Two independent approaches converge to the cloning of a new Leptosphaeria maculans avirulence effector gene, AvrLmS‐Lep2

Abstract Brassica napus (oilseed rape, canola) seedling resistance to Leptosphaeria maculans, the causal agent of blackleg (stem canker) disease, follows a gene‐for‐gene relationship. The avirulence genes AvrLmS and AvrLep2 were described to be perceived by the resistance genes RlmS and LepR2, respectively, present in B. napus ‘Surpass 400’. Here we report cloning of AvrLmS and AvrLep2 using two independent methods. AvrLmS was cloned using combined in vitro crossing between avirulent and virulent isolates with sequencing of DNA bulks from avirulent or virulent progeny (bulked segregant sequencing). AvrLep2 was cloned using a biparental cross of avirulent and virulent L. maculans isolates and a classical map‐based cloning approach. Taking these two approaches independently, we found that AvrLmS and AvrLep2 are the same gene. Complementation of virulent isolates with this gene confirmed its role in inducing resistance on Surpass 400, Topas‐LepR2, and an RlmS‐line. The gene, renamed AvrLmS‐Lep2, encodes a small cysteine‐rich protein of unknown function with an N‐terminal secretory signal peptide, which is a common feature of the majority of effectors from extracellular fungal plant pathogens. The AvrLmS‐Lep2/LepR2 interaction phenotype was found to vary from a typical hypersensitive response through intermediate resistance sometimes towards susceptibility, depending on the inoculation conditions. AvrLmS‐Lep2 was nevertheless sufficient to significantly slow the systemic growth of the pathogen and reduce the stem lesion size on plant genotypes with LepR2, indicating the potential efficiency of this resistance to control the disease in the field.


