In-silico identification and differential expressions of LepR4-syntenic disease resistance related domain containing genes against blackleg causal fungus Leptosphaeria maculans in Brassica oleracea

Journal Pre-proof In-silico identification and differential expressions of LepR4-syntenic disease resistance related domain containing genes against blackleg causal fungus Leptosphaeria maculans in Brassica oleracea

Mostari Jahan Ferdous, Mohammad Rashed Hossain, Jong-In Park, Arif Hasan Khan Robin, Sathishkumar Natarajan, Denison Michael Immanuel Jesse, Hee-Jeong Jung, Hoy-Taek Kim, Ill-Sup Nou PII:

S2452-0144(20)30012-1

DOI:

https://doi.org/10.1016/j.genrep.2020.100598

Reference:

GENREP 100598

To appear in:

Gene Reports

Received date:

2 December 2019

Revised date:

2 January 2020

Accepted date:

15 January 2020

Please cite this article as: M.J. Ferdous, M.R. Hossain, J.-I. Park, et al., In-silico identification and differential expressions of LepR4-syntenic disease resistance related domain containing genes against blackleg causal fungus Leptosphaeria maculans in Brassica oleracea, Gene Reports (2020), https://doi.org/10.1016/j.genrep.2020.100598

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© 2020 Published by Elsevier.

Journal Pre-proof In-silico identification and differential expressions of LepR4-syntenic disease resistance related domain containing genes against blackleg causal fungus Leptosphaeria maculans in Brassica oleracea a

a,b

a

a,b

Mostari Jahan Ferdous , Mohammad Rashed Hossain , Jong-In Park , Arif Hasan Khan Robin , Sathishkumar a

a

a

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Natarajan , Denison Michael Immanuel Jesse , Hee-Jeong Jung , Hoy-Taek Kim , Ill-Sup Nou

Department of Horticulture, Sunchon National University, Suncheon, Jeonnam, 57922, Republic of Korea

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a

a,*

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Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensing-2202, Bangladesh

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Correspondence: [email protected] (I-S.N.), Tel: +82-61-750-3249; Fax: +82-61-750-3208.

Author’s Email addresses:

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[email protected] (MJF); [email protected] (MRH); [email protected] (JIP); [email protected] (AHKR); [email protected] (SN); [email protected] (DMIJ); [email protected] (HJJ);

Running Title:

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[email protected] (HTK) and [email protected] (ISN).

Investigating Blackleg resistant genes in cabbage

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Journal Pre-proof Highlights No R-gene is known for blackleg (BL) resistance in cabbage (Brassica oleracea).

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Syntenic regions of B. rapa and B. napus R-genes may lead to R-gene in cabbage.

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B. napus blackleg resistant loci LepR4 is investigated in B. oleracea.

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Twelve putative R-genes are mined within LepR4 syntenic region in B. oleracea.

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Expression analysis revealed key genes having roles in BL resistance in cabbage.

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Abstract

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Blackleg, caused by Leptosphaeria maculans, is a devastating disease for which no resistant (R)

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gene is reported in cabbage (C-genome). All known R-genes for blackleg in Brassicaceae have

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been identified in A- or B-genome. However, identification of a few blackleg-resistant cabbage

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genotypes suggest that C-genome crops may also contain R-genes. Since, there is high ancestral synteny between these three genomes, we reasoned that mining the syntenic regions of known

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A- or B-genome R-genes may lead to the corresponding functional orthologues in C-genome.

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Here, we investigated the Brassica napus blackleg resistant-loci LepR4 and identified two syntenic blocks of 9.56 and 1 Mb in chromosomes C03 and C08, respectively, hosting a total of 1137 genes in C- genome. Among these, twelve genes have functional domains such NB-ARC, LRR, CC, TIR, MAP-kinase and S_TKc that have putatively roles in disease resistance. Higher expressions of leucine-rich repeat receptor-like kinase genes Bo3g110840 and Bo3g103150 against two L. maculans isolates in two resistant genotypes and of Serine/Threonine protein kinase genes Bo3g114980 and Bo3g130040 against both isolates in one resistant genotype and against one isolate in both resistant genotypes, respectively suggest their putative roles in 2

Journal Pre-proof conferring resistance to blackleg in cabbage. This approach is auxiliary to conventional mapping based approach and can be replicated for identifying all other A- or B-genome syntenic putative R-genes in C-genome which, upon validation by functional assays, will be helpful in enhancing our understanding and improving blackleg resistance in cabbage.

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Key words: Blackleg, Cabbage, Gene Expression, Leptosphaeria maculans, putative R gene.

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1. Introduction

Blackleg is one of the most devastating diseases of Brassica worldwide (Raman et al., 2013;

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Zhang and Fernando, 2018). While it is particularly devastating to canola (B. napus) (Fitt et al.,

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2006a), it can cause significant economic damage to cabbage (B. oleracea) as well. The first

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epidemic of the disease in cabbage was reported in Wisconsin, USA, and the disease then spread throughout North America and Europe (Dilmaghani et al., 2010). Globally, blackleg is

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caused by the heterothallic ascomycete fungal pathogen Leptosphaeria maculans (Desmaz.)

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Ces. et De Not. [anamorph: Phoma lingam (Tode ex Fr.) Desmaz.]. In Asian countries such as Korea, China, and Japan, the disease is caused by the comparatively less damaging species, Leptosphaeria biglobosa (Hao et al., 2015; Liu et al., 2014b; Zhang et al., 2014). However, the aggressive L. maculans strains have been reported to spread in L. biglobosa predominant areas of the world, raising concern for its introduction into Asian countries (Fitt et al., 2006b; Liu, 2008; Robin et al., 2017) where it would threaten cabbage and canola production. Since, cabbage is an economically important vegetable in these countries where around 38% of

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Journal Pre-proof world’s total cabbage is produced, safeguarding cabbage production against the disease is thus of paramount importance. The pathogen causes two distinct symptoms: leaf lesions and stem canker. Circular to oval ashygray spots with scattered small, dark pycnidia develop on cotyledons, leaves, stems, and petioles. These leaf lesions are found in high numbers in infected fields. Characteristic stem

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canker or blackleg symptoms appear when the fungus systemically attacks vascular tissue;

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turning it black, decaying the pith, girdling the stem at the soil line, and causing brown-black rot

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in the stem and root (Gabrielson, 1983; Henderson, 1918; Williams, 1992). The fungus survives as mycelia, pycnidia, and pseudothecia on infected crop residues (Li et al., 2007b), can

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reproduce both sexually and asexually, and can complete multiple disease cycles in a single

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growing season (Li et al., 2007a). High humidity and moderate temperatures during vegetative

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growth favor disease development (Ghanbarnia et al., 2009). There is extreme global diversity in the pathogenicity of L. maculans strains (Kutcher et al.,

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2011). Based on the pathogenicity reactions on differential cultivars (mainly, B. napus), L.

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maculans strains were initially grouped in three- (Chen and Fernando, 2006) and six-(Kutcher et al., 1993) different pathogenicity groups. Later, more refined race structures were elucidated via pathogenicity tests on differential sets of hosts with known blackleg resistant (R) genes (Balesdent et al., 2005; Dilmaghani et al., 2010; Kutcher et al., 2011; Liban et al., 2015). The R gene mediated sources of resistance against blackleg have been identified mainly in the B genome of B. nigra, B. carinata, and B. juncea (Balesdent et al., 2002; Chèvre et al., 1997; Christianson et al., 2006a) and also in the A genome of B. rapa (Leflon et al., 2007) and B. napus (Chèvre et al., 1997; Delourme et al., 2006; Rimmer and van den Berg, 1992). So far, at least 19 4

Journal Pre-proof race-specific R genes have been identified in the A and B genomes (reviewed in Raman et al., 2013; Zhang and Fernando, 2018). Several of these R genes have been used for developing resistant cultivars in Canada, Australia, and Europe (Raman et al., 2013; Rouxel et al., 2003; Sprague et al., 2006; Yu et al., 2008b, 2013b). In the C genome, however, the presence of such R genes conferring blackleg resistance has not been extensively investigated and R-genes are

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yet to be identified in C genome (Delourme et al., 2006; Rimmer and van den Berg, 1992). A few screens of L. maculans pathogenicity have identified several moderately resistant B. oleracea

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accessions (Ananga et al., 2006; Ferreira et al., 1992; Robin et al., 2017). Very recently, two

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Korean cabbage lines were identified to be highly resistant to L. maculans at cotyledon stage,

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indicating the presence of R-genes in C genome (Robin et al., 2017). Lack of isolates with

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individual Avr genes, however, made it difficult to determine which R gene(s) might be present in these two lines. The R gene(s) could also be identified via mapping and sequencing based

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approaches but those are often time, cost and resource demanding (Delourme et al., 2018;

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Miles and Wayne, 2008). Molecular analyses indicated that plant’s R genes contain domains

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such as NBS (nucleotide-binding site), LRR (leucine-rich repeat), CC (coiled-coil), TIR (toll/interleukin-1 receptor), RLP (Receptor like proteins) and RLK (receptor-like kinases) etc. that have roles in disease resistance (Ellis et al., 2000; Larkan et al., 2013; Liu et al., 2007; Meyers et al., 1999). Such genes of C-genome can be identified by mining the syntenic regions of known A or B genome R genes/QTLs in the C genome since these genomes share high ancestral synteny, common genetic evolutionary footprints, and higher rate of conserved genomic sequences (Chalhoub et al., 2014; Cheng et al., 2012, 2013; Liu et al., 2014a; Murat et al., 2015). This approach have been successfully employed to identify the genes for seed oil 5

Journal Pre-proof content, seed color and Sclerotinia stem rot resistance in Brassica napus using the corresponding known genes/loci of Arabidopsis (Ding et al., 2012; Stein et al., 2013; Wu et al., 2013; Zhao et al., 2012). In case of blackleg resistance, we have already investigated few major known A-or B-genome Rgenes such as Rlm1, LepR3/Rlm2, LepR1 and LepR2 in C genome (I-S Nou, unpublished data). In

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this study, we mined the syntenic region of the B. rapa blackleg resistant gene LepR4 (Yu et al.,

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2013a) in the C genome of B. oleracea, and identified the genes having putative disease

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resistance related domains via comprehensive in-silico and differential expression-analyses in

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the contrastingly resistant cabbage inbred lines against two L. maculans isolates.

