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1 Running head: Role of ABA and ET in rice resistance against C. miyabeanus 1 2 3 4 5 Corresponding Author: 6 Name: Monica Höfte 7 Address: Laboratory of Phytopathology, Faculty of Bioscience Engineering, Ghent 8 University, Coupure links, 653, B-9000 Gent, Belgium 9 Telephone: 32-9-2646017 10 e-mail: monica.hofte@ugent.be 11 12 13 Journal research area: Plants Interacting with Other Organisms 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Plant Physiology Preview. Published on February 3, 2010, as DOI:10.1104/pp.109.152702 Copyright 2010 by the American Society of Plant Biologists 2 Title: Abscisic acid-induced resistance against the brown spot pathogen Cochliobolus 30 miyabeanus in rice involves MAPK-mediated repression of ethylene signaling 31 32 33 Authors: David De Vleesschauwer1, Yinong Yang2, Casiana Vera Cruz3, and Monica Höfte1 34 35 Institution addresses: 1 Laboratory of Phytopathology, Faculty of Bioscience Engineering, 36 Ghent University, Coupure Links 653, B-9000 Ghent, Belgium 37 38 2 Department of Plant Pathology and Huck Institutes of Life 39 Sciences, 405C Life Sciences Bldg., Penn State University, 40 University Park, PA 16802, United States of America 41 42 3 Plant Breeding, Genetics and Biotechnology Division, International 43 Rice Research Institute, Los Banos, Laguna, Philippines 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 3 Footnotes 61 62 financial source: This work was supported by a specialization fellowship of the Flemish 63 Institute for the stimulation of Scientific-Technological Research in Industry (IWT, Belgium) 64 given to David De Vleesschauwer and by grants from the Special Research Fund of Ghent 65 University and the “Fonds voor Wetenschappelijk Onderzoek-Vlaanderen”. 66 67 corresponding author: Monica Höfte, e-mail: monica.hofte@ugent.be, fax: 32-92646238 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 4 Abstract 90 The plant hormone abscisic acid (ABA) is involved in an array of plant processes, including 91 the regulation of gene expression during adaptive responses to various environmental cues. 92 Apart from its well-established role in abiotic stress adaptation, emerging evidence indicates 93 that ABA is also prominently involved in the regulation and integration of pathogen defense 94 responses. Here, we demonstrate that exogenously administered ABA enhances basal 95 resistance of rice (Oryza sativa) against the brown spot-causing ascomycete Cochliobolus 96 miyabeanus. Microscopic analysis of early infection events in control and ABA-treated plants 97 revealed that this ABA-inducible resistance (ABA-IR) is based on restriction of fungal 98 progression in the mesophyll. We also show that ABA-IR does not rely on boosted expression 99 of SA-, JA-, or callose-dependent resistance mechanisms but, instead, requires a functional 100 Gα-protein. In addition, several lines of evidence are presented suggesting that ABA steers its 101 positive effect on brown spot resistance through antagonistic cross-talk with the ET-response 102 pathway. Exogenous Ethephon application enhances susceptibility, whereas genetic 103 disruption of ET signaling renders plants less vulnerable to C. miyabeanus attack, thereby 104 inducing a level of resistance similar to that observed on ABA-treated wild-type plants. 105 Moreover, ABA treatment alleviates C. miyabeanus-induced activation of the ET-reporter 106 gene EPB89, while de-repression of pathogen-triggered EBP89 transcription via RNAi-107 mediated knockdown of OsMPK5, an ABA-primed MAP kinase gene, compromises ABA-IR. 108 Collectively, these data favor a model whereby exogenous ABA enhances resistance against 109 C. miyabeanus at least in part by suppressing pathogen-induced ET action in an OsMPK5-110 dependent manner. 111 112 113 114 115 116 117 118 119 120 121 5 Introduction 122 To effectively combat invasion by microbial pathogens, plants have evolved sophisticated 123 mechanisms providing several strategic layers of constitutive and induced defenses. Pre-124 formed physical and biochemical barriers constitute the first line of defense and fend off the 125 majority of pathogens. However, should the pathogen overcome or evade these constitutive 126 defenses, recognition of pathogen-derived molecules by plant receptors leads to the activation 127 of a concerted battery of defenses designed to impair further pathogen spread. These inducible 128 defenses are regulated by the coordinated activity of an elaborate matrix of signal transduction 129 pathways in which the plant hormones salicylic acid (SA), jasmonic acid (JA), and ethylene 130 (ET) act as key signaling molecules (Lorenzo and Solano, 2005; Grant and Lamb, 2006; Adie 131 et al., 2007). In response to pathogen attack, plants produce a highly specific blend of SA, JA, 132 and ET, resulting in the activation of distinct sets of defense-related genes (Koornneef and 133 Pieterse, 2008; Bari et al., 2009). It is thought that this so-called signal signature, which varies 134 greatly in quantity, timing and composition according to the type of attacker encountered, 135 plays a primary role in the orchestration of the plant’s defense response and eventually 136 determines the specific nature of the defense response triggered (Rojo et al., 2003; De Vos et 137 al., 2005; Mur et al., 2006). 