| INTRODUC TI ON
Diseases of field crops are best controlled by the use of resistant varieties. Major race-specific resistance (R) genes, matching avirulence (AVR) genes in the pathogen, are extensively used in crop breeding programmes. However, the widespread and repeated use of cultivars with a single R gene exerts strong selection pressure on the pathogen populations, leading to the emergence of virulent isolates and breakdown of the resistance.
An example of such an arms race between the host crop and its pathogen is the breakdown of Brassica napus (oilseed rape, canola) resistance against Leptosphaeria maculans, the ascomycete causing stem canker (blackleg), a major disease of oilseed rape (Fitt et al., 2006;Sprague et al., 2006). The L. maculans infectious cycle starts with ascospores or conidia germination on cotyledon and leaves.
The fungus then asymptomatically colonizes petioles and stems, and finally develops a dry necrosis at the stem base responsible for the main yield losses. Both quantitative, also known as polygenic or adult plant resistance, and single, major gene resistance have been reported in B. napus (Delourme et al., 2006). To date, 18 R genes to L. maculans have been described, with only three cloned to date: LepR3, Rlm2, and Rlm9 (Delourme et al., 2004(Delourme et al., , 2006Larkan et al., 2013Larkan et al., , 2015Larkan et al., , 2020Long et al., 2011;Rimmer, 2006; Van de Wouw et al., 2009;Yu et al., 2005Yu et al., , 2008. Meanwhile, nine L. maculans avirulence genes have already been cloned: AvrLm1, AvrLm2, AvrLm3, AvrLm4-7, AvrLm5-9, AvrLm6, AvrLm10, AvrLm11, and AvrLm14 (Balesdent et al., 2013;Degrave et al., 2021;Fudal et al., 2007;Ghanbarnia et al., 2015Ghanbarnia et al., , 2018Gout et al., 2006;Parlange et al., 2009;Petit-Houdenot et al., 2019;Plissonneau et al., 2016;Van de Wouw et al., 2014). Map-based cloning of the first AvrLm genes (AvrLm1, AvrLm6, and AvrLm4-7; Fudal et al., 2007;Gout et al., 2006;Parlange et al., 2009) took many years due to the lack of a L. maculans genome sequence and their location in repeat-rich, gene-poor regions of the genome (Rouxel et al., 2011). The availability of a reference genome and a repertoire of effector genes facilitated rapid identification of several further AVR genes, including AvrLm11, AvrLm2, AvrLm5-9, and AvrLm14 (Balesdent et al., 2013;Degrave et al., 2021;Ghanbarnia et al., 2015Ghanbarnia et al., , 2018. B. napus is an allotetraploid (AACC, 2n = 38) species derived from the hybridization of the diploid parents, Brassica oleracea (CC,2n = 18) and Brassica rapa (AA,2n = 20). These related species have been explored as resistance sources to increase the R gene pool in oilseed rape. Four R genes to L. maculans (LepR1,LepR2,LepR3,and LepR4) have been genetically characterized in B. rapa subsp. sylvestris (Yu et al., 2005(Yu et al., , 2008(Yu et al., , 2013, which was used as a source of resistance in the 1990s (Crouch et al., 1994), leading to the release of B. napus varieties with "sylvestris-derived resistance". From 2000 to 2003, the B. napus cultivar Surpass 400 and related cultivars containing sylvestris-derived resistance were grown on large acreages across Australia before the resistance was overcome in the Eyre Peninsula region of South Australia (Li et al., 2004;Sprague et al., 2006). Based on the genetic analysis of virulent isolates on Surpass 400, Van de Wouw et al. (2009) reported that at least two avirulence genes, AvrLm1 and AvrLmS, conveyed avirulence towards Surpass 400, supporting the idea of an RlmS-AvrLmS interaction in the host plant. On the plant side, genetic analyses described either one or two resistance loci in Surpass 400 (Li & Cowling, 2003;Long et al., 2011;Yu et al., 2008), termed LepR3 (Yu et al., 2008), BLMR1 and BLMR2 (Long et al., 2011), or LepR3 and RlmS (Larkan et al., 2013). Larkan et al. (2013) mapped and cloned LepR3 from Surpass 400 and showed that LepR3 recognizes the AvrLm1 protein. LepR3 can therefore be considered a functional homologue of Rlm1, although LepR3 and Rlm1 reside on different chromosomes (A10 and A07, respectively). It was also demonstrated that a second resistance gene was present in Surpass 400, probably corresponding to the AvrLmS avirulence gene, that is, RlmS (Larkan et al., 2013). Yu et al. (2008)  Identification of R gene content in a cultivar and conclusions on the genetic control of the resistance has traditionally relied on phenotypic evaluation of the interaction based on inoculation tests with L. maculans isolates, for which the avirulence gene content may differ between studies. Due to epistatic effects between AVR genes, a single-gene control of a resistant phenotype toward a given isolate may hide a more complex R gene control due to the lack of adequate differential isolates. Cloning of the corresponding AVR genes can thus help us understand the relationships between R genes with different names. In the current study, we report on the cloning of the AVR gene matching RlmS and LepR2 using two independent approaches. The AVR gene recognized by LepR2, AvrLep2, was cloned following a standard map-based cloning approach, whilst the gene interacting with RlmS, AvrLmS, was cloned using a bulked-segregant sequencing (BSS) strategy.
The two approaches identified the same gene, now referred to as AvrLmS-Lep2, which shares all characteristics of L. maculans avirulence effector genes. Noticeably, the two strategies converged although the interaction phenotypes differed between laboratories, probably due to the environmental conditions. and avirulent on Surpass 400, respectively ( Figure 1) was used to generate a progeny for BSS. Phenotyping of the parental and progeny isolates on an extended B. napus differential set revealed that all isolates lacked AvrLm1 (data not shown) and that AvrLm6, AvrLm4-7, and AvrLmS segregated with the expected 1:1 ratio (Table 1). In addition, eight phenotypic classes were recovered in the progeny, with ratios fitting the hypothesis of independence between AvrLm6, AvrLm4-7, and AvrLmS (χ 2 = 8.25, p = 0.689;