2. Materials and Methods

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2.1. Plant materials and growth conditions

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Seeds of two susceptible, SCNU-59 and SCNU-72 (formerly, BN4059 and BN4072, respectively),

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and two resistant, SCNU-98 and SCNU-03 (formerly, BN4098 and BN4303, respectively), Korean cabbage (B. oleracea var. capitata) inbred lines, as identified by Robin et al., (2017), were sown in 32-cell trays using a commercial garden soil mixture in a plant growth room with 20±2°C, 16 h day length, and 400 µmol m−2 s−1 light intensity at bench level. 2.2. L. maculans isolates and inoculation Two L. maculans isolates 00-100s and 03-02s with multiple avirulence (Avr) gene profiles (Robin et al., 2017) collected from Agriculture and Agri-Foods (AAFC Saskatoon, Canada), were used to inoculate the cotyledons of 12-day-old cabbage seedlings. The isolates were cultured on 20% 6

Journal Pre-proof V8 agar at 22°C and 16 h photoperiod under fluorescent light. The spores were suspended in 10 mL sterile distilled water with a sterile plastic scraper prior to filtering with sterile Miracloth (EMD Millipore Corporation, USA). The spore suspensions were adjusted to 2.25×10 7spores mL1

to inoculate small wounds created in the center of the lobe of each cotyledon. Three

replicates were used for each treatment.

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2.3. LepR4-syntenic genes in B. oleracea

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The syntenic genomic region of LepR4, a 5.8-Mb segment of B. rapa linkage group A6, was

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identified in the B. oleracea genome (Ensemble plants database) using SyMap v4.2. All B.

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oleracea genes within that syntenic region were extracted and genes with a putative role in disease resistance were manually identified based on functional annotations. The presence of

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functional domains related to disease resistance were confirmed using the SMART database

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(http://smart.embl-heidelberg.de/) and the Conserved Domain Database (CDD) of NCBI (https://www.ncbi.nlm.nih.gov/cdd/). MEME suite version 5.0.3 (http://meme-suite .org/tools

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/meme) was used to identify conserved motifs in the proteins encoded by these genes. The

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exon–intron distribution of the genes were identified by comparing the coding sequence (CDS) with corresponding genomic sequence of the genes using GSDS2.0 Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/). 2.4. Microsynteny- and protein interaction network- analysis Homologues of the twelve LepR4-syntenic genes of B. oleracea (C genome) were identified in B. rapa (A genome), B. nigra (B genome), and Arabidopsis thaliana by BLAST search. The microsyntenic relationships of these genes were visualized using Circos v0.69 (http://circos.ca/).

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Journal Pre-proof The protein-protein interaction networks of the twelve LepR4-syntenic genes in Brassicaceae were generated using STRING (http://string-db.org) with default settings. 2.5. Total RNA extraction and cDNA synthesis Untreated (0 h), mock- (wounds inoculated with sterile water) and pathogen-inoculated (6 h, 24 h, and 48 h) cotyledons of resistant and susceptible cabbage lines were snap frozen in liquid

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nitrogen and stored at -80°C. Total RNA was extracted from the leaf samples using the ‘RNeasy

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mini kit’ (Qiagen, CA, USA). The purity and concentration of RNA were determined

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spectrophotometrically using a Nanodrop-2000 (Nanodrop Technologies, Wilmington, DE, USA).

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Synthesis SuperMix’ (Invitrogen, CA, USA).

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The total RNA was then converted to first-strand cDNA using ‘SuperScript-III First-Strand

2.6. Expression analysis using qRT-PCR

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Expression levels of LepR4-syntenic genes in B. oleracea were analyzed using qRT-PCR in a

were

designed

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primers

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Roche LightCycler® 96 System (Roche Applied Science, Penzberg, Germany). Gene-specific

https://primer3plus.com,

using

using

the

‘Primer3Plus’

sequence

data

online from

tool

available Ensembl

from Plants

(http://plants.ensembl.org/index.html). For each qRT-PCR reaction, a total volume of 10 μl was prepared containing 5 μl of 2 × qPCRBIO SyGreen Mix Lo-ROX (PCR Biosystems, London, UK), 1 μl each of the forward and reverse primers (10 pmoles), 2 μl of ultra-pure water, and 1 μl of 60 ng μl−1 cDNA as a template. qRT-PCR was performed with initial denaturation at 95°C for 5 min followed by 50 cycles of 95°C for 10 s, 58°C for 10 s and 72°C for 15 s. Three technical replicates of each biological sample were analyzed. Data were processed using LightCycler® 96

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Journal Pre-proof software version 1.1.2.1320 (Roche Diagnostics International Ltd., Mannheim, Germany). Data were normalized to the mean expression value of three B. oleracea actin genes, AF044573, JQ435879, and XM_013753106 as endogenous controls. The normalized relative expression levels were quantified using Livak’s comparative 2−ΔΔCt method. 2.7. Statistical analysis

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Significance tests were performed using analysis of variance (ANOVA) and statistically

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significant differences between treatments were tested using Tukey’s pairwise comparison

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using Minitab® 18 software package (Minitab Inc., State College, PA, USA).

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3. Results

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3.1. Syntenic regions of LepR4 in B. oleracea

Synteny analysis of the LepR4 region (the 5.8 Mb segment between genes Bra017959 and

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Bra024309 of B. rapa linkage group A06) identified four blocks from B. rapa that were syntenic

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to three blocks on chromosomes C03 and C08 of the B. oleracea. The B. rapa block 'A06: 22653778396 bp' was syntenic to the region 'C08: 24655389-25659268 bp' on B. oleracea chromosome C08. The other three B. rapa blocks were syntenic to three blocks on B. oleracea chromosome C03 (Figure 1A and Table 1). Among these three B. oleracea blocks, the smallest block C03: 42614672-43599496 bp (syntenic to B. rapa block ‘A06: 2372557-2717192 bp’) was contained within the largest C03 block (C03: 35372933-44937345 bp). Altogether, a total of 109 and 1028 genes were found within these syntenic regions on chromosome C08 and C03,

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Journal Pre-proof respectively (Figure 1B and Table 1). The list of all genes, along with their functional descriptions, is shown in Table S1. 3.2. Domain organization and motif structures of LepR4-syntenic disease resistance related domain containing genes of B. oleracea Domain analysis of the genes found in the LepR4-syntenic regions identified twelve genes that

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have functional domains associated with disease resistance (Table 2 and Figure 2). Seven

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functional domains, often related to disease resistance, were identified within these twelve

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genes: NB-ARC (nucleotide-binding adaptor shared by APAF-1, certain R gene products, and

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CED-4), LRR (Leucine Rich Repeat), CC (Coiled-Coil), TIR (Toll/Interleukin-1 Receptor), Pkinase (Protein kinase domain), S_TKc (Serine/Threonine protein kinase) and Pkinase_Tyr (Protein

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tyrosine kinase). Five genes Bo3g099380, Bo3g103150, Bo3g110840, Bo3g107230 and

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Bo8g077270 contained three domains, of which the former four contained LRR, Pkinase, and S_TKc domains while the last one contained NB-ARC, TIR, and LRR domains. Genes Bo3g134690

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and Bo3g130040 had Pkinase and S_TKc domains while Bo3g102880 coded for NB-ARC and LRR

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domains. The remaining genes contained only one domain: Bo8g077320 (coiled coil), Bo3g107530 (Pkinase), Bo8g077170 (Pkinase), and Bo3g114980 (Pkinase_Tyr) (Table 2). Analysis of conserved motifs encoded by these twelve genes identified a total of 13 statistically significant and highly conserved motifs, consisting of 11–50 amino acids (Figure 3 and Table 3). Among these, motifs 1, 2, 5, 9, and 12 were associated with STKc, Pkinase_Tyr, and Pkinase domains; motifs 3 and 6 with STKc and Pkinase_Tyr domains; motifs 7 and 10 with STKc domains; motifs 4, 8, and 11 with LRR domains, and motif 13 associated with NB-ARC domains (Figure 3 and Table 3). 10

Journal Pre-proof 3.3. Exon–intron distribution of the identified disease resistance related domain containing genes of B. oleracea Exon–intron structure analysis revealed that the number of exons in the twelve putative NBS, LRR, TIR, CC and RLK genes within the syntenic regions of LepR4 in B. oleracea ranged from a minimum of 1 to a maximum of 16 (Figure 3). The highest 16 exons were identified in the gene

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Bo8g077320 followed by 12, 11, and 10 in Bo3g107230, Bo3g099380, and Bo3g103150,

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respectively. Three genes, Bo8g077270, Bo3g134690, and Bo8g077170 were bi-exonic genes.

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The longest exon were exon-1 (2683 bp) of bi-exonic gene Bo3g134690 followed by exon-2 (2284 bp) of another bi-exonic gene Bo8g077270. The shortest gene Bo3g107530 (261 bp CDS

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and 86 AA) had only one exon and no intron (Figure 3). This exon–intron structure along with

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the motif distributions and domain organization indicate a high level of diversity among these

regions in B. oleracea.

