BnaMPK6 is a determinant of quantitative disease resistance against Sclerotinia sclerotiorum in oilseed rape

Journal Pre-proof BnaMPK6 is a determinant of quantitative disease resistance against Sclerotinia sclerotiorum in oilseed rape Zheng Wang, Feng-Yun Zhao, Min-Qiang Tang, Ting Chen, Ling-Li Bao, Jun Cao, Yu-Long Li, Yan-Hua Yang, Ke-Ming Zhu, Shengyi Liu, Xiao-Li Tan

PII:

S0168-9452(19)31535-3

DOI:

https://doi.org/10.1016/j.plantsci.2019.110362

Reference:

PSL 110362

To appear in:

Plant Science

Received Date:

18 June 2019

Revised Date:

4 November 2019

Accepted Date:

26 November 2019

Please cite this article as: Wang Z, Zhao F-Yun, Tang M-Qiang, Chen T, Bao L-Li, Cao J, Li Y-Long, Yang Y-Hua, Zhu K-Ming, Liu S, Tan X-Li, BnaMPK6 is a determinant of quantitative disease resistance against Sclerotinia sclerotiorum in oilseed rape, Plant Science (2019), doi: https://doi.org/10.1016/j.plantsci.2019.110362

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BnaMPK6 is a determinant of quantitative disease resistance against Sclerotinia sclerotiorum in oilseed rape 1,#

1,#

2,#

1

1

1

Zheng Wang , Feng-Yun Zhao , Min-Qiang Tang , Ting Chen , Ling-Li Bao , Jun Cao , 1 1 1 2, 1, Yu-Long Li , Yan-Hua Yang , Ke-Ming Zhu , Shengyi Liu *, Xiao-Li Tan *

1

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

2

The Oil Crops Research Institute (OCRI) of the Chinese Academy of Agricultural Sciences

These authors contributed equally to this work.

Highlights

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(CAAS), Wuhan, China Correspondence: E-mail: [email protected]；[email protected].

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• BnaMPK6 plays an important role in defense to S. sclerotiorum

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• The activation of ET signaling by BnaMPK6 may play a role in the defense. • Four BnaMPK6-encoding homologous loci were mapped in the B. napus genome, and it was revealed that the BnaA03.MPK6 locus makes an important contribution to quantitative disease

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resistance (QDR) against S. sclerotiorum in natural B. napus population.

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•BnaMPK6 is a previously unknown determinant of QDR against S. sclerotiorum in B. napus.

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Abstract

Sclerotinia sclerotiorum causes a devastating disease in oilseed rape (Brassica napus), resulting in major economic losses. Resistance response of B. napus against S. sclerotiorum exhibits a typical quantitative disease resistance (QDR) characteristic, but the molecular determinants of this QDR are largely unknown. In this study, we isolated a B. napus mitogen-activated protein kinase gene, BnaMPK6, and found that BnaMPK6 expression is highly responsive to infection by S. sclerotiorum and treatment with salicylic acid (SA) or jasmonic acid (JA). Moreover, 1

overexpression (OE) of BnaMPK6 significantly enhances resistance to S. sclerotiorum, whereas RNAi in BnaMPK6 significantly reduces this resistance. These results showed that BnaMPK6 plays an important role in defense to S. sclerotiorum. Furthermore, expression of defense genes associated with SA-, JA- and ethylene (ET)-mediated signaling was investigated in BnaMPK6-RNAi, WT and BnaMPK6-OE plants after S. sclerotiorum infection, and consequently, it was indicated that the activation of ET signaling by BnaMPK6 may play a role in the defense. Further, four BnaMPK6-encoding homologous loci were mapped in the B. napus genome. Using the allele analysis and expression analysis on the four loci, we demonstrated that the locus

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BnaA03.MPK6 makes an important contribution to QDR against S. sclerotiorum. Our data indicated that BnaMPK6 is a previously unknown determinant of QDR against S. sclerotiorum in

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B. napus.

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Keywords: BnaMPK6; Brassica napus; Quantitative disease resistance; Sclerotinia sclerotiorum

1. Introduction

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Sclerotinia sclerotiorum (Lib.) de Bary is a cosmopolitan and devastating necrotrophic fungal plant pathogen. It is capable of infecting over 400 plant species including many important crops, such as soybean, sunflower, peanut, and oilseed rape [1]. On oilseed rape (Brassica napus L.), an

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important oil crop in China that fulfills nearly 50% of vegetable oil requirements of the country, the pathogen causes rotting of pods, leaves and stems, resulting in major losses in yield and

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quality [2, 3]. Research on interactions of this pathogen with host plants is mainly focused on the pathogenicity of the pathogen to host plants [4-12]. However, the molecular bases controlling

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plant defense response to S. sclerotiorum infection remain poorly understood. It has been indicated that mitogen-activated protein kinases (MAPKs or MPKs) are involved in

plant defense response. For example, a well-studied pathway related to plant defense response is MEKK1-MKK4/MKK5- MPK3/MPK6 [13]. In this signaling pathway, flg22, a derived peptide from Pseudomonas aeruginosa flagellin that reveals the presence of the pathogen to the plant, is sensed by a LRR (Leucine-rich repeat) receptor kinase FLS2, which leads to the activation of MPK3 and MPK6, suggesting that the two MPKs play roles in plant immunity [13]. Our previous 2

study has shown that B. napus MPK3 (BnaMPK3) is a key regulator in defense responses to S. sclerotiorum. However, whether BnaMPK6 mediates defense-related signaling pathways and thus regulates resistance to S. sclerotiorum in oilseed rape remains unclear. In the model plant Arabidopsis thaliana, the role of MPK6 has been implicated in pathogen defense. Arabidopsis plants with AtMPK6 suppressed by RNAi were reported to be more susceptible to P. syringae [14]. However, pathogen resistance assays using the T-DNA insertion mutants showed that Arabidopsis mpk6 plants exhibit wild type susceptibility levels to the infection of the bacterial pathogen [15]. Recently, repeated work was done and showed that there

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is a slightly higher bacterial growth in two independent Arabidopsis mpk6 mutants [16]. AtMPK6 was also examined in the interaction of plants with the fungal pathogens such as Botrytis cinerea.

