Overexpression of Brassica napus NPR1 enhances resistance to Sclerotinia sclerotiorum in oilseed rape

Journal Pre-proof Overexpression of Brassica napus NPR1 enhances resistance to Sclerotinia sclerotiorum in oilseed rape Zheng Wang, Wen-Hua Zhang, Lu-Yue Ma, Xiao Li, Feng-Yun Zhao, Xiao-Li Tan PII:

S0885-5765(19)30330-3

DOI:

https://doi.org/10.1016/j.pmpp.2020.101460

Reference:

YPMPP 101460

To appear in:

Physiological and Molecular Plant Pathology

Received Date: 6 November 2019 Revised Date:

6 January 2020

Accepted Date: 12 January 2020

Please cite this article as: Wang Z, Zhang W-H, Ma L-Y, Li X, Zhao F-Y, Tan X-L, Overexpression of Brassica napus NPR1 enhances resistance to Sclerotinia sclerotiorum in oilseed rape, Physiological and Molecular Plant Pathology (2020), doi: https://doi.org/10.1016/j.pmpp.2020.101460. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Author Statement Zheng Wang: Conceptualization, Methodology, Formal analysis, Writing – original draft, Funding acquisition. Wen-Hua Zhang: Investigation, Data curation, Writing–review and editing. Lu-Yue Ma: Investigation, Resources. Xiao Li: Investigation. Feng-Yun Zhao: Formal analysis, Resources. Xiao-Li Tan: Validation, Project administration, Supervision.

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Overexpression of Brassica napus NPR1 Enhances Resistance to Sclerotinia sclerotiorum in

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Oilseed Rape

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Zheng Wang, Wen-Hua Zhang, Lu-Yue Ma, Xiao Li, Feng-Yun Zhao, Xiao-Li Tan*

4

Institute of Life Sciences, Jiangsu University, 301# Xuefu Road, Zhenjiang 212013, PR China

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*Correspondence: E-mail: [email protected].

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Abstract

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Sclerotinia sclerotiorum causes a devastating disease in oilseed rape (Brassica napus), an important oil

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crop, resulting in huge economic losses. Studies have shown that Arabidopsis thaliana

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NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1(NPR1), a key regulator of salicylic acid

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(SA) signaling, plays an important role in plant defense against pathogens. However, little is known

13

about the B. napus (Bna) NPR1 gene and its role in defense to S. sclerotiorum. In this study, we cloned

14

a new NPR1 homolog (BnaNPR1) from B. napus. The new cloned BnaNPR1 exhibits 68.35% identity

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with AtNPR1 in protein level, and its expression is strongly activated by the SA treatment that, in turn,

16

can enhance resistance to S. sclerotiorum. Further, transgenic Nicotiana benthamiana and B. napus

17

overexpressing BnaNPR1 showed significantly enhanced resistance to S. sclerotiorum. Further

18

experiments showed that after S. sclerotiorum infection, transgenic plants activated the expression of

19

genes associated with SA defense response but suppressed genes associated with JA signaling. These

20

results indicated that BnaNPR1 plays a positive role in resistance of B. napus against S. sclerotiorum,

21

which provides molecular evidence about the positive role of SA signaling in this resistance.

22

Interestingly, it was revealed that the induced expression of BnaNPR1 is suppressed during the S.

23

sclerotiorum infection. Thus, we propose that the strategies for utilization of BnaNPR1 to improve

24

resistance to S. sclerotiorum will be overexpression.

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Keywords: Brassica napus; NPR1; Sclerotinia sclerotiorum; Overexpression; SA signaling

27

2

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1. Introduction

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Oilseed rape (Brassica napus L.) is an economically important oil crop in China and fulfills nearly 50%

30

of vegetable oil requirements of the country [1]. However, sclerotinia disease caused by Sclerotinia

31

sclerotiorum (Lib.) has been the main limit factor for the production of B. napus. S. sclerotiorum is a

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necrotrophic fungal plant pathogen [2, 3]. The infection of S. sclerotiorum to B. napus causes rotting of

33

leaves, stems and pods, which results in serious crop losses. For example, in China, this pathogen

34

causes annual yield losses of 10–20%, and even the yield losses can reach 80% in severely infected

35

fields [2]. Genetic resistance to S. sclerotiorum exists in B. napus as well as in other plant species [2,

36

4-6]. However, the molecular basis of the genetic resistance remains poorly understood in oilseed rape.