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twelve putative disease resistance related domain containing genes within the LepR4-syntenic

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3.4. Chromosomal location, microsynteny and protein-protein interaction of LepR4-syntenic

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disease resistance related domain containing genes of B. oleracea Among the identified twelve NBS, LRR, TIR, CC and RLK domain containing genes, nine were located on chromosome C03 while three were located on C08 (Figure 4A). The nine genes on chromosome C03 were found in two separate clusters. The large cluster consisted of six genes, Bo3g099380, Bo3g102880, Bo3g103150, Bo3g107230, Bo3g107530, Bo3g110840, and Bo3g114980, within a 5.4 Mb segment (35629681-41005807) of C03 (Figure 4A). Two other genes, Bo3g130040 and Bo3g134690, formed the smaller cluster located 6.5 Mb away from the larger cluster in a 2 Mb (C3:49546291-47473965) segment. The three genes on chromosome 11

Journal Pre-proof C08 are located very close together within a 0.14 Mb segment (C8: 25212759-25355361). These potential disease resistance related domain containing gene rich regions in C03 and C08 may have significance in plant defense responses against blackleg. Microsynteny analysis of the twelve selected genes was conducted to investigate their evolutionary links and genetic relationships with B. rapa, B. nigra, and A. thaliana. We found

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that these twelve genes from B. oleracea were orthologous to 30 genes of B. rapa, distributed

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across all chromosomes except A07 (maximum 10 orthologous pair with A06), 22 genes from A.

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thaliana, and 28 genes from B. nigra (Figure 4B). These results suggest high microsynteny of these genes among these species.

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Protein–protein interaction network analysis of these twelve genes revealed that the LRR and

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MAP-kinase proteins strongly interacted with each other, while CC domain proteins did not

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show any direct interaction with LRR or MAP-kinase proteins (Figure S1). Both LRR and MAPkinases showed strong interactions with transcription factors, such as MADS box TFs, GATA Zn-

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finger containing TFs, and leucine zipper TF as well as serine/threonine protein kinases, G-

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proteins, GTP-binding proteins, cAMP-specific 3,5-cyclic phosphodiesterase, Caspase-1, Ras-2 kinase suppressor, Jun protein, and phosrestin. The MAP-kinase proteins interacted with cyclin dependent kinase inhibitors and phosducin proteins whereas the CC-domain containing protein only interacted with G-protein. 3.5. Expression profiling of the LepR4-syntenic disease resistance related domain containing genes of B. oleracea Expression of the twelve LepR4selected genes in cabbage seedlings varied significantly across resistant vs. susceptible genotypes at different time points after inoculation with L. maculans 12

Journal Pre-proof isolates 00-100s and 03-02s (P<0.001). Among the nine genes located on C03, significantly different expressions in resistant vs susceptible lines were observed for six genes, Bo3g114980, Bo3g130040, Bo3g134690, Bo3g103150, Bo3g110840, and Bo3g107530, while no such differences in expression levels were observed for the three genes located on C08 (Figure 5). Four genes, Bo3g114980, Bo3g134690, Bo3g103150, and Bo3g110840 showed significant

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differences in expression against both 00-100 s and 03-02 s isolates while genes Bo3g130040 and Bo3g107530 showed significant differential expression against only one of these two

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isolates (00-100 s and 03-02 s, respectively).

Gene Bo3g110840 was consistently highly expressed at all three time points (6 h, 24 h, and 48 h)

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against both isolates in SCNU-98, and at 6 h (~4 fold) and 24 h against isolate 03-02 s in SCNU-

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03 (Figure 5). Against isolate 03-02 s, the gene Bo3g103150 was highly expressed in SCNU-98 at

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6 h (~6 fold) and 24 h (~3 fold), and in SCNU-03 at 24 h (~3 fold). In response to 00-100 s, this gene was expressed at ~6-fold higher levels at 6 h in both resistant genotypes. Bo3g114980 was

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highly expressed at 6 h (~2 fold) and 24 h (~6 fold) in response to the isolate 00-100 s, and at 48

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h (~2 fold) against isolate 03-02 s in the resistant genotype SCNU-98 (Figure 5). In response to isolate 00-100 s, the gene Bo3g134690 showed ~3- and ~2-fold higher expressions in SCNU-98 at 6 h and 48 h, respectively. Against isolate 03-02 s, this gene showed a slightly increased expression in both resistant genotypes, SCNU-98 and SCNU-03 at 24 h, and ~2 fold higher expression in SCNU-98 at 48 h. Bo3g130040 was highly expressed in both resistant genotypes at 6 h (~20 and ~12 fold for SCNU-98 and SCNU-03, respectively) and 48 h (~3 fold in both genotypes) only against the isolate 00-100 s. Whereas, the gene Bo3g107530 showed ~5 fold higher expression in SCNU-03 13

Journal Pre-proof against the isolate 03-02 s at 24 h. None of the NB-ARC and CC domain containing genes showed such significant differential expression.

4. Discussion Here, we identified (i) putative disease resistance related functional domain containing genes

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within the syntenic region of B. napus blackleg resistant loci LepR4 in B. oleracea via in-silico

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analysis and (ii) their potential association with blackleg resistance in two resistant cabbage

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lines via differential expression analysis. Identification of sources of resistance in cabbage (Cgenome crop) is necessary for safeguarding the crop against blackleg, a disease that cause

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significant damage to the crop and constitute an eminent threat, particularly to the East Asian

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cabbage industry. Two cabbage lines were previously identified to be highly resistant against

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two L. maculans isolates (Robin et al., 2017). Specific R-gene(s) that may have conferred the resistance could not be determined due to lack of isolate differentials and mapping the R-genes

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were limited by time, resource and expense requirement. We thus resorted to identifying genes

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having putative disease resistance related domains such as NBS, LRR, TIR, CC and RLK etc. within the corresponding syntenic regions of known A-/B-genome R genes in C-genome, since resistance to phyto-pathogens are known to be manifested by these genes (Kourelis and van der Hoorn, 2018). Most of the known blackleg resistance genes are either dominant, including LepR1 on B. napus linkage group N2; Rlm1, Rlm3, Rlm4, Rlm7, and Rlm9 on N7; Rlm2, BlmR2, and LepR3 on N10 (Balesdent et al., 2002; Delourme et al., 2004; Yu et al., 2008b), or partially dominant, such as LepR2 on linkage group N2 (Yu et al., 2005). By contrast, only a few recessive resistance genes 14

Journal Pre-proof have been identified, including rjlm2 in B. napus lines carrying B genome chromosome additions derived from B. juncea (Saal et al., 2004), LMJR2 on LG J18 of B. juncea (Christianson et al., 2006a), and, very recently, LepR4 on A06 of B. napus (F. Yu et al., 2013; Yu et al., 2008b). In this study, we particularly focused on mining the LepR4 syntenic regions in C-genome since it is the first recessive blackleg resistance gene identified in the B. napus (A genome) and is

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known to confer broad spectrum resistance against a range of L. maculans isolates at cotyledon and adult stages (F. Yu et al., 2013). Such broad spectrum and durable resistance conferred by

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recessive resistance genes has been found in barley with mlo (Buschges et al., 1997), in

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Arabidopsis with RRSR-1 (Deslandes et al., 2002) and edr1 (Frye and Innes, 1998), and in B.

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napus by rjlm2 (Saal et al., 2004).

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The LepR4 locus of B. napus linkage group A06 was found to correspond to a 5.78 Mb region in

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chromosome A06 of B. rapa (A6:9074366-14856483) where four nucleotide binding site (NBS)leucine-rich repeat (LRR) encoding genes, Bra018037, Bra018057, Bra018198, and Bra019483

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were identified (Yu et al., 2013a). In the C genome, regions syntenic to LepR4 were found in two

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chromosomes; C03 and C08. Within these LepR4-syntenic regions, twelve genes were identified that have putative disease resistance related functional domains such as NB-ARC, LRR, CC, TIR and RLK etc.LepR4. These domains play important roles in gene-for-gene model of plants’ resistance where specific pathogen effector is recognized by corresponding plants R gene (DeYoung and Innes, 2007; Eitas and Dangl, 2010; Jones and Dangl, 2006). The NBS domain functions as a molecular switch in activating defenses; the ATP-bound form represents the “on” state while the ADP-bound form the “off” state (Takken et al., 2006; van Ooijen et al., 2008) while the LRR domains provide 15

Journal Pre-proof specificity in pathogen recognition, TIR and kinase domains play roles in signaling that ultimately induces downstream defense responses and subsequent activation of innate immune responses (DeYoung and Innes, 2007; Eitas and Dangl, 2010). Genome-wide NBS-LRR genes are now known for a number of crop species including rice, soybean, potato, sorghum, cucumber, melon, apple, papaya, grape and peach etc. (reviewed in

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Alamery et al., 2018; Bayer et al., 2019; Fu et al., 2019). In Brassica species, NBS-LRR genes have been identified by several studies (Alamery et al., 2018; Fu et al., 2019; Golicz et al., 2016; Yu et

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al., 2014). Alamery et al., (2018) reported maximum number of NBS-LRR genes in B. napus (641),

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B. oleracea (443) and B. rapa (249). A total of 176 and 97 NBS-encoding genes of B. napus were

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homologous with B. rapa and B. oleracea, respectively (Fu et al., 2019). In the recent

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pangenome of B. oleracea, a total of 213 RLP, 556 NBS-LRR and 901 RLK and genes have been identified using improved genome assembly, RGA candidate prediction and annotation (Bayer

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et al., 2019). A total 100 R genes (CNL=5 gene; TNL=22 gene; RLP=45 gene and RLK=28) have

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been listed for Brassica oleracea genome in ‘Plant R gene database’, PRGdb (http://prgdb.org/).

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Within the syntenic region of LepR4 loci in B. oleracea, we identified five leucine-rich repeat receptor like kinase genes, four mitogen-activated protein kinase genes and one gene for each of NBS-LRR, NBS-TIR-LRR and C-C domains (Table 2). These genes were found to be clustered in three locations across the syntenic region (Figure 4A). This clustering of putative LepR4-syntenic putative disease resistance related genes is consistent with the observation that plant R genes tend to remain clustered in the genome (Delourme et al., 2004b; Ellis et al., 2000; Michelmore and Meyers, 1998). Most R loci conferring resistance to blackleg in Brassica species were reported to be localized in two major 16

Journal Pre-proof clusters: one in a 35 cM region on chromosome A7 including Rlm1, Rlm3, Rlm4, Rlm7, and Rlm9 (Delourme et al., 2004a, 2006; Ferreira et al., 1995; Raman et al., 2012) and the other on chromosome A10 hosting LepR2, BlmR2, LepR3, and Rlm2 (Long et al., 2011; Yu et al., 2008a, 2005). Clustering of R genes has critical evolutionary implications where recombination events between closely located genes help to form new motif combinations leading to the generation

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of novel resistance specificities, thereby broadens plants adaptability against various phytopathogenic agents (Hulbert et al., 2001; Meyers, 2003). Such R gene clusters that provide

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resistance to multiple diseases have been observed for clubroot, blackleg and sclerotinia stem

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rot diseases in B. napus (Delourme et al., 2018; Kato et al., 2013; Li et al., 2015) and B. rapa

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(Kato et al., 2013) and for anthracnose, angular leaf spot and downy mildew diseases in

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cucumber (Wang et al., 2019).

