External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells.

Pathogens of plants and animals produce effector proteins that are transferred into the cytoplasm of host cells to suppress host defenses. One type of plant pathogens, oomycetes, produces effector proteins with N-terminal RXLR and dEER motifs that enable entry into host cells. We show here that effectors of another pathogen type, fungi, contain functional variants of the RXLR motif, and that the oomycete and fungal RXLR motifs enable binding to the phospholipid, phosphatidylinositol-3-phosphate (PI3P). We find that PI3P is abundant on the outer surface of plant cell plasma membranes and, furthermore, on some animal cells. All effectors could also enter human cells, suggesting that PI3P-mediated effector entry may be very widespread in plant, animal and human pathogenesis. Entry into both plant and animal cells involves lipid raft-mediated endocytosis. Blocking PI3P binding inhibited effector entry, suggesting new therapeutic avenues.

Facilitation of Symplastic Effector Protein Mobility by Paired Effectors Is Conserved in Different Classes of Fungal Pathogens.

It has been discovered that plant pathogens produce effectors that spread via plasmodesmata (PD) to allow modulation of host processes in distal uninfected cells. Fusarium oxysporum f. sp. lycopersici (Fol) facilitates effector translocation by expansion of the size-exclusion limit of PD using the Six5/Avr2 effector pair. How other fungal pathogens manipulate PD is unknown. We recently reported that many fungal pathogens belonging to different families carry effector pairs that resemble the SIX5/AVR2 gene pair from Fol. Here, we performed structural predictions of three of these effector pairs from Leptosphaeria maculans (Lm) and tested their ability to manipulate PD and to complement the virulence defect of a Fol SIX5 knockout mutant. We show that the AvrLm10A homologs are structurally related to FolSix5 and localize at PD when they are expressed with their paired effectors. Furthermore, these effectors were found to complement FolSix5 function in cell-to-cell mobility assays and in fungal virulence. We conclude that distantly related fungal species rely on structurally related paired effector proteins to manipulate PD and facilitate effector mobility. The wide distribution of these effector pairs implies Six5-mediated effector translocation to be a conserved propensity among fungal plant pathogens. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A new family of structurally conserved fungal effectors displays epistatic interactions with plant resistance proteins.

Recognition of a pathogen avirulence (AVR) effector protein by a cognate plant resistance (R) protein triggers a set of immune responses that render the plant resistant. Pathogens can escape this so-called Effector-Triggered Immunity (ETI) by different mechanisms including the deletion or loss-of-function mutation of the AVR gene, the incorporation of point mutations that allow recognition to be evaded while maintaining virulence function, and the acquisition of new effectors that suppress AVR recognition. The Dothideomycete Leptosphaeria maculans, causal agent of oilseed rape stem canker, is one of the few fungal pathogens where suppression of ETI by an AVR effector has been demonstrated. Indeed, AvrLm4-7 suppresses Rlm3- and Rlm9-mediated resistance triggered by AvrLm3 and AvrLm5-9, respectively. The presence of AvrLm4-7 does not impede AvrLm3 and AvrLm5-9 expression, and the three AVR proteins do not appear to physically interact. To decipher the epistatic interaction between these L. maculans AVR effectors, we determined the crystal structure of AvrLm5-9 and obtained a 3D model of AvrLm3, based on the crystal structure of Ecp11-1, a homologous AVR effector candidate from Fulvia fulva. Despite a lack of sequence similarity, AvrLm5-9 and AvrLm3 are structural analogues of AvrLm4-7 (structure previously characterized). Structure-informed sequence database searches identified a larger number of putative structural analogues among L. maculans effector candidates, including the AVR effector AvrLmS-Lep2, all produced during the early stages of oilseed rape infection, as well as among effector candidates from other phytopathogenic fungi. These structural analogues are named LARS (for Leptosphaeria AviRulence and Suppressing) effectors. Remarkably, transformants of L. maculans expressing one of these structural analogues, Ecp11-1, triggered oilseed rape immunity in several genotypes carrying Rlm3. Furthermore, this resistance could be suppressed by AvrLm4-7. These results suggest that Ecp11-1 shares a common activity with AvrLm3 within the host plant which is detected by Rlm3, or that the Ecp11-1 structure is sufficiently close to that of AvrLm3 to be recognized by Rlm3.

Regulation of effector gene expression as concerted waves in Leptosphaeria maculans: a two-player game.

