Introduction

Plant receptor-like kinases (RLKs) act as an important class of sentinels acting in both broad-spectrum, elicitor-initiated defense responses and as dominant resistance (R) genes in race-specific pathogen defense. Most defense-related RLKs are of the leucine-rich repeat (LRR) subclass although new data are highlighting other classes of RLKs as important players in defense responses (Goff and Ramonell 2007). Plant genomes contain a large number of genes encoding proteins having the basic receptor serine/threonine kinase structure, which have been termed RLKs because in most cases receptor function (i.e., specific ligand binding) has not been demonstrated. RLKs likely play a major role in signal perception and transduction, controlling a wide range of physiological responses in plants. Experimental evidences suggest that phosphorylation of specific Ser and Thr residues controls kinase activation and signal transduction to downstream components of brassinosteroid (BR) signaling pathway (Eckardt 2005). Serine/threonine protein kinases (STPKs) are potential targets for genetic manipulation for transgenic solution for crop improvement as they regulate cascades of molecular events in plant stress reactions (Coello et al. 2010).

Crop wild relatives are the possible progenitors of crops and species that can serve as gene donors to crops. They are in fact an important natural resource and deserve urgent attention for systematic conservation and substantially expanded use (Ford-Lloyd et al. 2011). Piper colubrinum Link., is a woody shrub which is a wild relative of cultivated black pepper (Piper nigrum L.). It is native to the northern part of South America. This species is important because of its resistance to the oomycete Phytophthora capsici, which causes the devastating disease ‘quick wilt’ in P. nigrum (Purseglove et al. 1981; Ravindran and Remashree 1998). P. capsici presents an oomycete worst- case scenario to growers as it has a broad host range, often produces long-lived dormant sexual spores, has extensive genotypic diversity and has an explosive asexual disease cycle. It is becoming increasingly apparent that novel control strategies are needed to safeguard food production from P. capsici and other oomycetes (Lamour et al. 2012). Ten resistance gene analogues were recently identified from P. colubrinum and clustering analysis revealed that NBS-containing R genes comprise a large gene family in Piper species (Lau et al. 2012).

In our previous work, a salicylic acid induced subtracted library was generated in P. colubrinum for discovery of candidate genes and regulatory elements as an essential step towards genetic improvement of cultivated pepper (Dicto and Manjula 2005; Mani and Manjula 2011; Mani et al. 2012). The serine/threonine protein kinase gene (PcSTPK) was identified in the subtracted library as a differentially over expressed partial clone (EB104037). The specific pathogen defense role of this gene needs to be explored to enable its efficient utilization for crop improvement programmes.

The analysis of gene functions in a non-model plant species like P. colubrinum is often hampered by the fact that stable genetic transformation to over express/downregulate gene expression is laborious and time-consuming, or, sometimes, even not achievable. The past several years have seen the complete sequencing of many plant genomes and the generation of large databases of sequence information as a result of which alternative approaches to traditional forward genetics can be implemented to identify the genes involved in a process of interest. Reverse genetics investigates the function of a gene or DNA sequence directly by altering the expression of the sequence of interest and then identifying the mutant phenotype that is produced. Most reverse genetics approaches described in plants to date rely on posttranscriptional gene silencing (PTGS; Watson et al. 2005). The mechanism of PTGS involves the sequence-specific degradation of RNA and several different techniques have been developed to harness PTGS phenomena for gene functional studies. One of these is virus-induced gene silencing (VIGS; Baulcombe 1999; Dinesh-Kumar et al. 2003). The observation that plants could overcome infection by viruses and then be rendered resistant to subsequent infection by closely related viruses was the first suggestion that PTGS was an innate antiviral defense in plants (Lindbo et al. 1993; Ratcliff et al. 1999; Soosaar et al. 2005). VIGS takes advantage of this defense system to silence endogenous RNA sequences that are homologous to a sequence engineered into the viral genome, which generates the double-stranded RNA that mediates silencing (Burch-Smith et al. 2006). VIGS can serve as an alternative to mutant collections or stable transgenic plants to allow the characterization of gene functions in a wide range of angiosperm species, albeit in a transient way. This strategy is nowadays widely used in plant genetics for gene knockdown due to its ease of use and the short time required to generating phenotypes (Lange et al. 2013; Demircan and Akkaya 2010; Martin et al. 2013). It is a virus vector technology that exploits RNA-mediated defense and the most widely used VIGS vectors are derived from the TRV. TRV provides the most robust results in terms of efficiency, ease of application, and absence of disease symptoms (Padmanabhan and Dinesh-Kumar 2009). Engineered TRV vectors carrying host-derived segments are transformed into Agrobacterium tumefaciens, which is then introduced into the plant (Liu et al. 2002; Tian et al. 2014).

Phytoene desaturase (PDS) is a rate-limiting enzyme in carotenoid synthesis that converts phytoene to colored ξ-carotene in a two-step desaturation reaction (Bartley and Scolnik 1995). Therefore, PDS gene is very important in carotenoid metabolism and photosynthesis. It was reported earlier that in VIGS experiments, leaf with PDS silencing was albinotic and this gene was used as a silencing marker (Kumagai et al. 1995; Wang et al. 2009).

