elicitor and fusarium-induced expression of npr1-like genes in banana

Elicitor and Fusarium-induced expression of NPR-1 like genes in banana 1

Rosita Endah*a, Getu Beyene*b, Andrew Kiggunduc, Noelani van den Bergd, Urte 2

Schlütera, Karl Kunerta, Rachel Chikwambaa&b 3

aPlant Science Department and Forestry and Agricultural Biotechnology Institute, 4

University of Pretoria 0002, South Africa 5

bCouncil for Scientific and Industrial Research (CSIR), Pretoria 0001, South Africa 6

cNational Agricultural Research Organisation (NARO), Uganda 7

dDepartment of Microbiology and Plant Pathology and Forestry and Agricultural 8

Biotechnology Institute, University of Pretoria 0002, South Africa 9

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*RE and GB have contributed equally to this work and should be both regarded as the 11

first author. 12

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Correspondence: 14

Dr. Rachel Chikwamba, Council for Scientific and Industrial Research (CSIR), 15

Biosciences Division, Building 20 P O Box 395, Pretoria 0001, South Africa 16

email: [email protected] 17

Tel.: (+27) 12 841 2177 18

Fax: (+27) 12 420 3960 19

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Abstract 21

NPR1 is an essential positive regulator of salicylic acid-induced PR gene 22

expression and systemic acquired resistance. Two novel full-length NPR1-like genes; 23

MNPR1A and MNPR1B, were isolated by application of the PCR and RACE 24

techniques. The two identified MNPR1-sequences differed greatly in their expression 25

profile using qRT-PCR following either elicitor or Foc treatment. MNPR1A was 26

greatly expressed after Foc treatment with higher and earlier expression in the Foc-27

tolerant cultivar GCTCV-218 than in the sensitive cultivar Grand Naine. In 28

comparison, MNPR1B was highly responsive to SA, but not to MeJA treatment, in 29

both the tolerant banana cultivar GCTCV-218 and the more sensitive cultivar Grand 30

Naine. Expression of the MNPR1 genes further directly related to PR gene expression 31

known to be involved in fungal resistance. Reduced sensitivity to Foc in GCTCV-218 32

might be partially attributed to the higher and an earlier expression of both MNPR1A 33

and PR-1 in this cultivar after Foc treatment. 34

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Keywords: NPR1; Banana; Musa; Fusarium oxysporum; systemic acquired 36

resistance; PR proteins 37

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Abbreviations: NPR1, non-expressor of pathogenesis-related genes 1; MNPR1A, 39

Musa non-expressor of pathogenesis-related genes 1A; MNPR1B, Musa non-40

expressor of pathogenesis-related genes 1A; SA, Salicylic acid; MeJA, methyl 41

jasmonate; Foc, Fusarium oxysporum Schlecht f. sp. cubense (Smith) Snyd; SAR, 42

systemic acquired resistance; PR proteins, pathogenesis-related proteins 43

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Article Outline 45

1. Introduction 46

2. Results 47

2.1. Identification and isolation of NPR1-like genes 48

2.2. Elicitor-induced MNPR1 and PR genes 49

2.3. Fusarium-induced MNPR1 and PR genes 50

3. Discussion 51

4. Materials and methods 52

4.1. Isolation of banana NPR1-like gene sequences 53

4.2. Sequence analysis 54

4.3. Plant material and treatment 55

4.4. RNA extraction and cDNA synthesis 56

4.5. Quantitative RT-PCR 57

4.6. Data analysis 58

Acknowledgements 59

References 60

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1. Introduction 70

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NPR1 is an essential positive regulator of SA-induced PR gene expression and 72

SAR [39]. The PR proteins are expressed in plants in response to infection by 73

pathogens such as fungi or viruses [16, 31, 38]. These include PR-1, whose biological 74

activity is still unknown but seemingly has antifungal activity, and PR-3, which 75

consists of various chitinases and lysozymes. NPR1 is localized in the cell cytoplasm 76

[21] and exists as an inactive oligomer. This oligomer has to be activated by the 77

perception of salicylic acid [21]. Redox changes in the cytoplasm results in the 78

dissociation of the NPR1 protein into monomeric active forms, which translocates to 79

the nucleus where they interact with members of the TGA family of transcription 80

factors [13]. NPRI-TGA transcription factor complexes are known to bind to SA-81

responsive elements in the PR-1 promoter, facilitating PR gene expression and the 82

deployment of SAR [27, 39]. The co-regulatory activity of NPR1 is facilitated by the 83

presence of the Bric-a-Brack Poxvirus and zinc finger (BTB/POZ) domain and the 84

ankyrin repeats found within their protein structure [6, 8, 28]. In addition to its role in 85

regulating SAR, a further function of NPR1 in cross-communication between SA- and 86

jasmonic acid-dependent defence signalling pathways has been found [24]. 87

Several studies have shown that over-expression of NPR1 provides resistance to 88

a variety of bacterial and fungal pathogens [11, 12, 19, 40]. Also there is evidence 89

from transgenic plants that SA and PR-1 are required in Arabidopsis for resistance 90

against pathogen infection [34], and that NPR1 is involved in resistance to Fusarium 91

head blight in wheat [18]. However, transgenic Oryza sativa (rice) plants, expressing 92

an Arabidopsis NPR1 gene displayed a lesion mimic cell death phenotype [14], while 93

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rice plants over-expressing a rice NPR1 homologue (NH1) had increased SA levels 94

and were more sensitive to light resulting in a dwarf phenotype [11]. 95

NPR1 is further functionally conserved in diverse plant species and full length 96

