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First published online September 1, 2006; 10.1104/pp.106.082107 Plant Physiology 142:1169-1179 (2006) © 2006 American Society of Plant Biologists Characterization of a Mitogen-Activated Protein Kinase Gene from Cucumber Required for Trichoderma-Conferred Plant Resistance1Department of Plant Sciences, Weizmann Institute of Science, Rehovot 76100, Israel (M.S., I.C.); and Department of Virology, Agricultural Research Organization, Volcani Center, Bet Dagan 50250, Israel (A.G.-O., D.L.)
The fungal biocontrol agent Trichoderma asperellum has been recently shown to induce systemic resistance in plants through a mechanism that employs jasmonic acid and ethylene signal transduction pathways. Mitogen-activated protein kinase (MAPK) proteins have been implicated in the signal transduction of a wide variety of plant stress responses. Here we report the identification and characterization of a Trichoderma-induced MAPK (TIPK) gene function in cucumber (Cucumis sativus). Similar to its homologs, wound-induced protein kinase, MPK3, and MPK3a, TIPK is also induced by wounding. Normally, preinoculation of roots with Trichoderma activates plant defense mechanisms, which result in resistance to the leaf pathogen Pseudomonas syringae pv lachrymans. We used a unique attenuated virus vector, Zucchini yellow mosaic virus (ZYMV-AGII), to overexpress TIPK protein and antisense (AS) RNA. Plants overexpressing TIPK were more resistant to pathogenic bacterial attack than control plants, even in the absence of Trichoderma preinoculation. On the other hand, plants expressing TIPK-AS revealed increased sensitivity to pathogen attack. Moreover, Trichoderma preinoculation could not protect these AS plants against subsequent pathogen attack. We therefore demonstrate that Trichoderma exerts its protective effect on plants through activation of the TIPK gene, a MAPK that is involved in signal transduction pathways of defense responses.
Trichoderma spp. are effective biocontrol agents for a number of soil-borne pathogens and are also known for their ability to enhance the plant-growth response. Trichoderma antagonizes plant pathogens by producing antibiotics, competing for nutrients in the rhizosphere and exhibiting mycoparasitism (Harman et al., 2004  ). Trichoderma asperellum (Trichoderma harzianum 203) can penetrate the roots of cucumber (Cucumis sativus) seedlings and colonize the epidermis and outer root cortex (Yedidia et al., 1999). Recently, it has been shown that these interactions induce systemic resistance (ISR) mechanisms in plants (Yedidia et al., 2003; Shoresh et al., 2005). Studies have shown that ISR induced by certain rhizospheric bacteria is not a salicylic acid (SA)-dependent phenomenon but rather requires components of the jasmonic acid (JA) signaling pathway followed by the ethylene signaling pathway (Pieterse et al., 2003). Analysis of signal molecules involved in defense mechanisms and application of specific inhibitors of plant hormones indicated the involvement of JA and ethylene in the protective effect conferred by Trichoderma spp. against the leaf pathogen Pseudomonas syringae pv lachrymans (Psl). Moreover, examination of local and systemic gene expression in cucumber plants revealed that T. asperellum (T203) modulates the expression of genes involved in the jasmonate/ethylene signaling pathways of ISR (Shoresh et al., 2005).
Plants are exposed to a wide variety of environmental stresses and they have developed a broad range of responses to resist these stresses. Mitogen-activated protein kinase (MAPK) pathways have been implicated in signal transduction for a wide variety of stress responses, and some may be involved in JA signaling pathways (Meskiene and Hirt, 2000
There are numerous examples for the involvement of MAPKs in signaling pathways of plant responses to pathogens. Direct proof of this involvement came from studies of parsley (Petroselinum crispum) cells treated with Pep13, a 13-amino acid oligopeptide fragment derived from an extracellular glycoprotein of Phytophtora sojae. By binding to a specific plasma membrane receptor, Pep13 activated plant defense responses, including activation of the MAPK MPK3a, at both the transcriptional and posttranslational levels (Ligterink et al., 1997
Plant RNA viruses have been shown to be an efficient tool for overexpression and knockdown expression of endogenous genes. This technology provides important new insights into the roles of specific genes in plant development and plant defense responses. Virus-induced gene silencing is a quick and efficient technique involving recombinant viruses for reverse genetics by down-regulation of target genes (Burch-Smith et al., 2004
Zucchini yellow mosaic virus (ZYMV) is a member of the Potyviridae family. ZYMV-AGII is a potyvirus-based vector system that has been successfully used for overexpression of various foreign genes in cucurbits (Arazi et al., 2001 In this study, we demonstrate that in cucumber, a MAPK is activated by inoculation of the roots with the biocontrol agent T. asperellum. Moreover, activation of this gene is necessary for the plant's Trichoderma-conferred defense against bacterial pathogens; silencing of this MAPK completely eliminates this protection. We present evidence suggesting that this MAPK is also involved in other plant stress responses, including that to wounding.
