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First published online April 25, 2008; 10.1104/pp.108.119511 Plant Physiology 147:802-815 (2008) © 2008 American Society of Plant Biologists The Wheat Mitogen-Activated Protein Kinases TaMPK3 and TaMPK6 Are Differentially Regulated at Multiple Levels during Compatible Disease Interactions with Mycosphaerella graminicola1,[W]Centre for Sustainable Pest and Disease Management, Department of Plant Pathology and Microbiology, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United Kingdom
Many race- or isolate-specific disease resistance responses of plants toward pathogens (incompatible interactions) invoke hypersensitive response (HR)-like programmed cell death (PCD) and the coordinated activation of mitogen-activated protein kinases homologous with Arabidopsis (Arabidopsis thaliana) AtMPK6 and AtMPK3 (or tobacco [Nicotiana tabacum] SIPK and WIPK), respectively. Resistance of wheat (Triticum aestivum) leaves to the necrotrophic fungal pathogen Mycosphaerella graminicola can also operate at an isolate/cultivar-specific level. We confirm here that resistance is achieved without any sign of HR-like PCD during the incompatible interaction. Instead, PCD is strictly associated with the compatible interaction and is triggered during disease symptom expression. A strong transcriptional activation of TaMPK3, the wheat homolog of Arabidopsis AtMPK3, was observed immediately preceding PCD and symptom development in the compatible interaction. Generation and use of TaMPK3- and TaMPK6-specific antibodies on western blots and in coupled immunoprecipitation-protein kinase assays demonstrated that the TaMPK3 protein also accumulated, and was subsequently posttranslationally activated, during the compatible interaction in parallel to PCD. In contrast, no increase in expression, protein levels, or posttranslational activation of TaMPK6 was observed at any stage of either compatible or incompatible interactions. However, the protein levels of TaMPK6 became markedly reduced during the compatible interaction coincident with the onset of TaMPK3 protein accumulation. These data highlight the emerging similarity between the signaling pathways triggered in a host plant during successful infection by a necrotrophic fungal pathogen and the resistance responses normally effective against biotrophs.
Plant disease resistance to pathogens takes many forms. The most widespread form is referred to as nonhost resistance and operates at various levels to prevent infection of entire species of plants by entire species of pathogens (Nürnberger and Lipka, 2005  ). Below this level of control is race- or isolate-specific resistance, which operates between individual races or isolates of a pathogen species and individual cultivars or genotypes of a plant species. Resistance of this type is frequently regulated via a gene-for-gene interaction between complementary pathogen avirulence (Avr) genes and plant resistance (R) genes (Flor, 1971; Hammond-Kosack and Jones, 1997; Nimchuk et al., 2001; Martin et al., 2003). The host resistant reactions triggered as a consequence of Avr-R interactions are multifaceted and often include a rapid and strictly localized form of programmed cell death (PCD), referred to as the hypersensitive response (HR; Heath, 2000; Beers and McDowell, 2001; Nimchuk et al., 2003; Greenberg and Yao, 2004). This is particularly effective in inhibiting the growth of biotrophic pathogens, which require living host tissue in order to complete their infection cycle. In contrast, few reports have addressed plant race- or isolate-specific resistance toward necrotrophic pathogens, which complete their infection cycle in dead and/or dying host tissues and that have been suggested to benefit from the HR (Govrin and Levine, 2000; Lincoln et al., 2002; van Baarlen et al., 2004; van Kan, 2006).
