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Brassica oilseed crops (canola/oilseed rape) are the world's third leading source of vegetable oil with Brassica napus L. (AACC, n = 19), Brassica rapa (AA, n = 10) and Brassica juncea (AABB, n = 18) being the main Brassica oilseed species cultivated worldwide (Singh, 2006). Over the past four decades blackleg disease (stem canker) has become a major limitation for canola production (Fitt et al., 2006). The Dothideomycete fungus Leptosphaeria maculans is the causal agent of the disease. The disease cycle initiates with spores that are released from infected plant debris. The spores germinate on cotyledons and leaves of young plants producing germ tubes that enter through stomata, leading to the subsequent intercellular growth of the hyphae. Except for the formation of lesions on the host leaves, L. maculans remains asymptomatic through most of its life cycle within the adult plant until it forms a canker at the crown of the stem, which results in lodging and significant yield loss (Howlett et al., 2001). The most efficient approach for controlling blackleg disease is deployment of plant resistance genes (Delourme et al., 2006).
There have been many genetically characterized Brassica genes described for resistance to L. maculans, though, as yet, none of these genes have been cloned. The majority of these genes have been mapped to the Brassica A genome, including Rlm1 on chromosome A07 and LepR3 on A10 (Ansan-Melayah et al., 1998; Balesdent et al., 2001, 2002; Delourme et al., 2004, 2006; Mayerhofer et al., 2005; Yu et al., 2005, 2008; Long et al., 2011; Raman et al., 2012). At least three R-genes are carried in the B genome (Chèvre et al., 1996, 1997; Balesdent et al., 2002; Christianson et al., 2006; Kutcher et al., 2010) and none have yet been reported for the C genome. These genes convey resistance to L. maculans isolates in a race-specific manner. The corresponding avirulence (Avr) genes are often located in genetic clusters in the L. maculans genome (Balesdent et al., 2002) and several have been mapped or cloned (Cozijnsen et al., 2000; Gout et al., 2006; Fudal et al., 2007; Parlange et al., 2009; Ghanbarnia et al., 2012). The recent release of both the B. rapa (AA) genome (Wang et al., 2011) and the L. maculans genome (Rouxel et al., 2011) sequences should help facilitate a greater understanding of this host-pathogen system.
The L. maculans resistance (LepR) genes, LepR1, LepR2 and LepR3 have been introgressed into B. napus through a resynthesis of B. napus from a B. rapa ssp. sylvestris × B. oleracea var. alboglabra interspecies cross (Crouch et al., 1994; Buzza & Easton, 2002). LepR1 and LepR2 were identified and mapped to linkage groups A02 and A10, respectively (Yu et al., 2005). The third major blackleg resistance gene, LepR3, is present in the B. napus cultivar ‘Surpass 400’ and has been mapped to linkage group A10, at a distance of 11.7 cM from the LepR2 locus (Yu et al., 2008). LepR3-initiated resistance in ‘Surpass 400’ is associated with a hypersensitive response (Li et al., 2004, 2007), a feature of many, although not all, plant resistance gene (R-gene)-mediated defense responses (Hammond-Kosack & Jones, 1996). LepR3 was first described as a single, dominant gene from field-based studies (Li & Cowling, 2003), although several recent reports suggest the presence of two independent R-genes in ‘Surpass 400’ (Van de Wouw et al., 2009; Long et al., 2011). It was demonstrated that two independent L. maculans Avr genes, AvrLm1 and AvrLmS, trigger defence responses in ‘Surpass 400’, thus it was inferred that ‘Surpass 400’ contained both Rlm1 and a second gene referred to as ‘RlmS’ (Van de Wouw et al., 2009).
Plant resistance responses to pathogens fall under two general categories based on the pathogen molecules that trigger the responses; ‘pattern-triggered immunity’ (PTI) where slowly-evolving pathogen-associated molecular patterns (PAMPS) trigger basal defense responses, or ‘effector-triggered immunity’ (ETI), in which specific pathogen effectors, targeted to disrupt PTI, either directly or indirectly trigger specific R-genes (Jones & Dangl, 2006). Though PTI and ETI are often described as separate pathways, the responses likely function as a coordinated network (Katagiri & Tsuda, 2010). A common feature of most plant R-genes is the presence of leucine rich repeat (LRR) motifs that play a major role in recognition of pathogen effectors by facilitating protein–protein interactions (McDowell & Woffenden, 2003).