However, it was shown that loss of function in the gene did not affect resistance of plants to the fungal pathogen [17]. These studies suggested that MPK6 positively regulates the defense to

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bacterial pathogens in Arabidopsis thaliana.

The defense role of MPK6 orthologs in other plant species has also been investigated in several

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important crop-pathogen interaction systems. In the tobacco, NbSIPK, Nicotiana tabacum orthologs of AtMPK6, participate in N-gene-mediated resistance to Tobacco mosaic virus [18, 19].

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In rice, OsMAPK6 null mutant, displaying small grains, dwarfism and erect leaves [20], enhance susceptible to Xanthomonas oryzae pv. oryzicola (Xoc) [21], an important bacterial pathogen of

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Oryza sativa. These reports have mainly focused on MPK6’s role in plant defense to bacterium or virus. However, studies on the biological functions of MPK6 in defense to fungal pathogens are limited, and there have been no reports to this date on the role of MPK6 in defense against S.

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sclerotiorum, a generalist fungal necrotroph.

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Plant immune response to generalist necrotrophs often is governed by a complex interplay of minor-effect genes, which results in a full continuum of resistance phenotypes in natural plant populations, designated as quantitative disease resistance (QDR) [22]. It has been shown that genetic resistance to S. sclerotiorum exists in oilseed rape as well as sunflower [23-25], and oilseed rape, as well as other host plants, show symptoms ranging from high susceptibility to relative tolerance to this pathogen [24, 26-28], corresponding to a typical QDR response. QDR is based on complex inheritance, involving numerous genes of small effect. A better understanding 3

of plant QDR genes function is critical for plant breeding, because QDR is frequent in crops and natural plant populations and is often more durable than R-mediated resistance [29]. However, our knowledge of the molecular bases of QDR to S. sclerotiorum in B. napus is very limited. In this study, we revealed the role of BnaMPK6 in defense responses against S. sclerotiorum using both gain- and loss-of-function approaches. Further, we used a natural population to identify elite alleles for resistance improvement to S. sclerotiorum in B. napus. Our data identify BnaMPK6 as a previously unknown determinant of QDR against the fungal pathogen S.

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sclerotiorum in B. napus. 2. MATERIALS AND METHODS 2.1. Plant and fungal materials

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The Asian semi-winter conventional B. napus cultivar Zhongshuang11 (ZS11), exhibiting a modest tolerance to S. sclerotiorum, was used in this study. ZS11 is an elite B. napus cultivar with

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high oil content and high seed production [37]. Plants were grown in a plant growth room under the following growth conditions: 20 ± 2 °C with a photoperiod of 16 h light and 8 h dark at a light

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intensity of 44 μmol/m2/s and 60%–90% relative humidity. Fresh sclerotia of the fungus S. sclerotiorum, collected from oilseed rape stems in the field in Zhenjiang, China, were germinated to produce hyphal inoculum on potato dextrose agar (PDA).

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2.2. Isolation of BnaMPK6 cDNA

Total RNA from B. napus leaf tissues was extracted using TRIzol reagent (Invitrogen). After

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removal of genomic DNA contamination by DNase I (TaKaRa), 200 ng of poly(A)+ mRNA was converted into cDNA by MMLV Reverse Transcriptase (Promega). The cDNA template was used

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for PCR analysis subsequently. The BnaMPK6 cDNA was obtained using the primers BnaMPK6-F1

(5′-ATGGACGGTGGAACGGGT-3′)

and

BnaMPK6-R1

(5′-TCAAACTATTTGCTGATACTCTGGA-3′), cloned into PMD18-T vector and then sequenced. The resulting plasmid pMD18-BnaMPK6 was used as templates for all experiments described below. Both the molecular mass and theoretical isoelectric point (pI) of the polypeptide were determined using the Compute pI/MW tool (https://web.expasy.org/cgi-bin/protparam/protparam). Multiple-aligned sequences were determined by MeGa5, and GENEDOC was used to manually 4

edit the results. 2.3. Plasmid construction for transgenic plant generation For construction of RNAi vector, a 115-bp cDNA fragment of BnaMPK6 was amplified with primers

BnaMPK6-RNAi-F:5′-CACCGGTGGAACGGGTCAAC-3′

BnaMPK6-RNAi-R:5′-ATGGCTAAGTGTCGCCGGAATAT-3′,

and

and

inserted

into

the

pENTR/D-TOPO vector, creating PENTR::BnaMPK6-Ri. Then, the target BnaMPK6-RNAi fragment in PENTR::BnaMPK6-Ri vector was transferred into the destination vector pHellsgate

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12 to generate the BnaMPK6-RNAi vector pHellsgate 12:: BnaMPK6-Ri using the Gateway LR recombinase (Invitrogen, Carlsbad, CA, USA). The inserted sequences were confirmed by

restriction enzyme digestion and sequencing. The resulting vector pHellsgate 12::BnaMPK6-Ri contains a neomycin phosphotransferase II (NPT II) gene in its T-DNA region for selection of

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transgenic plants by Kanamycin and was transformed into the A. tumefaciens GV3101 for plant transformation.

cDNA

was

amplified

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To construct a vector for the constitutive expression of BnaMPK6, the full-length BnaMPK6 with