In addition to the execution of HR-like cell death during resistance, many studies have also described the posttranslational activation of mitogen-activated protein kinases (MAPKs) homologous with Arabidopsis (Arabidopsis thaliana) AtMPK6/tobacco (Nicotiana tabacum) SIPK (herein referred to as MPK6 homologs belonging to the A2 subgroup; Ichimura et al., 2002
We are studying the interaction between wheat (Triticum aestivum) and its host-specific fungal leaf pathogen Septoria tritici (teleomorph Mycosphaerella graminicola), the causal agent of Septoria leaf blotch disease. Plant infection by M. graminicola exhibits characteristics shared by a number of related agriculturally important fungi (Goodwin, 2004
Although quantitative trait loci are also known to play roles in some forms of plant resistance to M. graminicola, specific interactions between wheat cultivars and fungal isolates occur and have been shown to conform to the gene-for-gene hypothesis (Kema et al., 2000
We recently described a M. graminicola-wheat seedling bioassay using attached leaves that allows the investigation of both host and pathogen physiology during a compatible disease interaction (Keon et al., 2000 This study describes and compares novel host signaling responses triggered during compatible and incompatible interactions between wheat and M. graminicola. The data we present demonstrate a strong correlation between the appearance of PCD markers and symptom expression only during the compatible interaction. Furthermore, we have identified three different levels of control on the wheat AtMPK6 and AtMPK3 homologs, referred to as TaMPK6 and TaMPK3, respectively, during these interactions. This includes transcriptional and posttranslational control and contrasting changes in steady-state protein levels. We discuss these findings in the context of the emerging similarity in host signaling responses triggered during successful attack by necrotrophic pathogens and those that occur during effective resistance toward biotrophs.
Selection of Experimental Cultivars and Fungal Isolates
The predominant fungal isolate used in this study is IPO323, which was selected for the M. graminicola genome sequencing project (http://genome.jgi-psf.org/Mycgr1/Mycgr1.home.html). IPO323 is avirulent on wheat cultivars possessing the Stb6 resistance gene, in accordance with the gene-for-gene hypothesis (Kema et al., 2000
Figure 1A presents a time course of visual symptom development for a single leaf inoculated with an incompatible or compatible fungal isolate and illustrates that, for up to 8 d, both interactions occurred without visible disease symptoms. Symptoms subsequently appeared in both cases by day 12 and were restricted to the fungal inoculated areas of the leaf. In the compatible wheat leaves, these symptoms appeared as gray sunken necrotic regions across the inoculated area. In contrast, leaves of Cadenza when inoculated with isolate IPO323 developed a patchy chlorosis (yellowing) during this incompatible interaction. Only very discrete and infrequent necrosis-type lesions were seen at much later stages, and very few asexual sporulation structures (pycnidia) developed subsequently. Mock-inoculated leaves showed no visible symptoms (data not shown). Real-time quantitative PCR analysis of fungal genomic DNA throughout the infection time course, which reports the levels of fungal biomass, confirmed the strong resistance phenotype of the incompatible interaction (Fig. 1B; Supplemental Fig. S1). We were unable to detect any increase in the levels of fungal biomass during the presymptomatic phases (up to day 12) of either interaction, and subsequent increases after this time point were restricted to the compatible interaction (Fig. 1B; Supplemental Fig. S1). Both the visible symptoms and the lack of sporulation of isolate IPO323 in the resistant genotype (Stb6) segregated in a 1:1 ratio in 40 progeny lines generated from a double haploid population derived from an F1 cross between Avalon and Cadenza (K. Kanyuka, J. Keon, and J.J. Rudd, unpublished data). This confirmed the presence of a single segregating R gene locus.