Significant higher expression of the gene Bo3g110840 at all tested time points and of the gene

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Bo3g103150 at initial time points against both isolates in both resistant genotypes suggest

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putative roles of these two genes in resistance against blackleg in these genotypes. Both these

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genes encoded leucine-rich repeat receptor like kinase protein. Kourelis and van der Hoorn (2018) performed a meta-analysis on 314 cloned plant R genes, of which 60 were RLKs/RLPs. RLKs has been reported to be involved in both pathogen specific dominant R gene mediated defense response e.g., gene Xa21 against bacterial blight in rice and broad-spectrum elicitorinitiated defense responses e.g., gene FLS2 against bacterial elicitor in Arabidopsis (GómezGómez and Boller, 2000; Goff and Ramonell, 2007; Liu et al., 2017; Sekhwal et al., 2015; Song et al., 1995). Among the blackleg resistant loci in Brassica species, only two loci namely, LepR3 and Rlm2 have been cloned so far; and both of these genes encoded leucine-rich repeat receptor 17

Journal Pre-proof like proteins (Larkan et al., 2015, 2013). In addition, very high expression of the gene Bo3g114980 against both isolates in the resistant genotype SCNU-98 and high expression of gene Bo3g130040 against isolate 00-100 s in both resistant genotypes suggests their genotype and isolate specific roles against blackleg. Both of these genes bore protein kinase and Serine/Threonine protein kinase domains and lacked the leucine-rich repeat receptor domains.

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Protein–protein interaction network analysis identified interactions of the key candidates

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Bo3g110840 and Bo3g114980 with other proteins that have roles in plants innate immunity,

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including G proteins and GTP binding proteins that are involved in signal transduction (Ma, 2007; Zhang et al., 2012b), caspase-1 which is involved in programmed cell death (Coll et al.,

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2011), cAMP which acts as a secondary messenger (Ma et al., 2009), and cyclin dependent

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kinases which play roles in cell-cycle control during host–pathogen interactions (Hamdoun et

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al., 2016; Wang et al., 2014).

The syntenic region of B. napus blackleg resistant loci LepR4 in B. oleracea were mined and

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genes having putative roles in blackleg resistance in cabbage is identified via in-silico- and

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differential expression-analysis. Mapping of the R-loci will be necessary to reveal if more than one locus is involved in these resistant lines and to reveal if the identified gene(s) fall within the map derived R-loci. Nonetheless, the genes identified in this study, upon testing against diverse isolates and validation by functional analyses, can be used for developing molecular markers by characterizing sequence variation pertinent to disease resistance and for introgression into elite cabbage cultivars lacking blackleg resistance via breeding and biotechnological means. Our approach of mining the syntenic regions of known A- or B-genome R gene in the C genome offers an auxiliary approach to the classical mapping based approaches of identifying R-genes. 18

Journal Pre-proof Replicating this approach for other known A-or B-genome R genes may lead to a set of genomewide disease resistance related genes in the C genome. These genes may also serve as reference genomic regions to be saturated with molecular markers and thereby, may assist in fine mapping of the putative R-genes via mapping based approaches. We are also working on mapping the QTLs for blackleg resistance using segregating populations. These will enhance our

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understanding of blackleg resistance in cabbage and will be helpful in attaining durable resistance against diverse and rapidly evolving fungal populations for areas where blackleg is

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already a threat or is predicted to become endemic in near future.

Supplementary Materials: The following supplementary information can be found online in the

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journal website.

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Figure S1. Protein–protein interaction network of the identified twelve NBS, LRR, TIR, CC and RLK genes within the LepR4-syntenic regions in B. oleracea. Input genes are shown in blue.

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associations.

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Lines between proteins represent possible interactions and thicker lines represent stronger

Table S1. List of primers used for qRT-PCR-based gene expression assay of the twelve LepR4syntenic NBS, LRR, TIR, CC and RLK genes in Brassica oleracea. The details of the genes are shown in Table 2. Table S2. List of genes within the syntenic LepR4 regions of the Brassica rapa in Brassica oleracea chromosomes. The syntenic maps and chromosomal positions are shown in Figure 1 and Table 1, respectively. The candidate disease resistance related-genes that were subjected to expression analysis are highlighted as yellow. (Provided in separate excel sheet online) 19

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Author Contributions: Conceptualization, ISN, JIP, AHKR, and HTK; in silico analysis, MJF, SN and MRH; protein network and microsynteny analysis, DMEJ; assisting in sample preparation and RNA extraction, HJJ and AHKR; all experiments, MJF; data analysis, interpretation, manuscript editing and review, MJF and MRH; supervision, ISN, JIP and HTK; project administration and

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funding acquisition, ISN. All authors read the article and approved the manuscript.

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Funding: This study was supported by the Golden Seed Project (Grant Number: 213007-05-4-

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CG100), Center for Horticultural Seed Development, Ministry of Agriculture, Food and Rural

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Affairs in the Republic of Korea (MAFRA). The funders had no role in the design of the study; in

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decision to publish the results.

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the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the

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Acknowledgments: We thank Nicholas J. Larkan and Hossein Borhan of Agriculture and Agri-

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Food Canada (AAFC), Saskatoon for providing the Leptosphaeria maculans isolates.

Conflicts of Interest: The authors declare no conflict of interest.

References Alamery, S., Tirnaz, S., Bayer, P., Tollenaere, R., Chaloub, B., Edwards, D., Batley, J., 2018. Genome-wide identification and comparative analysis of NBS-LRR resistance genes in Brassica napus. Crop Pasture Sci. 69, 79–93. https://doi.org/10.1071/CP17214 20

Journal Pre-proof Alamery, S.F., Sciences, F., 2015. Genome wide identification of NBS-LRR genes in Brassica and their association with disease resistance in Brassica napus. The University of Queensland. Ananga, A.O., Cebert, E., Soliman, K., Kantety, R., Pacumbaba, R.P., Konan, K., 2006. RAPD markers associated with resistance to blackleg disease in Brassica species 5, 2041–2048. Badawy, H.M.A., Hoppe, H., Koch, E., 1991. Differential reactions between the genus Brassica

of

and aggressive single spore isolates of Leptosphaeria maculans. J. Phytopathol. 131, 109–

ro

119.

-p

Balesdent, M.H., Attard, A., Kühn, M.L., Rouxel, T., 2002. New Avirulence Genes in the

re

Phytopathogenic Fungus Leptosphaeria maculans. Genet. Resist. 92, 1122–1133.

lP

Balesdent, M.H., Barbetti, M.J., Li, H., Sivasithamparam, K., Gout, L., Rouxel, T., 2005. Analysis of Leptosphaeria maculans race structure in a worldwide collection of isolates.

na

Phytopathology 95, 1061–1071.

ur

Barret, P., Guérif, J., Reynoird, J.P., Delourme, R., Eber, F., Renard, M., Chèvre, A.M., 1998.

Jo

Selection of stable Brassica napus-Brassica juncea recombinant lines resistant to blackleg (Leptosphaeria maculans). 2. A ‘to and fro’ strategy to localise and characterise interspecific introgressions on the B. napus genome. Theor. Appl. Genet. 96, 1097–1103. https://doi.org/10.1007/s001220050844 Bayer, P.E., Golicz, A.A., Tirnaz, S., Chan, C.K.K., Edwards, D., Batley, J., 2019. Variation in abundance of predicted resistance genes in the Brassica oleracea pangenome. Plant Biotechnol. J. 17, 789–800. https://doi.org/10.1111/pbi.13015 Bohman, S., Wang, M., Dixelius, C., 2002. Arabidopsis thaliana-derived resistance against 21

Journal Pre-proof Leptosphaeria maculans in a Brassica napus genomic background. Theor Appl Genet 105, 498–504. https://doi.org/10.1007/s00122-002-0885-5 Brun, H., Levivier, S., Somda, I., Ruer, D., Renard, M., Chèvre, A.M., 2000. A Field Method for Evaluating the Potential Durability of New Resistance Sources: Application to the Leptosphaeria maculans-Brassica napus Pathosystem. Phytopathology 90, 961–966.

of

https://doi.org/10.1094/PHYTO.2000.90.9.961

ro

Buschges, R., Hollricher, K., Panstruga, R., Simons, G., Wolter, M., Frijters, A., van Daelen, R.,

-p

van der Lee, T., Diergaarde, P., Groenendijk, J., Topsch, S., Vos, P., Salamini, F., Schulze-

re

Lefert, P., 1997. The barley Mlo gene: a novel control element of plant pathogen resistance.

lP

Cell 88, 695–705.

Chalhoub, B., Denoeud, F., Liu, S., Parkin, I.A.P., Tang, H., Wang, X., Chiquet, J., Belcram, H.,

na

Tong, C., Samans, B., 2014. Early allopolyploid evolution in the post-Neolithic Brassica

ur

napus oilseed genome. Science (80). 345, 950–953.