A MAPK Is Induced by Trichoderma Inoculation of Plants We recently demonstrated the involvement of the plant signal molecules JA and ethylene in the Trichoderma-induced plant defense response. We were therefore interested in analyzing the expression of genes involved in signal transduction during the plant-Trichoderma interaction. Because many MAPKs have been found to be involved in the plant defense response and their regulatory effects on the control of plant defense responses have been described, we decided to focus on these kinases. PCR with degenerate primers designed according to resistance-associated MAPK plant proteins allowed us to isolate a putative resistance-associated cucumber MAPK clone. We used real-time PCR analysis to examine the expression profile in reaction to Trichoderma inoculation of plant roots. The specificity of the primers was confirmed by having one clear peak of melting curve, indicating only one fragment is produced by this primer pair and by sequencing the PCR product. Six hours after Trichoderma inoculation, the MAPK mRNA levels in the roots increased, peaking at 4- to 5-fold the level in untreated plants (Fig. 1A ). This was followed by another, higher peak 24 h postinoculation (hpi). In leaves, the expression levels of the gene also began to rise 6 hpi and reached a peak at 24 hpi, which was about two and one-half times higher than in noninoculated plants (Fig. 1B). Expression of this MAPK in leaves exemplifies a systemic gene response to root inoculation. Our results suggest that this Trichoderma-induced MAPK (TIPK) participates in the Trichoderma-induced defense response.
TIPK Is a Homolog of Wound-Induced MAPKs Using RACE analysis, we obtained the full-length cDNA of TIPK. Further PCR and sequence analysis enabled us to deduce the genomic organization of the gene encoding TIPK in the cucumber genome (Fig. 2A ). When genomic DNA was digested with the appropriate restriction endonucleases and hybridized at high-stringent conditions with a TIPK cDNA probe, we observed unique hybridization signals, which were expected according to the deduced restriction map (Fig. 2B). This indicated that the gene encoding TIPK is apparently present as a single-copy gene in the cucumber genome. This MAPK is 84% identical and 93% similar to WIPK (tobacco), 82% identical and 92% similar to MPK3 (Arabidopsis [Arabidopsis thaliana]), and 81% identical and 91% similar to MPK3a (parsley; Fig. 3 ). The expressions of wipk and mpk3 have been shown to be wound induced. We therefore examined whether TIPK also responds to wounding. Wounding leaves with carborundum resulted in a rise in TIPK expression levels, starting from 10 min and peaking 1 h postwounding, with or without preinoculation with Trichoderma (Fig. 4A ). TIPK was also up-regulated in systemic (nonwounded) leaves 24 h postwounding (Fig. 4B). These results demonstrate the close functional relationship of TIPK with other wound-induced MAPKs.
Effect of Plant Hormones and Their Inhibitors on TIPK Expression Because both Trichoderma inoculation and wounding exert their effects via the JA and ethylene signal molecules, we examined whether plant hormones or their inhibitors might affect TIPK expression levels. Treatment of cucumber roots with JA or SA did not seem to affect the TIPK expression in roots (Fig. 5A ). The effect of JA and SA treatments was validated by examining the JA- and SA-inducible genes, lipoxygenase and chitinase, respectively (Fig. 5A). Because we identified a peak of TIPK expression at 24 hpi, we decided to focus on this time point for hormone treatment experiments; however, it may be that an activation of the gene occurred at a different time point under these treatments. We also examined whether inhibitors of JA production (diethyldithiocarbamic acid [DIECA]) or ethylene action (silver thiosulfate [STS]) would reduce the induction of TIPK expression in roots by Trichoderma inoculation. We noticed that treatments with JA, SA, and DIECA could repress TIPK expression when compared to control. However, using both inhibitors, induction of TIPK gene expression was similar to that in the control experiment (Fig. 5B). This suggests that TIPK may act prior to the hormones' actions.
