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Running head: Role of ABA and ET in rice resistance against C. miyabeanus 1
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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
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Journal research area: Plants Interacting with Other Organisms 14
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Plant Physiology Preview. Published on February 3, 2010, as DOI:10.1104/pp.109.152702
Copyright 2010 by the American Society of Plant Biologists
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Title: Abscisic acid-induced resistance against the brown spot pathogen Cochliobolus 30
miyabeanus in rice involves MAPK-mediated repression of ethylene signaling 31
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Authors: David De Vleesschauwer1, Yinong Yang2, Casiana Vera Cruz3, and Monica Höfte1 34
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Institution addresses: 1 Laboratory of Phytopathology, Faculty of Bioscience Engineering, 36
Ghent University, Coupure Links 653, B-9000 Ghent, Belgium 37
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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
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3 Plant Breeding, Genetics and Biotechnology Division, International 43
Rice Research Institute, Los Banos, Laguna, Philippines 44
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Footnotes 61
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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
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corresponding author: Monica Höfte, e-mail: monica.hofte@ugent.be, fax: 32-92646238 68
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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
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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
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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
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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
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this ABA/ET crosstalk, and describe how ABA might interfere with the postulated fungal 223
manipulation of the plant. 224
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Results 226
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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
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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
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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
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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