Jo

Chen, Y., Fernando, W.G.D., 2006. Prevalence of pathogenicity groups of Leptosphaeria maculans in western Canada and North Dakota, USA. Can. J. Plant Pathol. 28, 533–539. https://doi.org/10.1080/07060660609507331 Cheng, F., Mandáková, T., Wu, J., Xie, Q., Lysak, M.A., Wang, X., 2013. Deciphering the Diploid Ancestral Genome of the Mesohexaploid Brassica rapa. Plant Cell 25, 1541–1554. https://doi.org/10.1105/TPC.113.110486 Cheng, F., Wu, J., Fang, L., Wang, X., 2012. Syntenic gene analysis between Brassica rapa and other Brassicaceae species. Front. Plant Sci. 3, 198. 22

Journal Pre-proof Chèvre, A.M., Barret, P., Eber, F., Dupuy, P., Brun, H., Tanguy, X., Renard, M., 1997. Selection of stable Brassica napus-B. juncea recombinant lines resistant to blackleg (Leptosphaeria maculans).1. Identification of molecular markers, chromosomal and genomic origin of the introgression. Theor. Appl. Genet. 95, 1104–1111. https://doi.org/10.1007/s001220050669

of

Christianson, J.A., Rimmer, S.R., Good, A.G., Lydiate, D.J., 2006a. Mapping genes for resistance

ro

to Leptosphaeria maculans in Brassica juncea. Genome 41, 30–41.

-p

https://doi.org/10.1139/G05-085

re

Christianson, J.A., Rimmer, S.R., Good, A.G., Lydiate, D.J., 2006b. Mapping genes for resistance to Leptosphaeria maculans in Brassica juncea. Genome 49, 30–41.

lP

https://doi.org/10.1139/g05-085

na

Coll, N.S., Epple, P., Dangl, J.L., 2011. Programmed cell death in the plant immune system. Cell

ur

Death Differ. 18, 1247–1256. https://doi.org/10.1038/cdd.2011.37

Jo

Cristina, M.S., Petersen, M., Mundy, J., 2010. Mitogen-activated protein kinase signaling in plants. Annu. Rev. Plant Biol. 61, 621–649. Dangl, J.L., Jones, J.D.G., 2001. Plant pathogens and integrated defence responses to infection. Nature 411, 826–833. https://doi.org/10.1038/35081161 Delourme, R., Archipiano, M., Horvais, R., Tanguy, X., Rouxel, T., Brun, H., Renard, M., Balesdent, M.H., 2004a. A Cluster of Major Specific Resistance Genes to Leptosphaeria maculans in Brassica napus. Phytopathology 94, 578–583. Delourme, R., Chevre, A.M., Brun, H., Rouxel, T., Balesdent, M.H., Dias, J.S., Salisbury, P., Biop, 23

Journal Pre-proof U.M.R., Cedex, L.R., St-cyr, R. De, 2006. Major gene and polygenic resistance to Leptosphaeria maculans in oilseed rape ( Brassica napus ). Eur. J. Plant Pathol. 114, 41–52. https://doi.org/10.1007/s10658-005-2108-9 Delourme, R., Laperche, A., Bouchet, A., Jubault, M., Paillard, S., Nesi, N., 2018. Genes and Quantitative Trait Loci Mapping for Major Agronomic Traits in Brassica napus L., in: The

of

Brassica napus Genome. Springer International Publishing, pp. 41–85.

ro

https://doi.org/10.1007/978-3-319-43694-4

-p

Delourme, R., Pilet-Nayel, M.L., Archipiano, M., Horvais, R., Tanguy, X., Rouxel, T., Brun, H.,

re

Renard, M., Balesdent, M.H., 2004b. A Cluster of Major Specific Resistance Genes to Leptosphaeria maculans in Brassica napus. Phytopathology 94, 578–583.

lP

https://doi.org/10.1094/PHYTO.2004.94.6.578

na

Deslandes, L., Olivier, J., Theulières, F., Hirsch, J., Feng, D.X., Bittner-Eddy, P., Beynon, J., Marco,

ur

Y., 2002. Resistance to Ralstonia solanacearum in Arabidopsis thaliana is conferred by the

Jo

recessive RRS1-R gene, a member of a novel family of resistance genes. Proc. Natl. Acad. Sci. 99, 2404 LP-2409. https://doi.org/10.1073/pnas.032485099 DeYoung, B., Innes, R., 2007. Plant NBS-LRR proteins in pathogen sensing and host defense. Mol. Cell 7, 59–64. https://doi.org/10.1038/ni1410.Plant Dilmaghani, A., Balesdent, M.H., Rouxel, T., Moreno-Rico, O., 2010. First report of Leptosphaeria biglobosa (blackleg) on Brassica oleracea (cabbage) in Mexico. Plant Dis. 94, 791. Eitas, T.K., Dangl, J.L., 2010. NB-LRR proteins: Pairs, pieces, perception, partners, and pathways. 24

Journal Pre-proof Curr. Opin. Plant Biol. 13, 472–477. https://doi.org/10.1016/j.pbi.2010.04.007 Ellis, J., Dodds, P., Pryor, T., 2000. Structure, function and evolution of plant disease resistance genes. Curr. Opin. Plant Biol. 3, 278–84. Ferreira, M.E., Dias, J.S., Mengistu, A., Williams, P.H., 1992. Screening of Portuguese cole landraces (Brassica oleracea L.) with Leptosphaeria maculans and Xanthomonas

of

campestris pv. campestris. Euphytica 65, 219–227. https://doi.org/10.1007/BF00023086

ro

Ferreira, M.E., Rimmer, S.R., Williams, P.H. Osborn, T.C., 1995. Mapping loci controlling Brassica

-p

napus resistance to Leptosphaeria maculans under different screening conditions.

re

Phytopathology. 85(2), 213-217.

lP

Fitt, B.D.L., Brun, H., Barbetti, M.J., Rimmer, S.R., 2006. World-wide importance of phoma stem canker (Leptosphaeria maculans and L. biglobosa) on oilseed rape (Brassica napus). Eur. J.

na

Plant Pathol. 114, 3–15. https://doi.org/10.1007/s10658-005-2233-5

ur

Fitt, B.D.L., Huang, Y.-J., van den Bosch, F., West, J.S., 2006. Coexistence of related pathogen

Jo

species on arable crops in space and time. Annu. Rev. Phytopathol. 44. Frye, C.A., Innes, R.W., 1998. An Arabidopsis Mutant with Enhanced Resistance to Powdery Mildew. Plant Cell 10, 947 -956. https://doi.org/10.1105/tpc.10.6.947 Fu, Y., Zhang, Y., Mason, A.S., Lin, B., Zhang, D., Yu, H., Fu, D., 2019. NBS-Encoding Genes in Brassica napus Evolved Rapidly After Allopolyploidization and Co-localize With Known Disease Resistance Loci. Front. Plant Sci. 10, 1–18. https://doi.org/10.3389/fpls.2019.00026

25

Journal Pre-proof Gabrielson, R.L., 1983. Blackleg disease of crucifers caused by Leptosphaeria maculans (Phoma lingam) and its control. Seed Sci. Technol. Ghanbarnia, K., Dilantha Fernando, W.G., Crow, G., 2009. Developing rainfall-and temperaturebased models to describe infection of canola under field conditions caused by pycnidiospores of Leptosphaeria maculans. Phytopathology 99, 879–886.

of

Goff, K.E., Ramonell, K.M., 2007. The Role and Regulation of Receptor-Like Kinases in Plant

ro

Defense. Gene Regul. Syst. Bio. 1, 167–175.

-p

Hamdoun, S., Zhang, C., Gill, M., Kumar, N., Churchman, M., Larkin, J.C., Kwon, A., Lu, H., 2016.

re

Differential Roles of Two Homologous Cyclin-Dependent Kinase Inhibitor Genes in

lP

Regulating Cell Cycle and Innate Immunity in Arabidopsis. Plant Physiol. 170, 515–527. https://doi.org/10.1104/pp.15.01466

na

Hao, L., Song, P., Huangfu, H., Li, Z., 2015. Genetic diversity and differentiation of Leptosphaeria

ur

biglobosa on oilseed rape in China. Phytoparasitica 43, 253–263.

Jo

Hardie, D.G., 1999. Plant protein serine/threonine kinases: Classification and Functions. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 97–131. https://doi.org/10.1146/annurev.arplant.50.1.97 Harris, C.J., Slootweg, E.J., Goverse, A., Baulcombe, D.C., 2013. Stepwise artificial evolution of a plant disease resistance gene. PNAS 110, 21189–21194. https://doi.org/10.1073/pnas.1311134110 Henderson, M.P., 1918. The black-leg disease of cabbage caused by Phoma lingam (Tode) Desmaz. Phytopathology 8, 379–431. 26

Journal Pre-proof Hong, S.-K., Kim, W.-G., Shin, D.-B., Choi, H.-W., Lee, Y.-K., Lee, S.-Y., 2009. Occurrence of stem canker on rape caused by Leptosphaeria biglobosa in Korea. Plant Pathol. J. 25, 294–298. Howlett, B.J., 2004. Current knowledge of the interaction between Brassica napus and Leptosphaeria maculans. Can. J. Plant Pathol. 26, 245–252. Hulbert, S.H., Webb, C.A., Smith, S.M., Sun, Q., 2001. Resistance Gene Complexes: Evolution

of

and Utilization. Annu. Rev. Phytopathol. 39, 285–312.

ro

https://doi.org/10.1146/annurev.phyto.39.1.285

-p

Humpherson‐Jones, F.M., 1985. The incidence of Alternaria spp. and Leptosphaeria maculans in

re

commercial brassica seed in the United Kingdom. Plant Pathol. 34, 385–390.

lP

Jones, J.D.G., Dangl, J.L., 2006. The plant immune system. Nature 444, 323–329.

na

https://doi.org/10.1038/nature05286

Kato, T., Hatakeyama, K., Fukino, N., Matsumoto, S., 2013. Fine mapping of the clubroot

ur

resistance gene CRb and development of a useful selectable marker in Brassica rapa.

Jo

Breed. Sci. 63, 116–124.