NPR1 sequences from some of these have been deposited in the Genbank. In the 97

Arabidopsis genome six NPR1-related genes have been identified [17]. In addition 98

MpNPR1-1, has been recently cloned from Malus domestica (apple) [19] while in 99

Brasica juncea, two copies of the NPR1 gene have been identified [20]. In rice, three 100

homologous NPR1-like genes, OsNPR1/NH1, OsNPR2/NH2 and OsNPR3, have been 101

isolated [40]. OsNPR1 is induced not only after treatment with the rice pathogens 102

bacterial blight Xanthomonas oryzae pv. oryzaerice and blast Magnaporthe grisea, 103

but also by benzothiadiazole, methyl jasmonate (MeJA) and ethylene [40]. Despite 104

these reports, information about existence and expression of NPR1-like genes in 105

monocot plants is still very limited. 106

The aim of this study was therefore to isolate and characterize expression of 107

NPR1-like genes from banana following SA, MeJA and Fusarium treatment. 108

Fusarium wilt caused by Foc is one of the most destructive diseases known in banana 109

and a major threat to the international banana industry [26]. Results show that the two 110

newly isolated NPR1-like genes, MNPR1A and MNPR1B, greatly differed in their 111

expression due to elicitor and Foc treatment in two banana cultivars, a relatively 112

tolerant cultivar GCTCV-218 and a more-sensitive cultivar Grand Naine. 113

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2. Results 115

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2.1. Identification and isolation of NPR1-like genes 117

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PCR amplification using primer pairs designed to amplify the highly conserved 119

ankyrin repeat region of known NPR1 gene sequences yielded two distinct products of 120

approximately 570bp. By applying a combination of the 3’ RACE and 5’-end genome 121

walking techniques, full-length sequences were isolated. MNPR1A (GenBank 122

accession no. DQ925843) had a full length of 1927 bp and MNPR1B (GenBank 123

accession no. EF137717) had a full length of 2073 bp. The complete amino acid 124

sequences of the two gene sequences displayed 78% identity. Further, these full 125

length sequences had the highest identity with the rice NPR1 gene sequence, 63% for 126

MNPR1A and 65% for MNPR1B (table I). A preliminary phylogenetic analysis using 127

bootstrap consensus for neighbour joining, maximum parsimony and maximum 128

likelihood revealed that MNPR1A and MNPR1B grouped closely with other monocot 129

plants, such as rice and maize, for which NPR1 gene sequences have already been 130

identified (data not shown). 131

Detailed analyses of the two MNPR1 sequences revealed that the two sequences 132

harbour a BTB/POZ zinc finger domain and the ankyrin repeat domain (figure 1), 133

typical features of NPR1 genes which are highly conserved across many species. 134

However, there is a relative positional change and some amino acid dissimilarities 135

occur in these domains between the two isolated sequences. The BTB/POZ domain of 136

MNPR1A was identified at amino acid positions 58 to136 while the ankyrin repeats 137

were identified at amino acid positions 290 to 365 and 324 to 349, respectively (figure 138

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1). In comparison, the BTB/POZ domain of MNPR1B occupies amino acid positions 139

65 to 148 and the ankyrin repeats are found at positions 302 to 377 and 336 to 361. 140

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2.2. Elicitor-induced MNPR1 and PR gene expression 142

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SA and MeJA treatment induced MNPR1A and MNPR1B gene expression in both the 144

tolerant GCTCV-218 and the sensitive Grand Naine banana cultivars (figure 2A and 145

2B). However, MNPR1A expression was not significantly (P<0.01) different from 146

basal levels of expression in both banana cultivars at the start of the treatment (figure 147

2A). MNPR1B expression was significantly induced 1.3-fold (P<0.01) 12 h after SA 148

treatment in Grand Naine and in GCTCV-218, MNPR1B was significantly elevated by 149

3.2 fold (P<0.01) 24 h after treatment (figure 2B). In general, MeJA-induced 150

MNPR1B expression was much lower than SA-induced MNPR1B expression. When 151

plants of both cultivars were treated with MeJA, a 3.2-fold significant induction 152

(P<0.05) in MNPR1A expression was observed at 12 h after MeJA treatment, 153

followed by a decline in expression in GCTCV-219 when compared to expression at 154

the beginning of the treatment (figure 2C). Such a significant increase in expression 155

(3.5-fold) (P<0.05) at the same time point followed by a decline in expression was 156

also observed for MNPR1B in GCTCV-219 (figure 2D). In contrast, in Grand Naine 157

no significant induction of MNPR1A (figure 2C) and a 2.9-fold induction of MNPR1B 158

expression over 48 h (figure 2D) were found. 159

SA and MeJA treatments also induced PR-1 and PR-3 gene expression in 160

GCTCV-218 and Grand Naine (figure 3A and 3B). However, in contrast to SA-161

induced expression in Grand Naine, PR-1 expression in GCTCV-218 significantly 162

increased (P<0.01) at 12 h (1.8-fold), 24 h (5.6-fold) and 48 h (4-fold) after SA 163

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treatment. In Grand Naine, no significant increase in PR-1 expression occurred over 164

48 h. However, PR-3 expression increased after SA treatment in both banana cultivars 165

and expression was significantly higher (P<0.05) in GCTCV-218 at 24 h (1.8-fold) 166

and 48 h (1.5-fold) after SA treatment when compared to Grand Naine (figure 3B). 167

When PR-1 and PR-3 expression was measured in the two cultivars after MeJA 168

treatment, PR-1 expression significantly increased (10.9-fold) (P<0.05) 12 h post 169

