentage of disease severity (DS%) was calculated according to the formulae:

  DS% = {Σ (n x v) / N x S} × 100

Where, Σ = Summation; n = No. of wilted plants in each category; v = Numerical value of wilted plants observed; N= Total number of examined plants and S = Maximum numerical value/grade.

Determination of photosynthetic pigments and growth parameters

Chlorophyll a, b and β-Carotene content on the leaves of the two potato cultivars were determined spectrophotometrically 15 dpi according to Wintermans and De Mots (1965).

Differential display analysis:

Sampling, RNA extraction and cDNA synthesis

Samples of potato leaves (3^rd- 4^th leaves from the top) from Rs infected plants as well as control plants of both cultivars were collected at 3, 6 and 12 hrs post inoculation (hpi), and stored at -80 °C for further analysis. Total RNA was extracted from leave samples using RNeasy Mini Kit (QIAGEN, Germany) according to the manufacturer’s protocol. The obtained RNA was treated with DNase for 1 h at 37°C to remove any DNA residues. RNA concentration and purity were assayed spectrophotometrically (Jenway, Model 6305, Bibby Scientific Limited, UK).  RNA samples were reverse transcribed into cDNAs using Oligo (dT) primer and reverse transcriptase enzyme (MLV, Fermentas, USA) following manufacturer’s instructions.                          

Differential display real time-PCR (DDRT-PCR)

The synthesized cDNA was used as template for DDRT-PCR. The reaction (25μL) contained: 10× buffer (5μL), 5μL of 10pmol primer (NS2, WRKY1-F and PPO-F, Table 1), 2.5μL of 25mM MgCl₂, 2μL of 10mM dNTPs, 0.2μL Taq DNA polymerase (Promega, USA) and 1μL cDNA template. The PCR cycles were one cycle of 95 ^°C for 5 min, then 40 cycles (95^°C for 30 sec, 40°C for 1 min, 72 ^°C for 1min) and final extension of 72 ^°C for 10 min (Chin et al., 2000). The amplified cDNA was visualized on a 1.5% (w/v) agarose gel, stained with ethidium bromide and photographed using gel documentation system.

Table 1. Primers used in the molecular studies

Sequencing and gene identification:

Selected differentiated fragments were excised from the gel and purified using Gel Extraction Kit (BioLine, UK). The amplified DNA was sequenced by Macrogen Company (Macrogen Inc., Seoul, South Korea). Alignment analysis was done using BLASTN searches at the National Center for Biotechnology Information Web site (http://www.ncbi.nlm.nih.gov). Pair-wise and multiple DNA sequence alignments were carried out using CLUSTALW (1.82) (Thompson et al. 1994).  Bootstrap neighbor-joining tree was generated using MEGA version 6 (Tamura et al. 2013).

Quantitative Real-time (qRT-PCR) assay and data analysis

Total RNA was isolated from leaf samples taken at 1, 3, 6, 12 and 24 hpi using total RNA purification kit (Jena bioscience, Germany) according to the manufacturer’s protocol. RT-PCR of mRNA was carried out with the same method illustrated in differential display analysis. Two antioxidant POD and PPO and the differentially expressed StWRKY StDXS1 genes (Table 1), were confirmed and quantified by comparative qRT-PCR. The potato actin gene was used as internal housekeeping (reference) gene (Nicot et al., 2005).

The PCR Reaction (25 μl) consists of 12.5 μl of 2× Quantitech SYBR® Green Mix (Fermentaz, USA), 1 μl of 25 pM/μl of both forward and reverse primers (Table 1), 1 μl of template cDNA (150 ng), and 9.5 μl of RNase free water. The qRT-PCR was performed on Rotor-Gene 6000 system (Qiagen, ABI System, USA), with one cycle at 95 °C for 10 min followed by 40 cycles (95 °C for 15 sec, 60 °C for 30 sec and 72 °C for 30 sec). Data acquisition performed during the extension step. Relative expression level of each gene was performed using the comparative C_T method (2^-ΔΔCt method, Schmittgen and Livak, 2008). The expression levels of the target genes were normalized relative to actin gene and relative expression of untreated control plants of each cultivar at each time point were set as 1.