We report in this paper, full-length cloning of Ser/Thr Protein Kinase gene from the resistant wild Piper species, P. colubrinum, and corroboration of its pathogen defense role by TRV-induced VIGS. This is the first report of application of VIGS technique for elucidation of gene functions in Piper species and will open up newer possibilities for molecular studies in this important but underutilized non-model plant species.

Materials and methods

Plant material and growth conditions

In vitro cultures of P. colubrinum were established from leaf explants as described earlier (Mani and Manjula 2011) and the cultures were incubated at 25 ± 2 °C under 16/8-h photoperiod per day, with light provided by white fluorescent lighting at an intensity of 54 l mol m−2 s−1. Direct shoots derived from the leaves were rooted in basal MS solid medium and hardened in soilrite mixture (peat moss: vermiculite: perlite, 1:1:1, v/v/v—Keltech Energies Ltd, Bangalore, India) contained in sterile cups. The plants were maintained in a growth chamber (Conviron CMP6010) with the temperature adjusted to 24 °C, humidity of 70 % and under a 16/8-h light/dark regime. Young leaves (2nd or 3rd) of 2 month old plants were collected for RNA isolation and whole plants were used for VIGS experiments.

P. capsici inoculation

The virulent P. capsici strain obtained from Kerala Agricultural University (Thiruvananthapuram, India) was used to inoculate P. colubrinum plants. To prepare the fungal inocula, P. capsici was grown on potato dextrose agar medium at 28 °C overnight. P. capsici infection was carried out by the mycelium agar disc method on detached leaves (Godoy et al. 1990). Mycelial agar plugs of 5.0 mm diameter punched from growing margins of a 24 h old P. capsici culture was applied on the pin pricked abaxial surface of the leaves. Leaves were kept under 16 h photoperiod and 100 % humidity. The disease symptoms were observed every 24 h, over a period of 1 week. Plain agar plugs (without mycelia) applied at pin pricks on the abaxial side of leaves, served as control.

P. capsici infection scoring

P. capsici infections were scored based on the increase in the diameter of lesions. Leaf lesions >2 mm diameter which progressed with time were taken as lesions caused by P. capsici infection. Smaller dark spots (<2 mm diameter), which did not show an increase in size with time were taken as spots representing hypersensitive cell death response.

RNA isolation and rapid amplification of cDNA Ends (RACE) PCR

Total RNA was isolated from leaf of P. capsici challenged P. colubrinum plant by Trizol (Invitrogen, CA, USA) method. Approximately 1 μg of DNase (Sigma, USA) treated RNA was used for cDNA synthesis. The isolated RNA sample was checked for integrity on 2 % EtBr agarose gel and quantified by NanoDrop1000 spectrophotometer (Thermo Fischer Scientific, USA). PcSTPK was cloned full-length following 5′ and 3′ RACE protocol. RACE ready cDNA synthesis was carried out using SMARTScribe™ reverse transcriptase following set procedures (SMARTer™ RACE cDNA amplification kit, Clontech, CA, USA). The standard RACE PCR protocol was carried out using the P. colubrinum serine/threonine protein kinase (EB104037) gene specific primers (STPK1 for 3′ RACE and STPK 2 for 5′ RACE and universal primer UPM; Table 1). The PCR conditions were as follows: 5 cycles of 94 °C for 30 s, 72 °C for 3 min, 5 cycles of 94 °C for 30 s, 70 °C for 30 s, 72 °C for 3 min and 20 cycles of 94 °C for 30 s, 68 °C for 30 s, 72 °C for 3 min. The products were checked on 1.2 % EtBr-agarose gel. Primers were designed based on the sequences of the RACE products to amplify the complete ORF (forward primer PcSTPK F, reverse primer PcSTPK R; Table 1). The PCR was carried out using the Advantage 2 polymerase mix (Clontech, CA, USA). The PCR cycling parameters were: Initial denaturation at 95 °C for 4 min, followed by 35 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 3 min and a final extension of 72 °C for 5 min. The PCR product was cloned into the pGEM-T cloning vector (Promega, Madison, WI, USA) and subjected to sequencing (ABI PRISM® 3730 DNA Analyzer, Applied Biosystems, Foster City, CA, USA).).

Table 1 List if primers used in the present study
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BLAST program of NCBI was used for identity searches for PcSTPK sequence at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). ALIGN program of the European Bioinformatic Institute (EBI) server (www.ebi.ac.uk) was used to determine the identity of PcSTPK and its similarity with the amino acid sequences of known receptor-kinases from other taxa. Multiple alignment of the protein sequences were carried out using BioEdit sequence alignment editor version 7.0.5.3. Motif searches were performed by the PROSITE program using ExPASy proteomics server (www.us.expasy.org). A phylogenetic tree was constructed using the MEGA software version 4.0 by the neighbour joining (NJ) method.

Cloning of P. colubrinum PDS gene

One kbp PDS gene fragment was amplified from P. colubrinum by PCR using degenerate primers PcPDS F and PcPDS R (Table 1) from conserved regions of the PDS gene sequences of other plants. The amplified product was purified and cloned into pCR8/GW/TOPO cloning vector (Invitrogen, CA, USA) and transformed into DH5α E. coli cells. Recombinants were confirmed by colony PCR using degenerate PDS primers. Plasmids from putative recombinants were isolated and digested using EcoRI (New England Biolabs). Sequencing with TOPO vector specific GW1 & GW2 primers (Table 1), confirmed the PCR product as Phytoene desaturase.