MeJA treatment in GCTCV-218 (figure 3C) compared to the expression at 0 h. This 170

was followed by a sharp decline in PR-1 expression. Such an increase in PR-1 171

expression was not found for Grand Naine. In contrast, PR-3 expression significantly 172

increased following MeJA treatment and expression was 13.9-fold (P<0.05) higher at 173

48 h post MeJA treatment when compared to PR-3 expression at the beginning of the 174

experiment (figure 3D). In GCTCV-218, no increase in PR3 expression was found 175

after MeJA treatment. 176

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2.3. Fusarium-induced MNPR1 and PR gene expression 178

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Expression of both MNPR1A and MNPR1B was found due to Foc treatment in 180

both banana cultivars (figure 4A and 4B). However, increase in MNPR1A expression 181

was much higher (1.3-fold) in GCTCV-218 than in Grand Naine. In Grand Naine, 182

MNPR1A expression at 24 hours after infection was significantly 1.9-fold higher 183

(P<0.05) than MNPR1B expression (figure 4A) whereas in GCTCV-218 this increase 184

in expression at 12 hours after infection was 14.7–fold higher (P<0.05) (figure 4B) for 185

MNPR1A compared to MNPR1B. 186

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Both cultivars also expressed PR-1 and PR-3 due to Foc treatment (figure 4C 188

and 4D). However, Grand Naine expressed significantly more PR-3 than PR-1 with a 189

1.5-fold difference in expression (P<0.05) 24 hours after infection (figure 4C), 190

whereas GCTCV-218 expressed significantly more PR-1 than PR-3 with a 3.9–fold 191

difference in expression (P<0.05) (figure 4D) 12 hours after infection. However, this 192

increase in PR-1 expression in GCTCV-218 was followed by a sharp decline to near 193

basal levels. 194

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3. Discussion 195

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This is the first report on the isolation of NPR1-like gene sequences from banana 197

and their expression due to elicitor and fungal pathogen treatments. We have isolated 198

two distinct sequences; MNPR1A and MNPR1B, from Cavendish banana and both 199

have the typical features of other previously described NPR1-like gene sequences. 200

This includes two identifiable protein-protein interaction motifs; a zinc finger and 201

ankyrin repeat domains (ARD) [8, 13, 28]. Further, the two sequences share a 78% 202

similarity in their amino acid sequence but vary in their sequence from previously 203

described NPR1-like gene sequences [17]. Our preliminary data also show that the 204

two banana sequences also group more closely with other monocot NPR1 sequences 205

but less with known dicot sequences (Endah, unpublished results). 206

So far, we have no knowledge of the genomic origin of the two banana 207

sequences. Cultivated banana plants in the genus Musa, such as Grand Naine (AAA), 208

are derived from the wild diploid banana species M. acuminata and M. balbisiana 209

[22] contributing either the A or B genome, respectively. Cultivars resulting from this 210

hybridisation are either diploid (AA, AB, BB), triploid (AAB, AAA, ABB), or 211

tetraploid (AAAB, AABB, ABBB) [22]. In Brassica juncea, there is evidence that the 212

two versions of NPR1 originate from two individual parental genomes (B. rapa and B. 213

nigra) [20]. However, since Grand Naine (AAA) only contains the A genome, this 214

genome has very likely contributed both MNPR1 gene sequences. We currently 215

speculate that the two sequences could be part of a greater NPR1 gene family in 216

banana and are possibly involved in a variety of pathogen defence mechanisms like 217

other NPR1-like gene sequences [4, 17]. Alternatively, intra-specific and inter-218

specific hybridisation of subspecies belonging to the Musa genus might have 219

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contributed to the overall genome of Cavendish banana resulting in a very complex 220

genome [5, 10] in which the A genomes are not identical. 221

This study further showed that MNPR1A and MNPR1B are expressed in banana 222

after SA and MeJA elicitor treatment. This result is consistent with findings of other 223

research groups that NPR1 is expressed when plants sense SA, MeJA or pathogen 224

attack [8-9, 18, 25, 40]. However, in comparison to MNPR1A, MNPR1B was highly 225

responsive to SA-treatment in both banana cultivars and to a much smaller degree to 226

MeJA treatment. In a previous study with B. juncea plants, JA was ineffective in both 227

NPR1 and PR-1 expression [20]. However, in a recent study expression of the rice 228

OsNPR1SA was found after MeJA treatment by Yuan et al. [40]. In general, jasmonic 229

acid pathways have been shown to be activated during herbivore and pathogen attack 230

[32]. Further, there is evidence that NPR1 is also involved in cross-communication 231

between SA- and jasmonic acid-dependent defense signalling pathways [24]. 232

In our study, both MNPR1A and MNPR1B expression was associated with 233

greatly increased PR gene expression in the more Foc-tolerant cultivar GCTVV-218. 234

This increase was either gradual, as a response to SA treatment, or rapid followed by 235

a sharp decline as a response to MeJA treatment. A similar expression profile was 236

observed in the response of MNPR1B to MeJA treatment. In contrast, PR-3 was 237

highly responsive to MeJA treatment but only in the more Foc-sensitive cultivar 238

Grand Naine. Future research has therefore to show if MNPR1B, in comparison to 239

MNPR1A, is more prominently involved in PR-3 expression. 240

In this study there was also a clear difference in MNPR1A and MNPR1B 241

expression following Foc treatment. MNPR1A, but not MNPR1B, was more 242

responsive in both cultivars to treatment with Foc. Response to Foc treatment was 243

also earlier and of a higher magnitude in the more Foc-tolerant cultivar GCTCV-218 244