Expression of TIPK after Psl Infection Is Potentiated by Prior Trichoderma Inoculation The expression of TIPK in roots of Trichoderma-inoculated and Psl-challenged plants was higher than in plants subjected to only one of those treatments (Fig. 6 ). Moreover, whereas leaves of Trichoderma-inoculated plants did not differ in TIPK expression level from the controls, leaves of plants inoculated with Trichoderma and challenged with Psl expressed 3- to 4-fold higher TIPK mRNA levels than plants challenged only with Psl (Fig. 6). It should also be noted that while systemic expression of TIPK decreased in Trichoderma-inoculated plants 72 hpi (Fig. 1B) and remained low at 96 hpi (Fig. 6, +T–P treatment), the high levels of TIPK transcript in leaves of the +T+P treatment 96 hpi (Fig. 6) suggest that Trichoderma inoculation prior to pathogen challenge also results in prolonged induction of the gene. This indicates that the plant's interaction with Trichoderma enables a stronger defense reaction to subsequent pathogen attack.
Construction of AGII-TIPK and AGII-TIPK Antisense The TIPK cDNA and an antisense (AS) fragment of TIPK were inserted between the NIb and coat protein (CP) genes of the AGII virus vector using a polylinker-cloning site next to the NIa proteinase cleavage site in the NIb gene at the 3' end of AGII (Fig. 7A ). Inserted genes were designed to create an in-frame translational fusion with both flanking NIa processing sites. Proteolysis of the nascent AGII-TIPK polyprotein by NIa protease in trans was predicted to yield recombinant TIPK protein lacking the first Met and having an additional seven amino acid residues (VDTVMLQ) at the C terminus. AGII-AS was designed to produce a polyprotein that would be processed to produce all the viral proteins necessary for its reproduction in the plant, while the AS fragment embedded in the viral RNA genome was significant for the induction of TIPK silencing via virus-induced gene silencing. The presence of the intact TIPK and AS sequences was verified by reverse transcription (RT)-PCR of the viral progeny 14 and 17 d postinoculation (dpi; Fig. 7B) as well as by direct sequencing of the amplified products. As a control treatment, we used the AGII-green fluorescent protein (GFP) construct.
Expression of TIPK Is Modified by Infection with the Different AGII Constructs The AGII-GFP construct has been reported to be stable in cucumber plants for at least 60 dpi. Nevertheless, we verified the viral accumulation in these plants in our hydroponic system over the time interval needed for our experiments. The second leaves of systemic AGII-GFP-infected cucumbers were analyzed for GFP by visualization under UV light. Green fluorescence was observed in many regions of the leaves, but not in those that were mock inoculated (Fig. 8 ). Real-time PCR analysis using primers designed according to the CP gene demonstrated a consistent rise in virus quantity from 8 to 17 dpi, resulting in a 40-fold higher quantity on day 17 than on day 8 (data not shown). We then measured the endogenous RNA levels of TIPK in the AGII-GFP-, AGII-TIPK-, and AGII-AS-infected plants. The RNA levels of TIPK in the AGII-TIPK plants were 3- and 6-fold of controls, 14 dpi and 17 dpi, respectively (Fig. 9 ), whereas TIPK mRNA levels in the AGII-AS were 50% and 60% of controls, 14 dpi and 17 dpi, respectively (Fig. 9). Therefore, infection with the AGII constructs allowed us to modify endogenous TIPK RNA levels and produce TIPK-overexpressing and-underexpressing plants.
To verify the specificity of the AS construct, we cloned a fragment of another cucumber MAPK that had 97% and 96% identity to the MSK7 (alfalfa [Medicago sativa]; gi:298019) and MPK6 (Arabidopsis; gi:15224359) proteins, respectively, but only 87% identity to the TIPK protein. The RNA levels of this MAPK gene in the AGII-GFP and AGII-AS-infected plants had no significant difference at 14 dpi (means of relative RNA levels ± SE and the number of repeats were as follows: AGII-GFP 14 dpi, 1.00 ± 0.21, n = 5; AGII-AS 14 dpi, 0.93 ± 0.19, n = 6). At 17 dpi, we also observed no significant difference between AGII-GFP and AGII-AS plants (AGII-GFP 17 dpi, 0.59 ± 0.19, n = 6; AGII-AS 17 dpi, 0.70 ± 0.21, n = 5). Examination of TIPK expression in AGII-GFP plants treated with Trichoderma (Fig. 9, plants marked GFP + T) revealed the expected up-regulation of the gene at 14 dpi (which is 24 hpi) and reduction of the expression to basal level at 17 dpi (which is 96 hpi). These results are similar to TIPK expression observed in cucumber plants treated with Trichoderma (Fig. 1). In addition, we also compared the expression of TIPK in AGII-AS plants after Trichoderma inoculation (Fig. 9, plants marked AS + T) to AGII-GFP plants treated with Trichoderma (Fig. 9, plants marked GFP + T). While in the AGII-GFP plants treated with Trichoderma, TIPK expression increased 24 hpi (14 dpi), at this time point not only the TIPK expression was not elevated in AGII-AS plants post Trichoderma inoculation, but it was decreased (Fig. 9).