Construction of TRV: VIGS vectors

First, PcSTPK sequence was analyzed on a web-based computational tool (http://bioinfo2.noble.org/RNAiScan.htm) developed by Xu et al. (2006), to identify potential off-target silencing from the VIGS vector containing the insert. Target dataset were set to “plant” which includes a large library of transcript sequences from model and non-model plants. The query sequence fragment analyzed did not have hits in target dataset which suggests that the sequence selected might not produce an off-target gene silencing. TRV-RNA1 (pBIN) and TRV-RNA2 (pTV00) VIGS vectors were kindly offered by Dr. David Baulcombe (University of Cambridge, UK). To construct TRV:PcPDS vector, a PcPDS fragment of 600 bp from 1 kbp PcPDS PCR product was cloned into pTV00 by restriction digestion using HindIII (New England Biolabs) and SpeI (New England Biolabs) and ligated using T4 DNA ligase (Promega, Madison, WI, USA). To generate TRV:PcSTPK, 354 bp STPK fragment was amplified by PCR from P. colubrinum cDNA using gene specific primers TRVSTPK F and TRVSTPK R (Table 1). The amplified product was purified and cloned into pGEM-T easy vector and transformed into DH5α E. coli cells. Recombinants were confirmed by colony PCR using gene specific primers. Plasmid was isolated and sequence was confirmed by sequencing using pGEM-T easy vector specific primers Tvect F and Tvect R (Table 1). pGEM-T vector carrying STPK gene and pTV00 vector were digested using HindIII and SpeI and ligated using T4 DNA ligase.

The resulting construct TRV:PcPDS/TRV:PcSTPK was transformed into DH5α competent E. coli cells and plated on selective LB media containing 50 μg/ml kanamycin. Colonies were PCR screened for the presence of the modified constructs using primers TRV F and TRV R which span the multiple cloning site in TRV-RNA2. Plasmids were isolated from selected positive colony and the identity of the final constructs was verified by sequencing with the TRV F and TRV R primers.

Agroinfiltration

VIGS vectors carrying PcPDS, PcSTPK gene fragments as well as TRV-RNA1 and TRV-RNA2 were transformed into A. tumefaciens strains GV3103 by freeze–thaw method (Weigel and Glazebrook 2002). Agrobacterium tumifaciens GV3103 cultures containing TRV-RNA1, TRV-RNA2, TRV:PcPDS and TRV:PcSTPK were separately grown overnight at 28 °C in LB medium containing antibiotics (50 μg/ml kanamycin, 25 μg/ml rifampicin). Subsequently, bacterial cells (OD600 of 1.5) were harvested and resuspended in infiltration media (10 mM MgCl2, 10 mM 2-(N-morpholino)ethanesulfonic acid (MES; SIGMA), pH 5.7 and 200 µM acetosyringone (SIGMA), and incubated at room temperature for 3 h without shaking. Agrobacterium cultures containing the Agrobacterium mixtures of TRV-RNA1 and TRV-RNA2 or TRV:PcPDS or TRV:PcSTPK (1:1ratio) were infiltrated with a needle-less 1 ml syringe into lower leaves of four to five leaf stage plants following the protocol by Liu et al. (2002). Experimental and control plants were transferred to growth chamber and maintained under set conditions as mentioned before. Each experiment was repeated three times with three replicates each. Accumulation of virus in the upper uninoculated leaves was analyzed by RT PCR every 2 for 8 weeks. Leaves inoculated with empty vector (TRV-RNA1 + TRV-RNA2) served as control.

RT PCR for determining viral movement

Total RNA was extracted from fresh leaf tissue of silenced (TRV-RNA1 and TRV-RNA2:PcPDS/PcSTPK), non-silenced control (infiltrated with empty vector TRV-RNA1 and TRV-RNA2) and untreated plants (without agroinoculation) using TRIzol reagent following the manufacturer’s instructions and treated with RNasefree DNase. The first strand cDNA was generated using 1 µg of total RNA, oligo d(T) primer and M-MLV reverse transcriptase (Promega, Madison, WI, USA). To detect the presence of TRV virus, fragments of RNA1 and RNA2 of TRV were amplified by two pairs of specific primers (PBIN F and PBIN R, PTV F and PTV R, Table 1). The P. coubrinum β-actin gene (Table 1) served as an internal control. PCR products were separated on a 1 % TBE agarose gel and visualized by EtBr staining.

Real-time PCR analysis

New leaves were collected after 15, 21, 30 and 45 days of inoculation from control, TRV:PcPDS and TRV:PcSTPK infiltrated plants to determine the efficiency of silencing. All the qRT-PCR primers are listed in Table 1. First-strand cDNA synthesis was conducted using the M-MLV reverse transcriptase on purified RNA for each sample and all amplifications were performed on the ABI 7900 realtime PCR machine (Applied Biosystems, CA, USA) using the SYBR green PCR reagent (Applied Biosystems, CA, USA). cDNA equivalent to 1 µg of total RNA was used. The reaction was set up in a final volume of 20 μL containing 10 μL SYBR green PCR reagent, 2 μL of diluted cDNA and 300nM each of the designed primers and the conditions were: 50 °C for 2 min initially followed by 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min in a real-time PCR machine. The housekeeping gene β-actin cloned from P. colubrinum was used for normalisation and the relative expression level of the genes was determined by comparing the Ct values using 2− ΔΔCT method. Each experiment included three independent samples (biological replicates), each performed in triplicate (technical replicates). Statistical significance were analysed by calculating one way ANOVA followed by Bonferroni method using GraphPad prism (www.graphpad.com).