37

Plants are able to protect themselves from the pathogen infection through the deployment of various

38

induced defense responses. These defense responses are dependent on a complex network of

39

transduction pathways and are mediated by a number of signaling molecules, including salicylic acid

40

(SA) and jasmonic acid (JA) [7]. Based on studies on the interaction of the model plant Arabidopsis

41

thaliana with pathogens, a general defense model was proposed, in which SA-mediated defense

42

response provides protection from biotrophic pathogens, whereas JA-mediated defense response is

43

against necrotrophs [7]. In the case of the necrotrophic fungus S. sclerotiorum, the results of studies on

44

the host gene expression profiling appear to conform to the general defense model, in which S.

45

sclerotiorum infection induces expression of genes associated with JA defense response in B. napus,

46

while expression of genes associated with SA defense response are not induced [8, 9]. Further, another

47

study with Arabidopsis mutants showed that the impairment in SA signaling did not affect

48

susceptibility to the pathogen [6]. However, roles of SA signaling in defense against S. sclerotiorum

49

are recently challenging, since a study showed that the application of a biologically active analog of SA

50

enhanced resistance to the necrotrophic S. sclerotiorum in B. napus, suggesting a possible positive role

51

of SA signaling in this resistance [10]. This result from the B. napus-S. sclerotiorum pathosystem

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appears to conflict with the general defense model. However, its molecular evidences are still lacking.

53

SA signaling is important not only in plant defense against pathogens, but also in mediating a type of

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broad spectrum, systemic disease resistance known as systemic acquired resistance (SAR) [11, 12].

55

This signaling is dependent on the transcription coactivator “nonexpressor of PR1 genes 1” (NPR1), an

56

important regulator of plant immunity [13]. NPR1 was first cloned from A. thaliana [11, 14]. The

57

typical characteristic of AtNPR1 is that its protein sequence contains an N-terminal BTB/POZ domain, 3

58

a central ankyrin-repeat domain and a C-terminal transactivation domain [15]. AtNPR1 is the receptor

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of SA [16] and assist TGA transcription factors to activate the expression of PR1, the marker gene of

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SA-mediated defense response [12]. The mutation occurred in AtNPR1 block induction of genes related

61

to SA defense response in Arabidopsis plants and, consequently, resulted in enhanced susceptible to

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pathogens [17, 18]. Hence, AtNPR1 is the key positive regulator of SA defense response.

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In fact, researchers have used the Aribidopsis mutant npr1 to investigate the role of AtNPR1 in defense

64

to S. sclerotiorum. In a study, it was reported that npr1 mutant showed enhanced susceptibility to this

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pathogen [19], whereas another study reported that npr1 mutant did not showed increased susceptibility

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[6]. These results from the npr1 mutant are contradictory. Overexpression researches may give a new

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hint. However, the resistance to the pathogen is not yet assessed in Aribidopsis plants overexpressing

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AtNPR1.

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To date, some NPR1 homologs have been cloned from various crop species, and overexpression

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researches of these NPR1 homologs have been performed on many important pathosystems. For

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example, in Arabidopsis plants, overexpression of AtNPR1 was found to be able to enhance resistance

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to pathogens including Pseudomonas syringae, Peronospora parasitica and Erysihe cichoracearum [20,

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21]. In tobacco plants, expressing a gene encoding Malus hupehensis NPR1 resulted in enhanced

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resistance to Botrytis cinerea [22]. Recently, the B. juncea NPR1 homolog was cloned from this crop

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specie and found that its overexpression in this crop confers resistance to Alternaria brassicae and E.

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cruciferarum [23]. In B. napus, however, there has not been a specific report examining whether

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overexpression of B. napus NPR1 (BnaNPR1) affects resistance against S. sclerotiorum, the most

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important pathogen of this crop.

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In this study, a new NPR1 homolog (BnaNPR1) is cloned from B. napus, and its role in the crop for

80

regulating defense response and improving disease resistance against S. sclerotiorum is evaluated by

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using the overexpression approach. These analyses allowed us to i) identify the positive role of

82

BnaNPR1 in resistance against S. sclerotiorum, and ii) provide further molecular evidence about the

83

positive role of SA defense response in this resistance. S. sclerotiorum is the most important pathogen

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of oilseed rape in China as well as other regions of the world. Currently, breeding S.

85

sclerotiorum-resistant oilseed rape cultivars using traditional methodsis is difficult [2, 24, 25].