The majority of the plant R-proteins are predicted to be located intracellularly; however, there are several examples of extra-cytoplasmic LRR (eLRR)-containing R-proteins that are anchored to the plasma membrane via a transmembrane (TM) domain (Kruijt et al., 2005; Yang et al., 2012). One group of well-characterized eLRR R-genes are the tomato Cf genes that confer resistance against Cladosporium fulvum, the causal agent of tomato leaf mold disease. Cf genes encode a group of receptor-like proteins with recognition specificity for different avirulence proteins encoded by C. fulvum (Wulff et al., 2009). Hyphae of C. fulvum enter the intercellular space through stomata and are confined to the intercellular space (Thomma et al., 2005), which is analogous to the infection and growth of L. maculans hyphae within the plant host tissues. While three of the L. maculans effectors – AvrLm1, AvrLm4-7 and AvrLm6 – have been cloned (Gout et al., 2006; Fudal et al., 2007; Parlange et al., 2009) their function and subcellular location in the host remains to be determined. This paper provides insight into the molecular recognition of L. maculans by its host. Here we report high-resolution mapping of the LepR3 locus on linkage group A10 of B. napus, the investigation of collinearity between the LepR3 region of B. napus, B. rapa and Arabidopsis thaliana, and the cloning of the LepR3 gene, which encodes a receptor-like protein. We also demonstrate that the L. maculans avirulence gene AvrLm1 confers avirulence to both LepR3 and Rlm1. We believe this to be the first published report of the cloning of an R-gene from the important oilseed crop species Brassica napus.
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- Supporting Information
The region of the B. napus chromosome A10 in which the LepR3 locus is situated had previously been shown to share a high degree of low-resolution collinearity with chromosome 5 of A. thaliana (Parkin et al., 2002, 2005). The results of our study highlight the conserved nature of this collinear region at high resolution, with perfect marker order conservation. No change in marker order was detected over this region of A. thaliana (not shown), B. rapa or B. napus (Fig. 1). This close synteny allowed the development of targeted markers that reduced the size of the LepR3 interval to 0.4 cM (Fig. 1).
In a previous study of the LmR1 gene on linkage group A07 of B. napus (Mayerhofer et al., 2005) collinearity with A. thaliana chromosome 1 (At1) was exploited to identify a number of candidate genes associated with disease resistance, though this study was complicated by an inversion that differentiated B. napus and A. thaliana in this region. By contrast, the search for candidate genes for LepR3 was facilitated by the close collinearity over the interval between markers sN8502 and sR0685 on A10 of B. napus and the corresponding region between At5g13810 and At5g11700 on At5. Although none of the A. thaliana genes were likely homologues of LepR3, the collinear nature of the relationship between A. thaliana and Brassica spp. over this region allowed the retrieval of B. rapa BAC sequences related to the target interval, one of which harbored the LepR3 homologue. While this approach to finding a candidate gene was employed successfully in this study, the recent release of the B. rapa genome (Wang et al., 2011) makes this approach mostly obsolete for finding Brassica A genome genes. However, using A. thaliana as a bridging genome may still be useful for placing Brassica B and C genome BACs within target intervals, at least until sequencing of these genomes is completed.
The LepR3 gene discovered in this study is a member of the receptor-like protein family of genes first linked to disease resistance in plants with the cloning of the Cf-9 gene for resistance to Cladosporium fulvum in tomato (Jones et al., 1994). Both Cf-9 and the well-characterized tomato RLP Ve1 (Kawchuk et al., 2001) have been previously clustered in the same ‘superclade’ as At3g05650 (Fritz-Laylin et al., 2005); the best match to LepR3 in A. thaliana (this study). A large family of 56 RLP genes is found in the A. thaliana genome (Fritz-Laylin et al., 2005). Each of the two Brassica genomes in B. napus (AACC) have evolved through the whole-genome triplication of an ancestral Brassicaceae genome closely related to A. thaliana (Lagercrantz, 1998; Nelson & Lydiate, 2006; Ziolkowski et al., 2006) thus the RLP gene family in B. napus could number close to 300 members.
The nested PCR strategy employed in this study to clone LepR3 enabled us to efficiently amplify a unique fragment without interference from any other RLP locus in the genome and without knowing the precise sequence of the target, or the added time and expense of building and screening a large-insert genomic library. This method may also prove useful for the cloning of genes from other large gene families. However, this approach has the limitation of only being applicable for use in examining candidate genes detected in the B. rapa genome. A full B. napus reference genome sequence would likely be a better resource for this approach; however, a full pathological examination of the reference B. rapa var. ‘Chiifu’ for the presence of resistance genes for all of the major B. napus pathogens would be very useful.