Light microscopy

Lactophenol cotton blue staining was carried out using the protocol described by Chung et al. (2010). Pathogen inoculated leaf samples were cut into 1 × 1 cm2 segments, incubated first in an acetic acid: ethanol (1:3, v/v) solution overnight, followed by treating in an acetic acid: ethanol: glycerol (1:5:1, v/v/v) solution for at least 3 h. The samples were subsequently incubated overnight in a staining solution of 0.01 % (w/v) trypan blue (Loba Chemie, India) in lactophenol, rinsed 3–4 times with water and then stored in 60 % glycerol until examination. Specimens were transferred onto microscopic slides and examined under a 40× magnification in a compound microscope (Leica Microsystems DM750).

Quantification of H2O2

H2O2 was measured by ferrous oxidation using the Xylenol Orange assay (Gay et al. 1999). The Xylenol orange reagent was freshly prepared and remained stable for 6–8 h. One milliliter of solution [25 mm FeSO4 and 25 mm (NH4)2SO4, dissolved in 2.5 m H2SO4] was added to 100 ml of 125 μm xylenol orange and 100 mM sorbitol. Leaf discs (0.5 cm2) were floated on 1 ml distilled water for 10 min, followed by centrifugation at 5,000 g in a microcentrifuge (Eppendorf, Germany) for 1 min. The supernatant (100 μL) was incubated for 30 min in 1 ml of xylenol orange reagent. After incubation for 30 min at room temperature, the absorbance (A560) was measured in spectrophotometer (Shimadzu, Japan) against the blank containing 100 μL distilled water. H2O2 was determined at 560 nm using a standard H2O2 curve.

DAB staining

For in situ detection of H2O2, DAB staining was carried out using an adaptation of the method of Thordal-Christensen et al. (1997). Silenced and control leaves were infiltrated under gentle vacuum with 1 mg ml−1 DAB containing 0.05 % v/v Tween 20 and 10 mm sodium phosphate buffer pH 7.0. The reaction was terminated at 6–7 h post-inoculation, when a brown precipitate started to be visible in the leaves. Leaves were fixed and then boiled for 15 min in ethanol:acetic acid:glycerol (3:1:1) solution. Bleaching solution was replaced and leaves were incubated until the chlorophyll was completely bleached. Leaves were observed by light microscopy under bright field at 4× magnification (Leica Microsystems DM750).

Estimation of chlorophyll content

Chlorophyll extracted from 100 mg of leaf tissue in an acetone:DMSO mix was centrifuged and the supernatant was made up to a known volume. The absorbance was recorded at 663 and 645 nm using a UV–visible spectrophotometer (UV 2450, Shimadzu). Total chlorophyll was estimated (Hiscox and Israelstam 1979; Nageswara Rao et al. 2001) and the percentage reduction over control was calculated.

Statistical analysis

Results are presented as mean ± SD. The statistical significance of differences was analyzed by one-way ANOVA followed by post hoc (Bonferroni’s multiple comparison test) using GraphPad Prism (GraphPad Software, San Diego, CA; www.graphpad.com). Data with asterisks are significantly different (P < 0.05) according to the post hoc ANOVA statistical analysis.

Results

Full-length amplification and sequence analysis of PcSTPK

5′ RACE and 3′ RACE primers designed based on the sequence of an SSH fragment were used for amplification of ends of PcSTPK. Amplicon sizes of 2 kbp and 500 bp were obtained from 5′ and 3′ RACE reactions respectively (Supplementary Fig. 1a, b). Each of the products was cloned separately and sequenced. Full-length of the gene was deduced by aligning and assembling the sequences of 5′ RACE, 3′ RACE and the SSH fragment (EB104037) obtained earlier in our lab. Primers designed based on the sequences of 5′ RACE and 3′ RACE successfully amplified the PcSTPK as a single amplicon (Supplementary Fig. 1)

Analysis of the resultant nucleotide sequence showed that the full-length PcSTPK cDNA was 2170 bp in length and contained a 1725 bp ORF, with a 5′ UTR of 265 bp upstream of the start codon and a 3′ UTR of 177 bp downstream from the stop codon. The deduced PcSTPK protein consisted of 575 amino acid residues with a calculated molecular weight of 64.45 kDa and an isoelectric point (pI) of 5.95. The identities of the PCR products were confirmed by homology search at NCBI database. Homology search of amino acid sequence using the BLAST programme (NCBI) showed that PcSTPK shared significant identity with protein kinase sequences of other plant species like Lotus japonicus (71 % identity to accession number AB353300.1), Ricinus communis (78 % identity to accession number XM002521189.1), Nelumbo nucifera (69 % identity to accession numbers XP 010266065.1 and Oryza sativa (72 % identity to accession number NM001052241.1). The PcSTPK nucleotide sequence is deposited in the NCBI Gen Bank under Accession number KM582051.