| BSS statistics and validation of the BSS strategy
The Illumina whole-genome sequences (2 × 150 bp) were generated from the six bulks and the two parental isolates. After quality trimming, the average number of reads generated was in the range of 47-75 million and the average genome coverage depth ranged between 164× and 272× (Table S1). In total, 65,727 single nucleotide polymorphisms (SNPs) were identified between the parental isolates, excluding 19,650 SNPs in repetitive regions. The average number of SNPs found in the bulked progeny was 64,473 (Table S2). After quality filtering for quantitative trait locus (QTL) mapping in the bulked F I G U R E 1 Box plot of rating scores of interaction phenotypes between wild-type or transformed isolates of Leptosphaeria maculans and four Brassica napus genotypes. From top to bottom: susceptible check Bristol (Rlm2-Rlm9), Surpass 400 (LepR3-RlmS), Topas DH16516 (no R gene), Topas-LepR2 (LepR2). For each box, the red cross indicates the score mean and the black horizontal line indicates the score median. The rectangles comprise 75% (Q1-Q3) of the rating scores. Wild-type isolates are in bold; X82.14-i and WT50-j are five and six independent transformed isolates of X82.14 and WT50, respectively, with the candidate gene Lmb_jn3_08343    Samples with AvrLmS are shown in green and those with avrLmS in purple. The y axis limit was set to 400. (b) Circos plot of ∆(SNP-index) between Bulk 1/2 with AvrLmS (outer circle) and Bulk 5/6 with AvrLmS+AvrLm7 (inner circle) for the 19 scaffolds larger than 1 Mb. Confidence intervals of 99% (red) are shown. The y axis is bounded from −0.5 to 0.8 for the outer plots and from −0.9 to 0.9 for the inner plots. The shared quantitative trait locus (QTL) for AvrLmS is located on scaffold 9. Coordinates for QTLs are provided in Tables  within the AvrLep2 interval, previously generated RNA-Seq data produced from L. maculans-infected B. napus seedlings (Haddadi et al., 2016(Haddadi et al., , 2019 were mapped to the L. maculans genome. Genes within the AvrLep2 interval were manually annotated and a predicted secreted protein was identified as the AvrLep2 candidate.

| Two approaches, one "typical" avirulence effector gene candidate
The two cloning strategies identified the same candidate gene, Lmb_jn3_08343, which is 426 bp and contains one exon. In isolate v23.1.3, it is located in a typical AT-rich region of 285 kb containing one single gene (Figures 3 and S4 (Figures 1, 4, and S6) and on the RlmS line 72.1 (Figure 4). Therefore, Lmb_jn3_08343 encodes the avirulence effector protein corresponding to RlmS and is also able to elicit the LepR2 resistance response.
Second, two types of construct for transformation using the  (Table 3), confirming the identity and the specificity of the candidate gene as AvrLep2.

| Expression analysis
Previously generated RNA-Seq data from isolate v23. with AvrLep2 having a similar expression to AvrLm5-9 in both isolates ( Figure S7).  Table S9).

| Adult plant tests
Only one "AvrLep2" isolate (AI397) was able to produce relatively high infection in Topas-LepR2. In contrast, all three isolates that carried a deletion at the AvrLep2 locus (B16-13, B18-10, and TA B L E 3 Pathogenicity test for Leptosphaeria maculans isolates (wild-type and transformants) on Brassica napus lines carrying diverse blackleg resistance (R) genes

B18-11) produced severe and identical levels of infection in both
the control Topas DH16516 and Topas-LepR2 lines.

| A posteriori control of the consistency of phenotypic data
The sequenced reference isolate v23.1.3 had been previously shared between laboratories but here the interaction phenotypes  to be monogenic, based on field assays in Australia (Li & Cowling, 2003) and genetic mapping (Yu et al., 2008), though later mapping with defined isolates under controlled conditions suggested the occurrence of two genes in Surpass 400 (named as BLMR1 and BLMR2, Long et al., 2011;or LepR3 and RlmS, Larkan et al., 2013). The resistance gene LepR2 in the doubled-haploid line AD49 was described as limiting, but not preventing, hyphal growth of avirulent isolates, along with restricting sporulation on the infected plant tissues (Yu et al., 2005). In the same study, most field isolates inoculated (22 out of 32) were found to display large, nonsporulating lesions (scores between 3 and 6 on a 0-9 scale), while only eight isolates displayed typical HR (score <3) (Yu et al., 2005). Furthermore, in the study by Yu et al. (2005) the two most virulent isolates on LepR2 displayed scores between 6 and 6.5, but never reached the scores observed on the susceptible genotypes (scores >7.5  (Balesdent et al., 2002;Ghanbarnia et al., 2015;Plissonneau et al., 2018) that can be challenging to identify through cotyledon phenotyping. Another hypothesis to explain the vari-  transgenic isolates generated here could be further used to determine whether BLMR2, also obtained from Surpass 400, also recognizes the same avirulence gene using plant material (near-isogenic lines with or without BLMR2) recently described (Zhang et al., 2021). This work illustrates a first step toward the standardiza-