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(5′-CACCATGGACGGTGGAACGGGTCAA-3′)

primers

BnaMPK6M-F2

and

BnaMPK6M-R2

(5′-CCCTCGAGTCAAACTATTTGCTGA-3′) and subcloned into the TA cloning site of the

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pENTR/D-TOPO vector, creating the intermediate vector PENTR:: BnaMPK6. Then, the target BnaMPK6 cDNA in the PENTR:: BnaMPK6 was transferred into the vector pB2GW7.0 to generate the BnaMPK6-overexpressing vector pB2GW7.0::BnaMPK6 using the Gateway LR

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recombinase (Invitrogen, Carlsbad, CA, USA). The inserted sequence was confirmed by restriction enzyme digestion and sequencing. The resulting vector pB2GW7.0:: BnaMPK6

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contains a bar gene in its T-DNA region for selection of transgenic plants by herbicide Basta and was transformed into the Agrobacterium tumefaciens GV3101 for plant transformation. For the transformation in oilseed rape plants, the plants were grown in a protected field in

Zhenjiang, China, and transformed by in planta Agrobacterium-mediated transformation according to the procedure described by Wang et al. [38]. The transformants were examined as described in the Results section. 5

2.4. Plant inoculation and Chemical treatments Plant inoculation with S. sclerotiorum was performed as described previously [38, 39]. The experiment was in a randomized complete block design and was repeated three times. Six hours after inoculation and at intervals thereafter, the lesion size was determined as the area of the lesion after S. sclerotiorum infection. Chemical treatments with SA or MeJA

were performed as

described previously [40, 41]. The leaves were sprayed with SA (100 μM) and meJA (100 μM) solutions, respectively, on their leaves for 0, 3, 6, 9, or 12 h in the dark. For the ACC treatment,

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the solution was diluted to 100μM and used to treat the leaves. 2.5. Quantitative real-time PCR (qRT-PCR)

Total RNA extractions and cDNA synthesis were performed according to Cai et al. [42].

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

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Real-Time PCR System with three technical replicates for each gene using different cDNAs synthesized from three biological replicates. BnTIP41 (TIP41-like protein gene) was used as

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reference gene [40]. The relative expression level of the target gene was calculated using the comparative CT method (2-∆∆CT method) [43] by normalizing the PCR threshold cycle number (Ct

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value) of the target gene with that of the reference gene. The Ct value was calculated as follows: ∆∆Ct = (CTgene-CTTIP41)treat-(CTgene-CTTIP41)control. Primers used for qPCR are listed in

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Supplementary File S1. These primer sets were tested by dissociation curve analysis and verified for the absence of nonspecific amplification.

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2.6. Statistical analysis

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

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lesion size were subjected to one-way ANOVA of variance followed by a comparison of the means according to a significant difference test at P < 0.05. Using ΔCt values (target – reference), pairwise comparisons relating to PCR were conducted according to Student’s t-test at P < 0.001, 0.001

accessions were grown in the field in Wuhan, Hubei province. The field trials were designed in a randomized complete block design with three replicates. All the accessions were evaluated for resistance to S. sclerotiorum in a naturally-infested nursery. Thirty-six plants from each plot were rated with the disease index, which was performed as described previously (Supplementary File S2) [36], and for the disease incidence at approximately the beginning of physiological maturity. The trait distributions for the disease index and the disease incidence rate were showed in the supplementary File S3 and S4. Genomic DNA is from juvenile leaves of the 324 self-pollinated lines. The polymorphic SNPs

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of genomic homologous loci of B. napus MPK6 and their respective 2500bp upstream regions were genotyped by genomic DNA re-sequencing, and all the re-sequencing data were mapped on

the reference genome "Darmor-bzh" [44]. The SNP variations of 324 lines were acquired using Genome Analysis ToolKit (https://software.broadinstitute.org/gatk/). The genotypic matrix was

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showed in the supplementary File S5. Allele analyses of all SNPs in B. napus MPK6 loci were performed by analysis of variance (ANOVA), and the ratio of phenotypic variance between

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different groups, grouped by different SNP alleles, divided by the total variance was regarded as the SNP phenotypic contribution. Two-tailed ttest was performed to detect the significance

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between the two different allele groups.

2.8. Expression analysis of BnaMPK6 homologous genes

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To determine the changes in the expression of each of BnaMPK6 homologous genes, we analyzed RNA-seq data sets from two B. napus cultivars (Westar and ZY823 that comparatively exhibit susceptible and resistant phenotype to S. sclerotiorum) infected by the fungal pathogen S.

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sclerotiorum, available in Gene Expression Omnibus (GEO; accession number GSE81545) [47], for the expression of BnaMPK6 homologous genes. To investigate the expression level of

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BnaMPK6 homologous genes in different plant tissues, we extracted total RNA from different plant tissues of cv. ZS11. Then these total RNA were sequenced, and the sequenced reads were mapped to the reference genome, and expression quantity was calculated as FPKM [48], as described by Wang et al [34]. 3. Result 3.1. Isolation of BnaMPK6 from B. napus and its sequence analysis 7

To detect the function of BnaMPK6 in defense to S. sclerotiorum, its cDNA was cloned from a cDNA library of B. napus cv. ZS11. The cloned cDNA was predicted to encode a protein of 395 amino acid residues with a putative molecular weight of 44.91 kDa and a predicted isoelectric point of 5.21. The protein sequence is highly similar to MPK6 homologs from various plant species. Further, consistent with other plant MPK6s, the protein sequence shows the same family signature [49], including a conserved phosphorylation motif (TEY motif) and a common docking (CD) domain (Fig.1). Thus, these results showed that the cloned cDNA encodes a MPK6 homolog in B. napus. According to the nomenclature for the Brassica genus and plant MAPKs [50, 51], the

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new MPK6 gene was designated as BnaMPK6. 3.2. Expression pattern of BnaMPK6 during infection of S. sclerotiorum and activation of plant defense responses in B. napus

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To determine whether BnaMPK6 expression is triggered by S. sclerotiorum infection, we

used qRT-PCR to detect its expression in B. napus under S. sclerotiorum infection. The results

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showed that BnaMPK6 expression was significantly up-regulated up to about 5 fold (P < 0.001) within 48 h post inoculation (hpi) with this pathogen (Fig.2A) in the B. napus cv. ZS11, indicating

responses of B. napus.