Investigation of the LepR3 locus in the resistant ‘Surpass 400’ and the susceptible ‘Topas DH16516’ revealed three regions harboring insertion/deletion (indel) differences between the two loci (Figs 3, S2). However, these indels do not disrupt the expression of the two alleles as we were able to detect transcripts from both loci. It is not yet known what role, if any, the truncated form of the LepR3 transcript plays in the resistance response, though we speculate that it may be the product of post-transcriptional control of transcript abundance. While alternative splicing has been described in NBS-LRR R-genes (Gassmann et al., 1999; Marathe et al., 2002; Schornack et al., 2004; Ferrier-Cana et al., 2005) we are unaware of any reports of alternative splicing or ‘exon editing’ in RLPs.
Here we provide evidence that the L. maculans effector AvrLm1 confers avirulence to both LepR3 and Rlm1. It has already been demonstrated that the addition of AvrLm1 to an avrLm1, AvrLmS’ L. maculans isolate produces a stronger avirulence reaction on ‘Surpass 400’ than the untransformed isolate (Van de Wouw et al., 2009). The recognition of a single Avr gene by two plant R-genes is not unprecedented in the L. maculans-B. napus pathosystem, since the L. maculans Avr gene AvrLm4-7 was shown to trigger a defense response in both Rlm4 and Rlm7-carrying B. napus lines (Parlange et al., 2009), although in this case both of the R-genes are located in the same genetic cluster, and may be allelic variants (Delourme et al., 2004). Only three L. maculans Avr genes have been cloned to date, yet two of them have been shown to trigger resistance responses from two separate Brassica resistance loci. This is suggestive of the broad use of these Avr genes by the pathogen and possible chromosomal rearrangement and/or horizontal transfer of R-genes among the Brassica spp.
Are Rlm1 and LepR3 the same gene? Rlm1 occupies a different chromosomal location (A07 – Delourme et al., 2004) than LepR3 (A10 – this study) thus, genetically, they should be considered separate resistance loci. Van de Wouw et al. (2009) speculated that the LepR3 resistance locus may have been produced via the translocation of Rlm1 from A07 to A10 during the original Crouch et al. (1994) resynthesis used to create ‘Surpass 400’, or that ‘Surpass 400’ contained both Rlm1 and LepR3, in addition to RlmS. During our mapping of the LepR3 locus there was no evidence to support either of these hypotheses; all markers behaved as predicted by their syntenic location on chromosome A10 of B. rapa, including the LepR3 locus itself. Had the LepR3 gene been nonsyntenic (i.e. located on A07) in B. rapa, we would not have been able to identify the candidate RLP gene on A10 of the B. rapa genome sequence. Alternatively, if both Rlm1 (A07) and LepR3 (A10) were present in ‘Surpass 400’ we would have observed a 3R : 1S segregation ratio for the BC population (two independently-assorting loci) when characterizing the plants with ‘3R5′ (AvrLm1, avrLmS). Instead, we saw the expected 1R : 1S ratio, with all of the observed resistance being explained by the A10 LepR3 locus and no distortion of associated markers to suggest it was the product of a recent translocation. It is still possible that Rlm1 and LepR3 are homologous sequences located at two independent loci within the B. napus genome, as nonsyntenic transposition of genes has been observed in Brassica genomes (Cheung et al., 2009). However, the chromosomal regions of A07 and A10 harboring the two genes do not share any homology (Parkin et al., 2005) and while recombination between homoeologous A and C genome chromosomes is frequently observed in resynthesized material (Szadkowski et al., 2010), there are no reports of nonhomoeologous translocation events occurring within the A genome of B. napus. Indeed, the Rlm1 locus was previously mapped to the same A07 location in both B. rapa and in a resynthesized B. napus (Leflon et al., 2007). The relationship between the LepR3 and Rlm1 resistance loci will be elucidated through the future fine mapping and study of the genomic region surrounding the Rlm1 gene. Meanwhile, we may be able to determine if the protein products of the redundant R-genes recognize the same or different epitopes of AVRLM1 through the study of naturally occurring or induced mutants. In this way we may be able to identify a version of AvrLm1 that confers avirulence to LepR3 and not Rlm1, similar to AvrLm7, a mutated allele of AvrLm4-7 which triggers Rlm7 but not Rlm4 (Parlange et al., 2009). However, at present, we have no evidence for such a gene.
