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First published online January 11, 2006; 10.1104/pp.105.074906 Plant Physiology 140:681-692 (2006) © 2006 American Society of Plant Biologists Rewiring Mitogen-Activated Protein Kinase Cascade by Positive Feedback Confers Potato Blight Resistance1Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464–8601, Japan (C.Y., K. Kuchimura, S.K., K. Kawakita, N.D., H.Y.); Department of Upland Agriculture, National Agricultural Research Center of Hokkaido Region, Memuro, Hokkaido 082–0071, Japan (A.K.); and Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, United Kingdom (J.D.G.J.)
Late blight, caused by the notorious pathogen Phytophthora infestans, is a devastating disease of potato (Solanum tuberosum) and tomato (Solanum lycopersicum), and during the 1840s caused the Irish potato famine and over one million fatalities. Currently, grown potato cultivars lack adequate blight tolerance. Earlier cultivars bred for resistance used disease resistance genes that confer immunity only to some strains of the pathogen harboring corresponding avirulence gene. Specific resistance gene-mediated immunity and chemical controls are rapidly overcome in the field when new pathogen races arise through mutation, recombination, or migration from elsewhere. A mitogen-activated protein kinase (MAPK) cascade plays a pivotal role in plant innate immunity. Here we show that the transgenic potato plants that carry a constitutively active form of MAPK kinase driven by a pathogen-inducible promoter of potato showed high resistance to early blight pathogen Alternaria solani as well as P. infestans. The pathogen attack provoked defense-related MAPK activation followed by induction of NADPH oxidase gene expression, which is implicated in reactive oxygen species production, and resulted in hypersensitive response-like phenotype. We propose that enhancing disease resistance through altered regulation of plant defense mechanisms should be more durable and publicly acceptable than engineering overexpression of antimicrobial proteins.
The timely recognition of invading microbes and the rapid induction of defense responses are essential for plant disease resistance. At least two recognition systems are used by plants (Dangl and Jones, 2001  ; Parker, 2003). Plant defenses are often initiated by a gene-for-gene interaction between a dominant plant resistance (R) gene and a pathogen avirulence (Avr) gene, which provides race-specific resistance that is easily overcome by pathogen mutations. Plants also use a much less specific recognition system that identifies pathogen-associated molecular patterns (PAMPs), such as flagellin (Zipfel et al., 2004) and lipopolysaccharides (Zeidler et al., 2004), so-called general elicitors. Both animals and plants can recognize invariant PAMPs that are characteristic of pathogenic microorganisms. Perception of the peptide fragment of flagellin in Arabidopsis (Arabidopsis thaliana) depends on the Leu-rich repeat-type receptor kinase flagellin sensing 2 (FLS2; Gómez-Gómez and Boller, 2000). The fls2 mutant Arabidopsis is more susceptible to the bacterial pathogen Pseudomonas syringae pv tomato DC3000 than wild-type plants (Zipfel et al., 2004), suggesting that recognition of PAMPs by plant cells potentiates defense responses. Understanding these plant signaling systems creates an opportunity to manipulate these systems to enhance resistance in crops. FLS2- and R-gene products share similarities with components of the animal innate immune system, suggesting that some downstream signaling components are common between plants and animals (Dangl and Jones, 2001; Staskawicz et al., 2001).