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that BnaMPK6 responds highly to S. sclerotiorum infection and might be involved in the defense

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The plant hormones, such as salicylates (SA), jasmonates (JA) and ethylene (ET), function as signaling molecules to transduce the stress signals and then complex defense responses are activated. SA-, JA- and ET-mediated defense responses have been shown to play important roles

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in defense against S. sclerotiorum in oilseed rape [52, 53]. In order to determine whether activation of these defense responses affects the expression of BnaMPK6, the S.

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sclerotiorum-induced gene, we investigated its expression after treatments with SA, MeJA and 1-aminocyclopropane-1-carboxylic acid (ACC), the natural precursor of ET biosynthesis. The results showed that BnaMPK6 expression was rapidly induced by SA and MeJA. Especially in the SA induction, BnaMPK6 expression was significantly enhanced about 35 folds at 9 hour post treatment (hpt). In contrast, the expression of BnaMPK6 was not affected by ACC. The results suggested that BnaMPK6 may be a component of the defense response mediated by SA and JA in oilseed rape. 8

3.3. BnaMPK6-RNAi and BnaMPK6-overexpression result in altered resistance against S. sclerotiorum in B. napus In order to better understand the biological function BnaMPK6 in defense to S. sclerotiorum, we examined the effects of the loss or gain of BnaMPK6 function on this defense. We used the RNA Interfering (RNAi) approach to suppress BnaMPK6 expression. The BnaMPK6-RNAi transgenic plants were generated by cloning one 115-bp complementary DNA fragment and its reverse sequence into pHellsgate12 vector under the control of the CaMV (cauliflower mosaic virus) 35S promoter (Fig.3A). The transgenic lines were identified by the screening of kanamycin antibiotics

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and the detection of PCR, and BnaMPK6 expression was tested in these lines. Resultingly, we

acquired three independent BnaMPK6-RNAi lines (RNAi-66, -101 and -111) in which BnaMPK6

expression levels are significantly lower than those in the untransformed wild-type control (WT)

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(Fig.3B), and the three BnaMPK6-RNAi lines were used for further analysis. To generate transgenic lines with overexpression (OE) of BnaMPK6 in B. napus, we cloned the full-length

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cDNA of BnaMPK6 behind the CaMV 35S promoter (Fig.3C) to construct a plant transformation vector and transformed B. napus plants. Basta and PCR were used to screen transgenic lines and

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BnaMPK6 expression was tested in these lines. Finally, we acquired four independent BnaMPK6-overexpression lines (OE-54, -84, -105, and -135) which showed significantly higher expression levels of BnaMPK6 than WT (Fig.5D), and the OE-54, -84 and -135 transgenic lines

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were used for further analysis.

To examine whether altered expression of BnaMPK6 affected the resistance of BnaMPK6-OE

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and BnaMPK6-RNAi plants to S. sclerotiorum, the transgenic antibiotic- and PCR-positive T3 line plants were tested. Disease symptoms, such as chlorosis and necrosis, were noted at 36 h

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post-inoculation (hpt) in both transgenic line plants and WT. As shown in Fig.3E-G, more severe disease symptoms were observed on leaves of BnaMPK6-RNAi plants but less disease symptoms were seen on leaves of BnaMPK6-OE plants, compared with those in WT. Accordingly, investigation of lesion area showed that lesion sizes of all the three tested RNAi transgenic line plants were significantly larger (P < 0.05) than those of WT controls, whereas the three tested OE transgenic line plants were significantly smaller (P < 0.05) (Fig.3H-I). These results indicated that overexpression of BnaMPK6 results in an increased resistance, while downexpression of 9

BnaMPK6 results in a decreased resistance against S. sclerotiorum in oilseed rape. 3.4. BnaMPK6 positively regulates gene expression associated with ET signaling but not SA and JA signaling in response to S. sclerotiorum infection It has been showed that JA, ET and SA defense response contribute to basal resistance against S. sclerotiorum [52-54]. To examine whether the alteration of BnaMPK6 expression affects these defense responses under S. sclerotiorum infection, we detected transcription abundance of the plant defensin gene PDF1.2 and the pathogenesis-related gene PR1, well-known marker genes for the JA/ET-mediated and SA-mediated defense response, respectively, among BnaMPK6-OE, WT

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and BnaMPK6-RNAi, three kinds of line plants exhibiting significant difference in BnaMPK6 expression level, after the pathogen infection. The qRT-PCR analyses indicated that the two

marker genes exhibited enhanced expression in WT controls after S. sclerotiorum infection (Fig.4),

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which is in good agreement with the previous observation [52, 53]. However, the expression

change of BnPDF1.2 was significantly higher in the plants overexpressing BnaMPK6 and was

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lower in the RNAi plants down-expressing BnaMPK6 than in WT controls (Fig.4). Contrastingly, BnPR1 expression was reduced by 30 folds in the BnaMPK6-OE plants (Fig.4). Further, we found

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that BnPAL, the SA biosynthesis gene, exhibited similar expression pattern with BnPR1’s (Fig.4). The results suggested that BnaMPK6 activates the JA- or ET-mediated defense responses but suppress the SA-mediated defense responses in response to S. sclerotiorum infection.