While direct R-gene–Avr interactions have been demonstrated in a few plant–pathogen interactions (Jia et al., 2000; Deslandes et al., 2003; Dodds et al., 2006), other evidence has suggested that R-genes are not always triggered by direct interaction with pathogen effectors. The ‘guard hypothesis’ (reviewed in Jones & Dangl, 2006) suggests many R-genes are triggered indirectly by recognizing the disruption of other host cellular targets by pathogen effectors. In this scenario the R-protein is likely to be part of a multiprotein complex involved in monitoring the basal defense machinery of the cell and in triggering defense signaling by modifying the stability of the R-protein (reviewed in Belkhadir et al., 2004). Given that LepR3 codes for a protein with extracellular LRRs, it would seem likely that it would detect a fungal elicitor present outside of the cell, produced by the invading L. maculans during infection, suggesting a direct interaction. However, in studies with the well-characterized Cf RLP gene family of tomato, which is structurally similar to LepR3, no direct interaction between the RLPs and their corresponding AVR proteins has been detected. This suggests that the Cf proteins act as guards of the avirulence targets and interact with the AVR proteins in an indirect manner (Kruijt et al., 2005). For example, recognition of C. fulvum Avr2 effector by the Cf2 protein is mediated by the tomato cysteine protease Rcr3. Avr2 is a cysteine protease inhibitor that binds and inhibits Rcr3 (Rooney et al., 2005). However, unlike many fungal effectors, which are cysteine-rich, AVRLM1 only contains one cysteine residue. Cysteine-rich effectors are believed to withstand plant proteases, which are secreted into the host apoplastic space. Based on this, Gout et al. (2006) suggested that AVRLM1 may be localized to the host cytoplasm. Cloning of LepR3 and future determination of its protein interactions will help in elucidating the function and potential host target of AVRLM1.
Our results, demonstrating that AvrLm1 confers avirulence to LepR3, also offer a possible explanation as to the rapid loss of effective LepR3 resistance in B. napus material, including ‘Surpass 400′, in some parts of Australia soon after it was deployed (Sprague et al., 2006). Given that Rlm1 varieties were already in use in Australia before the release of ‘Surpass 400′ (Rouxel et al., 2003) and that the ‘breakdown’ of resistance was coincident with a large decrease in AvrLm1 allele frequency (Van de Wouw et al., 2010) it is possible that some populations of L. maculans had been driven towards a high proportion of virulent avrLm1 pathotypes because of previous exposure to Rlm1. This could have effectively enriched entire populations of the pathogen for virulence on LepR3, drastically reducing the gene's effective lifespan in these areas.
Van de Wouw et al. (2009) showed the L. maculans avirulence gene ‘AvrLmS’ triggers a second AvrLm1-independent R-gene in ‘Surpass 400’ referred to as ‘RlmS’. Our results, demonstrating that the avrLm1 isolate ‘99-53’ induces a resistance response from ‘Surpass 400’ but not Rlm1 lines or LepR3-transgenic ‘NLA8’ plants, corroborates the presence of both AvrLmS in L. maculans and RlmS in ‘Surpass 400’. This is also supported by the findings of a recent survey of western Canadian L. maculans isolates where 97.9% of the ‘avrLm1’ isolates tested still produced a resistance response on ‘Surpass 400’ (Kutcher et al., 2010). It also shows that the AvrLmS does not complement LepR3. In light of this evidence we believe the correct designation for the resistance genotype of ‘Surpass 400’ should be ‘LepR3, RlmS’. Mapping of the RlmS resistance locus and study of its interaction with AvrLmS isolates is currently in progress. In another recent report, ‘Surpass 400’ resistance was once again investigated and two resistance loci (named ‘Blm1’ and ‘Blm2’) were mapped to B. napus chromosome A10 (Long et al., 2011) using the L. maculans isolate ‘87-41’, which we have shown also carries AvrLm1 (Table 1). The Blm1 locus reported in that study corresponds to the same B. rapa BAC (KBrB080E24) described here for cloning the LepR3 gene. As the Blm1 resistance locus was mapped from the same cultivar, corresponds to the same genomic location and produces a resistance response to an AvrLm1 isolate we believe that it is LepR3.
Our results highlight the need to consider redundancy in R-gene specificities when using differential isolate sets to determine the R-gene content of Brassica lines. Rlm1 and LepR3 cannot currently be distinguished by differential phenotypic reactions; one needs to either determine the chromosomal linkage of the resistance phenotype or amplify and sequence the LepR3/lepR3 locus in order to discriminate between the presence of the two genes. We should not rely on a literal interpretation of the ‘gene-for-gene’ hypothesis, where any given avirulence protein only ever interacts with one specific R-protein.