Kobe, B., Deisenhofer, J., 1995. A structural basis of the interactions between leucine-rich repeats and protein ligands. Nature 374, 183–186. https://doi.org/10.1038/374183a0 Kourelis, J., van der Hoorn, R.A.L., 2018. Defended to the Nines: 25 Years of Resistance Gene Cloning Identifies Nine Mechanisms for R Protein Function. Plant Cell 30, 285–299. https://doi.org/10.1105/tpc.17.00579 Kutcher, H.R., Cross, D., Nabetani, K., Kirkham, C.L., 2011. A large-scale survey of races of L.

27

Journal Pre-proof maculans occurring on canola in western Canada. Kutcher, H.R., Van Den Berg, C.G.J., Rimmer, S.R., 1993. Variation in pathogenicity of Leptosphaeria maculans on Brassica spp. based on cotyledon and stem reactions. Can. J. Plant Pathol. 15, 253–258. Kuusk, A., Happstadius, I., Zhou, L., Steventon, L.A., Giese, H., Dixelius, C., 2002. Presence of

of

Leptosphaeria maculans group A and group B isolates in Sweden. J. Phytopathol. 150, 349–

ro

356.

-p

Larkan, N.J., Lydiate, D.J., Parkin, I.A.P., Nelson, M.N., Epp, D.J., Cowling, W.A., Rimmer, S.R.,

re

Borhan, M.H., 2013. The Brassica napus blackleg resistance gene LepR3 encodes a

lP

receptor-like protein triggered by the Leptosphaeria maculans effector AVRLM1. New Phytol. 197, 595–605. https://doi.org/10.1111/nph.12043

na

Larkan, N.J., Ma, L., Borhan, M.H., 2015. The Brassica napus receptor-like protein RLM2 is

ur

encoded by a second allele of the LepR3 / Rlm2 blackleg resistance locus. Plant Biotechnol

Jo

J 1–10. https://doi.org/10.1111/pbi.12341 Leflon, M., Brun, H., Eber, F., Delourme, R., Lucas, M.O., Vallée, P., Ermel, M., Balesdent, M.H., Chèvre, A.M., 2007. Detection, introgression and localization of genes conferring specific resistance to Leptosphaeria maculans from Brassica rapa into B. napus. Theor. Appl. Genet. 115, 897–906. https://doi.org/10.1007/s00122-007-0616-z Li, H., Kuo, J., Barbetti, M.J., Sivasithamparam, K., 2007a. Differences in the responses of stem tissues of spring-type Brassica napus cultivars with polygenic resistance and single dominant gene-based resistance to inoculation with Leptosphaeria maculans. Botany 85, 28

Journal Pre-proof 191–203. Li, H., Sivasithamparam, K., Barbetti, M.J., 2007b. Soilborne ascospores and pycnidiospores of Leptosphaeria maculans can contribute significantly to blackleg disease epidemiology in oilseed rape (Brassica napus) in Western Australia. Australas. Plant Pathol. 36, 439–444. Li, J., Zhao, Z., Hayward, A., Cheng, H., Fu, D., 2015. Integration analysis of quantitative trait loci

of

for resistance to Sclerotinia sclerotiorum in Brassica napus. Euphytica 205, 483–489.

ro

https://doi.org/10.1007/s10681-015-1417-0

-p

Liban, S.H., Cross, D.J., Kutcher, H.R., Peng, G., Fernando, W.G.D., 2015. Race structure and

re

frequency of avirulence genes in the western Canadian Leptosphaeria maculans pathogen

lP

population , the causal agent of blackleg in brassica species. Plant Pathol. 1–9. https://doi.org/10.1111/ppa.12489

na

Liu, J., Liu, X., Dai, L., Wang, G., 2007. Recent Progress in Elucidating the Structure, Function and

ur

Evolution of Disease Resistance Genes in Plants. J. Genet. Genomics 34, 765–776.

Jo

https://doi.org/10.1016/S1673-8527(07)60087-3 Liu, P.L., Du, L., Huang, Y., Gao, S.M., Yu, M., 2017. Origin and diversification of leucine-rich repeat receptor-like protein kinase (LRR-RLK) genes in plants. BMC Evol. Biol. 17, 1–16. https://doi.org/10.1186/s12862-017-0891-5 Liu, S., Liu, Y., Yang, X., Tong, C., Edwards, D., Parkin, I.A.P., Zhao, M., Ma, J., Yu, J., Huang, S., Wang, X., Wang, J., Lu, K., Fang, Z., Bancroft, I., Yang, T., Hu, Q., Wang, X., Yue, Z., Li, H., Yang, L., Wu, J., Zhou, Q., Wang, W., King, G.J., Pires, J.C., Lu, C., Wu, Z., Sampath, P., Wang, Z., Guo, H., Pan, S., Yang, L., Min, J., Zhang, D., Jin, D., Li, W., Belcram, H., Tu, J., Guan, M., 29

Journal Pre-proof Qi, C., Du, D., Li, J., Jiang, L., Batley, J., Sharpe, A.G., Park, B., Ruperao, P., Cheng, F., Waminal, N.E., Huang, Y., Dong, C., Wang, L., Li, J., Hu, Z., Zhuang, M., Huang, Y., Huang, J., Shi, J., Mei, D., Liu, J., Lee, T., Wang, J., Tang, X., Liu, W., Wang, Y., Zhang, Y., Lee, J., Kim, H.H., Denoeud, F., Xu, X., Liang, X., Hua, W., Wang, X., Wang, J., Chalhoub, B., Paterson, A.H., 2014. The Brassica oleracea genome reveals the asymmetrical evolution of polyploid

of

genomes. Nat. Commun. 5, 1–11. https://doi.org/10.1038/ncomms4930

ro

Liu, Z., 2008. Strategies for managing Leptosphaeria maculans and Leptosphaeria biglobosa to

-p

decrease severity of phoma stem canker epidemics on winter oilseed rape. University of

re

Hertfordshire.

Liu, Z., Latunde-Dada, A.O., Hall, A.M., Fitt, B.D.L., 2014. Phoma stem canker disease on oilseed

na

Plant Pathol. 140, 841–857.

lP

rape (Brassica napus) in China is caused by Leptosphaeria biglobosa ‘brassicae.’ Eur. J.

ur

Long, Y., Wang, Z., Sun, Z., Fernando, D.W.G., McVetty, P.B.E., Li, G., 2011. Identification of two

Jo

blackleg resistance genes and fine mapping of one of these two genes in a Brassica napus canola cultivar “Surpass 400”. Theor. Appl. Genet. 122, 1223–1231. https://doi.org/10.1007/s00122-010-1526-z Ma, Q.-H., 2007. Small GTP-binding Proteins and their Functions in Plants. J. Plant Growth Regul. 26, 369–388. https://doi.org/10.1007/s00344-007-9022-7 Ma, W., Qi, Z., Smigel, A., Walker, R.K., Verma, R., Berkowitz, G.A., 2009. Ca2+, cAMP, and transduction of non-self perception during plant immune responses. Proc. Natl. Acad. Sci. 106, 20995 -21000. https://doi.org/10.1073/pnas.0905831106 30

Journal Pre-proof Marcroft, S.J., Wratten, N., Purwantara, A., Salisbury, P.A., Potter, T.D., Barbetti, M.J., Khangura, R., Howlett, B.J., 2002. Reaction of a range of Brassica species under Australian conditions to the fungus, Leptosphaeria maculans, the causal agent of blackleg. Aust. J. Exp. Agric. 42, 587–594. Martin, G.B., Bogdanove, A.J., Sessa, G., 2003. Understanding the functions of plant disease

of

resistance proteins. Annu. Rev. Plant Biol. 54, 23–61.

ro

https://doi.org/10.1146/annurev.arplant.54.031902.135035

-p

Mayerhofer, R., Good, A.G., Bansal, V.K., Thiagarajah, M.R., Stringam, G.R., 1997. Molecular

re

mapping of resistance to Leptosphaeria maculans in Australian cultivars of Brassica napus.

lP

Genome 40, 294–301.

Mendes-Pereira, E., Balesdent, M.-H. ne, Brun, H., Rouxel, T., 2003. Molecular phylogeny of the

na

Leptosphaeria maculans – L. biglobosa species complex. Mycol. Res. 107, 1287–1304.

ur

https://doi.org/10.1017/S0953756203008554

Jo

Mengistu, A., Rimmer, S.R., Koch, E., Williams, P.H., 1991. Pathogenicity grouping of isolates of Leptosphaeria maculans on Brassica napus cultivars and their disease reaction profiles on rapid-cycling Brassicas. Plant Dis 75, 1279–1282. Meyers, B.C., 2003. Genome-Wide Analysis of NBS-LRR-Encoding Genes in Arabidopsis. PLANT CELL ONLINE 15, 809–834. https://doi.org/10.1105/tpc.009308 Meyers, B.C., Dickerman, A.W., Michelmore, R.W., Sivaramakrishnan, S., Sobral, B.W., Young, N.D., 1999. Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily. Plant J. 20, 317–332. 31

Journal Pre-proof Michelmore, R.W., Meyers, B.C., 1998. Clusters of resistance genes in plants evolve by divergent selection and a birth-and-death process. Genome Res. 8, 1113–1130. https://doi.org/10.1101/gr.8.11.1113 Miles, C.M., Wayne, M., 2008. Quantitative Trait Locus (QTL) Analysis. Nat. Educ. 1(1): 208. Mithen, R.F., Lewis, B.G., Heaney, R.K., Fenwick, G.R., 1987. Resistance of leaves of Brassica

of

species to Leptosphaeria maculans. Trans. Br. Mycol. Soc. 88, 525–531.

ro

https://doi.org/https://doi.org/10.1016/S0007-1536(87)80036-0

-p

Morillo, S.A., Tax, F.E., 2006. Functional analysis of receptor-like kinases in monocots and dicots.

re

Curr. Opin. Plant Biol. 9, 460–469. https://doi.org/10.1016/j.pbi.2006.07.009

lP

Murat, F., Louis, A., Maumus, F., Armero, A., Cooke, R., Quesneville, H., Crollius, H.R., Salse, J., 2015. Understanding Brassicaceae evolution through ancestral genome reconstruction.