138 Over the past decade, it has become increasingly clear that a plant’s resistance to attack is 139 not brought about by the isolated activation of parallel, linear hormonal circuits, but rather is 140 the consequence of a complex regulatory network that connects the individual pathways, 141 enabling each to assist or antagonize the others (Grant and Jones, 2009; Pieterse et al., 2009). 142 In addition to differential signal signatures, such pathway crosstalk provides the plant with a 143 powerful regulatory potential to fine-tune its immune response to different types of attackers. 144 Thus, some exceptions notwithstanding (Thaler et al., 2004; Asselbergh et al., 2007), it is 145 commonly accepted that SA promotes resistance against pathogens with a biotrophic lifestyle, 146 whereas JA and ET act as positive signals in the activation of defenses against necrotrophic 147 pathogens and herbivorous insects (Thomma et al., 2001; Rojo et al., 2003; Glazebrook, 148 2005). Additionally, the primary mode of interaction between the SA and JA/ET signaling 149 pathways appears to be mutual antagonism with corresponding trade-offs between biotroph 150 resistance, on the one hand, and resistance to necrotrophic pathogens and insect herbivores, 151 on the other hand (Bostock, 2005; Stout et al., 2006; Spoel et al., 2007). However, this is 152 likely an oversimplified model as synergistic actions of SA and JA/ET have been reported as 153 well (Van Wees et al., 2000; Mur et al., 2006; Adie et al., 2007; Truman et al., 2007). 154 6 Although mechanistic explanations of antagonistic and cooperative crosstalk are scarce, a 155 number of transcription factors and effector proteins have been characterized that are critical 156 in the circuitry controlling signal sensitivity and transduction in induced defense. For 157 instance, SA repression of JA signaling requires the activation of proteins such as NPR1 and 158 WRKY70 that activate expression of SA-responsive genes while repressing JA-dependent 159 genes (Spoel et al., 2003; Li et al., 2004; Li et al., 2006). Besides transcription factors, 160 crosstalk between the SA and JA signaling pathways may also be mediated by fatty acid-161 derived signals and/or glutaredoxin genes (Kachroo et al., 2003; Ndamukong et al., 2007). 162 Other important effectors that contribute to differential response activation include mitogen-163 activated protein kinases (MAPKs). Arabidopsis MPK4 is one such kinase and has been 164 shown to regulate SA/JA crosstalk by simultaneously repressing SA biosynthesis and 165 promoting the perception of or response to JA, thereby functioning as a molecular switch 166 between these mutually antagonistic pathways (Brodersen et al., 2006). On the other hand, 167 fine-tune regulation of the antagonism and cooperation between JA and ET depends on the 168 balance of activation by both hormones of ERF1 and MYC2, two opposing transcription 169 factors that differentially regulate divergent branches of the JA signaling pathway involved in 170 the response to necrotrophic pathogen attack and wounding, respectively (Berrocal-Lobo et 171 al., 2002; Lorenzo et al., 2003; Lorenzo et al., 2004). 172 In contrast to the overwhelming amount of information with respect to SA, JA, and ET 173 serving as important regulators of induced disease resistance, the role of abscisic acid (ABA) 174 in plant defense is less well understood, and even controversial. Most comprehensively 175 studied as a global regulator of abiotic stress adaptation, ABA has only recently emerged as a 176 key determinant in the outcome of plant-pathogen interactions. In most cases, ABA behaves 177 as a negative regulator of disease resistance. Exogenous application of ABA increases the 178 susceptibility of various plant species to bacterial and fungal pathogens (Mohr and Cahill, 179 2003; Thaler et al., 2004; Achuo et al., 2006; Asselbergh et al., 2007; Mohr and Cahill, 2007), 180 while disruption of ABA biosynthesis was shown to confer resistance to the necrotroph 181 Botrytis cinerea (Audenaert et al., 2002) and virulent isolates of the bacterial speck pathogen 182 Pseudomonas syringae pv tomato DC3000 in tomato (Thaler and Bostock, 2004), and the 183 oomycete Hyaloperonospora parasitica in Arabidopsis (Mohr and Cahill, 2003). Moreover, 184 an intriguing study by de Torres-Zabala and coworkers (2007) revealed that P. syringae 185 hijacks the ABA biosynthetic and response machinery to inflict disease in Arabidopsis, 186 suggesting that ABA is a susceptibility factor for this bacterium. This detrimental effect of 187 ABA on pathogen resistance is likely explained by its well-documented ability to counteract 188 7 SA- and JA/ET-dependent basal defenses (Asselbergh et al., 2008; de Torres-Zabala et al., 189 2009; Ton et al., 2009). 