The mitogen-activated protein kinase (MAPK) cascade is one of the major and evolutionally conserved signaling pathways utilized to transduce extracellular stimuli into intracellular responses among eukaryotes (Ligterink et al., 1997
Exposure of potato plants to only an avirulent Phytophthora infestans (late blight pathogen) causes multiple defense responses, including the oxidative burst, nitric oxide production, and accumulation of antifungal sesquiterpenoid phytoalexins (lubimin and rishitin) dependent upon de novo synthesis of the enzymes involved in their production (Doke, 1983
PVS3 Promoter Is Induced in Potato Tubers and Leaves by the Inoculation with Pathogens
PVS, the key enzyme of the phytoalexin synthesis of potato, is encoded by a multiple-gene family (PVS1 to 4; Yoshioka et al., 1999
Transgene activation to trigger the defense responses should occur only at the time and the site of pathogen challenge and not under other circumstances. In the field, disease caused by P. infestans can be initiated on the leaves. The virulent pathogen-inducible gene promoter in potato leaves is indispensable to drive the transgene that cues cell death. As shown in Figure 2B, PVS3 was significantly induced in leaves in both incompatible and compatible interactions of P. infestans, but not by wounding. We tested whether PVS3 promoter is useful to drive the StMEK1DD in response to P. infestans. The PVS3 promoter was fused to the  -glucuronidase (GUS) reporter gene, and the temporal and spatial expression patterns of GUS were monitored in the transgenic potato plants. No GUS activity was detected in leaves, roots, and growth points of unstimulated transgenic plants (data not shown). These results suggest that PVS3 promoter is suitable for generation of disease-resistant transgenic potato plants. To investigate the adaptable range of the PVS3 promoter, we monitored GUS activities in the PVS3::GUS transgenic potato leaves under biotical or physical stimuli. Inoculation with mock (Fig. 3B) and nonpathogen Escherichia coli (Fig. 3C), and Alternaria alternata Japanese pear (Pyrus serotina Rehd.) pathotype strain 15A (Fig. 3G), did not induce the GUS activity because these microbes cannot invade inside the plant cells. Importantly, wounding of the potato plants also did not activate the PVS3 promoter (Fig. 3D). In contrast, the promoter was strongly activated in transgenic potato plant by inoculation with the virulent P. infestans (Fig. 3, A and F) and early blight pathogen A. solani strain A-17 (Fig. 3E) in the restricted sites where pathogens tried to infect. These results confirm that the PVS3 promoter is specifically and locally activated by pathogens and thus suitable to drive StMEK1DD.
PVS3 Promoter Is Controlled by MAPKs
We investigated the control mechanism of the PVS3 promoter by Agrobacterium (Agrobacterium tumefaciens) infiltration (agroinfiltration). PVS3 promoter was not activated by inoculation with Agrobacterium-carrying vector (control; Fig. 3H). In contrast, the promoter was activated in response to Agrobacterium-carrying Avr9/Cf-9, Avr-R interaction (Thomas et al., 2000
To investigate this possibility, we employed virus-induced gene silencing (VIGS) in N. benthamiana using a potato virus X (PVX) vector (Lu et al., 2003
To determine the 5' boundary of the region that is important for the activity of the PVS3::GUSint fusion in response to StMEK1DD and INFESTIN (INF1; Kamoun et al., 1997 ), elicitor derived from P. infestans in N. benthamiana, a series of 5' deletions of the PVS3 promoter was constructed. GUS activity driven by each construct was measured in the transient gene expression assay 24 h after treatment with effectors. The promoter region of PVS3, pPVS3-1 (2,334 bp), conferred responsiveness to the StMEK1DD and INF1 (Fig. 5). Deletion to pPVS3-3 (–1,287) resulted in a clear reduction of the effector-responsive GUS activity. Further deletions from pPVS3-4 to pPVS3-10 did not affect the GUS activity. A 50-bp promoter region of PVS3 (positions –1,337 to –1,287) was shown to play an important role in transcriptional activation in response to both StMEK1DD and INF1 (Fig. 5). Both StMEK1DD and INF1 elicitor activate the SIPK and WIPK (Katou et al., 2003; Sharma et al., 2003), indicating that the PVS3 promoter is controlled by these MAPKs.
Potato Ortholog of Tobacco SIPK, StMPK1, Is Activated by the Inoculation with a Virulent Isolate of P. infestans
Previous studies have shown that a 51-kD MAPK is activated in potato tuber tissue by treatment with an elicitor, hyphal wall components (HWC) prepared from P. infestans or SA and arachidonic acid, which are known to induce various defense responses in potato plants (Katou et al., 1999 To gain a better understanding of the involvement of the MAPK cascade in the defense responses of potato leaves, we investigated the MAPK activity after the inoculation with a virulent or an avirulent isolate of P. infestans. In-gel kinase assay using myelin basic protein (MBP) as a substrate revealed that activation of the 51-kD MAPK (StMPK1) was rapidly induced in response to both virulent and avirulent P. infestans, which clearly precedes the expression of PVS3 (Figs. 2B and 6). These results suggest that components of the innate immunity system are induced not only in incompatible but also in compatible interactions.