Activity of the antioxidant enzymes

Peroxidase (POD) and polyphenol oxidase (PPO) activity were determined in potato leaves after 1, 3, 6, 12 and 24 hpi. The 3^rd to 4^th leaves from the top of five plants (replicates) per treatment at each specific sampling time were collected for enzyme analysis.

Potato leaf samples (1g) were homogenized in 5 ml of a pre-chilled 50 mM phosphate buffer (pH 7.0) containing 1 mM EDTA, and 2% polyvinyl pyrrolidone (PVPP). The homogenate was centrifuged at 12,000×g for 20 min at 4 °C (Universal 32R, Hettich Zentrifugen model D-78532, Germany). The collected supernatant was stored at -20 °C and used for enzyme activity assays (Ghareeb et al., 2019).

POD activity was assayed by mixing 100 μl of enzyme extract with 1.5 ml of 0.05 pyrogallol and reaction was started by adding 0.5 ml of 1% H₂O₂. The increase in the absorbance was measured spectrophotometrically at 420 nm (Jenway, Model 6305, Bibby Scientific Limited, UK). POD activity was expressed as ΔOD₄₂₀ min⁻¹ g⁻¹ of fresh weight (FW) (Hammerschmidt and Kuc, 1982). PPO activity was measured using the method of Mayer et al. (1965). The PPO reaction solution contained 200 μl of enzyme extract, 1.5 ml of 100 mM phosphate buffer (pH 6.5) and 200 μl of 10 mM catechol. The increase in absorbance were recorded at 495 nm. Enzyme activity was presented as ΔOD₄₉₅ min⁻¹ g⁻¹ FW.

Statistical analysis

Data of disease severity, photosynthetic pigments, gene expression and enzymes activity were analyzed with GraphPad PRISM version 8.4.3 for Windows (GraphPad Software, San Diego, California USA) and Tukey’s HSD test was used to assess differences among means at each time point at a p ≤ 0.05. Data were expressed as mean ± SE.

RESULTS

Susceptibility of the potato cultivars to R. solanacearum

Plants were artificially inoculated with Rs and wilting symptoms were evaluated at 25 days post inoculations (dpi). Potato cv. Lady Bedford reaching a mean disease severity of 24.3% at 25 dpi and were significantly more tolerant to Rs than the susceptible cv. Diamant (83.8% wilt severity) (Fig.1A).

Disease progress on cv. Diamant was more rapid than in cv. Lady Bedford. The cv. Diamant plants inoculated with Rs started developing a distinct yellow coloration at 7-10 dpi as compared to the non-infected healthy plants and infected cv. Lady Bedford plants. At 25 dpi the susceptible cv. Diamant plants showed clear wilting symptoms while the plants of partially resistant cv. Lady Bedford remained healthy, although there was occurrence of wilt signs on the lower leaves (Fig.1B).

Fig. 1. Comparison of two different potato cultivars (Lady Bedford and Diamant) in response to R. solanacearum(Rs2)infection.

* Different letters indicate significant differences between cultivars according to the Tuckey HSD test (P < 0.05). Values are the mean of five replicates ± SE.

Photosynthetic pigments content:

Figure (2) showed that, there was no significant difference between the non-infected control of both cultivars for total chlorophyll, chlorophyll a, chlorophyll b and the carotene contents. A significant decrease in all pigment contents were observed on Rs inoculated plants of the two potato cultivars. The susceptible cv. Diamant exhibited the highest decrease in pigment contents at 15 days after inoculation with approximately 42.02, 42.49, 41.40 and 56.16% decreasing compared with the non-infected control for total chlorophyll, chlorophyll a, chlorophyll b and the carotene, respectively, comparing with 27.11, 20.56, 35.97 and 31.65% decreasing in the same pigments in the partially resistant cv. Lady Bedford.

Fig. 2. Total chlorophyll, chlorophyll a, chlorophyll b and the carotene contents in the leaves of two potato cultivars (the partially resistant cv. Lady Bedford and the susceptible cv. Diamant) 15 days after inoculation with R. solanacearum.