190 In contrast, some studies describe a positive role of ABA in activation of defense 191 responses and pathogen resistance. For instance, ABA primes for callose accumulation and 192 thereby enhances basal resistance in response to Blumeria graminis f. sp. hordei and activates 193 induced resistance in response to the necrotrophic fungi Alternaria brassicicola and 194 Plectosphaerella cucumerina (Ton and Mauch-Mani, 2004; Wiese et al., 2004; Flors et al., 195 2008). In the case of bacterial leaf pathogens, ABA plays a crucial role in the activation of 196 stomatal closure that, as part of the SA-regulated innate immune system, represents a major 197 barrier to bacterial infection (Melotto et al., 2006). Furthermore, a recent study in Arabidopsis 198 uncovered a new role for ABA in defense against insects (Bodenhausen and Reymond, 2007). 199 ABA thus appears to play a complex and ambivalent role in the plant’s defense response, 200 acting as either a positive or negative regulator of disease and pest resistance by interfering at 201 multiple levels with biotic stress-response pathways. 202 Rice is the most important staple food crop in the world, only rivaled in importance by 203 maize and wheat. However, despite its emergence as a pivotal scientific model for 204 monocotyledonous plants, surprisingly little is known about the effector responses and 205 hormonal signal transduction pathways underlying rice disease resistance. This is particularly 206 true for rice brown spot disease, caused by the ascomycete Cochliobolus miyabeanus 207 (anamorph: Bipolaris oryzae). One of the most devastating rice diseases in rainfed 208 ecosystems, brown spot adversely affects the yield and milling quality of the grain (Dela Paz 209 et al., 2006). In 1942, an epidemic of the disease was one of the major factors contributing to 210 the great Bengal famine, which reportedly claimed the lives of no less than 2 million Indians 211 (Stuthman, 2002). Nowadays, brown spot is as prevalent as ever with recent studies by Savary 212 et al. (2000a,b) showing that among the many diseases occurring in rice fields, brown spot, 213 along with sheath blight (Rhizoctonia solani), accounts for the highest yield loss across all 214 production situations in South and Southeast Asia. Although the genetic and molecular basis 215 of the rice-C. miyabeanus interaction is still poorly understood, it is known that like other 216 Cochliobolus species, the fungus employs a varied arsenal of phytotoxins to trigger host cell 217 death (Xiao et al., 1991). 218 Here, we show that pretreatment of rice with ABA renders leaves more resistant to C. 219 miyabeanus attack and present results supporting ABA-mediated repression of pathogen-220 induced ET action as a core resistance mechanism. In addition, we provide novel evidence 221 regarding the role of the ABA-inducible MAP kinase gene OsMPK5 as a pivotal regulator of 222 8 this ABA/ET crosstalk, and describe how ABA might interfere with the postulated fungal 223 manipulation of the plant. 224 225 Results 226 227 Exogenous ABA treatment induces resistance against Cochiobolus miyabeanus in rice 228 Six rice cultivars, including four indica and two japonica lines, were screened with two C. 229 miyabeanus strains, both of which were isolated from diseased rice in field plots at the 230 International Rice Research Institute in the Philippines (Table I). With the exception of 231 japonica cultivar CR203, isolate Cm988 was highly virulent on all cultivars tested, causing 232 typical ellipsoidal light- or dark-brown lesions with a grey sporulating center, often 233 surrounded by chlorotic tissue. On most cultivars, these susceptible-type lesions coalesced 234 within 96 h postinoculation (hpi), killing large areas of affected leaves (Fig. 1B, no ABA 235 treatment). By contrast, in case of infection by strain Cm963, fungal development was 236 restricted to a few dark-brown necrotic spots, representing a genetically resistant reaction (Ou, 237 1985). Owing to its differential response to Cm988 and Cm963 and its widespread use as a 238 pathogen-susceptible control in numerous other studies, indica cultivar CO39 was chosen for 239 further analysis. 