Multiple alignment of PcSTPK with amino acid sequences of RLKs from different taxa is shown in Fig. 1, which suggests that PcSTPK shares significant sequence homology with other receptor-like protein kinase class. PROSITE analysis of deduced protein sequence of PcSTPK identified residues 295–549 in the sequence as the protein kinase domain, of which 412–424 (IVHRDLKTANLLM) were identified as serine/threonine protein kinase active-site signature. Signal P software predicted no signal peptides and target P software predicted the protein as non-secretory protein. PcSTPK forms a part of a distinct clade in phylogeny analysis, sharing higher similarity to receptor-like protein kinase class than other classes like SNF-1 and CDPK of ser/thr protein kinases (Fig. 2). Sequence and phylogenetic analyses of PcSTPK reveals its cytoplasmic localization, with sequence similarity to receptor-like protein class.

Fig. 1
figure 1

Multiple alignment of PcSTPK. Multiple alignment of PcSTPK amino acid sequence with sequences from different taxa viz. Sorghum bicolour (XP 002460599.1), Brachypodium distachyoni (XP 003578561.1), Oryza sativa Japonica Group (NP 001063830.1), Zea mays (DAA62681.1), Lotus japonicus (BAI63585.1), Ricinus communis (XP002521235.1), Arabidopsis thaliana (BAD93724.1), Aegilops tauschii (EMT07451.1), Ricinus communis (XP 002533015.1), Piper colubrinum (KM582051). The conserved region representing identical/similar residues are coloured. The multiple alignment was carried out using the Bioedit Sequence Alignment Editor version 7.0.5.3 (Hall 1999)

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Fig. 2
figure 2

Phylogenetic analysis of PcSTPK. The tree was constructed from amino acid sequences obtained from NCBI for protein kinases with the Neighbor-joining method using the MEGA program version 4.0 (Tamura et al. 2007) Accession numbers for each sequence is shown. PcSTPK is underlined in red (STPK P. colubrinum KM582051). (Color figure online)

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Regulation of PcSTPK transcripts in response to pathogen attack

qRT-PCR analysis was conducted to study the expression profile of PcSTPK transcripts in leaf tissues in a period of 0–72 h inoculation with P. capsici. The expression of PcSTPK gene was upregulated upon P. capsici inoculation and the maximum accumulation of STPK transcripts were observed in inoculated leaves after 12 h, which was not significantly (P > 0.05) altered up to 24 h. But beyond this time point, the transcript levels were significantly reduced (P < 0.05; Fig. 3).

Fig. 3
figure 3

qRT-PCR analysis of STPK expression in P. colubrinum leaves in a period of 0-72 h after P. capsici inoculation. STPK gene was significantly upregulated 12–24 hpi. Wounded (pin pricked) leaves were used as control and set as the calibrator. Relative expression was calculated by the 2−ΔΔCT method. Data are the mean ± SD from three independent experiments. Data with asterisks are significantly different (P < 0.05) according to the post hoc ANOVA statistical analysis

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VIGS in P. colubrinum

Recombinant TRV infects P. colubrinum

The ability of TRV vector to cause systemic infection in P. colubrinum was assessed in order to employ it in VIGS experiments. Twenty-one days post-agroinfiltration, total RNA was prepared from the upper un-infiltrated leaves, followed by RT-PCR analysis with primers derived from the RNA1 and RNA2 sequences of TRV. Amplicons corresponding to RNA1 and RNA2, of size 210 and 345 bp respectively, were detected only in those leaves infiltrated with Agrobacterium-containing TRV clones (Fig. 4b, Lanes 3 and 4) and were absent in the control agroinfiltrated plants. TRV infection however did not result in any visible symptoms. The results clearly show that recombinant TRV can efficiently replicate and spread systemically in P. colubrinum plants.

Fig. 4
figure 4

Agroinfiltration with TRV empty vector. a Control plants of P. colubrinum maintained in the growth chamber for VIGS experiments. b Amplification of viral transcripts (TRV-RNA1 and TRV-RNA2) from upper uninfiltrated leaves of empty vector control- infiltrated plants by RT-PCR. Lane M—Marker (NEB 100 bp ladder), Lane 1—TRV-RNA1 Positive control, Lane 2—TRV-RNA2 Positive control, Lane 3—TRV-RNA1 amplification, Lane 4—TRV-RNA2 amplification

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Silencing of PcPDS by VIGS

A mixture of Agrobacterium cultures containing TRV-RNA2 carrying P. colubrinum PDS (TRV2: PcPDS), and TRV-RNA1 was infiltrated onto the lower leaves of plants. Plants infected with TRV2:PcPDS developed a yellow coloration in the upper newly formed leaves 21 days post-agroinfiltration (Fig. 5a), which persisted for at least 4 months (Fig. 5c). The presence of viral particles in the PDS silenced leaves was confirmed by RT-PCR (Fig. 5d) after 21 days of infiltration in each experiment. Real-time PCR was performed to quantify PDS silencing. In TRV:PcPDS infected plants, the PDS transcripts were reduced by more than 50 % compared with the TRV infected controls (Fig. 5e), and the efficiency of PcPDS VIGS was about 83 % (Supplementary Table 1). P. colubrinum actin gene served as an internal control for RNA quality and RT-PCR amplification. Total chlorophyll was extracted from control and PDS downregulated leaves. The concentration of total chlorophyll was reduced by 58 % in PDS silenced leaves compared with empty vector control leaves (Fig. 5f). The fact that TRV effectively caused the VIGS of PDS in P. colubrinum suggests that other nuclear genes could be targeted for silencing using TRV:VIGS vector.