Characteristic Features of PCD Are Associated with Symptom Expression during Compatible But Not Incompatible Interactions
Previous work showed that physiological hallmarks of "apoptosis-like" PCD appeared during disease symptom development in a compatible interaction between Riband wheat and M. graminicola isolate IPO323. These included DNA laddering and translocation of cytochrome c from mitochondria to the cytosol (Keon et al., 2007
Previous studies also demonstrated a rapid loss of host cell membrane integrity associated with symptom development during a compatible interaction. This was shown to result in large increases in both apoplastic electrolytes and metabolites (Keon et al., 2007 ). Therefore, we tested whether the symptoms observed during the incompatible interaction also involved a loss of host cell membrane integrity via the electrolyte leakage assay. In the compatible interaction, no increase in electrolyte leakage occurred until the first appearance of visible symptoms (9–11 d after fungal inoculation). The subsequent increase thereafter was dramatic and peaked by day 14 (Fig. 2C). In contrast, and despite the development of chlorotic symptoms, there was no measurable loss of host membrane integrity at any stage of the incompatible interaction, including the very early stages (<12 h after inoculation; data not shown). An analysis of the integrity of leaf RNA also highlighted that, in contrast to the compatible interaction, no obvious degradation of total RNA was observed during the appearance of chlorotic symptoms during the incompatible interaction (Fig. 2D). These data collectively demonstrate the strict association of the appearance of PCD markers with symptom expression only during the compatible disease interaction.
A preliminary wheat Affymetrix microarray experiment using leaf tissue collected prior to symptom development in both compatible and incompatible interactions identified a single MAPK as being transcriptionally up-regulated relative to mock-inoculated controls (J.J. Rudd, unpublished data). This gene encoded an extracellular signal-regulated kinase (ERK)-like MAPK previously referred to as WCK-1 (GenBank accession no. AY079318) and shown to be transcriptionally activated in a fungal elicitor-treated cell culture (Takezawa, 1999
The relative mRNA levels of TaMPK3 and TaMPK6 were studied across an infection time course taken at 1, 4, and 8 d after fungal inoculation preceding visible symptom development. Both experimental wheat cultivars were inoculated separately with both fungal isolates in order to generate reciprocal specific interactions for each. No significant change in the relative expression of TaMPK6 was detected during this analysis. In contrast, increases in levels of the TaMPK3 transcript were detected during the course of both compatible and incompatible interactions. However, for each cultivar-fungal isolate combination tested, this increase was strongest during the compatible interaction at 8 d after inoculation (Fig. 3B; Supplemental Fig. S2). Transcriptional activation of TaMPK3 was also seen at the same time point during incompatible interactions but was notably weaker (Fig. 3B; Supplemental Fig. S2).
The strong transcriptional activation of TaMPK3 preceding disease symptom expression led us to test whether ERK-type MAPKs became posttranslationally activated at any point during either compatible or incompatible interactions with M. graminicola. Protein extracts from fungus-inoculated leaves were subjected to western-blot analysis using an anti-active MAPK antibody (anti-ERK-PTEYP) that specifically detects the dual phosphorylation on Thr and Tyr residues that is an essential feature of their posttranslational activation. Figure 4 shows that at least two (as indicated by two discrete bands) ERK-type MAPKs were strongly activated in this experiment at 14 d after fungal inoculation during the compatible interaction. This was detected coincident with the appearance of disease symptoms and PCD markers (Fig. 2). Only a very slight increase in MAPK activity was detected at the corresponding time point of the incompatible interaction between Cadenza and isolate IPO323 (Fig. 4). No MAPK activation was detected at any tested earlier stage of either compatible or incompatible interactions within the first few hours after fungal inoculation (Supplemental Fig. S2).