To determine whether TIPK plays a role in the Trichoderma-induced defense response, we challenged AGII-TIPK- and AGII-AS-infected plants with Psl and determined the levels of Psl proliferation in the plants' leaves. As a control, we used AGII-GFP-infected plants. Some samples varied greatly from the average of their group with respect to pathogen multiplication. Because the distribution of AGII virus in leaves was not uniform (Fig. 8), we speculate that when the pathogenic bacteria penetrate the leaf in a region where the AGII virus exists, the latter can affect the bacterial propagation based on its transgenic construct. On the other hand, when the bacteria and the virus do not colocalize, the pathogen will behave as in control leaves. For example, in the group of AGII-AS transformants, one plant had 150-fold fewer bacteria than the average, another transformant in this group had 5-fold less bacteria. We assume that in these transformants, the virus and the Psl did not colocalize, or the viral infection was not efficient. In the AGII-TIPK transformants, we had four plants with levels of bacteria that were more similar to the AGII-GFP control plants. This could also result from noncolocalization of the virus and the Psl or inefficient viral infection. Nevertheless, the AGII-TIPK transformants group was significantly different from the AGII-GFP plants (Fig. 10 ). In the AGII-AS plants treated with Trichoderma, we had five plants with 5- to 10-fold higher Psl counts than all other plants. In these plants, the virus either colocalized with the bacteria perfectly, or the efficiency of the AS inhibition was greater. In either case, it fits well with our hypothesis that inhibition of TIPK expression decreases plant defense even in the presence of Trichoderma.
Psl multiplication was significantly lower at 72 dpi in the AGII-TIPK (29 x 106, compare with µ/g fresh weight [FW]) plants than in AGII-GFP control plants (181 x 106, compare with µ/g FW; Fig. 10). In fact, Psl multiplication in AGII-TIPK plants was similar to that observed in AGII-GFP plants that had been inoculated with Trichoderma prior to pathogen challenge (37 x 106, compare with µ/g FW). This demonstrates that overexpression of the TIPK gene can confer the same level of protection as Trichoderma inoculation. Although Psl multiplication was higher in AGII-AS plants (303 x106, compare with µ/g FW) than in AGII-GFP control plants (181 x 106, compare with µ/g FW), these differences were not statistically significant (Fig. 10). More importantly, the significantly higher Psl multiplication in plants infected with AGII-AS and preinoculated with Trichoderma (940 x 106, compare with µ/g FW) than in AGII-GFP control plants (Fig. 10) demonstrates that reduced TIPK expression abolishes the protective effect normally conferred by Trichoderma.
In recent years, it has become clear that MAPK signaling pathways are involved in plant resistance. We isolated and characterized a MAPK gene that is activated by root inoculation with the biocontrol fungus T. asperellum (Fig. 1). Sequence analysis demonstrated that the TIPK is homologous to MPK3a, WIPK, and MPK3 (Fig. 3), genes which have been shown to be up-regulated by pathogenic bacterial and fungal elicitors (Ligterink et al., 1997 ; Zhang et al., 2000; Schenk et al., 2003; Mayrose et al., 2004). Moreover, similar to these genes, TIPK is also induced by wounding. The transcriptional response is fast and transient, with transcript levels starting to accumulate 10 min after wounding and reaching a maximum at 1 h (Fig. 4A). This fast and transient accumulation is very similar to the reaction of AtMPK3, LeMPK3, and WIPK to mechanical stress and wounding (Seo et al., 1995; Mizoguchi et al., 1996; Mayrose et al., 2004). The kinetics of the accumulation of TIPK mRNA in response to Trichoderma root inoculation was slower than that in response to wounding, reaching a maximum at 24 hpi (Fig. 1A). This demonstrates that TIPK is induced with distinct activation kinetics by different stimuli and suggests that TIPK may be a convergence point for different stimuli and that different induction kinetics may play a role in the types of downstream processes it mediates.