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than in the more Foc-sensitive cultivar Grand Naine. Similar observations were also 245

made on the expression of the two pathogenesis-related genes PR-1 in GCTCV-218 246

and PR-3 in Grand Naine. Less sensitivity to Foc in GCTCV-218 might be partially 247

attributed to a higher and an earlier expression of both MNPR1A and PR-1 in this 248

cultivar after Foc treatment. This response of PR-1 has also been reported for 249

GCTCV-218 after treatment with Foc [36]. There is evidence that necrotrophic 250

pathogens, such as Fusarium, elicit the jasmonic acid/ethylene-dependent pathway, 251

whereas biotrophic pathogens elicit a SA-dependent pathway [23, 33]. Recent 252

analysis in Arabidopsis further revealed that resistance to Fusarium oxysporum 253

requires, besides the ethylene, jasmonic acid, and SA signalling pathways also the 254

NPR1 gene [7]. Since we did not observe a pronounced response by MNPR1A to SA 255

or MeJA treatment in comparison to the high response that was found for MNPR1B 256

after SA treatment, we currently speculate that MNPR1A might be more responsive to 257

the ethylene dependent pathway when treated with Foc. 258

This study has provided first evidence for the existence of a possible NPR1 gene 259

family in banana. We have also shown that the two newly identified MNPR1-260

sequences differ greatly in their expression profile following either elicitor or Foc 261

treatment. Expression of the two gene sequences further related to the expression of 262

two specific PR genes known to be involved in fungal resistance. However, the exact 263

function of the two genes, MNPR1A and MNPR1B, in plant defence response is yet to 264

be elucidated in further studies. As a first step, we are currently investigating if 265

transformed plants over-expressing either MNPR1A or MNPR1B are more resistant to 266

Foc treatment. 267

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4. Materials and methods 268

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4.1. Isolation of banana NPR1-like gene sequences 270

271

For the isolation of NPR1-like gene sequences from banana, PCR primers 272

(forward primer 5'-GAGCTTTTGGATCTCGCACTTGCAGA-3'; reverse primer 5'-273

CCGAGCTCCACTGTTTTGGAGAGTGCT-3') were designed using Primer 3 274

software based on sequence information available for the rice NPR1 gene (GenBank 275

accession no. AY92398). Double-stranded cDNA synthesized from Cavendish banana 276

(Grand Naine) roots was used as a PCR template. For amplification by PCR, a primer 277

annealing temperature of 55 ˚C was used in a standard PCR reaction. 278

A combination of both 5' and 3' Rapid Amplification of cDNA Ends (RACE) 279

and genome walking were applied to isolate full-length cDNA clones of NPR1-like 280

banana sequences. For isolation of MNPR1A, both 5' and 3' RACE was performed 281

using the GeneRacer™ kit according to the manufacturer’s instruction (Invitrogen, 282

USA) along with gene-specific primers. Two nested gene-specific forward primers 5'-283

TGGTGATGACTTGCGGGGAAGATT-3' and 5'-284

TTGCCATGGACATTGCTCGAGTTG-3' and two reverse nested primers 5’-285

AATCTTCCCCGCAAGTCATCACCA-3' and 5'-286

TGCGGGTCTCTTCTTTCAGCTTGC-3' were used to amplify the 3'- and 5'-ends, 287

respectively, of the MNPR1A gene. Both ends were joined by amplifying with 288

forward 5'-CGGCGCGATATGGAAGACAA-3' and reverse 5'-289

GCAGGAGTCAGCAAAAGGAAGC-3' primers that flank the coding region and a 290

portion of un-translated regions (UTRs) of the MNPR1A gene. Similarly, 3' RACE 291

was performed to isolate the 3' end of MNPR1B using two nested gene specific 292

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primers 5'-TGATGGCACATCGGAGTTCACC-3' and 5'-293

GCATCTGGCACGAATGAGAGCA-3'. The 5' RACE, 5' nested, 3' RACE and 3' 294

nested primers were provided with the GeneRacer™ kit (Invitrogen, USA) that were 295

used together with the gene specific primers. The 5' end of MNPR1B was amplified 296

from genomic DNA by genome walking using a series of gene specific and adapter 297

specific primers from a library generated by digestion with different restriction 298

enzymes (EcoRV, PvuII, SmaI, ScaI and StuI) and ligation of adapters according to 299

the method described by Siebert et al. (1995). The coding region and portions of 300

UTRs of MNPR1B were then amplified from a cDNA template using forward 5'-301

TTGGACGACGGCGGTACACG-3' and reverse 5'-302

CAGCATGATCTAGTGGTGTGTCATGG-3' primers. All amplified PCR products 303

were T/A cloned into the pCR4-TOPO cloning vector (Invitrogen) and sequenced 304

using M13 forward and reverse primers. 305

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4.2. Sequence analysis 307

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Sequencing of the inserts was performed by using the BigDye Terminator 309

Cycle Sequencing FS Ready Reaction Kit, v 3.1(Perkin Elmer, Applied Biosystems, 310

USA) in an ABI PRISM 3100 automatic DNA-Sequencer (Applied Biosystems). 311

The BLASTN and BLASTP programs [1] were used for gene sequence similarity 312

searches. Amino acid sequences of selected monocot and dicot NPR1-like sequences 313

were aligned using Clustal W [35] and ExPASy [15] was utilized for the prediction of 314

amino acid features and identification of conserved domains of MNPR1A and 315

MNPR1B. 316

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4.3. Plant material and treatment 318