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Engineering resistance by genetic transformation is pursued as an important strategy to control diseases

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caused by this devastating pathogen. Interestingly, in the induced expression experiment, the 4

88

down-expression of BnaNPR1 was observed in B. napus infected with S. sclerotiorum, which will raise

89

the exploitation value of BnaNPR1 overexpression. Thus, we propose that the strategies for utilization

90

of BnaNPR1 to improve resistance to S. sclerotiorum will be overexpression.

91

2. Materials and methods

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2.1. Plant and fungal materials

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The B. napus cultivar NY12 was used in this study. Plants were grown in a plant growth room. The

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growth condition is set up as described previously [3]. Fresh sclerotia of the fungus S. sclerotiorum,

95

collected from oilseed rape stems in the field in Zhenjiang, China, were germinated to produce hyphal

96

inoculum on potato dextrose agar (PDA) [3].

97

2.2. Isolation of BnNPR1 cDNA

98

Total RNA isolation from leaf tissues treated with SA and cDNA synthesis were performed as

99

described previously [26]. The cDNA was used as template for subsequent PCR. According to the

100

sequence

101

(5′-CTTGTCTCTTGGAGTTTTCAC-3′) and BnaNPR1-R1 (5′-GAATGAGCCAACAATAGACAG-3′)

102

were designed for the PCR amplification of the BnaNPR1 cDNA. The amplified BnaNPR1 cDNA was

103

cloned

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pMD18-BnaNPR1 was used as template for all experiments described below.

105

2.3. SA treatment and plant inoculation

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SA treatment was performed as described previously [26]. Plant inoculation with S. sclerotiorum was

107

performed as described previously [27, 28]. The experiment was in a randomized complete block

108

design and was repeated three times. Twelve hours after inoculation and at intervals thereafter, the

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lesion size was determined as the area of the lesion after S. sclerotiorum infection.

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2.4. Vector construction for transgenic plant generation

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To construct a vector for overexpression of BnaNPR1 expression, the vector pCAMBIA1300-35S-Nos

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was generated as described previously [3]. Then, a 1,740 kb full-length BnaNPR1 cDNA was PCR

113

amplified

114

(5’-ggtaccATGGAGACCATTGCCGGA-3’),

115

(5’-ggatccTCACCGACGCCGGTGAGAGGGTT-3’), and inserted into the KpnI/BamHI sites of

116

pCAMBIA1300-35S-Nos, creating a BnaNPR1-overexpressing vector 1300-35S- BnaNPR1-NOS. The 5

of a

into

B.

rapa

PMD18-T

from

NPR1

vector

its

homolog (XM_009141648.1), the

(Invitrogen)

cDNA

and

clone

then

with

sequenced.

the

primers

The

primers

BnaNPR1-F1

resulting

plasmid

BnaNPR1-F2 BnaNPR1-R2

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inserted sequences were confirmed by restriction enzyme digestion and sequencing. The resulting

118

vector 1300-35S-BnaNPR1-NOS contains a hygromycin-resistant gene in its T-DNA region for

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selection of transgenic plants by hygromycin. The vector was transformed into Agrobacterium

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tumefaciens (GV3101) for plant transformation. For the transformation in oilseed rape plants, the plants

121

were grown in a protected field in Zhenjiang, China, and transformed by in planta

122

Agrobacterium-mediated transformation according to the procedure described by Wang et al. [3]. The

123

transformants were examined as described in the Results section.

124

2.5. Abiotic stress treatments

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Various abiotic stress treatments were performed as described previously [26]. In order to eliminate the

126

influence of circadian rhythms on gene expression, the seedlings were pretreated for 48 hours in the

127

dark. For drought, salt, and heavy metal stress treatments, the seedlings were placed in PEG4000

128

(15 %), NaCl (400 mM), or K2Cr2O7 (500 µM) solutions, respectively, for 0, 1, 3, 6, or 12 hours in the

129

dark. For the cold stress treatments, the seedlings in pots were grown at 4 °C for 0, 1, 3, 6, or 12 hours

130

in the dark [26].