* Different letters indicate significant differences between cultivars according to the Tuckey HSD test (P < 0.05). Values are the mean of five replicates ± SE

DDRT-PCR analysis

In the present experiments leaves of 3, 6 and 12 hpi were selected for analysis to isolate expressing genes involved in potato wilting defense. The DDRT-PCR profiles of the potato samples revealed considerable variation of gene expression in response to the infection with Rs. Many up-regulated and down-regulated genes were observed in both cultivars at different times post inoculation with Rs (see Fig. 3). Three randomly clear differentiated cDNA fragments that are present in inoculated plants of the partially resistant cv. Lady Bedford but not in the susceptible cv. Diamant or control plants were selected for sequencing and identification. The partial nucleotide sequences of the selected genes were aligned, compared with other genes available in the GenBank database and the phylogenetic trees were constructed.

cDNA fragment No.1 (446 bp, Fig. 3A) was up-regulated and amplified by NS2 primer from cv. Lady Bedford after 12 hpi and identified as 1-D-deoxyxylulose 5-phosphate synthase gene and showed 93% similarity to Solanum tuberosum 1-D-deoxyxylulose 5-phosphate synthase (DXS1) mRNA, complete cds, from S. tuberosum (GenBank accession No. NM_001288201). The cDNA fragment No.2 (475 bp, Fig. 3B) amplified from cv. Lady Bedford by PPO-F primer was highly expressed after 3 hpi and showed 98% homologous to S. tuberosum tuber polyphenol oxidase PPO (POT33 allele) mRNA(GenBank accession No. U22922.1). The cDNA fragments No.3 (630 bp, Fig. 3C) amplified by WRKY1-F primer was boosted up in cv. Lady Bedford after 3 hpi and showed 97% homologous to S. tuberosum StWRKY mRNA (GenBank accession No.  AY615273, NM_001288675 and AJ278507).

Fig. 3. Differential display (DD-PCR) using primers NS2 (A), and PPO-F (B) and WRKY-F (C) of two potato cultivars infected with R. solanacearum and non-infected plants (controls). M:1000bp DNA marker, LBC: Lady Bedford control, lanes (3h, 6h and 12h) samples collected after 3, 6 and 12 hpi respectively of cv. Lady Bedford; DC: Diamant control, lanes (3, 6 and 12h) samples collected after 3, 6 and 12 hpi respectively, of cv. Diamant.

Phylogenetic analysis

The phylogenetic analysis of the three sequenced genes including StDXS1, PPO and StWRKY of S. tuberosum and other plant species was performed to estimate the evolutionary distances between these genes and other orthologs.  Neighbor-Joining tree was performed using Mega 6 software. StDXS1 showed similarity with the other ones isolated from S. tuberosum, S. lycopersicum, S. pennellii and S. pennatisectum, with different identity (Fig. 4). Data in Figure 5 revealed that PPO gene showed similarity with their alleles listed in gene bank isolated from S. tuberosum, S. lycopersicum, S. pennellii, Capsicum annuum and S. melongena. The gene was grouped with S. tuberosum genes. StWRKY gene showed similarity with their alleles listed in gene bank isolated from S. tuberosum, S. pennellii, S. lycopersicum, Coffea eugenioides, Nicotiana tomentosiformis, Citrus sinensis, Chrysanthemum morifolium, Santalum album, S. pinnatisectum, Gossypium turneri and G. raimondii with different identity (Fig. 6). The gene was grouped with Solanum tuberosum genes.

Fig. 4.Phylogenetic tree showing evolutionary relationship of Solanum tuberosum 1-D-deoxyxylulose 5-phosphate synthase (StDXS1) mRNA of potato plants and the others presented in GenBank.

Fig. 5.Phylogenetic tree showing evolutionary relationship of Solanum tuberosum PPOgene of potato plants and the others presented in GenBank.

Fig. 6. Phylogenetic tree showing evolutionary relationship of Solanum tuberosum StWRKY gene of potato plants and the others presented in GenBank.

Expression analysis of candidate defense response genes against R. solanacearum

Expression timing patterns of peroxidase (POD), polyphenol oxidase (PPO), StWRKY and StDXS1 genes were analyzed using RT-qPCR in potato leaf samples of partially resistant cv. Lady Bedford and susceptible cv. Diamant at 1, 3, 6, 12, and 24 hrs post inoculation (hpi) with Rs.