Differences in TaMPK6 and TaMPK3 Protein Levels Contrast Markedly during the Course of the Compatible Interaction
In order to determine whether any of the MAPK activities stimulated during the compatible disease interaction corresponded to either TaMPK3 or TaMPK6, we generated kinase-specific peptide antibodies recognizing the N terminus of either protein. The resulting antisera had the correct specificity on western blots against the recombinant His-tagged TaMPK3 and TaMPK6 proteins (Fig. 5A
). Neither antiserum cross-reacted with an ERK-like MAPK from M. graminicola, MgFus3 (Fig. 5A; Cousin et al., 2006
The specificity of both antisera was then tested against extracts from mock-inoculated leaves (Fig. 5B, lane 1) and from leaves collected 14 d after challenge with a compatible fungal isolate (Fig. 5B, lane 2). Both antisera recognized a single specific band (shown by peptide competition) of the correct size in the mock-inoculated leaf extract (Fig. 5B, lane 1). The TaMPK3-N antiserum also strongly recognized its antigen in the leaf extract generated 14 d after inoculation with a compatible fungal isolate (Fig. 5B, lane 2). However, the TaMPK6-N antiserum no longer detected its specific target in the same extract. This suggested that the TaMPK6 protein levels had fallen beyond detectable levels in this extract. To test this further, we analyzed a more detailed time course following fungal inoculation to determine the overall MAPK activity levels (using the anti-PTEYP antibody) and the TaMPK3 and TaMPK6 protein levels (using their specific antisera) during both compatible and incompatible interactions. Figure 5C shows the results of this analysis for a time course ranging from 30 min to 18 d after fungal inoculation. Protein levels of TaMPK3 were found to be relatively low during the early stages of the compatible interaction but began to increase at approximately 7 d after inoculation. A subtle increase in overall MAPK activity was detected at the same time point. A more significant increase in MAPK activation became obvious by day 11 and then increased strongly for up to 18 d after inoculation. This pattern was mirrored by the corresponding levels of the TaMPK3 protein in the same extracts (Fig. 5C, top). In contrast, the TaMPK6 protein was relatively abundant in extracts that contained no appreciable MAPK activity and low TaMPK3 protein levels (up to 11 d after inoculation). However, from that point on the protein levels of TaMPK6 began to diminish to undetectable levels by 18 d after inoculation. No significant change in overall MAPK activity or increase in TaMPK3 protein levels was observed across the time course of the incompatible interaction (Fig. 5C, bottom). In addition, there was no dramatic decrease in levels of the TaMPK6 protein, which was clearly detectable at all time points. These data demonstrate that the strong activation of ERK-like MAPKs in wheat leaves specifically during the compatible disease interaction with M. graminicola is accompanied by inverse changes in the levels of the TaMPK3 and TaMPK6 proteins.
M. graminicola infection of wheat leaves is typified by the appearance of localized leaf lesions that do not spread appreciably from the initial inoculation site. The ability to inoculate discrete regions of a single leaf with fungal spores permits the study of both the immediate local host responses and also the responses that might occur beyond these regions, for example in immediately adjacent leaf areas (Fig. 6A ). Therefore, we tested whether changes in MAPK activity levels, and TaMPK3 and TaMPK6 protein levels, occurred outside the fungus-inoculated leaf region at both 7 d (before symptoms) and 12 d after inoculation with compatible or incompatible fungal isolates (Fig. 6B). MAPK activation (anti-PTEYP) again appeared strongest during the compatible interaction at day 12 and was restricted to the fungus-inoculated (local or L) region exhibiting disease symptoms. No increase in MAPK activity was detectable in the adjacent (Ad) noninoculated leaf areas. Protein levels of TaMPK3 were also highest in the immediate local region 12 d after inoculation with a compatible fungal isolate; however, some increase in protein level was also detected in the adjacent noninoculated region specifically at this time point (Fig. 6B).
A slight activation of MAPKs was also detected at the 12-d incompatible time point and was restricted to the local region while also appearing to correlate positively with the amount of TaMPK3 protein detected in this region (Fig. 6B). This slight increase in MAPK activity and possibly TaMPK3 protein levels may be explained by the subtle chlorotic symptom development in these areas at this time point in Cadenza (Fig. 1A). In all cases in which MAPK activity and TaMPK3 protein levels were high, a corresponding and noticeable reduction in TaMPK6 protein levels was apparent. This was evident in the compatible fungus-inoculated local regions and was not seen in the adjacent regions. These data demonstrate that the changes in TaMPK3 and TaMPK6 protein levels and MAPK activity states are strongly affected in the fungus-inoculated areas (local areas) that subsequently undergo PCD and develop into restricted lesions as part of the compatible disease interaction.