| Plant genotypes and inoculation tests
Isolates were grown on 20% V8-agar medium to produce conidia.
Conidia (10 µl, 10 7 spores/ml) were inoculated onto wounded cotyledons of 10 to 12 10-day-old seedlings per plant genotype. The Four different isolates were inoculated on each plant. Symptoms were scored two or three times at 12-21 dai using a 1-6 scale, with scores 1-3 and 4-6 corresponding to avirulent and virulent phenotypes, respectively (Balesdent et al., 2005). To compare the interaction phenotypes of progeny isolates with those of parental isolates, the nonparametric Kruskall-Wallis test was applied, with a p value threshold set up at 0. 05, using XLSTAT v. 2013.4.03. The phenotypes of the progeny selected for BSS were confirmed in an independent inoculation test.

| DNA extraction and bulk preparation
Isolates were grown on Fries liquid medium for 7 days as previously established (Fudal et al., 2007) (Danecek et al., 2011) and variants with a phred-scaled quality score <30 were excluded.
Indels were removed using GATK SelectVariants. The public reference genome repeat annotation was used to exclude SNPs occurring within repeats. SNPs that were heterozygous or monomorphic in parental isolates were also excluded. Finally, VCF files were converted to tabular format with GATK VariantsToTable.

| Phenotyping of mapping population
For mapping AvrLep2, an F 1 population produced from the parental isolates v23.1.3 and 00-100, previously used to map the AvrLm5-9 locus (Ghanbarnia et al., 2018), was shown to also be segregating for the AvrLep2 phenotype under the controlled growth chamber conditions used at AAFC Saskatoon (Larkan et al., 2013). The B. napus line Topas-LepR2 (Larkan et al., 2016) and the LepR2 line 1135 (Yu et al., 2012) were used to determine the phenotypic response of the parental isolates and progeny to LepR2. B. napus cotyledons were inoculated as described previously (Chen & Fernando, 2006). Each L. maculans isolate was tested on 12 seedlings of the differential lines and 12 seedlings of Topas as susceptible control. The disease reactions were scored 14 dai and rated using the 0-9 scale described by Williams (1985). Paired-end Illumina sequencing and assembly of parental isolates was previously described by Ghanbarnia et al. (2015).

| Expression analysis by RNA-Seq
Expression of AvrLep2 was examined from infection time course data previously generated (Haddadi et al., 2016 progeny were selected to screen KASP markers spanning the whole L. maculans genome (Rouxel et al., 2011). A linkage map of AvrLep2 was constructed using the MAP function of QTL IciMapping v. 3.2 software (Li et al., 2008). Minimum LOD (logs of the odds ratios of linkage vs. no linkage) scores of 6.0 (maximum recombination fraction of 0.6) were used to group loci. After initial linkage between markers and the AvrLep2 locus was established, additional KASP markers targeted to the AvrLep2 interval were designed based on genomic polymorphisms to enrich the map. Cloning, transformation, and functional validation of AvrLep2 was performed as described previously (Ghanbarnia et al., 2015). For functional validation two constructs were produced. First, the open reading frame for the AvrLep2 candidate gene (426 bp) was amplified and transferred to the fungal transformation vector pLM4 (Ghanbarnia et al., 2015) under the control of the AvrLm1 promoter. In addition, an AvrLep2 candidate gene amplicon, including the native promoter region showing infection damage. Results were plotted using the ggplot2 (Wickham, 2016) and reshape2 (Wickham, 2007) packages in R v.