The obtained results in Fig. (7) revealed that the infected plants of both cultivars showed a significantly higher expression level of POD and PPO genes compared to the non-infected control plants. Expression level of POD increased significantly from 1 hpi in the partially resistant cultivar Lady Bedford (1.41-fold), and from 3 hpi in the susceptible cultivar Diamant (1.28-fold). Levels of POD activity were gradually increased in both cultivars until 12 hpi and decline at 24 hpi. The highest expression level was recorded at 12 hpi in both cultivars, with approximately 3.96-fold and 2.35-fold in the resistant and susceptible cultivars, respectively (Fig. 7A).

 On the other hand, PPO expression level was not significantly different between infected and non-infected plants of the two cultivars at 1 hpi. A significant increase was observed in both cultivars from 3 hpi. PPO activity gradually increased in both cultivars until reaching a maximum expression of 4.36-fold and 2.46-fold, at 24 hpi, respectively, in the resistant and susceptible cultivars (Fig. 7B). A significant difference in POD and PPO activity were observed between both cultivars when they compared at each sampling point of the experiment.

Fig. 7. Relative gene expression of POD (A), PPO (B), StWRKY (C) and StDXS1 (D) genes in the leaves of two potato cultivars (the partial partially resistant cv. Lady Bedford and the susceptible cv. Diamant) after 1, 3, 6, 12 and 24 hours post inoculation (hpi) with R. solanacearum. LB-Rs and D-Rs are plants inoculated with R. solanacearum of cv. Lady Bedford and cv. Diamant, respectively.The expression level of the target genes were normalized relative to actin gene and relative expression of untreated control plants at each time were set as 1.

* Each value represents mean ± SE (n = 3).

* Asterisks indicate significant differences with control using Tuckey HSD test (ns, non-significant, *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Compared with the non-infected control plants, a significant Increase of StWRKY transcripts was observed early as 3 hpi in partially resistant cv. Lady Bedford (1.82- fold) and from 6 hpi in the susceptible cv. Diamant (1.25-fold). The maximal expression level was recorded at 24 hpi in the resistant cultivar (2.53-fold) and in the susceptible cultivar (1.84-fold) (Fig. 7C). high expression levels of stWRKY were maintained between 3 and 24 hpi.

For StDXS1 gene, a significant raise in the expression level was recorded in the two cultivars from 3 hpi. The highest expression level of StDXS1 gene was recorded at 12 hpi in the partially resistant cv. Lady Bedford (2.52-fold) and in the susceptible cv. Diamant (1.86-fold). Levels of StDXS1 transcripts were decrease on both cultivars at 24 hpi to reach values similar to those recorded at 3 hpi (Fig. 7D). Generally, the expression level of the four studied genes was significantly lower in the susceptible cultivar than that recorded in the resistant cultivar.

POD and PPO enzyme activities:

The results in Fig. 8 showed that the differences in peroxidase (POD) and polyphenol oxidase (PPO) enzyme activities between the uninoculated plants of the two tested cultivars were not significant (P< 0.05). After inoculation with Rs, the POD and PPO enzyme activities significantly increased in the both cultivars during 1 to 24 hpi. POD activity in the partially resistant cv. Lady Bedford was increased gradually from 1 hpi and the maximum activity (2.38-fold compared with control plants) was recorded at 12 hpi, then reduced at 24 hpi but still maintained a relatively higher level than control. While, POD activity in the susceptible cv. Diamant was raised from 3 hpi and peaked at 6 hpi (1.67-fold) and decline at 12 and 24 hpi to reach values close to those recorded in the control plants (Fig. 8A).

PPO enzyme activity was continuously increased in both cultivars and reaches a peak of 2.16-fold higher than that in un-inoculated control plants in the partially resistant cv. Lady Bedford at 24 hpi, while in the susceptible cv. Diamant, maximum activity was observed at 12 hpi and it was approximately 1.70-fold that in the control plants and decline later (Fig. 8B).

Fig. 8. Activities of peroxidase, POD, (A) and polyphenol oxidase, PPO, (B) enzymes in the leaves of two potato cultivars after inoculation with R. solanacearum. (LB-C), cv. Lady Bedford non-infected plants; (LB-Rs), cv. Lady Bedford infected plants; (D-C), cv. Diamant non-infected plants; (D-Rs), cv. Diamant infected plants.

* Values are the mean of five replicates ± SE.