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PDF1.2 serves as a common marker for both JA- and ET-mediated defense responses [55]. To analyze whether JA or ET defense response was activated by BnaMPK6, we investigated the

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expression of genes associated with these defense responses among the three kind plants, exhibiting different expression level of BnaMPK6, after S. sclerotiorum infection. Genes were

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selected to represent the well-characterized JA or ET defense response, including four JA-responsive genes including VSP1 (VEGETATIVE STORAGE PROTEIN1), VSP2, JR1 (JASMONATE RESPONSIVE1) and MYC2, encoding a basic helix-loop-helix transcription factor that plays an important role in the JA pathway, and JA biosynthesis gene AOS, encoding the allene oxide synthase [56]; ET signaling marker gene EIN3, encoding an important transcription factor that specifically responses to ET [57-66], and key ET biosynthesis genes, represented by ACS (1-amino-cyclopropane-1-carboxylic acid synthase) genes because ACS is the rate-limiting 10

enzyme in ethylene biosynthesis pathway [67]. There are three types of ACS isoforms in plants [67]. Here, the Type I ACS genes BnACS1, BnACS2 and BnACS6, the Type II gene BnACS8 and the Type III gene BnACS7 were selected to test whether BnaMPK6 regulates the expression of these ET biosynthesis genes under S. sclerotiorum infection. The qRT-PCR analyses showed that the expression change of BnVSP1, BnVSP2, BnJR1, BnMYC2 and BnAOS was significantly lower in the plants overexpressing BnaMPK6 than in WT controls (Fig.4). Contrastingly, these genes generally exhibited higher expression change in the RNAi plants down-expressing BnaMPK6 (Fig.4). These results suggested that BnaMPK6

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negatively regulate JA defense response in response to S. sclerotiorum infection. On the other hand, the expression change of BnEIN3, the ET signaling marker gene, exhibited significantly

higher in the BnaMPK6-OE plants when compared with that in WT. For the three types of ACS

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isoforms, transcripts of Type I ACS genes BnACS1, BnACS2 and BnACS6 and Type III ACS genes BnACS7 accumulated approximately 35, 309, 53, and 29 folds, respectively, over their basal levels

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in the untransformed WT controls in response to S. sclerotiorum infection (Fig.4). However, when observed on the transgenic plants, we found that their transcripts were further elevated to about 67,

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1684, 339 and 81 folds, respectively, in the BnaMPK6-OE plants, whereas were suppressed in BnaMPK6-RNAi plants exhibiting about 5, 52, 6 and 15 folds, respectively, over their basal levels (Fig.4). In contrast, the Type II gene BnACS8 showed an inverse expression pattern when

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comparing other four BnACSs (Fig.4). These results showed that BnaMPK6 generally positively regulates gene expression associated with ET signaling but not JA signaling when S. sclerotiorum

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infection, suggesting that BnaMPK6, the MPK6 homolog positively regulating resistance to S. sclerotiorum, positively regulates ET defense response in response to S. sclerotiorum infection.

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3.5. Mapping of BnaMPK6-encoding loci in B. napus genome and their allele analyses in QDR to S. sclerotiorum in natural population

For the allotetraploid species B. napus, its nuclear genome consists of two genomes, A and C,

originating from a hybridization between B. rapa (2n = 20, AA) and B. oleracea (2n = 18, CC). Consequently, every orthologous gene of Arabidopsis thaliana is usually represented by multiple homologous gene loci in the B. napus genomic DNA, and among these homologs there might be a divergence of roles in contributions to their corresponding phenotypes. 11

We first mapped the BnaMPK6-encoding loci in B. napus genome. We did a BLAT search for the

coding

sequence

(CDS)

of

BnaMPK6

in

B.

napus

genomic

database

(http://www.genoscope.cns.fr/blat-server/cgi-bin/colza/webBlat). The CDS was mapped into four gene loci. The four loci are BnaA03g20470D located in the genomic region from 9728779-9730523 on the chromosome (chr) A03, BnaC03g24480D located in the genomic region of 13735500-13737663 of the chrC03, BnaC04g03080D in the region of 2249100-2251001 of the chrC04, and BnaA05g03550D in the region of 1937140-1939069 of the chrA05, respectively. We analyzed the allelic diversity in the four BnaMAPK6-encoding genes using DNA sequence

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comparison. As shown in Supplementary File S6, the four DNA sequences exhibited highly similar in exons but tremendous variation in introns. Interestingly, further observation showed that BnaA03g20470D and BnaC03g24480D exhibited coincident exon/intron structure, both

containing 6 exons and 5 introns, whereas BnaA05g03550D and BnaC04g03080D exhibited

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coincident exon/intron structure, both containing 5 exons and 4 introns. Further, we found that compared to BnaMPK6 cDNA sequence, BnaA03g20470D and BnaC03g24480D shared common

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insertions in sites 2278, 2281 and 2282 in the figure of Supplementary File S6, whereas BnaA05g03550D and BnaC04g03080D shared common base deletions of sites from 61 to 67.

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Further, sequence comparison in the amino acid sequence level showed that the protein sequences encoded by the four gene loci are highly similar to BnaMPK6. These data suggested that they are

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four BnaMPK6-encoding loci in B. napus genome (Supplementary File S7). Thus, the four loci are named BnaA03.MPK6, BnaC03.MPK6, BnaC04.MPK6 and BnaA05.MPK6, respectively.