Effector genes, encoding molecules involved in disease establishment, are concertedly expressed throughout the lifecycle of plant-pathogenic fungi. However, little is known about how effector gene expression is regulated. Since many effector genes are located in repeat-rich regions, the role of chromatin remodeling in their regulation was recently investigated, notably establishing that the repressive histone modification H3K9me3, deposited by KMT1, was involved in several fungal species including Leptosphaeria maculans. Nevertheless, previous data suggest that a second regulatory layer, probably involving a specific transcription factor (TF), might be required. In L. maculans, a Dothideomycete causing stem canker of oilseed rape, we identified the ortholog of Pf2, a TF belonging to the Zn2Cys6 fungal-specific family, and described as essential for pathogenicity and effector gene expression. We investigated its role together with KMT1, by inactivating and over-expressing LmPf2 in a wild-type strain and a ∆kmt1 mutant. Functional analyses of the corresponding transformants highlighted an essential role of LmPf2 in the establishment of pathogenesis and we found a major effect of LmPf2 on the induction of effector gene expression once KMT1 repression is lifted. Our results show, for the first time, a dual control of effector gene expression.

Improving sustainable crop protection using population genetics concepts.

Growing genetically resistant plants allows pathogen populations to be controlled and reduces the use of pesticides. However, pathogens can quickly overcome such resistance. In this context, how can we achieve sustainable crop protection? This crucial question has remained largely unanswered despite decades of intense debate and research effort. In this study, we used a bibliographic analysis to show that the research field of resistance durability has evolved into three subfields: (1) "plant breeding" (generating new genetic material), (2) "molecular interactions" (exploring the molecular dialogue governing plant-pathogen interactions) and (3) "epidemiology and evolution" (explaining and forecasting of pathogen population dynamics resulting from selection pressure[s] exerted by resistant plants). We argue that this triple split of the field impedes integrated research progress and ultimately compromises the sustainable management of genetic resistance. After identifying a gap among the three subfields, we argue that the theoretical framework of population genetics could bridge this gap. Indeed, population genetics formally explains the evolution of all heritable traits, and allows genetic changes to be tracked along with variation in population dynamics. This provides an integrated view of pathogen adaptation, in particular via evolutionary-epidemiological feedbacks. In this Opinion Note, we detail examples illustrating how such a framework can better inform best practices for developing and managing genetically resistant cultivars.

Genome-wide mapping of histone modifications during axenic growth in two species of Leptosphaeria maculans showing contrasting genomic organization.

Leptosphaeria maculans 'brassicae' (Lmb) and Leptosphaeria maculans 'lepidii' (Lml) are closely related phytopathogenic species that exhibit a large macrosynteny but contrasting genome structure. Lmb has more than 30% of repeats clustered in large repeat-rich regions, while the Lml genome has only a small amount of evenly distributed repeats. Repeat-rich regions of Lmb are enriched in effector genes, expressed during plant infection. The distinct genome structures of Lmb and Lml provide an excellent model for comparing the organization of pathogenicity genes in relation to the chromatin landscape in two closely related phytopathogenic fungi. Here, we performed chromatin immunoprecipitation (ChIP) during axenic culture, targeting histone modifications typical for heterochromatin or euchromatin, combined with transcriptomic analysis to analyze the influence of chromatin organization on gene expression. In both species, we found that facultative heterochromatin is enriched with genes lacking functional annotation, including numerous effector and species-specific genes. Notably, orthologous genes located in H3K27me3 domains are enriched with effector genes. Compared to other fungal species, including Lml, Lmb is distinct in having large H3K9me3 domains associated with repeat-rich regions that contain numerous species-specific effector genes. Discovery of these two distinctive heterochromatin landscapes now raises questions about their involvement in the regulation of pathogenicity, the dynamics of these domains during plant infection and the selective advantage to the fungus to host effector genes in H3K9me3 or H3K27me3 domains.

Large-scale transcriptomics to dissect 2 years of the life of a fungal phytopathogen interacting with its host plant.

BACKGROUND: The fungus Leptosphaeria maculans has an exceptionally long and complex relationship with its host plant, Brassica napus, during which it switches between different lifestyles, including asymptomatic, biotrophic, necrotrophic, and saprotrophic stages. The fungus is also exemplary of "two-speed" genome organisms in the genome of which gene-rich and repeat-rich regions alternate. Except for a few stages of plant infection under controlled conditions, nothing is known about the genes mobilized by the fungus throughout its life cycle, which may last several years in the field. RESULTS: We performed RNA-seq on samples corresponding to all stages of the interaction of L. maculans with its host plant, either alive or dead (stem residues after harvest) in controlled conditions or in field experiments under natural inoculum pressure, over periods of time ranging from a few days to months or years. A total of 102 biological samples corresponding to 37 sets of conditions were analyzed. We show here that about 9% of the genes of this fungus are highly expressed during its interactions with its host plant. These genes are distributed into eight well-defined expression clusters, corresponding to specific infection lifestyles or to tissue-specific genes. All expression clusters are enriched in effector genes, and one cluster is specific to the saprophytic lifestyle on plant residues. One cluster, including genes known to be involved in the first phase of asymptomatic fungal growth in leaves, is re-used at each asymptomatic growth stage, regardless of the type of organ infected. The expression of the genes of this cluster is repeatedly turned on and off during infection. Whatever their expression profile, the genes of these clusters are enriched in heterochromatin regions associated with H3K9me3 or H3K27me3 repressive marks. These findings provide support for the hypothesis that part of the fungal genes involved in niche adaptation is located in heterochromatic regions of the genome, conferring an extreme plasticity of expression. CONCLUSION: This work opens up new avenues for plant disease control, by identifying stage-specific effectors that could be used as targets for the identification of novel durable disease resistance genes, or for the in-depth analysis of chromatin remodeling during plant infection, which could be manipulated to interfere with the global expression of effector genes at crucial stages of plant infection.