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Tissue cultured banana plants (cv Grand Naine and GCTCV-218) were 320

hydroponically grown in 250 mL cups in a green house following the method of Van 321

den Berg et al. [36]. Once plants had attained a five leaf stage and had developed a 322

healthy root system, they were challenged with an inoculum of Foc (2.5 x 103 323

condia/mL), 5 mM SA or 5 µM MeJA. Unless stated otherwise, the entire root system 324

was harvested at time points 0, 12, 24, and 48 h post treatment and flash frozen in 325

liquid nitrogen and stored at -80oC. Three plants were used for each time point for 326

every treatment, and for sample collection roots of the three plants were pooled 327

together. The experiment was repeated once. 328

Pathogen infection of the banana plants was done as described in Van den Berg 329

et al. [36]. The entire root system of the control plants was slightly wounded and 2.5 330

mL of sterile distilled water was added to each cup. Control samples were harvested 331

in the same manner as described above. 332

Treatment with SA was performed following a modified method of Anderson et 333

al. [2]. Both the roots and leaves of each plant were sprayed with a 5 mM salicylic 334

acid salt solution until imminent run-off. Plants were kept in a closed Perspex box 335

until time for collection of samples. Control plants were sprayed in the same way with 336

sterile distilled water. Treatment with MeJA was carried out by taping cotton balls 337

containing 400 µL of a 5 µM MeJA solution in ethanol on the roof of a sealed 338

Perspex box in the which banana plants were kept. All SA and MeJA treated samples 339

were collected and stored as described above. 340

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4.4. RNA Extraction and cDNA synthesis 342

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Total RNA was extracted from root material of Grand Naine and GCTCV-218 344

using the Qiagen RNeasy plant mini kit (Qiagen, Valencia, CA) according to the 345

manufacturer’s instructions. Traces of DNA in the RNA samples were eliminated by 346

treating each RNA sample with DNase 1 (Fermentas Life Sciences, Hanover, MD). 347

First strand cDNA was subsequently synthesized from the DNA-free RNA samples 348

by random hexamer primering (Fermentas Life Sciences, Hanover, MD) using the 349

first strand cDNA synthesis kit according to the manufacturer’s instruction (Promega, 350

USA). The quality of the cDNA was verified by amplifying a 170 bp actin fragment 351

(data not shown) with banana actin forward 5’-ACCGAAGCCCCTCTTAACCC-3’ 352

and reverse 5’-GTATGGCTGACACCATCACC-3’ primers [37]. 353

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4.5. Quantitative RT-PCR 355

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Four genes (MNPR1A, MNPR1B, PR-1 and PR-3) were used for expression 357

studies in Cavendish banana plants. The Musa 25s rRNA was used as an endogenous 358

control. Primer 3 was used to design primers from MNPR1A and MNPR1B gene 359

sequences while primer sequences for the amplification of PR-1, PR-3 and Musa 25s 360

rRNA PCR products were obtained from Van den Berg et al. [36]. Primers for 361

MNPR1A were 5’-GTCGGCATTGTACCAACACA-3’ (forward primer) and 5’-362

CAGTGCAGGAGTCAGCAAAA-3’ (reverse primer); MNPR1B 5’-363

AGGTTTGCCCGAACAAGAAG-3’ (forward primer) and 5’-364

TGAGAGGCAACAACTCAGAGAG-3’ (reverse primer). 365

Quantitative real time PCR (qRT-PCR) was performed using the LightCycler® 366

480, 384-well PCR plates and the LightCycler® 480 SYBR Green I Master kit 367

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(Roche Diagnosits, Germany) following the manufacturer’s instructions. All reactions 368

were conducted in triplicate with each PCR reaction consisting of 1 µL of the diluted 369

template (1/10), 1 µM primers, and 5 µL Lightcycler® 480 SYBR-Green I master 370

mix. The reaction volume was adjusted to 10 µL with nuclease-free water. Non-371

template control (NTC) reactions contained water instead of cDNA as template. 372

Cycling consisted of an initial denaturation phase of 10 min at 95°C an amplification 373

phase of 45 cycles each consisting of a denaturation step at 94°C for 5 s, annealing at 374

63°C for 5 s and extension at 72°C for 10 s. Individual PCR products were analysed 375

by melting-point analysis during which samples were heated from 65°C for 10 s to 376

95°C and the decline in fluorescent signals of each individual sample was assessed. 377

378

4.6. Data analysis 379

380

QRT-PCR data was analysed as previously described in the Applied 381

Biosystems, User Bulletin No. 2 [3]. The significance of differences for all treatments 382

and between the two cultivars was analysed by One-way ANOVA and the Tukey 383

Highest Square Difference (HSD) test at p<0.05 using the Statistica software [30]. 384

385

Acknowledgements 386

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We thank Dr. Pamela Ronald for providing the accession number of the rice NPR1 388

gene sequence. This work was financially supported by the Rockefeller Foundation. 389