The TaMPK3- and TaMPK6-specific antibodies were then used in coupled immunoprecipitation-protein kinase assays using the myelin basic protein as an artificial substrate to determine whether any change in the corresponding protein activity occurred at any stage of compatible or incompatible interactions. Figure 7A demonstrates that immunoprecipitation-protein kinase assays using the TaMPK3 antibody identified a strong activation of the protein during the compatible interaction, peaking at 14 d after fungal inoculation. This occurred in parallel to disease symptom development, the appearance of PCD markers, and its own protein accumulation (Figs. 5C and 6B). In contrast, TaMPK3 was not posttranslationally activated during the incompatible interaction (Fig. 7A). Immunoprecipitation-protein kinase assays with the TaMPK6 antibody failed to detect any change in its activity during either interaction, with the exception of a notable decrease in the activity level detected at the 16-d compatible time point (Fig. 7A). This probably occurs as a consequence of the diminishing levels of TaMPK6 protein during the compatible interaction (Fig. 5C). No change in either TaMPK6 or TaMPK3 activity was seen during any of the early stages (within hours) of compatible or incompatible interactions.
The specificity of the immunoprecipitation-TaMPK3 activation assay was further demonstrated by peptide competition, in which addition of an excess of the TaMPK3 peptide, but not the TaMPK6 peptide, to plant extracts from compatible leaves undergoing PCD blocked the immunoprecipitation of protein kinase activity (Fig. 7B). Finally, in order to ensure that the TaMPK6 antibody was able to immunoprecipitate its activated protein target, we tested whether both kinases would respond to leaf infiltration with hydrogen peroxide (H2O2), a known activator of MPK6 and MPK3 homologs in various plant species (Kovtun et al., 2000 ; Kroj et al., 2003). A strong activation of both kinases was detected at 10 min after leaf infiltration with 10 mM H2O2 (Fig. 7C), confirming the ability of the TaMPK6 antiserum to immunoprecipitate activated TaMPK6. These data identify TaMPK3 as an ERK-type MAPK posttranslationally activated in parallel to PCD specifically during the course of the compatible disease interaction with M. graminicola.
Isolate-Specific Cultivar Resistance in the Absence of HR-Like Cell Death
Race- or isolate-specific plant resistance toward pathogens is widely regarded to operate at the level of a gene-for-gene interaction between pathogen Avr genes and cognate host R genes. In many well-established systems, particularly with respect to defense against biotrophic pathogens, HR-like cell death has frequently appeared as a feature of incompatible interactions (Stakman, 1915
There is convincing genetic evidence that isolate-specific resistance of wheat toward M. graminicola operates at the gene-for-gene level (Kema et al., 2000
This work and our previous studies have shown the compatible disease interaction to be associated with a PCD response of the host leaf exhibiting various apoptosis-like features and similarities to HR-like cell death. The latter point is particularly emphasized by the strict localization of the response to the area of initial fungal inoculation (Keon et al., 2007
The execution of host PCD during a compatible interaction is the opposite of what one immediately associates with a gene-for-gene resistance mechanism, as these features are more characteristic of incompatible interactions. With respect to plant HR-like responses occurring during a successful pathogen infection, M. graminicola infection of wheat leaves shares features that have previously only been described for a small number of broad host range, cell-penetrating, necrotrophic fungi (Govrin and Levine, 2000
Intriguingly, during our gene expression studies, TaMPK3 mRNA levels strongly accumulated immediately preceding symptom development in the compatible interaction, while the protein was subsequently posttranslationally activated in parallel to the development of symptoms and the appearance of PCD markers. The strong association between posttranslational activation of TaMPK3 and a compatible interaction is clearly distinct from the MAPK signaling responses observed in several model systems for studying race-specific disease resistance (summarized in Fig. 8
). For example, the disease resistance reactions triggered toward Cladosporium fulvum and Psuedomonas syringae pv tomato by tomato (Solanum lycopersicum) and toward Tobacco mosaic virus by tobacco were each shown to involve simultaneous activation of MPK6 and MPK3 homologs only during the incompatible interaction. These responses were triggered via pathogen recognition through the Cf-9 (Romeis et al., 1999