Transgenic Potato Plants Harboring PVS3::StMEK1DD Show Resistance to Virulent Isolates of P. infestans and A. solani We produced transgenic potato plants carrying the StMEK1DD allele expressed from the PVS3 promoter. The transgenic plants and tubers developed normally (Fig. 7, A and B). The transgenic potato leaves as well as tubers showed high resistance to a virulent P. infestans (Fig. 7, C and F) and displayed an HR-like cell death phenotype (Fig. 7, D and F) accompanied by accumulation of hydrogen peroxide (H2O2) around the infected cell (Fig. 7E). We also examined whether PVS3::StMEK1DD transgenic potato plants show resistance to the necrotrophic pathogen A. solani. Six days after inoculation, typical disease symptoms appeared on the wild-type potato leaves. In contrast, transgenic potato leaves showed an HR-like cell death phenotype (Fig. 7G) and accumulated H2O2 around infected tissue (Fig. 7H). These results suggest that the transgenic potato plants provoke oxidative burst and show high resistance not only to the biotrophic pathogen P. infestans but also to the necrotrophic pathogen A. solani.
Transgenic Potato Plants Indicate Elevation of MAPK Activity and Up-Regulation of Defense-Related Genes during Compatible P. infestans-Potato Interactions
To investigate whether the HR-like phenotype correlated with introduced StMEK1DD expression, we analyzed RNA and protein extracts from leaves of transgenic plants in compatible P. infestans-potato interactions. The induction of StMEK1DD and rapid elevation of MAPK activity compared to wild-type plants was observed within 1 h (Fig. 8, A and B). The transcript level of StMEK1DD was decreased 24 h after pathogen inoculation (Fig. 8A) in agreement with profile of StMPK1 activity (Fig. 8B). These data suggest that the switch off of the gene resulted from localized cell death induced by the pathogen attack because HR-like cell death was observed 24 h after inoculation (Fig. 7D). In-gel kinase assay detected only StMPK1 activity; however, immunoprecipitation analyses using specific antibodies showed that both StMPK1 and StWIPK are activated in response to P. infestans (data not shown). A likely explanation for this is that basal levels of StMPK1 in unstimulated plant cells are much higher (10-fold) than those of StWIPK (Zhang and Klessig, 1998
To examine whether the activation of endogenous MAPKs by introduced StMEK1DD provokes the expression of defense-related genes, expression profiles for Phe ammonia-lyase (PAL) and hsr203J (HR marker) were determined (Fig. 8C) because it was reported that their expression is regulated by a MAPK cascade (Yang et al., 2001 ; Yoshioka et al., 2003; Takahashi et al., 2004). These genes were up-regulated in transgenic leaves in comparison with wild-type potato leaves. In addition, the expression pattern of StrbohA to D (plant NADPH oxidases) was also investigated. Oxidative burst is proposed to be responsible for HR cell death correlated with defense responses. StrbohC and StrbohD were drastically induced in the infected transgenic plants.
Ever since the initial discovery of the molecules and genes involved in disease resistance in plants, attempts have been made to engineer disease resistance in economically important crop plants. Genetic engineering has proved to be a powerful tool for controlling plant diseases and to be an alternative to economically costly and environmentally undesirable chemical control. To date, transgenic disease-resistant plants include constitutively overproducing  -thionin (Carmona et al., 1993) and the Ustilago maydis killer toxin (Park et al., 1996), and expressing genes encoding enzymes that are involved in the biosynthesis of antimicrobial compounds (Hain et al., 1993) have been produced. Other approaches have been based on the overexpression of genes encoding proteins that are produced during the natural defense responses, such as PR-1a (Alexander et al., 1993), chitinase (Broglie et al., 1991), or osmotin (Liu et al., 1994). An additional possibility involves the production of proteins that generate a signaling event and trigger the permanent onset of an array of defense responses, such as H2O2-generating Glc oxidase (Wu et al., 1995, 1997). Constitutive expression of the Arabidopsis NIM1/NPR1 gene, which is a mediator of systemic acquired resistance, results in varying degrees of resistance to different pathogens (Cao et al., 1998; Friedrich et al., 2001). Furthermore, enhanced disease resistance mutants have been identified (Bowling et al., 1994; Frye et al., 2001). Most plants possessing constitutive expression of a defense pathway show reduced yield or other deleterious phenotypes. Recently, R genes, which confer broad-spectrum late blight resistance in cultivated potato, were identified from wild-type potato (Song et al., 2003; van der Vossen et al., 2003). It remains to be seen if the use of these broad-spectrum late blight R genes is durable under conditions of large-scale agricultural production because races of the pathogen that were able to overcome these genes emerged within a few years after market introduction (Turkensteen, 1993).