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510

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Table I. Homology between MNPR1A and MNPR1B and NPR1-like amino acid 512

sequences (% identity) in plants. 513

514

515

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Figure legends 517

518

Fig. 1. Multiple alignment of MNPR1A and MNPR1B with selected plant NPR1-like 519

amino acid sequences. Amino acid sequences were aligned by Clustal W multiple 520

alignment software (Thompson et al., 1994). Identical amino acids are represented 521

with dots. Vertical rectangles represent conserved cysteine residues and horizontal 522

rectangles represent BTB/POZ domain (filled) conserved ankyrin repeat domain in 523

both MNPR1A and MNPR1B. Accession numbers used in the alignments are 524

Capsicum annum (ABG38308.1), Lycopersicum esculentum (AAT57637.1), 525

Nicotiana. tabacum (ABH04326.1), Oryza sativa (NP_001042286.1), Hordeum. 526

vulgare (CAJ19095.1), Arabidopsis thaliana (AAM65726.1), and Brassica napus 527

(AAM88865.2). 528

529 Fig. 2. Relative gene expression levels in roots of plants of Cavendish banana 530

cultivars GCTCV-218 and Grand Naine. Gene expression was determined for 531

MNPR1A and MNPR1B after treatment with 5 mM SA (A) and (B) and 5 µM MeJA 532

(C) and (D), respectively. Samples were collected at 0 h and 12, 24 and 48 hours after 533

treatment with the pathogen or the respective elicitor. The experiment was repeated 534

once; the relative expression was determined by quantitative RT-PCR and expressed 535

relative to a ‘calibrator’, the expression level at 0 h. The relative expression ratios 536

were plotted on the graph. Results are means ± SEM of six individual plants. 537

*Significant difference at P<0.05. 538

25

25

Fig. 3. Relative gene expression levels in roots of plants of Cavendish banana 539

cultivars GCTCV-218 and Grand Naine. Gene expression was determined for PR-1 540

and PR-3 after treatment with 5 mM SA (A) and (B) and 5 µM MeJA (C) and (D), 541

respectively. Samples were collected at 0 h and 12, 24 and 48 hours after treatment 542

with the pathogen or the respective elicitor. The experiment was repeated once; the 543

relative expression was determined by quantitative RT-PCR and expressed relative to 544

a ‘calibrator’, the expression level at 0 h. The relative expression ratios were plotted 545

on the graph. Results are means ± SEM of six individual plants. *Significant 546

difference at P<0.05. 547

548

Fig. 4. Relative gene expression of MNPR1A and PR-1 (dark bars) and MNPR1B and 549

PR-3 (white bars) in Foc-infected roots of Cavendish banana cultivar Grand Naine 550

and GCTCV-218. Samples were collected for analyses at 0 h and 12, 24 and 48 hours 551

after treatment. Relative gene expression of MNPR1A and MNPR1B (A and B) and 552

PR-1 and PR-3 (C and D) was determined and compared in each of the cultivars. The 553

experiment was repeated once; the relative expression was determined by quantitative 554

RT-PCR and expressed relative to a ‘calibrator’, the expression level at 0 h. The 555

relative expression ratios obtained from the only wounded control plants at each time 556

point was subtracted from those of the infected and wounded samples to obtain the 557

effect due to infection only. The expression ratios due to infection were plotted on the 558

graph. Results are means ± SEM of six individual plants. *Significant difference at 559

P<0.05. 560

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Figure 1 564

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A. thaliana M D T T I D G F A D S Y E I S S T S F V A T D N T D S S I V Y L A A E Q V L T G P D V S A L Q L L S N S F E S V F D S P D - - - D F Y S D A K L V L S D - - - - 73MNPR1A . E D N Y L T A . P A F S V . D N . R S V H F A G - - - - - G A S P D - - - P A A . . E . . R R . . D N L G A A . E . . - - D F E L F A . . R I A V E - - - D G 67MNPR1B . E P S Y L T A . T A F S G . D N . S C V H F S G . - A A A A A . P D S A P P A A E . E G . R R . . D H L G . A . Q . . - - D F E . L A . . R I A V G P P G D G 77C. annum . . - S R T A . S . . N D . . G S . S I C C I G G - - M T E S F S P . T S P - - A E I T S . K R . . E I L . . I . . . S S P D F . . F A . . . . . V P - - - - - 70L. esculentum . . - S R T A . S . . N D . . G S . S I C C M . E - - - - - - - - S . T S . - - A . . N S . K R . . E T L . . I . . A S A P D F . . F A . . . . L A P - - - - - 64N. tabacum . . N S R T A . S . . N D . . G S . S I C C I G G G - M T E S F S P . T S P - - A E I T S . K R . . E T L . . I . . A A S P E F . Y F A . . . . . I P G - - - - 73O. sativa . E P P T S H V T N A F S D . D - . A S V E E G G - - - - - - A D . D - - - - - A . . E . . R R . . D N L A A A . R . . E - D F A . L A . . R I A V P G G - - - 64H. vulgare . E A P S S H V T T . F S D C D - . V S M E . - - - - - - - - A . P D - - - - - A . . E . . R R . . D N L A A A . R . . . - D F A . L A . . R F A V P G - - - - 61

A. thaliana - - - G R E V S F H R C V L S A R S S F F K S A L A A A K K E - - - - K D S N N T A A V K L E L K E I A K D - - - - Y E V G F D S V V T V L A Y V Y S S R V R P 142MNPR1A G A P A . . . G V . . . . . . . . . P . . R E V F . E R E G - - - - A L A P - - - - - . R . . . W K L V S G - - - - F V . . Y . A L . . . . G . L . R G . . A . 134MNPR1B G S T P . . . A V . . . . . . . . . I V . R E E F . R R G R G - - T A A A P - - - - - . R M . . . . L V . . - - - - F . . . Y . A L . A . . G . L . T G . . A . 146C. annum - - I . K . I P V . . . I . . . . . P . . . N V F C G K E R K - - - - - - - - - - - - T . . . . . . L M . E - - - - . . A S Y . A L . N . . . . L . . G K . . . 132L. esculentum - - G . K . I P V . . . I . . . . . P . . . N V F C G K D S S - - - - - - - - - - - - T . . . . . . L M . E - - - - . . . S . . A . . S . . . . L . . G K . . . 126N. tabacum - - A . K . I P V . . . I . . . . . P . . . N L F C G K . E K N S - - - - - - - - - - S . V . . . . V M . E - - - - . . . S Y . A . . S . . . . L . . G K I . . 137O. sativa G G G . G D L L V . . . . . . . . . P . L R G V F . R R A A A - - A A G G G G E D G G E R . . . R . L L G G G G E E V . . . Y E A L R L . . D . L . . G . . G D 142H. vulgare - - - A P D L C V . . . . . . . . . P . L R A L F K R R A A A - - A G S A G G A E G - D R V . . R . L L G G - - - E V . . . Y E A L R L . . D . L . . G . . C D 132