Fig. 5
figure 5

Tobacco rattle virus (TRV)-induced PDS silencing in P. colubrinum. a Newly formed leaves of P. colubrinum plant infiltrated with TRV:PcPDS showing yellowing after 21 dpi. b Empty vector control infiltrated plant showing normal phenotype. c Persistence of PDS downregualtion 4 months post infiltration. d RT PCR amplification of TRV viral particles from new leaves of empty vector control and PDS downregulated plants. Lane M- Marker (NEB 100 bp ladder), Lane 1- Positive control, Lanes 2 and 3- leaves from PDS down regulated plant, Lane 4-Empty vector infiltrated plant, Lane 5- Uninfiltrated plant. e Real-time PCR of PDS gene expression in empty vector infiltrated and PDS downregulated plant. PDS expression was significantly (52.4 %) downregulated by silencing of P. colubrinum PDS gene. f Comparison of total chlorophyll content in control and PDS downregulated leaves. Total chlorophyll was reduced 58 % in silenced leaves. Relative expression was calculated by the 2−ΔΔCT method. Data are the mean ± SD from three independent experiments. Data with asterisks are significantly different (P < 0.05) according to the post hoc ANOVA statistical analysis. (Color figure online)

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PcSTPK silencing by VIGS

To validate the role of PcSTPK by VIGS, TRV:PcSTPK VIGS vector containing 354 bp PcSTPK insert was infiltrated into young leaves of P. colubrinum maintained in a growth chamber. 15, 30 and 45 days after infiltration, the upper newly formed leaves were collected and checked for viral presence. RT-PCR analysis using TRV-RNA1 indicated systemic movement and multiplication of viral particles in the infiltrated plants (Fig. 6a). Real-time PCR analysis was performed to quantify STPK mRNA levels. A time course study revealed that STPK transcript levels were significantly (P < 0.05) downregulated (40–64 %) at 15–45 dpi, with a maximum down regulation of 64 % observed in silenced leaves at 30 days of post infiltration (Fig. 6b), and an efficiency of 93 % (Supplementary Table 2).

Fig. 6
figure 6

PcSTPK silencing by VIGS. a RT-PCR confirmation of viral movement in control and TRV:PcSTPK infiltrated plants using TRV-RNA1 primers. Lanes 1 and 2 - Empty vector control, Lanes 3 and 4- Leaves from TRV:PcSTPK infiltrated plant, Lane 5- uninfiltrated leaf, Lane M- Marker (NEB 100 bp ladder). Expected amplicon of 210 bp was observed in empty vector control and TRV:PcSTPK infiltrated plants. b Real-time PCR of STPK gene expression in systemic leaves of empty vector infiltrated and STPK downregulated plant. Leaves samples were taken 15, 30 and 45 days of post infiltration (dpi). Maximum downregulation of 64 % observed at 30 dpi. Relative expression was calculated by the 2−ΔΔCT method. Data are the mean ± SD from three independent experiments. Data with asterisks are significantly different (P < 0.05) according to the post hoc ANOVA statistical analysis

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Increased susceptibility of TRV: PcSTPK infiltrated leaves of P. colubrinum to P. capsici

In a susceptible reaction (P. nigrum × P. capsici), primary necrotic spots develop into water-soaked lesions within 24–48 h (Fig. 7a; image on the right). In an incompatible reaction as in P. colubrinum × P. capsici interaction, fungal development is restricted to 1 or 2 few small, dark brown spots [non-spreading lesions, Fig. 7a image on the left; 7b (control)]. At 30 dpi, newly emerged leaves from both empty vector infiltrated control plants and STPK silenced plants were challenged with P. capsici. Disease symptoms on leaves were monitored 12–72 h after inoculation with virulent pathogen P. capsici. Control leaves showed very small dark spots representing points of HR (Fig. 7b), with no visible symptoms of disease initiation and progression. STPK down regulated leaves on the contrary, were marked by the development of larger lesions which progressed in size with time. Disease symptoms of P. capsici infection were observed in 93 % of the silenced leaves (Supplementary Table 2). The lesions observed were water soaked and necrotic, typical of P. capsici foliar symptoms (Fig. 7b). Staining with lactophenol blue displayed very sparse germination of fungal mycelia on the leaf epidermis in control leaves (Fig. 7c). Microscopic examinations revealed profuse hyphae producing copious chlamydospores in STPK silenced leaves. There was also a significant increase in the number of spots on the leaf showing hyphal development (Fig. 7c).