na

Genome Biol. 16, 262. https://doi.org/10.1186/s13059-015-0814-y

ur

Neik, T.X., Barbetti, M.J., Batley, J., 2017. Current Status and Challenges in Identifying Disease

Jo

Resistance Genes in Brassica napus. Front. Plant Sci. 8. https://doi.org/10.3389/fpls.2017.01788 Pang, E.C.K., Halloran, G.M., 1996. The genetics of blackleg [Leptosphaeria maculans (Desm.) Ces. et De Not.] resistance in rapeseed (Brassica napus L.). Theor. Appl. Genet. 93, 941– 949. https://doi.org/10.1007/BF00224097 Parlange, F., Daverdin, G., Fudal, I., Kuhn, M., Balesdent, M., Blaise, F., Grezes-besset, B., Rouxel, T., 2009. Leptosphaeria maculans avirulence gene AvrLm4-7 confers a dual recognition specificity by the Rlm4 and Rlm7 resistance genes of oilseed rape , and circumvents Rlm432

Journal Pre-proof mediated recognition through a single amino acid change. Mol. Microbiol. 71, 851–863. https://doi.org/10.1111/j.1365-2958.2008.06547.x Piliponytė-Dzikienė, A., Andriūnaitė, E., Petraitienė, E., Brazauskienė, I., Statkevičiūtė, G., Brazauskas, G., 2015. Genetic diversity and occurrence of Leptosphaeria spp. on Brassica oleracea and B. napus in Lithuania. J. Plant Pathol. 97(2).

of

Plieske, J., Struss, D., Röbbelen, G., 1998. Inheritance of resistance derived from the B-genome

ro

of Brassica against Phoma lingam in rapeseed and the development of molecular markers.

-p

Theor. Appl. Genet. 97, 929–936. https://doi.org/10.1007/s001220050973

re

Raman, H., Raman, R., Larkan, N., 2013. Genetic Dissection of Blackleg Resistance Loci in

lP

Rapeseed ( Brassica napus L .), in: Plant Breeding from Laboratories to Fields. InTech, pp. 85–120.

na

Raman, R., Taylor, B., Marcroft, S., Stiller, J., Eckermann, P., 2012. Molecular mapping of

ur

qualitative and quantitative loci for resistance to Leptosphaeria maculans causing blackleg

6

Jo

disease in canola ( Brassica napus L .) 405–418. https://doi.org/10.1007/s00122-012-1842-

Rimmer, S.R., van den Berg, C.G.J., 1992. Resistance of oilseed Brassica spp. to blackleg caused by Leptosphaeria maculans. Can. J. Plant Pathol. 14, 56–66. https://doi.org/10.1080/07060669209500906 Robin, A.H.K., Larkan, N.J., Laila, R., Park, J.I., Ahmed, N.U., Borhan, H., Parkin, I.A.P., Nou, I.S., 2017. Korean Brassica oleracea germplasm offers a novel source of qualitative resistance to blackleg disease. Eur. J. Plant Pathol. 149, 611–623. https://doi.org/10.1007/s1065833

Journal Pre-proof 017-1210-0 Rouxel, T., Penaud, A., Pinochet, X., Brun, H., Gout, L., Delourme, R., Schmit, J., Balesdent, M.-H., 2003. A 10-year Survey of Populations of Leptosphaeria maculans in France Indicates a Rapid Adaptation Towards the Rlm1 Resistance Gene of Oilseed Rape. Eur. J. Plant Pathol. 109, 871–881. https://doi.org/10.1023/A:1026189225466

of

Saal, B., Brun, H., Glais, I., Struss, D., 2004. Identification of a Brassica juncea-derived recessive

ro

gene conferring resistance to Leptosphaeria maculans in oilseed rape. Plant Breed. 123,

-p

505–511. https://doi.org/10.1111/j.1439-0523.2004.01052.x

re

Sekhwal, M.K., Li, P., Lam, I., Wang, X., Cloutier, S., You, F.M., 2015. Disease resistance gene

lP

analogs (RGAs) in plants. Int. J. Mol. Sci. 16, 19248–19290. https://doi.org/10.3390/ijms160819248

na

Shoemaker, R.A., Brun, H., 2001. The teleomorph of the weakly aggressive segregate of

ur

Leptosphaeria maculans. Can. J. Bot. 79, 412–419. https://doi.org/10.1139/b01-019

Jo

Sjodin, C., Glimelius, K., 1988. Screening for Resistance to Blackleg Phoma lingam ( Tode ex Fr .) Desm . within Brassicaceae 332, 322–333. Snowdon, R.J., Winter, H., Diestel, A., Sacristán, M.D., 2000. Development and characterisation of Brassica napus-Sinapis arvensis addition lines exhibiting resistance to Leptosphaeria maculans. Theor. Appl. Genet. 101, 1008–1014. https://doi.org/10.1007/s001220051574 Sprague, S.J., Balesdent, M.-H., Brun, H., Hayden, H.L., Marcroft, S.J., Pinochet, X., Rouxel, T., Howlett, B.J., 2006. Major gene resistance in Brassica napus (oilseed rape) is overcome by changes in virulence of populations of Leptosphaeria maculans in France and Australia., in: 34

Journal Pre-proof Fitt, B.D.L., Evans, N., Howlett, B.J., Cooke, B.M. (Eds.), Sustainable Strategies for Managing Brassica napus (Oilseed Rape) Resistance to Leptosphaeria. Springer Netherlands, Dordrecht, pp. 33–40. https://doi.org/10.1007/1-4020-4525-5_3 Takken, F.L., Albrecht, M., Tameling, W. Il, 2006. Resistance proteins: molecular switches of plant defence. Curr. Opin. Plant Biol. 9, 383–390.

of

https://doi.org/10.1016/j.pbi.2006.05.009

ro

van Ooijen, G., Mayr, G., Kasiem, M.M.A., Albrecht, M., Cornelissen, B.J.C., Takken, F.L.W., 2008.

-p

Structure-function analysis of the NB-ARC domain of plant disease resistance proteins. J.

re

Exp. Bot. 59, 1383–1397. https://doi.org/10.1093/jxb/ern045

lP

Wang, S., Gu, Y., Zebell, S.G., Anderson, L.K., Wang, W., Mohan, R., Dong, X., 2014. A Noncanonical Role for the CKI-RB-E2F Cell-Cycle Signaling Pathway in Plant Effector-

na

Triggered Immunity. Cell Host Microbe 16, 787–794.

ur

https://doi.org/https://doi.org/10.1016/j.chom.2014.10.005

Jo

Wang, Y., Tan, J., Wu, Z., VandenLangenberg, K., Wehner, T.C., Wen, C., Zheng, X., Owens, K., Thornton, A., Bang, H.H., Hoeft, E., Kraan, P.A.G., Suelmann, J., Pan, J., Weng, Y., 2019. STAYGREEN, STAY HEALTHY: a loss-of-susceptibility mutation in the STAYGREEN gene provides durable, broad-spectrum disease resistances for over 50 years of US cucumber production. New Phytol. 221, 415–430. https://doi.org/10.1111/nph.15353 West, J.S., Kharbanda, P.D., Barbetti, M.J., Fitt, B.D.L., 2001. Epidemiology and management of Leptosphaeria maculans (phoma stem canker) on oilseed rape in Australia, Canada and Europe. Plant Pathol. 50, 10–27. 35

Journal Pre-proof Williams, P.H., 1992. Biology of Leptosphaeria maculans. Can. J. Plant Pathol. 14, 30–35. Wu, J., Cai, G., Tu, J., Li, L., Liu, S., Luo, X., Zhou, L., Fan, C., Zhou, Y., 2013. Identification of QTLs for resistance to Sclerotinia stem rot and BnaC. IGMT5. a as a candidate gene of the major resistant QTL SRC6 in Brassica napus. PLoS One 8, e67740. Yang, J., Liu, G., Zhao, N., Chen, S., Liu, D., Ma, W., Hu, Z., Zhang, M., 2016. Comparative

of

mitochondrial genome analysis reveals the evolutionary rearrangement mechanism in

ro

Brassica. Plant Biol. 18, 527–536. https://doi.org/10.1111/plb.12414

-p

Yi, H., Richards, E.J., 2007. A Cluster of Disease Resistance Genes in Arabidopsis Is Coordinately

re

Regulated by Transcriptional Activation and RNA Silencing. Plant Cell Online 19, 2929–2939.

lP

https://doi.org/10.1105/tpc.107.051821

Yu, F., Gugel, R.K., Kutcher, H.R., Peng, G., Rimmer, S.R., 2013. Identification and mapping of a

na

novel blackleg resistance locus LepR4 in the progenies from Brassica napus × B. rapa subsp.

ur

sylvestris. Theor. Appl. Genet. 126, 307–315. https://doi.org/10.1007/s00122-012-1919-2

Jo

Yu, F., Lydiate, D.J., Rimmer, S.R., 2008a. Identification and mapping of a third blackleg resistance locus in Brassica napus derived from B . rapa subsp . sylvestris. Genome 6, 64– 72. https://doi.org/10.1139/G07-103 Yu, F., Lydiate, D.J., Rimmer, S.R., 2008b. Identification and mapping of a third blackleg resistance locus in Brassica napus derived from B. rapa subsp. sylvestris. Biotechnol. Tissue Cult. Mol. Breed. 51, 64–72. https://doi.org/10.1139/g07-103 Yu, F., Lydiate, D.J., Rimmer, S.R., 2005. Identification of two novel genes for blackleg resistance in Brassica napus. Theor Appl Genet 110, 969–979. https://doi.org/10.1007/s00122-00436

Journal Pre-proof 1919-y Yu, J., Tehrim, S., Zhang, F., Tong, C., Huang, J., Cheng, X., Dong, C., 2014. Genome-wide comparative analysis of NBS-encoding genes between Brassica species and Arabidopsis thaliana. BMC Genomics 15, 1–18. https://doi.org/10.1186/1471-2164-15-3 Yu, J., Zhao, M., Wang, X., Tong, C., Huang, S., Tehrim, S., Liu, Y., 2013. Bolbase : a

of

comprehensive genomics database for Brassica oleracea. BMC Genomics 14, 1.

ro

https://doi.org/10.1186/1471-2164-14-664

-p

Zhang, H., Gao, Z., Zheng, X., Zhang, Z., 2012. The role of G-proteins in plant immunity. Plant

re

Signal. Behav. 7, 1284–1288. https://doi.org/10.4161/psb.21431

lP

Zhang, X., Fernando, W.G.D., 2018. Insights into fighting against blackleg disease of Brassica

na

napus in Canada. Crop Pasture Sci. 69, 40–47. https://doi.org/10.1071/CP16401 Zhang, X., White, R.P., Demir, E., Jedryczka, M., Lange, R.M., Islam, M., Li, Z.Q., Huang, Y.J., Hall,

ur

A.M., Zhou, G., 2014. Leptosphaeria spp., phoma stem canker and potential spread of L.