A. thaliana P P K G V S E C A D E N - C C H V A C R P A V D F M L E V L Y L A F I F R I P E L V T L Y Q R H L L D V V D K V V I E D T L V I L K L A N I C G K A C M K L L D 221MNPR1A L T . E . C M . V . . E - . R . E . . . . V . . . . V . . . . A S . V . Q . S . . . S . F . . . . . . I L . . . S . D . I . . . . S V . . L . D S S . A . . F N 213MNPR1B L . . A . C A . V . . E - . R . E . . . . . . . . . A . . . . A S S V . Q . A . . . S . F . . . . . G I L . . M A . D . I P . . . S V . K L . D S S . A N . . S 225C. annum S . . D . C V . V . N E - . F . . . . . . . . A . L V Q . . . A S . T . Q . S . . . D K F . . . . . . I L N . A A A D . V M M V . S V . . . . . . . . E R . . S 211L. esculentum A S . D . C V . V . N E - . L . . . . . . . . A . . V Q . . . A S . T . Q . S Q . . D K F . . . . . . I L . . A . A D . V M M V . S V . . . . . . . . E R . . S 205N. tabacum S . . D . C V . V . N E - . S . . . . . . . . A . L V . . . . I S . T . Q . S . . . D K F . . . . . . I L G . A A A D . V M M V . S V . . . . . . . . E R . . S 216O. sativa L . . A A C L . V . . D - . A . . G . H . . . A . . A Q . . F A . S T . Q V A . . T N . F . . R . . . . L . . . E V D N L . L . . S V . . L . N . S . . . . . E 221H. vulgare L . . T A C A . V . . G G . A . . G . H . . . S . . A Q . . F A . S T . Q V G . . A S . F . . . . . . L L . . . E A D N L P L V . S V . . L . N . S . V . . F E 212

A. thaliana R C K E I I V K S N V D M V S L E K S L P E E L V K E I I D R R K E L G L E V P K V K - - - - K H V S N V H K A L D S D D I E L V K L L L K E D H T N L D D A C 297MNPR1A K . I . . A . . . D L . I I T . . . T M T P D I . . Q . M . S . L N . . T V G . E S I N F S D . . . K R I . G . . . N . . V D . . R M . . . . G N . T . . . . . 293MNPR1B K . I D V V . . . D L . T I T . . . K T . P D I . . Q . M . L . L N F . . V G . E S S S F P D . . . K R I . R . . . . . . V D . . R M . . . . G N . T . . . . . 305C. annum S . I . . . . . . . . . I I T . D . A . . N D I . . Q . T . S . T . . D . Q G . V N H G F P D . . . K R I . R . . . . . . V . . L R M . . . . G . . T . . . . Y 291L. esculentum . . I D . . . . . . . . I I T . D . . . . H D I . . Q . T . S . A . . . . Q G . E S N G F P D . . . K R I . R . . . . . . V . . L R M . . . . G . . T . . . . Y 285N. tabacum S . I . . . . . . . . . I I T . D . A . . H D I . . Q . T . S . A . . . . Q G . E S N G F P D . . . K R I . R . . . . . . V . . L Q M . . R . G . . T . . . . Y 296O. sativa . . L D M V . R . . L . . I T . . . . . . P D V I . Q . . . A . L S . . . I S . E N . G F P N . . . R R I . R . . . . . . V . . . R M . . T . G Q . . . . . . F 301H. vulgare . . L . R V . R . D L . . I T . D . A . . L D V I . Q . . . S . I T . . . A S . E D N G F P N . . . R R I L S . . . . . . V . . . R . . . . . G Q . . . . . . F 292

A. thaliana A L H F A V A Y C N V K T A T D L L K L D L A D V N H R N P R G Y T V L H V A A M R K E P Q L I L S L L E K G A S A S E A T L E G R T A L M I A K Q A T M A V E 377MNPR1A . . . Y . . . . . D S . V T . E . . D . E . . . I . . . . I . . . . . . . I . . . . . . . K I . V . . . T . . . R P . D L . . D . . K . V Q . S . R H . K S M . 373MNPR1B . . . Y . . . . . D S . I T . E . . D . A . . . . . . . D F . . . . . . . I . . . . . . . K I . V . . . T . . . R P . D L . . D . . K . . Q . . . R L . K S . . 385C. annum . . . Y . . . . . D A . . T S E . . D . A . . . . . . Q . . . . . . . . . . . . . . . . . K I . V . . . T . . . R P . D L . S D . . K . . Q . . . R R . R L . D 371L. esculentum . . . Y . . . . . D A . . T A E . . D . S . . . . . . Q . . . . H . . . . . . . . . . . . K I . V . . . T . . . R P . D L . S D . K K . . Q . . . R L . R L . D 365N. tabacum . . . Y . . . . . D A . . T A E . . D . A . . . . . . Q . S . . . . . . . . . . . . . . . K I . V . . . T . . . R P . D L . S D . . K . . Q . . . R L . R L . D 376O. sativa . . . Y . . E H . D S . I T . E . . D . A . . . . . . . . . . . . . . . . I . . R . R . . K I . V . . . T . . . R P A D V . F D . . K . V Q . S . R L . K Q G D 381H. vulgare . . . Y . . E H . D S . I T . E . . D I A . . . . . L . . . . . . . . . . I . . R . R D . K I V V . . . T . . . R P . D F . F D . . K . V Q . . . R L . K H G D 372