Fig. 7
figure 7

Enhanced susceptibility of PcSTPK downregulated P. colubrinum leaves to infection by P. capsici. All observations were made in a period of 24–72 hpi with P. capsici. a Foliar symptoms of P. capsici incompatible (P. colubrinum × P. capsici) and compatible (P. nigrum × P. capsici) interactions. 24 h post P. capsici infected P. colubrinum leaves showed hypersensitive cell death response where as P. nigrum leaves were suceptible for infection. b Photographs of P. capsici infected leaves of empty vector control plant and STPK downregulated plant. Control leaves showed very small dark spots representing points of HR, with no visible symptoms of disease initiation and progression. STPK down regulated leaves on the contrary, were marked by the development of larger lesions which progressed in size with time. c Micrographs of lactophenol blue staining. Control leaves showed very sparse germination of fungal mycelia on the leaf epidermis where as in STPK silenced showed profuse hyphae producing copious chlamydospores. d DAB staining for the detection of H2O2 production. After infection with P.capsici, dark-brown sites stained with DAB were observed in the empty vector control leaves, but distinctly reduced in the STPK silenced leaves. e Quantification of H2O2 accumulation in empty vector control and STPK silenced leaves. Data are the mean ± SD from three independent experiments. Data with asterisks are significantly different (P < 0.05) according to the post hoc ANOVA statistical analysis. (Color figure online)

Full size image

To investigate the role of the PcSTPK gene in the regulation of oxidative burst, H2O2 generated was quantified by Xylenol Orange assay and visualized by DAB staining in leaves of control and STPK silenced plants at 48 hpi with P. capsici. DAB staining at 48 hpi showed abundant dark brown spots as well as diffused staining concentrated around the leaf veins and around infection sites in control leaves (Fig. 7d). H2O2 quantification in leaves of STPK-silenced plants displayed significantly lower (50 % of control; P < 0.05) accumulation of H2O2, which was also evident microscopically (Fig. 7d, e). These data indicate that the PcSTPK gene is involved in the early events of cell death and defense response of pepper plants.

Discussion

Over the past few years, protein kinases have been identified after non race- and race-specific elicitation for each step in the induction of plant defence responses. They participate in the direct perception of elicitors and Avr products, mediate signaling required for the induction of defence mechanisms, including the activation of transcription factors and systemic responses, and function as negative regulators of defence responses (Romeis 2001; Zhang et al. 2010). Protein phosphorylation/dephosphorylation are major signaling events induced by biotic and abiotic stress as well as developmental pathways in higher plants (Afzal et al. 2008; Cao et al. 2011a, b). Serine/threonine protein kinases (EC 2.7.11.1) are host immune receptors which are known to play a role in Effector-triggered immunity (ETI) of plants, through phosphorylation of the OH group of serine or threonine residues leading to a functional change of the target protein (Hardie 1999; Romeis 2001). Elucidation of physiological roles of specific protein kinases in response to abiotic and biotic stress in plants is very crucial for better understanding and identification of key players in the defense pathway, and protein kinases are perceived as suitable targets for genetic manipulation for crop improvement (Ramonell and Somerville 2002; Sheen 2010).

The present study includes the successful cloning of a pathogen inducible serine/threonine protein kinase gene from P. colubrinum. Sequence analysis of PcSTPK revealed its cytoplasmic location and it was predicted as a member of the receptor-like protein class as indicated by phylogenetic analysis. Recent studies have highlighted the importance of receptor-like cytoplasmic kinase (RLCK) in mediating pathogen triggered immunity as well as effector triggered immunity (Kim and Hwang 2011; Liu et al. 2011). This is the first report of identification of a RLCK from Piper species, which attains more relevance as it is identified from a highly resistant, wild species like P. colubrinum, and is highly perceptive to the fungal pathogen P. capsici, a highly devastating pathogen infecting P. nigrum. Wild species have been widely used as genetic resources for introgression of useful traits into cultivated species by wide hybridization (Li et al. 2008), and P. colubrinum is one such species which is yet to be fully explored for its potential as a gene donor for P. nigrum genetic improvement programmes. An interspecific hybrid between P. colubrinum and P. nigrum was developed by Vanaja et al. (2008) having partial resistance to the dreaded disease Phytophthora foot rot.

Lack of reports on genetic manipulation in P. colubrinum, and our own experience so far prompt us to believe that the plant is recalcitrant to genetic transformation. New techniques need to be developed to promote genetic studies in P. colubrinum and our group had earlier optimized gene silencing by hairpin-mediated RNAi strategy (Mani and Manjula 2011). However, the scope for application of this technology for downstream applications including gene functional evaluation is limited because of the absence of a good transformation system and ensuing lack of reproducibility. The ability to generate gene knockdown phenotypes without having to genetically manipulate the plant genome is one of the advantages of VIGS compared to other gene suppression methods like mutagenesis and RNA interference (RNAi; Senthil-Kumar and Mysore 2011).

In this paper, we describe the successful optimization of VIGS as a transient silencing strategy and demonstrate the utility of this method as a tool for gene functional evaluation in P. colubrinum. Most marker genes used in VIGS experiments are related to chlorophyll synthesis because of the visible phenotypic effects that result from silencing. The PDS gene is a commonly used marker gene for VIGS which is known to cause loss of chlorophyll and carotenoids when silenced (Rotenberg et al. 2006). Photo bleaching occurs due to failure of chlorophyll synthesis, which if strong and persistent, can eventually lead to death of the leaves due to shortage of nutrients (Estevez et al. 2000; Gao et al. 2011). PDS down regulation in P. colubrinum led to mild photobleaching, though PDS transcripts were significantly reduced. The silencing phenotype observed in P. colubrinum is less pronounced than that observed in many other plants including N. benthamiana (Ruiz et al. 1998), Petunia (Reid et al. 2009), Tomato (Liu et al. 2002), etc. The location and amount of silencing within a plant can be highly variable in VIGS (Chen et al. 2004). The difference in phenotype observed may be due to genetic differences between plants with respect to viral resistance or antiviral silencing machinery, as host RNA-dependent RNA polymerase (RDRP) and Dicer-like enzymes are implicated in viral accumulation and antiviral silencing (Donaire et al. 2009). Useful viruses for VIGS must replicate quickly while suppressing the plant’s suppressing machinery, and there may be a balance between viral proliferation and gene silencing (Broderick and Jones 2014). This might explain the decreased symptoms of PTGS in P. colubrinum that may be the consequence of high disease resistance and resulting strong antiviral defense manifested by the plant. PDS gene therefore can be considered an efficient marker for VIGS in the plant, as it does not produce a detrimental effect on the leaf survival.