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To investigate the link between the cloned BnaMPK6 cDNA and the four loci, the BnaMPK6 cDNA was compared with the coding sequence (CDS) of every BnaMPK6 homologous gene

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acquired from B. napus genomic database, respectively. The results showed that base difference rate between the BnaMPK6 cDNA and the CDS of BnaA03.MPK6, BnaA05.MPK6, BnaC03.MPK6 or BnaC04.MPK6 was, respectively, 7/1188, 114/1185, 52/1188 and 117/1185 except for the base insertions/deletions, and suggested that the BnaMPK6 cDNA may be from the BnaA03.MPK6 locus (Supplementary File S8). The base difference between the BnaMPK6 cDNA and the BnaA03.MPK6 CDS should be caused by the cultivar difference; the cultivar (ZS11) used

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in the study is an Asian semi-winter conventional B. napus cultivar, whereas the reference genome in B. napus genomic database was from the winter-type cultivar ‘Darmor-bzh’. In order to investigate diversity and elite alleles to S. sclerotiorum we performed allele analyses of the four BnaMPK6-encoding loci in a set of 324 B. napus accessions collected from different geographic locations [34, 36]. The set of accessions were tested in a naturally-infested nursery. QDR of these accessions against S. sclerotiorum infection were evaluated using two phenotypic traits, disease incidence rate (DIR) and disease index (DI). These accessions exhibited a variation of DIR from 19% to 100% and a variation of DI from 7.14 to 100, corresponding to a typical

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QDR response. Total 218 SNPs were detected in the four BnaMPK6 homologous genes in the

natural population, and allele analyses results showed that BnaA03.MPK6 made the most

contribution to S. sclerotiorum when compared with the other three genes (Supplementary File S9).

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There were 7 SNPs and 3 SNPs with phenotypic contribution rate more than 10% for DIR and DI

in BnaA03.MPK6, respectively, however, the largest contribution rate was only 5.01% in other

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three loci. We investigated the DIR and DI of different group divided by alleles of BnaA03g20470D_chrA03_9736593, it contributed most variations for the two traits. According to

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the different alleles in this SNP, the set of B. napus accessions was divided into three different subgroups (the accessions missing this SNP is not counted). The three different subgroups included two homozygote subgroups, the CC subgroup and the TT subgroup (the allele in this

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SNP was homozygous CC or TT in the subgroup), and one heterozygote subgroup, the CT subgroup (the allele in this SNP was heterozygous AT in the subgroup). The results showed that

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for the CC subgroup (n=198), the phenotypic values of DI and DIR were 58.62 and 64.20%, respectively. For the TT subgroup (n=83), the DI and DIR were 38.27 and 44.07%, respectively.

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For the heterozygous CT subgroup (n=20), both phenotypic values were in the middle of the two homozygote subgroups. The significance test (two-tail T test) showed that the P values between the two AA and TT subgroups were 6.43×10-10 in the phenotypic trait DI and 3.91×10-10 in the DIR, respectively, reaching extremely significant levels (图等位分析 A 和 B). Further, we found that there were only 83 accessions with the allele TT of disease resistance and 198 accessions with the susceptible allele AA in the natural B. napus population. This indicated that the TT type of this SNP had great utilization in the B. napus resistance breeding. 13

3.6. Expression analysis of BnaMPK6 homologous genesIn order to determine whether BnaMPK6 homologous genes function in defense responses to the necrotrophic S. sclerotiorum, we determined the changes in the expression of each of these genes using transcriptomes of susceptible (Westar) and tolerant (ZY821) B. napus cultivars infected with this pathogen [47]. As shown in Fig. 6, the expression change of BnaA03.MPK6 was significantly higher in the tolerant cultivar than in the susceptible one after infection with S. sclerotiorum. Contrastingly, for other three homologous genes, no significant expression difference was observed between the tolerant and susceptible cultivars. These data indicated that in these BnaMPK6 homologous genes, only

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BnaA03.MPK6 was differentially regulated between the resistant cv. and the susceptible one in response to S. sclerotiorum infection, suggesting a defense role for the BnaMPK6-encoding loci on the chromosome A03.

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Next, transcriptome analysis was carried out to compare the expression level of BnaMPK6 homologous genes in different plant tissues. As shown in Supplementary File S10, the FPKM

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(Reads Per Kilobase of exon model per Million mapped reads) of BnaA03.MPK6 was significantly higher in stem, leaf and silique, suggesting that BnaA03.MPK6 is a major functional gene locus in

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the three tissues, whereas its homologous copies, such as BnaC03.MPK6 and BnaC04.MPK6, exhibit higher expression level in bud, speal, blossomy petal and pistil. It has been known that during infection, S. sclerotiorum firstly establishes an ascospore colonization on the leaves, stems

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or siliques of B. napus, and then necrotic lesions develop on these tissues [1, 72]. Thus, the tissue-specific expression analysis may provide an expression support to the biological functions

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associated with QDR to S. sclerotiorum for BnaA03.MPK6. 4. Discussion

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In this study, we identify the mitogen-activated protein kinase BnaMPK6 as a previously

unknown determinant of QDR to S. sclerotiorum in B. napus. Our data showed that the expression of BnaMPK6 was not only highly response to S. sclerotiorum infection, but it was also induced by SA and JA, two signaling molecules associated with defense against this pathogen [53]. Further, overexpression of BnaMPK6 in oilseed rape resulted in enhanced resistance to S. sclerotiorum, the RNAi transgenic plants reduced the resistance, and the expression of defense responses was generally in an inverse pattern between the BnaMPK6-OE and the BnaMPK6-RNAi plants. 14