During the past few years, efforts have been made to generate transgenic plants that express the introduced gene under controlled conditions only. Successful approaches satisfy for this point; disease-resistant transgenic tobacco was produced by expressing pathogen elicitor-related genes fused to plant-inducible promoter (Keller et al., 1999
Plants have evolved the complex and sophisticated defense systems to withstand a variety of pathogens (Greenberg and Yao, 2004
Perception of pathogen by plant cells triggers rapid defense responses via a number of signal transduction pathways. The interaction between transcription factors and cis-acting regulatory sequences presented in plant promoters is a key step involved in the regulation of plant gene expression. cis-Acting elements within the promoters of many of these genes have recently been defined, and investigators have started to isolate their cognate trans-acting factors. To identify the cis-acting elements and cognate trans-acting factors is a first step of elucidation of signal transduction mechanism. Transient expression experiments of the N. benthamiana-Agrobacterium system suggest that cis-element, which is activated by both StMEK1DD and INF1, exists in a 50-bp region of the PVS3 promoter (positions –1,337 to –1,287; Fig. 5). These results suggest that MAPK cascade is involved in the induction process of PVS3 promoter by INF1. INF1 is an elicitor derived from P. infestans and is known to activate SIPK and WIPK in N. benthamiana (Sharma et al., 2003
Figure 9 shows a hypothetical mechanism of enhanced immune response in the transgenic potato plants in response to pathogen attack. In the absence of pathogens, the transgenic plant displayed normal phenotype similar to that of wild type because transgene was not induced and little increase in MAPK activity can be detected in transgenic plant. In contrast, infection with virulent pathogen induces endogenous MAPKs (StMPK1 and StWIPK) to some extent. Following the activation of MAPK, the PVS3 promoter is induced, and then StMEK1DD driven by PVS3 promoter is expressed. This creates a positive feedback genetic circuit because StMEK1DD induces phosphorylation of MAPKs and enhancing its own induction, resulting in long-lasting activation of MAPKs. The kinetics of SIPK activation in response to abiotic stresses is transient, whereas biotic elicitors that induce cell death result in prolonged activation (Zhang and Klessig, 1998
Plant Growth Conditions Potato plants (Solanum tuberosum) and Nicotiana benthamiana were grown at 20°C or 25°C, respectively, with 70% humidity under a 16-h photoperiod and an 8-h-dark period in biotron or environmentally controlled growth cabinets.
Races 0 and 1.2.3.4 of Phytophthora infestans were maintained on susceptible potato (cv Irish cobbler) tubers. Suspensions of Phytophthora zoospores were prepared as described previously (Yoshioka et al., 2003
GUS activity was assayed in tissue extracts by fluorometric quantitation of 4-methylumbelliferone (4-MU) produced from the glucuronide precursor using a standard protocol (Jefferson et al., 1987
Histochemical localization of GUS activity in situ was performed by vacuum infiltration with a solution consisting of 50 mM sodium phosphate and 0.5 mg of 5-bromo-4-chloro-3-indolyl glucuronide mL–1, and incubated for 16 h at 37°C. Leaf discs containing the inoculum were excised and then fixed on the filter paper by immersion in a 3:1 solution of ethanol:acetic acid. The fixed samples were stained carefully with 0.1 µg mL–1 trypan blue solution to avoid washing spores away, and then examined by microscopy for plant responses and growth of pathogens. Alternatively, samples were fixed with lactophenol, then destained and viewed in 2.5 g mL–1 chloral hydrate solution (Wilson and Coffey, 1980
A 230-bp fragment of NbSIPK and a 178-bp fragment of NbWIPK, each starting from the ATG codon, were subcloned into a PVX vector pGR106 (Ratcliff et al., 2001
Approximately 6.0 x 105 recombinant plaques of a potato genomic library (CLONTECH) were screened using a 32P-labeled PVS1 cDNA probe (Yoshioka et al., 1999