A gene-for-gene interaction involving a 'late' effector contributes to quantitative resistance to the stem canker disease in Brassica napus.

The control of stem canker disease of Brassica napus (rapeseed), caused by the fungus Leptosphaeria maculans is based largely on plant genetic resistance: single-gene specific resistance (Rlm genes) or quantitative, polygenic, adult-stage resistance. Our working hypothesis was that quantitative resistance partly obeys the gene-for-gene model, with resistance genes 'recognizing' fungal effectors expressed during late systemic colonization. Five LmSTEE (stem-expressed effector) genes were selected and placed under the control of the AvrLm4-7 promoter, an effector gene highly expressed at the cotyledon stage of infection, for miniaturized cotyledon inoculation test screening of a gene pool of 204 rapeseed genotypes. We identified a rapeseed genotype, 'Yudal', expressing hypersensitive response to LmSTEE98. The LmSTEE98-RlmSTEE98 interaction was further validated by inactivation of the LmSTEE98 gene with a CRISPR-Cas9 approach. Isolates with mutated versions of LmSTEE98 induced more severe stem symptoms than the wild-type isolate in 'Yudal'. This single-gene resistance was mapped in a 0.6 cM interval of the 'Darmor_bzh' × 'Yudal' genetic map. One typical gene-for-gene interaction contributes partly to quantitative resistance when L. maculans colonizes the stems of rapeseed. With numerous other effectors specific to stem colonization, our study provides a new route for resistance gene discovery, elucidation of quantitative resistance mechanisms and selection for durable resistance.

A two genes - for - one gene interaction between Leptosphaeria maculans and Brassica napus.

Interactions between Leptosphaeria maculans, causal agent of stem canker of oilseed rape, and its Brassica hosts are models of choice to explore the multiplicity of 'gene-for-gene' complementarities and how they diversified to increased complexity in the course of plant-pathogen co-evolution. Here, we support this postulate by investigating the AvrLm10 avirulence that induces a resistance response when recognized by the Brassica nigra resistance gene Rlm10. Using genome-assisted map-based cloning, we identified and cloned two AvrLm10 candidates as two genes in opposite transcriptional orientation located in a subtelomeric repeat-rich region of the genome. The AvrLm10 genes encode small secreted proteins and show expression profiles in planta similar to those of all L. maculans avirulence genes identified so far. Complementation and silencing assays indicated that both genes are necessary to trigger Rlm10 resistance. Three assays for protein-protein interactions showed that the two AvrLm10 proteins interact physically in vitro and in planta. Some avirulence genes are recognized by two distinct resistance genes and some avirulence genes hide the recognition specificities of another. Our L. maculans model illustrates an additional case where two genes located in opposite transcriptional orientation are necessary to induce resistance. Interestingly, orthologues exist for both L. maculans genes in other phytopathogenic species, with a similar genome organization, which may point to an important conserved effector function linked to heterodimerization of the two proteins.

Epigenetic control of effector gene expression in the plant pathogenic fungus Leptosphaeria maculans.