Moreover, the allele analysis yielded an additional insight that the elite alleie could be used for resistance improvement to S. sclerotiorum in B. napus. These results explored in detail a clear biological function of BnaMPK6 in the defense against S. sclerotiorum in oilseed rape. Plant QDR to necrotrophs, such as S. sclerotiorum, is governed by a complex interplay of minor-effect genes, which renders identification of the underlying genetic components challenging. To data, genes conferring resistance to S. sclerotiorum in natural crop populations remain poorly documented in B. napus. For example, several genes have been shown to play roles in this resistance by using molecular biological methods, such as gene overexpression (gain-of-function)

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and gene RNAi (loss-of-function) [38, 41, 73, 74], but whether these genes are associated with the phenotype variation in this resistance never were determined in natural population. This limits the

application of these genes in marker assisted breeding. On the other hand, based on a large body

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of mapping information, early and recent studies have predicted several candidate resistance genes/loci for resistance to S. sclerotiorum in various crops, such as sunflower, soybean, bean and

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B. napus [75-81]. However, direct molecular evidences that demonstrate functional roles of these candidate loci/genes in this resistance are still lacking. It has been suggested that many identified

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trait-associated loci have nonfunctional spurious associations [82, 83]. Thus, these candidate loci/genes need be functionally determined by using molecular biological method. Here, our allele analysis data combined with the function identification suggested that BnaMPK6 could be

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developed as a functional molecular marker in marker assistant breeding for the improvement in resistance to S. sclerotiorum in oilseed rape.It has been well-known that whole-genome

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triplication (WGT), occurred in Brassica species after the divergence from Arabidopsis, and later allopolyploidy resulted in that every ortholog gene of Arabidopsis has multiple highly

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homologous copies in allotetraploid B. napus. However, the expression and biological functions of homologous

genes

are possibly subject

to

subfunctionalization,

and

even

produce

neofunctionalization with the constant evolution of polyploid species [84]. In the transcriptome analysis, compared with BnaA03.MPK6 predominantly expressing in stem, leaf and silique, other three homologous genes mainly express in other different tissues. Besides, among the four encoding-MPK6 homologous genes, only BnaA03.MPK6 exhibited different expression between the tolerant cultivar and the susceptible one after infection with S. sclerotiorum. Further, the allele 15

analysis suggested that BnaA03.MPK6 locus exhibited a more contribution to the phenotypic traits when compared with other three homologous loci. Together, these data suggested that subfunctionalization

is

produced

in

these

MPK6-encoding

homologous

copies.

The

subfunctionalization phenomena of the homologous genes have been found in other polyploid species. For example, the homologous genes TaWLHS1 [85] and TaMBD2 [86] have different expression pattern in different tissues in wheat, an allohexaploid species. Previous studies have shown that Arabidopsis MPK6 (AtMPK6), as well as its orthologs in other plant species, mainly function in defense to the bacterial pathogens. For example,

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Arabidopsis mpk6 plants are more susceptible to bacterial Pst [14, 16], but exhibit wild type

susceptibility levels under the infection of the fungal pathogen B. cinerea [17]. Recently, it was showed that overexpression of AP2C1, encoding a Ser/Thr phosphatase that can dephosphorylate

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AtMPK6, leads to impaired OG- and flg22-induced phosphorylation in Arabidopsis, and thus it

was suggested that OG- and flg22-induced defense responses effective against the fungal pathogen

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B. cinerea are mainly dependent on a greater contribution of AtMPK6 [17]. However, clear genetic evidence to demonstrate a role of MPK6 in the defense to fungal pathogens is still lacking.

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In the study, our new data showed BnaMPK6, an MPK6 homologous gene from the important oil crop oilseed rape, plays an important role in the defense against an agriculturally important plant fungal pathogen, S. sclerotiorum, which enlarges the understanding of MPK6 functions in defense

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to fungal pathogens.

It has been suggested that plants can activate distinct defense responses tailored to different

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types of pathogens. On the other hand, appropriate regulation of defense responses is also important for plant fitness, as activation of defense responses has deleterious effects on plant

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tissues [87]. Our results showed that SA and JA can activate the expression of BnaMPK6, and in return BnaMPK6 negatively regulates the expression of genes associated with SA and JA signaling in response to S. sclerotiorum infection, which implied that BnaMPK6 responds to the balance between these defense responses after the pathogen infection. Contrastingly, BnaMPK6 positively regulates the expression of genes associated with ET signaling in response to S. sclerotiorum infection. Previously, studies on Arabidopsis showed that the mutant eto3, affected in the regulation of ET signaling [88], is significantly affected in resistance to S. sclerotiorum, and the 16

block in ET signaling caused by ein2 results in enhanced susceptibility to the necrotrophic fungus [26, 54]. Thus, our results suggested that ET-mediated defense response, but not SA- or JA-mediated defense response, plays a role in the resistance conferred by BnaMPK6. Likewise, resistance to the necrotrophic B. cinerea requires ET defense response, as the mutant ein2 showed enhanced susceptibility [89]. However, for another necrotrophic fungal pathogen, Alternaria brassicicola, the mutation ein2, which blocks ethylene signaling, has no effect on resistance [89, 90]. In summary, our study revealed that BnaMPK6 play an important role in defense to S.

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sclerotiorum, and also give an indication that the activation of ET signaling by BnaMPK6 may play a role in the defense. Our allele analysis data combined with the function identification

suggested that BnaMPK6 is a new actor of plant QDR against a fungal pathogen in natural crop

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populations. This may be important for breeders to understand the genetics of resistance available

Conflict of interest

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ACKNOWLEDGEMENTS

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The authors declare no conflict of interest.

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in germplasm to develop varieties with greater resistance.