The fact that TaMPK3 is strongly transcriptionally up-regulated during the immediate presymptomatic phase of compatible interactions with M. graminicola suggests that it may have responded to a "general elicitation" caused by the presence of the fungus growing slowly within the leaf, despite the fact that no visible symptoms of infection are seen. The transcriptional activation of MPK3 homologs has been reported in various systems following exposure to a range of both biotic and abiotic stimuli (Zhang and Klessig, 1998b ; Liu et al., 2003; Ahlfors et al., 2004; Mayrose et al., 2004; Reyna and Yang, 2006), and a number of studies have specifically demonstrated the transcriptional activation of MPK3 homologs in association with HR-mediated incompatible disease resistance reactions (Zhang and Klessig, 1998a; Mayrose et al., 2004). This contrasts remarkably with the responses of wheat leaves during interactions with M. graminicola, in which transcriptional activation of TaMPK3 is strongest preceding the PCD responses that feature in the compatible disease reaction.
It is noteworthy that many of the pathogen signals shown to activate plant MAPKs transcriptionally and posttranslationally are often described as "general elicitors" or pathogen-associated molecular patterns (Ligterink et al., 1997
A limited number of studies have addressed MAPK function in monocot plants during interactions with fungal pathogens. For example, OsMAPK5a, the rice (Oryza sativa) homolog of TaMPK3, was shown to play an important role in disease lesion formation induced by the hemibiotrophic rice blast fungus and was also shown to be transcriptionally activated during this interaction (Xiong and Yang, 2003 Clear differences were detected in the comparative levels and activities of the TaMPK3 and TaMPK6 proteins during the compatible interaction. The TaMPK3 protein is initially present at relatively low levels in leaves but accumulates prior to the appearance of first disease symptoms and its own subsequent posttranslational activation. This series of events is remarkably similar to what has been reported for WIPK in tobacco leaves following N gene-mediated recognition of Tobacco mosaic virus and the activation of HR. The difference is that these responses were features of an incompatible reaction and therefore the opposite of what we have described here (summarized in Fig. 8).
Intriguingly, in the wheat-M. graminicola pathosystem, the levels of the TaMPK6 protein fell dramatically in extracts that began to accumulate active TaMPK3. The two most obvious explanations for this are (1) that TaMPK6 is specifically degraded in plant cells in the period leading up to the execution of PCD, or (2) that the degradation of TaMPK6 is a general feature of this response, but instead TaMPK3 is somehow stabilized or continually replenished via its strong transcriptional activation. Precedent exists for the selective degradation of plant signaling proteins during pathogen attack. For example, studies on the interaction of barley (Hordeum vulgare) with the biotrophic stem rust fungus identified a specific proteolysis of the RPG1 receptor-like kinase that preceded the execution of HR-mediated disease resistance responses (Nirmala et al., 2007
The various changes imposed upon the activities and levels of the TaMPK6 and TaMPK3 proteins were largely restricted to the immediate fungus-inoculated leaf areas and much more pronounced in the areas undergoing PCD during the compatible interaction. This again suggests some form of functional relationship between the two events. The one exception to this was an increase in the level of inactive TaMPK3 protein, which was detected in the leaf area adjacent to the developing lesion of the compatible interaction (Fig. 6B). This may suggest that the strictly localized lesions that form during the compatible interaction signal to uninoculated surrounding leaf areas in a similar, or identical, manner to how the development of HR-like lesions has been shown to trigger local and systemic acquired resistance reactions in many other pathosystems (Durrant and Dong, 2004
The differences in protein levels between TaMPK6 and TaMPK3 throughout the course of the compatible interaction in the M. graminicola-wheat pathosystem also has implications for the upstream activators of the cascade(s). In many dicot systems, MPK6 and MPK3 homologs are activated simultaneously, often via the same MKK, in response to pathogen signals (Yang et al., 2001
PCD events in plants have been shown to be triggered in response to the application of fungal toxins, and many of these responses have been demonstrated to be host and/or cultivar specific (Wolpert et al., 2002
Small proteinaceous toxins were also recently shown to play key roles in determining the outcome of interactions between wheat leaves and the necrotrophic fungi Pyrenophora tritici-repentis and Stagonospora nodorum. The ability of these fungi to cause full disease has been shown to be positively influenced by the host sensitivity toward the proteinaceous fungal toxin ToxA, which has been horizontally transferred between the two species (Friesen et al., 2006
Plant and Fungal Material and Handling Mycosphaerella graminicola isolates IPO323 and IPO88004 were used in all experiments. The isolates were stored at –80°C in 50% (v/v) glycerol. Fungal spores for plant inoculation were harvested from 7-d-old cultures growing (budding) on yeast peptone dextrose plates (Oxoid) at 15°C.