131

2.6. Quantitative Real-Time PCR (qRT-PCR)

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Total RNA extractions and cDNA synthesis were performed according to wang et al. [25]. Quantitative

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PCR was performed using SYBR green real-time PCR master mix in an ABI 7300 Real-Time PCR

134

System with three technical replicates for each gene using different cDNAs synthesized from three

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biological replicates. B. napus TIP41-like protein (BnTIP41) gene was used as reference gene [26]. The

136

relative expression level of the target gene was calculated using the comparative CT method (2-∆∆CT

137

method) [29] by normalizing the PCR threshold cycle number (Ct value) of the target gene with that of

138

the

139

(CTgene-CTTIP41)treat-(CTgene-CTTIP41)control. Primers used for qPCR are listed in Supplementary File S1.

140

These primer sets were tested by dissociation curve analysis and verified for the absence of nonspecific

141

amplification.

142

2.7. Statistical analysis

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Statistical analysis was performed using the SPSS program (SPSS Inc.). The data relating to lesion size

144

were subjected to one-way ANOVA of variance followed by a comparison of the means according to a

145

significant difference tested at P < 0.05. Using ∆Ct values (target–reference), pairwise comparisons

146

relating to PCR were conducted according to Student’s t-test at P < 0.001, 0.001

reference

gene.

The

Ct

value

was

calculated

as

follows:

∆∆Ct

=

147

<0.05 under the assumption that variances are unequal.

148

3. Results

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3.1. Cloning of BnaNPR1 and its sequence analysis

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By a homology cloning approach, a full-length cDNA was cloned from a B. napus cDNA library. The

151

library was constructed from mRNAs of leaf tissues treated with SA. The full-length cDNA contains

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1890 nt (nucleotide) with an entire open reading frame (ORF) of 1740 bp, 5′-untranslated region (UTR)

153

of 61 bp and 3′-UTR of 89 bp. The ORF encodes a protein of 579 amino acid residues with a calculated

154

molecular mass of 64.55 kDa and a predicted pI of 6.00. A BLASTP search in the National Center for

155

Biotechnology Information (NCBI) database showed that the deduced protein sequence exhibits 68.35%

156

identity with AtNPR1 (ABR46027.1). To investigate the typical domain structure of the protein, the

157

CDD analysis was performed in NCBI's Conserved Domain Database, and one BTB/POZ and two

158

ANK conserved domain, the typical characteristics of NPR1, were revealed in the protein sequence

159

(Fig. 1a). Further, a sequence alignment was performed between the protein and AtNPR1. The result

160

showed that the protein contains similar domains or motifs with AtNPR1 (Fig. 1b). Based on above

161

results, the cloned cDNA was designated as BnaNPR1 and submitted to the Genbank with accession

162

number MN646953.

7

163 164

Fig. 1 Sequence analysis of BnaNPR1. (A) The Conserved Domain analysis the BnaNPR1 protein. (B) The

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sequence alignment of BnaNPR1 with AtNPR1. Identical amino acids are shown in black boxes, and similar amino

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acids are shown in gray boxes. The BTB/POZ, ankyrin repeat domains and NPR1_like_C are indicated by black

167

bars below the alignment. Several important motifs are also indicated, such as the IκB phosphodegron motif

168

indicated by the short black bar, LENRV hinge region in the green box, NIMIN1/2bindingsite in the brow box, and

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NLS1 in the red box. The positions of important amino acids in the NLS of AtNPR1 are indicated by red stars

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below the alignment. Bna: Brassica napus; At: Arabidopsis thaliana.

171 172

3.2. Exogenous application of SA strongly activates BnaNPR1 expression and enhances resistance

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to S. sclerotiorum in B. napus 8

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The study with A. thaliana has showed that the expression of AtNPR1 is induced by SA, and ensures a

175

quick activation of SA-mediated defense response (Cao et al., 1998). To examine whether the new

176

cloned BnaNPR1 could be induced by SA, we treated leaves of B. napus with SA and then investigated

177

the expression of BnaNPR1. The results showed that expression of BnaNPR1 was rapidly induced at 3

178

h, peaking within 6 h post treatment (hpt) (19.5-fold) (Fig. 2A). The results showed that BnaNPR1 is

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highly responsive to SA in B. napus.

180

In B. napus, exogenous application of benzothiadiazole (BTH), a functional analog of SA in activating

181

SAR, results in enhanced resistance to S. sclerotiorum [10]. Here, we tested whether the treatment with

182

SA, the BnaNPR1-inducer, can enhance resistance to S. sclerotiorum in B. napus. Six h before SA

183

treatment, leaves of B. napus were inoculated with S. sclerotiorum. Lesions area was measured at 36 h post

184

inoculation. The results showed that necrotic lesions that formed on leaves treated with SA were

185

significantly smaller than on mock-treated leaves (Fig. 2B). Thus, these results suggested that the

186

activation of SA defense response strongly induces BnaNPR1 expression and confers resistance to S.