However, we also observed systemic expression of the gene in leaves post Trichoderma inoculation (Fig. 1B) and postwounding (Fig. 4B). Moreover, the systemic expression post Trichoderma inoculation was much higher and more prolonged when plants were inoculated with Trichoderma prior to pathogen challenge (Fig. 6). The potentiation effect of Trichoderma on plant defense-related gene expression has been recently demonstrated (Yedidia et al., 2003 The expression of TIPK in wounded plant leaves post Trichoderma root inoculation did not differ from that in wounded leaves from noninoculated plants (Fig. 4A), and no potentiation effect was observed postwounding in plants preinoculated with Trichoderma (data not shown). This demonstrates that TIPK potentiation post Trichoderma inoculation is specific to the plant's response to pathogen challenge.
We have recently demonstrated the involvement of the JA signaling pathway in the Trichoderma-induced plant response (Shoresh et al., 2005
If signaling through the MAPK cascade is the primary or only route by which Trichoderma-interaction information can be transmitted to trigger a defense response, then interfering with the cascade by reducing TIPK expression should reduce the plant's resistance to subsequent pathogen challenge, even in the presence of Trichoderma. Therefore, we modified the expression levels of TIPK by employing an attenuated potyvirus vector, ZYMV-AGII (Arazi et al., 2001
Introducing the coding region of TIPK into the AGII viral genome allowed us to create TIPK-overproducing plants. In these plants, 14 d after AGII-TIPK infection, the level of TIPK RNA was 3-fold higher than in control AGII-GFP plants, and 17 dpi, the levels of TIPK were 6-fold that in controls (Fig. 9). When AGII-GFP plants were inoculated with Trichoderma, we observed a 3-fold elevation in TIPK mRNA levels (Fig. 9), which is on the same order of magnitude as in TIPK-overproducing plants. Challenging the latter with a bacterial pathogen resulted in pathogen resistance that was at least as effective as with the Trichoderma root treatment of AGII-GFP control plants (Fig. 10). In a recent study, transformation of rice plants with the gene MK1, the pepper homolog of WIPK, resulted in expression of the transgene at both the RNA and protein levels, as well as increased resistance to rice blast disease (Lee et al., 2004
Southern analysis of DNA digested by several restriction enzymes revealed that TIPK is present as a single-copy gene (Fig. 2), thus simplifying the interpretation of the AS experiments. Similarly, the TIPK homologs WIPK and MPK3a have been shown to be single-copy genes (Seo et al., 1995
When we infected cucumber plants with AGII virus harboring an AS fragment of TIPK, a reduction of approximately 50% in TIPK mRNA levels was obtained (Fig. 9). It is important to note that the primers used to detect these mRNA levels did not overlap with the AS fragment cloned into the AGII-AS construct, thus enabling us to detect endogenous mRNA levels of TIPK gene. Virus-derived siRNA has been shown to accumulate in plants infected with Potyvirus (Xie et al., 2004
Plant Material
Seeds of cucumber (Cucumis sativus L. cv Kfir) from Gedera Seeds were used in this experiment. Plant growth medium (PGM) was prepared according to Yedidia et al. (1999)
Seeds were surface sterilized in 2.0% (v/v) NaOCl for 2 min and thoroughly washed with sterile distilled water. Seeds (25/box) were placed on a sterile gauze sheet, which was then placed in an axenic hydroponic growth system (Yedidia et al., 1999
Trichoderma asperellum (Trichoderma harzianum strain T203) was grown on potato (Solanum tuberosum) dextrose agar (Difco). Synthetic medium for T. asperellum was prepared according to Yedidia et al. (1999)
Inoculum was added under aseptic conditions to the PGM of 7-d-old seedlings to a final concentration of ±105 germinated spores/mL (Yedidia et al., 1999 Plants were harvested at 1, 3, 6, 9, 24, 48, and 72 hpi. The induced expression of defense-related genes was examined in roots and leaves. These experiments were repeated twice, and each time point represents approximately 20 plants/experiment.