Persistence of VIGS for longer duration is influenced by age of the plant, viral titer, and environmental conditions that favour virus multiplication (Fu et al. 2006). Our results provide evidence that VIGS can be maintained for 5 months in P. colubrinum, during which the plants show persistence of PDS silencing phenotype. Ongoing experiments will furnish further evidence on the exact duration of silencing effect by VIGS. The observation of longer duration of VIGS is highly relevant in P. colubrinum which is predominantly vegetatively propagated and hence transmittance of VIGS through stem cuttings is a potential application for adopting VIGS as a tool to study gene function in plant responses to stress and development.

PcSTPK transcripts were significantly downregulated by VIGS and the down regulation was systemic. A notable observation was the increased susceptibility of STPK downregulated leaves to P. capsici infection. This is concurrent to our knowledge that serine/threonine protein kinase is one of the important proteins responsible for defense signal transduction. The serine/threonine kinase domain is the major constituent of the tomato Pto gene which not only interacts with the avirulence proteins from Pseudomonas syringae, but also functions as a signal transduction mediator (Martin et al. 1993). The STK domain is also contained in the rice Xa21 gene which confers resistance to Xanthomonas oryzae pv oryzae (Song et al. 1995). Overexpression of an SNF1-type serine/threonine protein kinase of wheat (Triticum aestivum L.) TaSnRK2.4, was reported to confer enhanced multistress tolerance in Arabidopsis (Mao et al. 2010). The cloning of genes from wild relatives and the use of these genes in transgenic studies is an efficient way for modern genetic improvement. In a recent report, Cao et al. (2011a, b) demonstrated that a putative serine/threonine protein kinase Stpk-V cloned from a wild relative species of wheat confers strong durable resistance in cultivated wheat to powdery mildew.

Most pathogen infections are accompanied by production of activated oxygen species including superoxide (O ·−2 ), hydroxyl radical (OH·) and hydrogen peroxide (H2O2). These are recognized and can either intensify the damage or trigger multiple host defence responses (Vranova et al. 2002; Huckelhoven and Kogel 2003). Controlled modulation of active oxygen species (AOS) production/dissociation determines the host response to specific pathogen. AOS perform variety of phytochemical roles: anti-microbial action, inactivation of enzymes, signaling leading to activation of genes involved in biosynthesis (PR-proteins, phenolics and phytoalexins), promotion of lignin precursors and cell wall proteins cross-linking, oxidative burst during HR (Heath 1998; Vranova et al. 2002). In our experiments, deep brown polymerization product occurred at sites of DAB reaction with H2O2. In control plants, accumulation and extensive spread of H2O2 suddenly follows the contact with pathogen (6–12 h). Moreover, more diffuse reaction products were visualized, denoting possible spread of H2O2 signal from infected sites to surrounding cells. This supports H2O2 involvement and HR in signaling and mediation of plant defense responses. In leaves of TRV:PcSTPK infiltrated plants, however, site of H2O2 accumulation was restricted to the area surrounding the lesions, with no sign of diffusion to neighboring cells when observed 72 h after infection. Our study suggests the tight connection between oxidative burst and exhibition of hypersensitive reaction in P. colubrinum × P. capsici incompatible reaction, an observation coinciding with previous similar reports in other plants (Sedlarova et al. 2004). Contrarily, STPK down regulation resulted in increased foliar susceptibility to P. capsici, which was correlated to localized production of H2O2 around the infection sites, representing areas of necrosis. Phytophthora spp. are hemi-biotrophic pathogens, having a lifestyle that features a biotrophic phase, followed by a switch to necrotrophy (Judelson and Blanco 2005; Jupe et al. 2013). It is known that some necrotrophic pathogens may produce ROS to induce cell death that facilitates subsequent spread of the pathogen (Gonner and Schlosser 1993; Tiedemann 1997). Thus, similar to previous observations, H2O2 accumulation may be regarded as a nonspecific stress response of the host tissue resulting from cell disorganization caused by fungal colonization and deprivation of available nutrients (Shetty et al. 2003; Wang et al. 2007).

In summary, the paper describes the functional role played by PcSTPK in pathogen defense, as evidenced by the observation of increased susceptibility as a result of its transient down regulation, and the implication of H2O2 in limiting the spread of the oomycete, P. capsici in control non-silenced plants. This study demonstrates that VIGS can be used for reverse genetics or functional genomics studies in P. colubrinum. This approach also ensures that the gene validation can proceed without laboring with stable transformation in this recalcitrant host. The future challenge of this study will be the biochemical and genetic characterization of regulators, phosphorylation targets and other interacting partners involved in the defense signaling event.