187

sclerotiorum in B. napus.

188 189

Fig. 2 Exogenous application of SA strongly activates BnaNPR1 expression and enhances resistance to S.

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sclerotiorum in B. napus. (A) Exogenous application of SA strongly activates BnaNPR1 expression in B. napus.

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Relative expression levels of BnaNPR1 in B. napus were determined by real-time quantitative PCR at 0, 3, 6, 9,

192

and 12 h post-treatment (hpt) with salicylic acid (SA). Values are means of three replicates. The error bars show

193

the standard deviation. The significances of the gene expression differences between each time point and the 0-h

194

time point are indicated (***P < 0.001, **0.001 < P < 0.01 or *0.01 < P < 0.05). (B) Exogenous application of SA

195

enhances resistance to S. sclerotiorum in B. napus. Lesions area was measured at 36 h post inoculation. Error bars

196

indicate standard deviations. The difference in lesion size between the Mock and the SA treatnent is significant (P

197

< 0.05).

198 199

3.3. Transient expression of BnaNPR1 enhances resistance of N. benthamiana to S. sclerotiorum 9

200

In order to rapidly estimate the possible role of BnaNPR1 in defense to S. sclerotiorum prior to the

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time-consuming stably transgenic experiment, the gene was transiently expressing in N. benthamiana

202

plants by injecting their leaves with Agrobacterium containing the pCAMBIA1300-35S-BnaNPR1-Nos

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vector that contains the cauliflower mosaic virus (CaMV) 35S promoter, BnaNPR1 cDNA and the

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CaMV Nos terminator in its T-DNA (Fig. 3A). Three days after the injection, the leaves of N.

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benthamiana were inoculated with S. sclerotiorum, and at 36 h post inoculation the size of necrotic

206

lesions

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pCAMBIA1300-35S-BnaNPR1-Nos vector had higher expression level of BnaNPR1 and smaller lesion

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area than the control leaves treated with Mock, showing that overexpression of BnaNPR1 resulted in

209

enhanced resistance to S. sclerotiorum in tomato. These primary results of transient expression led us to

210

conduct stably transgenic experiment in B. napus.

10

was

measured.

As

shown

in

Fig.

3B–D,

the

leaves

treated

with

211 212

Fig. 3 Nicotiana benthamiana transiently expressing BnaNPR1 enhances resistance to S. sclerotiorum. (A)

213

Diagram of T-DNA of the pCAMBIA1300-35S-BnaNPR1-Nos vector used in this analysis. p35S, cauliflower

214

mosaic virus 35S promoter; Hyg(R), the Hygromycin resistance gene; NosT, terminator. (B) Expression of

215

BnaNPR1 in N. benthamiana enhanced resistance to S. sclerotiorum. The leaves a, b and c were treated with the

216

pCAMBIA1300-35S-BnaNPR1-Nos vector solution. The leaves d and e were treated with mock solution. (C)

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Relative expression of BnaNPR1 in the treatment with Mock or the pCAMBIA1300-35S-BnaNPR1-Nos vector. *

218

indicates statistically significant difference between the Mock and the pCAMBIA1300-35S-BnaNPR1-Nos vector

219

treatment is significant (P < 0.05). (D) Lesion area was measured 36 h post-inoculation. Means and standard errors

220

are

221

pCAMBIA1300-35S-BnaNPR1-Nos vector treatment is significant (P < 0.05). The experiment was repeated three

222

times with similar results.

shown.

*

indicates

statistically

significant

difference

between

the

Mock

and

223 224

3.4. Transgenic Brassica napus plants overexpressing BnaNPR1 show enhanced resistance to S. 11

the

225

sclerotiorum

226

In order to further functionally characterize BnaNPR1 in B. napus, we generated stably transgenic

227

B. napus lines overexpressing BnaNPR1 and estimated their resistance to S. sclerotiorum. The

228

pCAMBIA1300-35S-BnaNPR1-Nos vector was transformed into B. napus plants. Five

229

independent transgenic lines (OE-3, -6, -16, -28, and -72) were acquired by hygromycin and PCR

230

screening. The qRT-PCR analysis showed that BnaNPR1 expression levels in lines OE-3 and

231

OE-72 are significantly higher than those in the untransformed control (CK). Thus, the two

232

transgenic lines OE-3 and OE-72 were used for further analysis.