A. thaliana C N N I P E Q C K H S L K G R L C V E I L E Q E D K R E P I P R D V P P S F A V A A D E L K M T L L D L E N R V A L A Q R L F P T E A Q A A M E I A E M K G T C 457MNPR1A Y F K S T . E G Q A . P . S . . . I . . . . . A E R . D . Q V G E A S A F L . I . G . D . R G R . . Y . . . . . T . . R L . . . M . . R V . . D . . R V D . . L 453MNPR1B Y L R S I . E G E A . P . S . . . I . . . . . A E R . D . Q V G E A S V . L . M . G . D . R G R . . Y . . . . . . . . R L . . . M . . R V . . D . . Q V D . . S 465C. annum F I K S T . E G . S A P . D . . . I . . . . . A E R . D . L L G E A S V . L . M . G . D . R . K . . Y . . . . . G . . K L . . . M . . K V . . D . . Q V D . . S 451L. esculentum F T K S T . E G . S A P . D . . . I . . . . . A E R . D . L L G E A S L . L . M . G . D . R . K . . Y . . . . . G . . K L . . . M . . K V . . D . . Q V D . . S 445N. tabacum F S K S . . E G . S A S . D . . . I . . . . . A E R . D . L L G E A S V . L . M . G . D . R . K . . Y . . . . . G . . K L . . . M . . K V . . D . . Q V D . . S 456O. sativa Y F G V T . E G . P . P . D . . . I . . . . . A E R . D . Q L G E A S V . L . M . G E S . R G R . . Y . . . . . . . . R I M . . M . . R V . . D . . Q V D . . L 461H. vulgare Y F G N T . E G . P . P N D K . . I . . . . E A E R . D . Q L G E A S V . L . L . G . C . R G K . . Y . . . . . . . . R I M . . I . . R V . . D . . Q V D . . L 452

A. thaliana E F I V T S L E P D R L T G T K R T S P G V K I A P F R I L E E H Q S R L K A L S K T V E L G K R F F P R C S A V L D Q I M N C E D L T Q L A C G E D D T A E K 537MNPR1A . . T L G . A T - S H S . . N Q . . A A D L N E T . . T . K . . . L A . M R . . . R . . . . . . . . . . . . . . . I N K . . D - D G S . D F . Y L Q H . A S . G 531MNPR1B . . T L G . T S - N . S . . N Q . . A M D L N E . . . K . K . . . L A . M R . . . R . . . . . . . . . . . . . E . I N K . . D - D . . . E I T G L G H H . S . E 543C. annum . . P L A . I R - K K M A D A Q . . T V D L N E . . . K M K . . . L N . . M . . . R . . . . . . . . . . . . . E . . N K . . D A D . . S E I . Y M G N . . P . E 530L. esculentum . L P L A . M R - K K I A D A Q . . T V D L N E . . . K M K . . . L N . . R . . . R . . . . . . . . . . . . . E . . N K . . D A D . . S E I . Y M G N . . V . E 524N. tabacum . . P L A . I S - K K M V N A Q . . T V D L N E . . . . . K . . . L N . . R . . . R . . . . . . . . . . . . . E . . N K . . D A D . . S E I . Y M G N . . . . E 535O. sativa . . N L G . G A - N P P P E R Q . . T V D L N E S . . I M K . . . L A . M T . . . . . . . . . . . . . . . . . N . . . K . . D - D E - . D P V S L G R . . S A E 538H. vulgare . . T L G . C T - N P P P E - - I . T V D L N D T . . K M K D . . L A . M R . . . . . . . . . . . . . . . . . N . . . K . . D - D E - P E . . S L G R . A S S E 527

A. thaliana R L Q K K Q R Y M E I Q E T L K K A F S E D N L E L G N S S - L T D S T S S T S K S T G G K R S N R K F S H R R R 593MNPR1A - - - . R M . S L . L . D A . P R . . . . . K E . F N K . A - . S S . S . . . . V G - - - I V P T Q R - - - - - - 575MNPR1B - - - . R R . F Q . L . . V . S . . . . Q . K E . F D R . A - . S S . S . . S . T . I D K V C P . K . M R - - - - 592C. annum . Q L . . . . . . . L . . I . T . . . T . . K E . F A K T N - V S S . C . . . . . G V D K P N K L P F R K - - - - 582L. esculentum . Q L . . . . . . . L . . I . S . . . T . . K E . F A K T N - M S S . C . . . . . G V D K P N N L P F R K - - - - 576N. tabacum . Q L . . . . . . . L . . I . T . . . T . . K E . F D K T N N I S S . C . . . . . G V D K P N K L P F R K - - - - 588O. sativa - - - . R K . F H D L . D V . Q . . . H . . K E . N D R . G - . S S . S . . . . I G A I - - - - R P R R - - - - - 582H. vulgare - - - R . R . F H D L H D . . L . . . . . . K E . F A R . A T . S A . S . . . P T V A R N L T G R P R R - - - - - 576

























27

27

587 Figure 2 588 589

590 591 592

28

28

593 Figure 3 594 595 596

597 598 599 600

29

29

601 Figure 4 602 603 604

605 606 607 608

elicitor and fusarium-induced expression of npr1-like genes in banana

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