240 In a first attempt to unravel the signaling network(s) orchestrating rice defense against C. 241 miyabeanus, we examined the effect of various signaling molecules and so-called plant 242 defense activators on brown spot disease development. To this end, five-week-old CO39 243 seedlings were sprayed until runoff with the respective compounds and, three days later, 244 inoculated with the virulent strain Cm988. Consistent with previous reports (Ahn et al., 2005), 245 treatment with 0.1 mM JA yielded no significant protection against C. miyabeanus (Fig. 1A), 246 even though this concentration is high enough to induce JA-responsive JIOsPR10 247 transcription (Jwa et al., 2001). Higher concentrations of JA also failed to trigger induced 248 resistance (data not shown), suggesting that JA is not a major signal for activation of defenses 249 against C. miyabeanus. Intriguingly, pretreatment with 0.5 mM Ethephon, an ET-releasing 250 plant growth regulator, rendered plants more vulnerable to brown spot disease compared to 251 non-induced controls. The disease-promoting effect of Ethephon strikingly contrasted with the 252 enhanced resistance observed in response to exogenously administered ABA. Supplying 253 plants with 0.1 mM ABA 3 d prior to inoculation induced high levels of protection, as shown 254 by a dramatic decrease in size, type and number of brown spot lesions in ABA-supplied 255 9 leaves (Fig. 1B). On the other hand, foliar application of the synthetic SA analog BTH (0.5 256 mM) or soil drench treatment with 150 μM BABA, a non-protein amino acid and potent 257 elicitor of broad-spectrum disease resistance in dicot plants (Ton et al., 2005; Flors et al., 258 2008), resulted in a rather weak and statistically not significant reduction in disease severity 259 compared with control plants. Collectively, these data uncover ABA as a powerful activator 260 of induced resistance against C. miyabeanus and suggest that ET acts as a negative signal in 261 the signaling circuitry underlying rice defense against this ascomycete. 262 263 ABA-induced resistance against C. miyabeanus is based on restriction of fungal 264 progression in the mesophyll 265 To gain more insight into the nature of ABA-inducible brown spot resistance (ABA-IR), 266 we next analyzed fungal development and cellular defense reactions in mock- and ABA-267 treated CO39 leaf sheaths following challenge with virulent Cm988. Regardless of ABA 268 treatment, conidial attachment and germination occurred within 6 hpi, followed by normal 269 hyphal growth and appressorium-mediated penetration attempts (Fig. 2A). Interestingly, at 270 some interaction sites, invading hyphae differentiated into subcuticular finger-shaped 271 multicell complexes (Fig. 2B), resembling the extracellular infection structures, so-called 272 stroma, frequently formed by Venturia inaequalis and Bipolaris sorokiniana (Ortega et al., 273 1998; Schafer et al., 2004). Further ramification of hyphal tissue occurred predominantly but 274 not exclusively intercellular (Figs. 2C, D), giving rise to a dense network that eventually 275 penetrated all host tissue types. Epidermal and mesophyll tissue necrotization was closely 276 associated with successful fungal infestation, whereby necrotization usually preceded fungal 277 growth, suggesting the involvement of C. miyabeanus-secreted phytotoxins. Comparing 278 control inoculated and ABA-treated plants, we found no marked differences in 279 abovementioned infection events, except for a drastic reduction of fungal spreading in the 280 mesophyll tissue of ABA-supplied leaf sheaths. By 36 hpi, fungal spreading in control 281 inoculated leaves amounted to approximately 1,400 μm, corresponding to 20-25 mesophyll 282 cells spanned by the fungus, as compared with 300 μm in ABA-pretreated sheath cells. 283 Together, these observations suggest that restriction of fungal proliferation during the 284 mesophyll-based growth phase, rather than a preinfectional, epidermis-based resistance 285 reaction, is the cause for the reduced disease susceptibility in ABA-treated plants. 286 287 288 289 10 ABA-IR against C. miyabeanus acts through a callose-independent mechanism 290 Recent evidence has implicated ABA as a positive signal in priming of callose 291 biosynthesis upon pathogen recognition, which suggests a putative mechanism explaining the 292 role of ABA in defense activation (Ton and Mauch-Mani, 2004; Flors et al., 2008). Callose 293 deposition is a hallmark of basal defense to attempted fungal and bacterial penetration and 294 may serve to fortify cell walls in order to inhibit pathogen penetration of the cell. To ascertain 295 the role of callose in the case of C. miyabeanus, we studied the deposition of this compound 296 and its effect on resistance in mock- and ABA-treated leaves stained with aniline blue. 297 Deposition of callose, visualized by an intense yellow-green fluorescence under UV light, was 298 detectable as early as 8 hpi in epidermal control cells in close contact to the invading h