233

To estimate whether overexpression of BnaNPR1 affects the resistance of B. napus to S. sclerotiorum,

234

hygromycin- and PCR-positive transgenic plants of T3 generation were tested. Plant leaves from

235

transgenic lines and CK were inoculated with S. sclerotiorum at the five-true-leaf stage, and then the

236

necrosis lesion sizes were investigated at 36 h post-inoculation (hpi). As shown in Fig. 4B-C, less

237

disease symptoms were seen on leaves of BnaNPR1-OE plants, compared with those in WT.

238

Accordingly, investigation of lesion area showed that lesion sizes of the two tested OE transgenic line

239

plants were significantly smaller (P < 0.05) than those of CK plants (Fig. 4D-E). These results suggest

240

that overexpression of BnaNPR1 significantly enhances resistance to S. sclerotiorum in oilseed rape.

241 242

Fig.4 Overexpression of BnaNPR1 results in enhanced resistance to S. sclerotiorum. (A) Validation of

243

BnaNPR1-overexpressing lines at transcription levels revealed by real-time quantitative PCR (qRT-PCR). The

244

significances of the gene expression differences between each BnaNPR1-overexpressing line and CK are indicated 12

245

(*P < 0.05). (B) and (C) Disease responses of inoculated plants at 42 hour post-inoculation (hpi) with S.

246

sclerotiorum. (D) and (E) Lesion area measurements in CK and BnaNPR1-overexpressing plants 36 hpi with S.

247

sclerotiorum. Data presented are the means ± standard deviation from three independent experiments and * above

248

the columns indicate significant differences at p < 0.05 level between CK and the transgenic plants. CK, the

249

untransformed control; OE-3 and 72 are two independent BnaNPR1-overexpressing transgenic lines.

250 251

3.5. Transgenic Brassica napus plants overexpressing BnaNPR1 affects S. sclerotiorum-induced

252

expression of genes associated with SA and JA signaling

253

NPR1 is known to be an important regulator of defense responses mediated by SA and JA. To

254

investigate if overexpression of BnaNPR1 affects SA and JA defense responses in the interaction of B.

255

napus with S. sclerotiorum, the expression of genes associated with these defense responses was

256

investigated in BnaNPR1-OE and CK plants under the pathogen infection. Four genes associated with

257

SA defense response were selected. They are PAL, ICS1, PR1, WRKY70 and PAD4. Phenylalanine

258

ammonialyase gene (PAL) and Isochorismate synthase gene (ICS1) are two key biosynthesis genes of

259

SA [30-34] PATHOGENESISRELATED1 (PR1) is the well-known marker of SA defense response,

260

WRKY70 encodes a transcription factor that is downstream of NPR1, Phytoalexin deficient 4 (PAD4)

261

encodes a protein that is upstream of NPR1. The results of the qRT-PCR showed that in BnaNPR1-OE

262

plants, the expression of this SA marker gene PR1 was significantly increased when compared with

263

that in CK plants (Fig. 5). BnaWRKY70 showed similar expression profile to BnaPR1’s (Fig. 5).

264

However, the expression of BnaPAD4, BnaICS1 and BnaPAL had not significant differences between

265

BnaNPR1-OE and CK plants. These data suggested that the overexpression of BnaNPR1 increase the

266

SA defense responses at the downstream of SA and PAD4 under the S. sclerotiorum infection.

267

In contrast, the expression of BnaPDF1.2, the well-known marker of JA defense response, was

268

significantly lower in the plants overexpressing BnaNPR1 than in CK plants (Fig. 5). Also, the JA

269

biosynthesis gene AOS (the allene oxide synthase gene) [35] and the JA-responsive gene VSP1

270

(VEGATATIVE STORAGE PROTEIN1) [36] exhibited similar expression pattern to BnPDF1.2 (Fig. 4).

271

These result suggested that overexpressing of BnaNPR1 inhibits the JA defense response under the S.

272

sclerotiorum infection.

13

273 274

Fig. 5 Changes in expression of defense genes associated with SA and JA defense response in CK, and

275

BnaNPR1-overexpressing plants under the S. sclerotiorum infection. Samples were collected at 12 hour

276

post-inoculation (hpi) with S. sclerotiorum for total RNAs isolation. Expressions of these genes were quantified by

277

real-time PCR, and then change of gene expression (folds of change relative to the level before inoculation) was

278

calculated. Values are means of three replicates, and error bars indicate standard deviations. The significances of

279

the gene expression differences between each transgenic line and CK are indicated (Student’s t-test, ***P < 0.001,

280

**0.001 < P < 0.01 or *0.01 < P < 0.05). CK, the untransformed control; OE-3 and 72 are two independent 14

281

BnaNPR1-overexpressing transgenic lines.