Plants were grown in a hydroponic growth system for 12 d and then transferred to 50-mL tubes in closed chambers, five plants per tube. Each tube contained hydroponic medium ± the hormone. Hormone concentrations (both JA and SA) were: 0 (control), 0.5, 1, and 2 mM. Plants were harvested at 24 h postexposure to the hormones (five plants per treatment at each time point).
DIECA (Sigma), a potent inhibitor of jasmonate biosynthesis (Menke et al., 1999
STS, an inhibitor of ethylene action (Abeles et al., 1992 The treatments and the controls were: –T–inhibitor; –T+inhibitor; +T–inhibitor; +T+inhibitor.
Plants were grown in a hydroponic growth system for 11 d. The roots were inoculated with germinated Trichoderma (T203) spores (as already described). After 96 h, leaves were wounded by rubbing with carborundum and then harvested from each treatment at the following time points: 0 min, 10 min, 30 min, 1 h, 2 h, 6 h, and 24 h postwounding, with 10 plants/time point.
For RNA analysis, roots and leaves were harvested and placed immediately in liquid nitrogen and then stored at –70°C until use (1–2 weeks). Total RNA was extracted using the EZ-RNA Total RNA Isolation kit (Biological Industries). RNA was treated with RNase-free DNase I in 40 mM Tris-HCl, pH 7.9, 10 mM NaCl, 6 mM MgCl2, and 1 mM CaCl2 for 30 min at 37°C (Roche). This was followed by a phenol/chloroform and chloroform extraction and a subsequent ethanolic precipitation.
Degenerate primers designed according to several known MAPK plant genes were used to isolate the MAPK gene using the Expand High Fidelity PCR system (Roche). Primer sequences used to clone TIPK were: forward 5'-GG(C/T)GCTTA(T/C)GG(T/A/C)AT(T/G)GT(C/T)TGT-3', reverse 5'-ACC(A/G)AC(A/T)GACCA(A/T)ATATCAA-3'. Primer sequences used to clone MPK6 were: forward 5'-C(A/C/T)TT(T/C)AA(T/C)GATGT(G/T)TA(C/T)AT(T/C)GC(A/G)TA-3', reverse 5'-TCTGA(A/T)GG(T/A)GT(G/T/A)CC(A/T)AT(C/G)A(G/A)CTCCA-3'. PCR fragments were cloned in pGEM-T Easy Vector (Promega) and both strands were sequenced.
A 5'/3' RACE kit (Roche) was used to isolate 5' and 3' sequences according to the manufacturer's instructions. The gene-specific primers used were: reverse 5'-CAAGGCACTCTAATTCGAAGATGCGGT-3', forward 5'-TGTGACAAGATGGTACAGAGCACCTGA-3'. The universal Genome Walker kit (CLONTECH) was used to isolate 5' upstream sequences to the gene. The gene-specific primer used was: 5'-AGGCATAATCGGAGGACGATATTTGGA-3'. Comparison of TIPK cDNA to genomic sequences of homologous genes was performed to design primers surrounding the postulated introns, and the corresponding introns were isolated. The GenBank accession number of the TIPK sequence is DQ118734. Accession number of the second cucumber MAPK is DQ841553.
Genomic DNA of cucumber was digested with EcoRI, SacI, PstI, and XhoI, separated on an agarose gel, and blotted onto a nylon membrane. A fragment of 1.2 kb from the cDNA was labeled with 32P and used as a probe. ULTRAhyb (Ambion) was used as a hybridization solution at 42°C. Membrane was washed according to manufacturer's instructions with one modification: stringent washing was done at 45°C.
After treatment with DNase I, 1 µg of total RNA was used for a RT reaction using Superscript II (Invitrogen) according to the manufacturer's instructions.
The size of all amplified fragments was 200 bp, and the annealing temperature of all primers was 60°C. The sequences of the primers used were: TIPK, forward 5'-CCGTCATGCATTCATTTTCAGAA-3', reverse 5'-TCCGCTCCAACCAAAGTTTATC-3'; 18S, forward 5'-GTTGCTTTAAGGACTCCGCCA-3', reverse 5'-AGGGGTACCTCCGCATAGCTAG-3' (gi|7595414); MPK6, forward 5'-CCAGATACTTCGTGGATTGAAG-3', reverse 5'-AGACATCAATAGCTGCAGTG-3'.