For plant infection, the second leaf of 17-d-old wheat (Triticum aestivum) seedlings (cultivar Avalon or cultivar Cadenza) were attached, adaxial side up, to Perspex sheets using double-sided tape. The inoculation procedure was as described previously (Keon et al., 2007
Full-length MAPK-encoding genes were obtained by reverse transcription (RT)-PCR on wheat leaf cDNA using the following primers. For TaMPK6, a first round of PCR was done with the primers MPK6FwdEST (5'-AAATCCACCGCACGGGCTTTC-3'; binds in the 5' untranslated region of the TaMPK6 transcript) and MPK6XhoRev (5'-CTCGAGCTGGTAATCAGGGTTGAACGTGATG-3'). The PCR product was cloned into vector pGEM-T Easy (Promega) and sequenced. A second round of PCR was then done to enable in-frame cloning via BamHI and XhoI into expression vector pET-28b(+) (Novagen) using MPK6BamsiteFwd (5'-ACGCGCGGGATCCGGCGGAGATGGAC-3') and MPK6XhoRev (as above). TaMPK3 was amplified in a single round of RT-PCR using primers MPK3BamFwd (5'-GGATCCGATGGACGGCGCTCCGGTGGCCGA-3') and MPK3XhoRev (5'-CTCGAGGTATCGGAAGTTGGGGTTCAACTC-3') for BamHI and XhoI cloning into the expression vector. MgFus3 was also cloned into the expression vector (via BamHI and HindIII) following a single round of PCR with primers MgFus3BamFwd (5'-GGATCCGATGTCGAGAACCGCACAGCAACAG-3') and MgFus3HindRev (5'-AAGCTTCCGCATGATCTCTTCGTAAATCAG-3'). Recombinant protein expression was driven in BLR(DE3) pLysS cells (Novagen) by 0.1 M isopropylthio-β-galactoside, and proteins were purified from cell extracts using His-Select Nickel Affinity Gel (Sigma) according to the supplier's guidelines.
Peptides corresponding to the N-terminal regions of TaMPK3 and TaMPK6 were used to immunize rabbits. For TaMPK3-N, the following amino acid sequence was used corresponding to residues 2 to 15 of the mature protein (GenBank accession no. AY079318): 5'-DGAPVAEFRPTMTHG-3'. For TaMPK6-N, amino acids 1 to 12 (accession no. AY173962) were used: 5'-MDAGGAQPPDSE-3'. Immunizations were performed by Eurogentec.