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maculans on oilseed rape crops in C hina. Plant Pathol. 63, 598–612.

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Tables

Table 1. Syntenic regions of B. rapa LepR4 in B. oleracea. Synteny maps are shown in Figure 1. Position in B. oleracea chromosomes (bp)

Number of genes*

A06: 22653-778396

C08: 24655389-25659268

109

A06: 2758351-5778365

C03: 35372933-44937345

793

A06: 2372557-2717192

C03: 42614672-43599496

A06: 835652-2215650

C03: 47000600-49935309

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Position in B. rapa chromosome A06 (bp)

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235

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*Segment C03: 42614672-43599496 overlapped with another segment (C03: 35372933-44937345) of C-genome.

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The total number of genes in the entire syntenic region is thus less than the sum of numbers shown here.

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Journal Pre-proof Table 2. List of genes with domains related to disease resistance within the LepR4-syntenic regions in B. oleracea.

Ensembl ID Bo8g077320 Bo3g107230 Bo3g099380 Bo3g103150 Bo3g102880 Bo3g130040 Bo3g110840 Bo3g114980 Bo8g077270 Bo3g134690 Bo8g077170 Bo3g107530 The primers

Chromosomal DNA CDS location (strand) (bp) (bp)

Bolbase Hit

AA

Arabidopsis Hit /e-value

Trembl ID Description

C8:253512024160 1800 599 Bol037407 AT3G48860.2/0 Q8RWD5 Coiled-coil domain25355361 (+) containing protein SCD2 C3:377662222965 1977 658 Bol019438 AT5G62710.1/0 Q8GX94 Leucine-rich repeat protein 37769186 (-) kinase family protein C3:356296813240 1827 608 Bol009214 AT5G65240.1/0 C0LGX1 Leucine-rich repeat protein 35632943 (+) kinase family protein C3:368272343165 1824 607 Bol025511 AT5G63710.1/0 Q9FFP2 Leucine-rich repeat protein 36830398 (-) kinase family protein C3:366693423980 3210 1069 Bol004493 AT4G36150.1/0 O65507 Disease resistance protein 36673321 (+) (TIR-NBS-LRR class) family C3:474724331533 1113 370 Bol041350 AT3G45640.1/0 Q5IV18 Mitogen-activated protein 47473965 (+) kinase C3:395048542535 2199 732 Bol025933 AT3G47090.1/0 Q9SD64 Leucine-rich repeat protein 39507388 (+) kinase family protein C3:41005111697 513 170 Bol025933 AT3G47090.1/3.07E-67 Q9SD64 Mitogen-activated protein 41005807 (-) kinase C8:253064752943 2748 915 Bol037412 AT5G17680.2/1.13E-162 Q9FN83 Disease resistance protein 25309417 (+) (TIR-NBS-LRR class) C3:495462912520 3021 1006 Bol024895 AT3G47580.1/0 Q9SD64 Leucine-rich repeat protein 49549404 (+) kinase family protein C8:25212759399 312 103 Bol037418 AT1G53510.1/8.63E-37 B9N7T1 Mitogen-activated protein 25213157 (+) kinase 18 C3:37989412261 261 86 Bol005997 AT1G53510.1/4.53E-35 B9N7T1 Mitogen-activated protein 37989672 (+) kinase 18 designed for these genes are shown in S1 Table. *Only the domains related to disease resistance are listed. NB-ARC =

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a n

r u

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P l

f o

Functional Domains* coiled coil LRR, Pkinase, S_TKc LRR, Pkinase, S_TKc LRR, Pkinase, S_TKc NB-ARC, LRR Pkinase, S_TKc LRR, Pkinase, S_TKc Pkinase_Tyr, S_TKc TIR, NB-ARC, LRR Pkinase, S_TKc Pkinase Pkinase nucleotide-binding adaptor

shared by APAF-1, certain R gene products, and CED-4; LRR = Leucine Rich Repeat; Pkinase = Protein kinase domain; Pkinase_Tyr = Protein tyrosine kinase; S_TKc = Serine/Threonine protein kinase.

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Journal Pre-proof Table 3: List of significant motifs along with the motif statistics and associated domains identified from the identified NBS, LRR, TIR, CC and RLK genes within the LepR4-syntenic regions in B. oleracea. Motif ID

Motif sequence

E-value

Sites

Width

Associated domain

Motif-1

NPKIIHRDLKPSNILLBDDLEPHVSDFGLAKL

3.00E-42

6

32

STKc (IRAK, MAPK), Pkinase_Tyr, Pkinase

Motif-2

THVTTGVRGTIGYIAPEYLLGGKPSEKTDVYSF

1.20E-38

5

33

STKc (IRAK, MAPK), Pkinase_Tyr, Pkinase

Motif-3

RHRNLVKLLTACSSIDYQGNEFRALVYEFMPNGSLDIWLHPEKVEGIHRP

1.40E-34

3

50

Motif-4

JGNLSNLQTLNLSRNSLLGSIPASLTNLSNLLYLYLRGNILSGEIPREJF

1.70E-27

7

50

Motif-5

ATDGFSESNLIGQGGFGKVYKGVLPDETKVVVKKLLDLKRPGGDKAFQRE

5.10E-20

4

Motif-6

DPDAEEVEAIIEVALRCTZASPEDRPAMSEVVKML

1.30E-18

5

Motif-7

RLRIAJGVARGLEYLHEHC

2.90E-16

Motif-8

LVALDLESNGFTGEJPSSJGKLSRLESLNLSNNSLSGEJP

3.60E-09

Motif-9

AVHKNLLRLIGFCTTPTERLLVYPYMQNLSVAYRLREJKRGEPVLDW

2.30E-08

Motif-10

GILLLELVTGKRPTDESF

1.70E-06

Motif-11

CSWSKVTCDDQ

Motif-12

MKKINNVFEHISDPKRILCEVKLLRLLDHEN

Motif-13

IVGVWGMGGRGKTTJAKC

35

LRR STKc (IRAK, MAPK), Pkinase_Tyr, Pkinase STKc (IRAK), Pkinase_Tyr

-p

19

STKc_IRAK

40

LRR

3

47

STKc (IRAK, MAPK), Pkinase_Tyr, Pkinase

5

18

STKc (IRAK)

5.10E-03

4

11

LRR

1.40E-02

3

31

STKc (IRAK, MAPK), Pkinase_Tyr, Pkinase

1.3e-002

2

18

NB-ARC

re

P l

a n

f o

ro 50

STKc (IRAK, MAPK), Pkinase_Tyr

5 7

Motif distribution across genes is shown in Figure 3. LRR= Leucine Rich Repeat; STKc = Serine/Threonine kinases; IRAK = Interleukin-1 Receptor Associated

r u

Kinases and related STKs; MAPK = Mitogen-Activated Protein Kinase and similar MAPKs; Pkinase = Protein kinase domain; Pkinase_Tyr = Protein tyrosine kinase; NB-ARC = Nucleotide-binding adaptor shared by APAF-1, certain R gene products, and CED-4.

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Journal Pre-proof Figure legends

Figure 1. Microsynteny maps showing the syntenic regions (A) and the positions of genes (B) in the LepR4syntenic regions of B. rapa blackleg resistant loci LepR4 in B. oleracea chromosomes. In Figure B, the location of the LepR4 region on A06 and the syntenic region on

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C03 & C08 are indicated by the scale (in Mb) of the chromosome lengths.

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Figure 2: Functional domains of the twelve genes related to disease resistance in LepR4-

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syntenic regions of B. oleracea. Domain names are labeled for each protein and also presented

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in the legend.

Figure 3: Occurrence and distribution of protein motifs and exon-intron structures of the

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identified NBS, LRR, TIR, CC and RLK genes within the LepR4-syntenic regions in B. oleracea.

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The length and position of motifs in the protein sequences are indicated using colored blocks.

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The amino acid sequence of each motif is shown in Table 3. Exons and introns are represented by light blue boxes and blue lines, respectively.

Figure 4: Chromosomal positions (A) of the twelve NBS, LRR, TIR, CC and RLK genes within the LepR4-syntenic regions in B. oleracea. Microsynteny analysis (B) of these twelve genes from B. oleracea (C genome) in B. rapa (A genome), B. nigra (B genome), and Arabidopsis thaliana. Each colored line indicates a specific gene with colors corresponding to the gene name in the legend.

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Figure 5: Expressions of the twelve LepR4-syntenic NBS, LRR, TIR, CC and RLK domain containing genes of B. oleracea in response to the Leptosphaeria maculans isolates 03-02 s and 00-100 s in the cotyledons of resistant (SCNU-98 and SCNU-03) and susceptible (SCNU-59 and SCNU-72) B. oleracea lines as analyzed by qRT-PCR. Twelve days old cotyledons were inoculated with fungal isolates and total RNA were extracted from cotyledons collected at 0 h

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(untreated) and 6 h, 24 h, 48 h (mock and pathogen inoculated). Error bars represent standard

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deviation of the means of three independent replicates. Different letters above the bars

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indicate statistically significant differences based on Tukey’s pairwise comparisons. Details of

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gene specific primers are shown in Table S2.

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==()==

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6