The specificity of the primers to the genes they were designed for was tested by using melting curve analysis of the PCR reaction (standard protocol of the real-time PCR machine), as well as sequence analysis of the PCR product amplified. The primers described above are those that passed these tests. PCR was carried out in 96-well plates (20 µL/well) in a reaction buffer containing 1x SYBR Green PCR Master mix (Perkin-Elmer Applied Biosystems), 350 nM primers (for each forward and reverse primer), and 1/40 of the RT reaction for TIPK detection or 1/1,000 for 18S detection. Quantitative analysis was performed using the GeneAmp7000 Sequence Detection system (Perkin-Elmer Applied Biosystems) with PCR conditions of 95°C for 15 s and 60°C for 1 min for 40 cycles. The absence of primer-dimer formation was examined in no-template controls. Specificity of primers to cucumber genes was examined by using Trichoderma DNA and reverse-transcribed RNA as templates. The 18S ribosomal cDNA was used as a control reference. Each sample was examined in triplicate, using relative quantification analysis. This method normalizes the expression of the specific gene versus the control reference with the formula 2–
For gel visualization of quantitative PCR, we used the same conditions and primers but with a standard PCR instrument for 20 cycles (for TIPK) or 18 cycles (for 18S) and ran 10 µL on the gel. Gels were then blotted and hybridized with a probe of TIPK cDNA or 18S DNA, respectively, using standard protocols (Sambrook et al., 1989
ZYMV-AGII (AGII) is a potyvirus-based vector system that has recently been developed for the expression of foreign genes in cucurbits (Arazi et al., 2001
Potted squash (Cucurbita pepo L. cv Ma'ayan) was grown in a greenhouse. Particle bombardment was used to propel microprojectiles containing plasmid with the AGII-TIPK, AGII-AS, or AGII-GFP constructs into the fully expanded cotyledons of each plant as described in Gal-On et al. (1997)
The second leaf from each plant was harvested. RNA extraction and RT were conducted as already described. The PCR was performed using AGII polylinker flanking primers: 5'-AAGGGAGCGGATACAAGTGA-3' and 5'-TGATGAGACGCTCGTGTGTT-3'. PCR conditions were: 95°C for 15 s, 56°C for 30 s, and 72°C for 1 min, for 40 cycles.
Psl was grown in Tryptic Soy Broth (Difco) overnight at 30°C. Bacterial cells were pelleted at 5,000 rpm and resuspended in sterile saline-phosphate buffer (5 mM, pH 7.2). Challenge was performed 48 hpi to the PGM. Psl bacterial suspension (20 µL; optical density 0.5) containing 0.01% (v/v) surfactant (Tween 20) was applied to the surface of the second leaf and gently smeared with a sterile tip. Bacterial inoculation was performed under aseptic conditions. Psl multiplication in the leaves was assessed 72 h postbacterial challenge. These experiments were repeated three times. Leaves were weighed and each leaf was homogenized in a sterile solution of 10 mM phosphate-saline buffer (1 mL/leaf). Ten-fold dilutions were plated onto Pseudomonas-selective King's B agar supplemented with 1 mL/L of 9 mg/mL basic fuchsin, 200 mg/mL cycloheximide, 10 mg/mL nitrofurantoin, and 23 mg/mL nalidixic acid. After incubation at 28°C for 2 d, the number of Psl colony-forming units per gram of infected tissue was determined. We also harvested the upper leaf for real-time analysis and to confirm the AGII virus's presence via RT-PCR analysis (as already described).
Statistical analysis was performed using STATISTICA 7 software. Data were subjected to one-way ANOVA analysis and Tukey-Kramer honestly significant difference for comparison of means. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers DQ118734 and DQ841553. Received April 13, 2006; accepted August 15, 2006; published September 1, 2006.
1 This work was supported by the U.S.-Israel Binational Agricultural Research and Development Fund (grant no. 3507–04), by the Dr. Alexander and Myrna Strelinger Endowment Fund, and by the Fienberg Graduate School of the Weizmann Institute of Science (postdoctoral fellowship to M.S.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Michal Shoresh (ms534{at}cornell.edu). www.plantphysiol.org/cgi/doi/10.1104/pp.106.082107 * Corresponding author; e-mail ms534{at}cornell.edu; fax 315–787–2320.
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and 
, Inoculated with Trichoderma; 
and 
, control, mock-inoculated plants.
= 0.05).