For cytochrome c assays, crude cytosolic and microsomal fractions were generated as described previously (Krause and Durner, 2004
For cytochrome c release assays, approximately 100 µg of protein was separated on 15% SDS-PAGE gels and blotted onto Hybond ECL nitrocellulose (Amersham Pharmacia). Blots were probed with a 1:2,000 dilution of monoclonal anti-cytochrome c antiserum (BD Biosciences clone 7H8.2C12) and subsequently a 1:5,000 dilution of anti-mouse IgG horseradish peroxidase conjugate (Sigma). Blots were developed using chemiluminescence (Amersham ECL-plus). For the investigation of ERK-type MAPK activity during plant infection, western blots were probed with Phospho-p44/42 MAP Kinase (Thr-202/Tyr-204) antibody according to the supplier's guidelines (Cell Signaling Technology). For western blots using the MAPK-specific antibodies, 1:1,000 dilutions of TaMPK3-N (affinity purified) and TaMPK6-N were used against His-tagged recombinant proteins or leaf extracts. To detect recombinant protein expression, an anti-His(C-term) antibody (Invitrogen) was used according to the supplier's guidelines to detect the C-terminal His tag generated following expression in pET-28c(+).
Leaf extracts containing 100 µg of soluble protein were immunoprecipitated with Protein A-Sepharose CL-4B (Amersham Biosciences) bound antiserum for 1 h at 4°C. All subsequent washing and radioactive steps were as described previously (Ligterink et al., 1997
All assays were performed in triplicate. Two inoculated leaf segments (approximately 5 cm) were added to 10 mL of deionized water (bathing solution). Leaves were then vacuum infiltrated (4 x 30 s) at 25 mbar pressure with complete release of vacuum in between. The infiltrated leaves were allowed to stand in the bathing solution for 1 h at room temperature. The leaves were then removed, the bathing solution was vortexed, and the ionic strength was monitored for conductivity as an indication of host membrane integrity.
Genomic DNA was isolated from 100 mg of infected leaf tissue (2- x 6-cm leaf segments) harvested on various days after inoculation, using a DNeasy Plant Mini Kit (Qiagen), following the supplier's instructions. Real-time quantitative PCR was performed in order to monitor levels of fungal biomass in infected leaf tissues using a Cy5-labeled probe to quantify the presence of the cytochrome b gene of M. graminicola (Fraaije et al., 2005
Total RNA was isolated from leaf tissues infected by M. graminicola using the Trizol procedure (Invitrogen), following the supplier's protocol and incorporating the suggested additional procedures for polysaccharide-containing tissues. Further purification of the total RNA was achieved by precipitation from a solution of 4 M lithium chloride. Total RNA was used for all real-time RT-PCR analyses. Where appropriate, RNA species were analyzed by agarose gel electrophoresis using MOPS buffer and formaldehyde. For RT-PCR analysis, first-strand cDNA was synthesized from total RNA using the SuperScript III First_Strand Synthesis System for RT-PCR (Invitrogen). Five micrograms of total RNA primed with oligo(dT)20 was used in a 20-µL reaction, following the supplier's instructions. The resulting cDNA was analyzed using the QuantiTect SYBR Green PCR Kit (Qiagen), following the supplier's instructions. Then, 0.5 µL of cDNA was used in a 20-µL PCR, with an annealing temperature of 56°C. Primers were added at a final concentration of 0.25 µM. PCR was run and analyzed using an ABI 7500 Real Time PCR System, with β-tubulin acting as the endogenous control.
The following materials are available in the online version of this article.
We thank Gert Kema (Plant Research International) for the provision of M. graminicola isolates IPO323 and IPO88004. We gratefully acknowledge the help of Bart Fraaije and Hans Cools for real-time PCR analyses and John Lucas for critical reading of the manuscript. Received March 20, 2008; accepted April 18, 2008; published April 25, 2008.
1 This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom to Rothamsted Research and by non-BBSRC funds provided for preliminary Affymetrix microarray analyses and antibody production.  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: Jason J. Rudd (jason.rudd{at}bbsrc.ac.uk).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.108.119511 * Corresponding author; e-mail jason.rudd{at}bbsrc.ac.uk.
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