282 283

3.6. Inducing expression analysis of BnaNPR1 under S. sclerotiorum and abiotic stresses

284

SA defense response occurs mainly through the coactivator NPR1. To further assess the defensive role

285

of BnaNPR1 to S. sclerotiorum, we detect whether BnaNPR1 responds to this pathogen. We

286

investigated its expression in B. napus plants after the inoculation with S. sclerotiorum. The results

287

showed that the expression of BnaNPR1 was induced at 12 h (2.0-fold) post-inoculation (hpi). However, the

288

inducing expression of BnaNPR1 is rapidly suppressed afterwards during the infection (Fig. 6A). In

289

addition, we detect the expression pattern of BnaNPR1 under various abiotic stresses (Fig. 6B). Upon

290

4°C treatment (cold stress), expression levels of BnaNPR1 rapidly increase at 1 hour post-treatment (hpt)

291

(4-fold) and sharply decline at later time points. Upon PEG4000 treatment (Simulated drought stress),

292

BnaNPR1 is significantly induced at 1 and 3 hpt. Under other abiotic stresses, including NaCl treatment

293

(salt stress) and K2Cr2O7 treatment (Heavy metal stress), down-regulation of BnaNPR1 expression was

294

observed. These results showed that BnaNPR1 also responds to various abiotic stresses.

15

295 296

Fig. 6 Expression analysis of BnaNPR1 under the S. sclerotiorum stress and various abiotic stresses. (A)

297

Expression analysis of BnaNPR1 under the S. sclerotiorum stress. (B) Expression analysis of BnaNPR1 under

298

various abiotic stresses. The significances of the gene expression differences between each time point and 0 hpi or

299

hpt are indicated (Student’s t-test, ***P < 0.001, **0.001 < P < 0.01 or *0.01 < P < 0.05). hpi, hour

300

post-inoculation with S. sclerotiorum; hpt, hour post-treatment with various abiotic stresses. Cold stress means 4°C

301

treatment; Salt stress means NaCl treatment; Drought stress is simulated by PEG4000 treatment; Heavy metal

302

stress means K2Cr2O7 treatment.

303 304

4. Discussion

305

In this study, we provided new data that overexpression of BnaNPR1, a new NPR1 homolog, results in

306

enhanced resistance to S. sclerotiorum, the most important pathogen of B. napus. These new data from

307

BnNPR1 gain-of-function plants, which indicate a positive effect on resistance to S. sclerotiorum, are

308

complementary to those of the negative effect obtained from the AtNPR1 loss-of-function mutant 16

309

which exhibits reduced resistance to the pathogen [19]. In other agriculturally important pathosystems,

310

such as B. juncea-E.cruciferarum or Alternaria brassicae, Oryza sativa-Magnaporthe grisea, Triticum

311

aestivum-Fusarium spp. and Vitis vinifera- Golovinomyces cichoracearum pathosystems, the NPR1

312

homologs from various plant species were also indicated to play positive role in defense [23, 37-39].

313

These data indicated that the NPR1 homologs are involved in broad spectrum of disease resistance.

314

SA-marker gene expression is in an inverse pattern between the B. napus transgenic lines

315

overexpressing BnaNPR1 and the Arabidopsis npr1 mutant. In the Atnpr1 mutant, induction of PR1 is

316

suppressed whereas in BnaNPR1-OE plants, the induction is enhanced, indicating SA defense response

317

activation in NPR1-OE plants and inhibition in npr1 mutant. Similarly, many observations showed that

318

the overexpression of AtNPR1 increased expression PR genes in tomato, grape, tobacco, and rice

319

[39-42]. Exceptionally, NPR1 overexpression in carrot plants did not enhance the expression of PR

320

genes under normal conditions [43]. Moreover, down-regulation of JA marker genes was observed in

321

BnaNPR1 transgenic plants after the S. sclerotiorum infection. Similar observations also occurred in

322

the B. juncea plants overexpressing BjNPR1 under normal conditions [23]. The future study would

323

focus on knock-down of BnaNPR1 that will have new insights into the precise functions of the

324

BnaNPR1 gene in regulating defense responses to S. sclerotiorum in B. napus.