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

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

17

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Figure legends Fig.1 Sequence alignment of BnaMPK6 and its homologs. The amino acid sequence of BnaMPK6 was aligned with those of the 8 closest matching proteins from a BLAST search. Identical amino acids are shown in black boxes, and similar amino acids are shown in grey boxes. The highly conserved domain TEY and the CD-domain are shown in grey square frame. Species abbreviations are as follows: Bna: Brassica napus; Bo: Brassica oleracea var. oleracea; Br: Brassica rapa; At: Arabidopsis thaliana; Bd: Brachypodium distachyon; Os: Oryza sativa; Th:

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Tarenaya hassleriana; Pc: Petroselinum crispum; Rs: Rhinolophus sinicus.

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Fig.2 Expression pattern of BnaMPK6 during infection of S. sclerotiorum and activation of plant defense responses in B. napus. A, Response of BnaMPK6 to S. Sclerotiorum infection in B. napus.

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Relative expression levels of BnaMPK6 in B. napus were determined by real-time quantitative PCR at 0, 12, 24, 36 and 48 h post S. sclerotiorum inoculation (hpi). B, Expression of BnaMPK6 during activation of defense responses in B. napus. Relative expression levels of BnaMPK6 in B. napus were determined by real-time quantitative PCR at 0, 3, 6, 9 and 12 h post various chemical treatments

(hpt).

SA,

salicylic

acid;

MeJA,

methyl

jasmonate;

ACC,

1-aminocyclopropane-1-carboxylicacid. Values are means of three biological replicates. The error bars show the standard deviation. The significances of the gene expression differences between 25

each time point and the 0-h time point are indicated (Student’s t-test, *** P < 0.001, ** 0.001

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0.01 or * 0.01< P <0.05).

Fig.3 BnaMPK6-RNAi and BnaMPK6-overexpression result in altered resistance against S.

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sclerotiorum in B. napus. A, Diagram of T-DNA of the plasmid used in this RNAi analysis. CaMV35S, cauliflower mosaic virus 35S promoter; NOS, terminator. NosP, NOS promoter; NosT, Nos Terminator; NPTII, kanamycin resistance gene; attR1 and attR2 are Gateway vectors recombination site. LB, left border; RB: right border. B, Validation of BnaMPK6-RNAi lines at transcription levels revealed by real-time quantitative PCR (qRT-PCR). C, Diagram of T-DNA of the plasmid used in this overexpression analysis. CaMV35S, cauliflower mosaic virus 35S promoter; Bar, biolaphos resistance gene; attR1 and attR2 are Gateway vectors recombination site. 26

LB, left border; RB: right border. D, Validation of BnaMPK6-overexpressing lines at transcription levels revealed by real-time quantitative PCR (qRT-PCR). E-G, Disease responses of wild-type (WT), BnaMPK6-overexpressing and BnaMPK6-RNAi plants 48 hour post-inoculation (hpi) with S. sclerotiorum. H-J, Lesion area measurements in WT, BnaMPK6-overexpressing and BnaMPK6-RNAi plants 48 hour post-inoculation (hpi) with S. sclerotiorum. Data presented are the means ± standard deviation from three independent experiments and * above the columns

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indicate significant differences at p < 0.05 level between WT and the transgenic plants.

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Fig.4 BnaMPK6 positively regulates gene expression associated with ET signaling but not SA and JA signaling in response to S. sclerotiorum infection. Changes in expression of the genes involving in SA, JA and ET defense response and biosynthesis in the BnaMPK6-overexpressing transgenic

line

54

(OE-54),

the

untransformed

wild-type

(WT)

control

and

the

BnaMPK6-RNA-interfering transgenic line 66 (RNAi-66) plants after 36 hour post-inoculation (hpi) with S. sclerotiorum. Samples were collected at the indicated time for total RNAs isolation. 28

Expressions of these genes were quantified by real-time PCR, and then change of gene expression (folds of change relative to the level before inoculation) was calculated. Values are means of three biological replicates, and error bars indicate standard deviations. These experiments were repeated with the BnaMPK6-overexpressing lines 84 and 135, and the BnaMPK6-RNAi lines 101 and 111,

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and results are similar.

Fig.

5

The

allele

analysis

of

the

largest

phenotypic

contribution

SNP,

BnaA03g20470D_chrA03_9736593, for disease index (left) and disease incidence rate (right).

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Fig. 6 Expression analysis of BnaMPK6 homologous genes in B. napus cultivars infected with S. sclerotiorum. The Bar charts, generated based on transcriptomic data (GEO accession number

GSE81545), shows changes in expression of four BnaMPK6-encoding homologous genes in

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leaves of susceptible (Westar) and tolerant (ZY821) B. napus cultivars after 24 hour

post-inoculation with S. sclerotiorum. Values are means of three biological replicates, and the

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error bars show the standard deviation. The significances of the gene expression differences

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between Westar and ZY821 are indicated (Student’s t-test, * <0.05).

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Supplementary File S1 Primers used for qPCR.

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Supplementary File S2 The disease index used to evaluate the symptom severity at maturity.

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Supplementary File S3 The trait distributions for the disease index in the natural population. Supplementary File S4 The trait distributions for the disease incidence rate in the natural population.

Supplementary File S5 The genotypic matrix of the natural population. Supplementary File S6 The DNA sequence comparison of BnaMPK6 homologous loci. Supplementary File S7 Protein sequence alignment of four BnaMPK6-encoding loci in B. napus. 31

Identical amino acids are shown in black boxes, and similar amino acids are shown in gray boxes. Supplementary File S8 The sequence comparison between the BnaMPK6 cDNA and the coding sequence (CDS) of every BnaMPK6 homologous gene. Supplementary File S9 The allele analysis of SNPs in four BnaMPK6-encoding loci in the natural population. Supplementary File S10 The expression level of BnaA03.MPK6 and its three homologous genes

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in different tissues. The Bar charts were generated based on the transcriptome analysis.

32