Potato plants (cv Sayaka carrying R1 and R3) were transformed with PVS3::GUS or PVS3::StMEK1DD construct. The PVS3 promoter up to –2,648 bp that includes 30 nucleotides of PVS3 open reading frame was amplified by PCR and introduced into the HindIII and SpeI sites of pBluescript SK (–) (Stratagene). S-Tag and StMEK1DD fusion or GUS gene were amplified by PCR and cloned into the SpeI and SmaI sites of pBluescript SK (–). This HindIII and SmaI cDNA fragment was amplified and Nos-terminator of pBI121 (CLONTECH) was amplified with SmaI and EcoRI sites, and introduced into the HindIII and EcoRI sites of pGreen0029 (Hellens et al., 2000
The plasmid pPVS3-1 was constructed by inserting the PVS3 promoter fragment into the EcoR1-ClaI sites of the pGreen0229 binary vector (Hellens et al., 2000
To visualize H2O2 in the infection site of P. infestans or A. solani, 3,3'-diaminobenzidine (DAB) staining was performed as described by Thordal-Christensen et al. (1997)
In-gel kinase assays were performed as described previously (Katou et al., 2003
Total RNA samples were prepared from wild-type or transgenic plants and used for RT-PCR as templates. Gene-specific primers of each sequence were as follows: PVS1 (176 bp; 5'-CATCGATTGTTTTGTACATCT-3', 5'-AATAATGATACAAAAAAAAATTAAGG-3'), PVS2 (132 bp; 5'-TATCAATTCACCAAGGAACACT-3', 5'-GAAGTAATTAAATTTAAATATTATCAA-3'), PVS3 (326 bp; 5'-TTGTCTGCTGCTGCTTGTGG-3', 5'-TCTCCATGAGTCCTTACATG-3'), PVS4 (131 bp; 5'-CATCCCTTAAAATTATAAGTATTC-3', 5'-AATAATGATACAAAATAAATTAAGG-3'), StMEK1DD transgene (527 bp; 5'-ATGAAAGAAACCGCTGCTGCTAAATT CGAA-3', 5'-ATATCGTGACACCTAACGACGTTAGGGTTG-3'), and EF-1
Total RNA was extracted from wild-type or transgenic plants. Total RNA (10 µg) was fractionated by electrophoresis on a 1.0% agarose-formaldehyde gel. The separated RNA was transferred from the agarose gel to a nylon membrane (Hybond N+; Amersham). The membrane was incubated for 2 h at 42°C in 50% (v/v) formamide, 5x Denhardt's solution, 5x sodium chloride/sodium phosphate/EDTA (SSPE; 1x SSPE; 10 mM NaH2PO4, pH 7.7, 180 mM NaCl, 1 mM EDTA), 0.5% SDS, and denatured salmon sperm DNA 200 µg mL–1. Hybridization was performed overnight under the same conditions with the addition of 32P-labeled fragments of the probe. The probes were labeled with [ Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AB062138, AB022598, AB022719, AB022720, AB023816, L04680, U20187, AJ005588, AB198716, and AB198717 for StMPK1, PVS1, PVS2, PVS3, PVS4, TEAS, HVS, PEAS, StrbohC, and StrbohD, respectively.
We deeply thank David C. Baulcombe of Sainsbury Laboratory for providing PVX vector pGR106; Philip M. Mullineaux and Roger P. Hellens of John Innes Centre for pGreen binary vectors; Klaus Hahlbrock of Max-Planck-Institute for potato Phe ammonia-lyase cDNA; Joseph Chappell of University of Kentucky for EAS cDNA; Sophien Kamoun of Ohio State University for INF1 elicitor; Nam-Hai Chua of Rockefeller University for pER8 vector; the Leaf Tobacco Research Center, Japan Tobacco, for N. benthamiana seeds; Takashi Tsuge of Nagoya University for A. solani strain A-17 and A. alternata Japanese pear pathotype strain 15A; and Motoyuki Mori of NARCH for valuable suggestions. We also thank Naoki Ikeda, Miki Yoshioka, and the Radioisotope Research Center, Nagoya University, for technical assistance. Received November 30, 2005; returned for revision November 30, 2005; accepted December 16, 2005.
1 This work was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists; by the Ministry of Education, Science and Culture of Japan (Grant-in-Aid for Scientific Research [S], grant no. 14104004); and by the Research for the Future Program of the Japan Society for the Promotion of Science (Grant-in-Aid for Scientific Research on Priority Area [A]).  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: Hirofumi Yoshioka (hyoshiok{at}agr.nagoya-u.ac.jp). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.074906. * Corresponding author; e-mail hyoshiok{at}agr.nagoya-u.ac.jp; fax 81–52–789–5525.
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-32P]ATP (4,000 Ci mmol–1) for 1 h. The reaction was stopped by washing the gel in 5% trichloroacetic acid and 1% sodium pyrophosphate. The gel was washed extensively with this solution, dried under vacuum, and autoradiographed with an intensifying screen. 