325

Resistance towards necrotrophic pathogens was usually suggested to be independent of the SA defense

326

response [7]. Recently, a study suggested a positive role for SA-mediated response in defense of B.

327

napus to necrotrophic S. sclerotiorum by using the pharmacological experiments [10]. However, the

328

evidences reported in the study were never at the gene level. In this study, the results based on

329

BnaNPR1, a key regulatory gene of SA signaling, provided an important molecular evidence to support

330

the view of the positive role of SA in resistance to S. sclerotiorum. In agreement with our result of

331

BnaNPR1 in enhanced resistance necrotrophic S. sclerotiorum, overexpression of BjNPR1 in B. juncea

332

confers resistance to another necrotrophic fungus, Alternaria brassicae [23]. Similarly, in the case of B.

333

cinerea, another necrotrophic pathogen, SA defense response is involved in the restriction of disease

334

development in Arabidopsis and tomato (Solanum lycospersum) [44, 45]. In contrast, resistance to

335

Alternaria brassicicola, a necrotrophic pathogen, is independent on SA defense response, as several

336

mutants with defects in SA signaling did not reduce resistance to the pathogen [46, 47].

337

Surprisingly, the induced expression of BnaNPR1 was rapidly suppressed during the interaction of B.

338

napus with S. sclerotiorum. This suggests that BnaNPR1 has limited defense role in the actual situation. 17

339

One possible explanation is that S. sclerotiorum inhibits expression of BnaNPR1 to suppress SA

340

defense response. Many studies have reported that S. sclerotiorum can secrete effector protein

341

manipulate and diminish plant defense responses for successful host invasion [48-50]. Similarly,

342

Magnaporthe oryzae can secrete effector protein to manipulate the rice defense system for the infection

343

[51]. Botrytis cinerea was also reported to use SlNPR1, a Solanum lycopersicum NPR1 homlog, to

344

regulate the tomato defense system for enhancing the disease [52]. PaNPR2, an Persea Americana

345

orthologous gene of AtNPR1, was not induced under the infection of Phytophthora cinnamomi [53],

346

and host defense signaling was suppressed by the pathogen [54]. Considering the down-expression of

347

BnaNPR1 during the S. sclerotiorum infection, we propose that the strategies for utilization of

348

BnaNPR1 to improve resistance to S. sclerotiorum will be overexpression. In addition, considering that

349

BnaNPR1 is also responsive to various abiotic stresses, the theme of future research will be the role

350

exploration of BnaNPR1 to combined S. sclerotiorum and abiotic stresses in B. napus.

351

5. Conclusions

352

In summary, this study reports that transgenic B. napus overexpressing BnaNPR1 significantly

353

enhanced resistance to S. sclerotiorum, and argues that the disease resistance of BnaNPR1 transgenic B.

354

napus exposed to S. sclerotiorum may owe to the protection conferred by SA-mediated defense

355

response, thereby providing molecular evidence for the view from previous studies on SA [10]. Further,

356

down-expression of BnaNPR1 during the interaction of BnaNPR1 with S. sclerotiorum will raise the

357

exploitation value of BnaNPR1 overexpression. Thus, BnaNPR1 may serve as an important candidate

358

gene for improving disease resistance by genetic engineering.

359

Acknowledgements

360

This work was supported by National Natural Science Foundation of China (No. 31771836) and

361

National Key Research and Development Program of China (2018YFD0201003).

362

The authors declare no conflict of interest.

363

This article does not contain any studies with human participants or animals (other than insects)

364

performed by any of the authors.

365

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22

Highlights 1. A new NPR1 homolog (BnaNPR1) is cloned from Brassica napus. 2. B. napus plants transformed with BnaNPR1 enhance resistance to Sclerotinia sclerotiorum, the most important pathogen of the crop. 3. BnaNPR1 positively regulates SA defense response, but negatively regulates JA signaling in the interaction of B. napus with S. sclerotiorum. 4. This study provides molecular evidences supporting the positive role of SA signaling in Sclerotinia resistance. 5. Reduced expression of BnaNPR1 in response to S. sclerotiorum indicates that the strategies for

utilization of BnaNPR1 to improve Sclerotinia resistance will be overexpression.

Conflict of Interest All the authors declare no conflict of interest. This article does not contain any studies with human participants or animals (other than insects) performed by any of the authors.