Plant pathogens secrete an arsenal of small secreted proteins (SSPs) acting as effectors that modulate host immunity to facilitate infection. SSP-encoding genes are often located in particular genomic environments and show waves of concerted expression at diverse stages of plant infection. To date, little is known about the regulation of their expression. The genome of the Ascomycete Leptosphaeria maculans comprises alternating gene-rich GC-isochores and gene-poor AT-isochores. The AT-isochores harbor mosaics of transposable elements, encompassing one-third of the genome, and are enriched in putative effector genes that present similar expression patterns, namely no expression or low-level expression during axenic cultures compared to strong induction of expression during primary infection of oilseed rape (Brassica napus). Here, we investigated the involvement of one specific histone modification, histone H3 lysine 9 methylation (H3K9me3), in epigenetic regulation of concerted effector gene expression in L. maculans. For this purpose, we silenced the expression of two key players in heterochromatin assembly and maintenance, HP1 and DIM-5 by RNAi. By using HP1-GFP as a heterochromatin marker, we observed that almost no chromatin condensation is visible in strains in which LmDIM5 was silenced by RNAi. By whole genome oligoarrays we observed overexpression of 369 or 390 genes, respectively, in the silenced-LmHP1 and -LmDIM5 transformants during growth in axenic culture, clearly favouring expression of SSP-encoding genes within AT-isochores. The ectopic integration of four effector genes in GC-isochores led to their overexpression during growth in axenic culture. These data strongly suggest that epigenetic control, mediated by HP1 and DIM-5, represses the expression of at least part of the effector genes located in AT-isochores during growth in axenic culture. Our hypothesis is that changes of lifestyle and a switch toward pathogenesis lift chromatin-mediated repression, allowing a rapid response to new environmental conditions.

Crystal structure of the effector AvrLm4-7 of Leptosphaeria maculans reveals insights into its translocation into plant cells and recognition by resistance proteins.

The avirulence gene AvrLm4-7 of Leptosphaeria maculans, the causal agent of stem canker in Brassica napus (oilseed rape), confers a dual specificity of recognition by two resistance genes (Rlm4 and Rlm7) and is strongly involved in fungal fitness. In order to elucidate the biological function of AvrLm4-7 and understand the specificity of recognition by Rlm4 and Rlm7, the AvrLm4-7 protein was produced in Pichia pastoris and its crystal structure was determined. It revealed the presence of four disulfide bridges, but no close structural analogs could be identified. A short stretch of amino acids in the C terminus of the protein, (R/N)(Y/F)(R/S)E(F/W), was well-conserved among AvrLm4-7 homologs. Loss of recognition of AvrLm4-7 by Rlm4 is caused by the mutation of a single glycine to an arginine residue located in a loop of the protein. Loss of recognition by Rlm7 is governed by more complex mutational patterns, including gene loss or drastic modifications of the protein structure. Three point mutations altered residues in the well-conserved C-terminal motif or close to the glycine involved in Rlm4-mediated recognition, resulting in the loss of Rlm7-mediated recognition. Transient expression in Nicotiana benthamiana (tobacco) and particle bombardment experiments on leaves from oilseed rape suggested that AvrLm4-7 interacts with its cognate R proteins inside the plant cell, and can be translocated into plant cells in the absence of the pathogen. Translocation of AvrLm4-7 into oilseed rape leaves is likely to require the (R/N)(Y/F)(R/S)E(F/W) motif as well as an RAWG motif located in a nearby loop that together form a positively charged region.

A game of hide and seek between avirulence genes AvrLm4-7 and AvrLm3 in Leptosphaeria maculans.

Extending the durability of plant resistance genes towards fungal pathogens is a major challenge. We identified and investigated the relationship between two avirulence genes of Leptosphaeria maculans, AvrLm3 and AvrLm4-7. When an isolate possesses both genes, the Rlm3-mediated resistance of oilseed rape (Brassica napus) is not expressed due to the presence of AvrLm4-7 but virulent isolates toward Rlm7 recover the AvrLm3 phenotype. Combining genetic and genomic approaches (genetic mapping, RNA-seq, BAC (bacterial artificial chromosome) clone sequencing and de novo assembly) we cloned AvrLm3, a telomeric avirulence gene of L. maculans. AvrLm3 is located in a gap of the L. maculans reference genome assembly, is surrounded by repeated elements, encodes for a small secreted cysteine-rich protein and is highly expressed at early infection stages. Complementation and silencing assays validated the masking effect of AvrLm4-7 on AvrLm3 recognition by Rlm3 and we showed that the presence of AvrLm4-7 does not impede AvrLm3 expression in planta. Y2H assays suggest the absence of physical interaction between the two avirulence proteins. This unusual interaction is the basis for field experiments aiming to evaluate strategies that increase Rlm7 durability.

Transposable element-assisted evolution and adaptation to host plant within the Leptosphaeria maculans-Leptosphaeria biglobosa species complex of fungal pathogens.

BACKGROUND: Many plant-pathogenic fungi have a tendency towards genome size expansion, mostly driven by increasing content of transposable elements (TEs). Through comparative and evolutionary genomics, five members of the Leptosphaeria maculans-Leptosphaeria biglobosa species complex (class Dothideomycetes, order Pleosporales), having different host ranges and pathogenic abilities towards cruciferous plants, were studied to infer the role of TEs on genome shaping, speciation, and on the rise of better adapted pathogens. RESULTS: L. maculans 'brassicae', the most damaging species on oilseed rape, is the only member of the species complex to have a TE-invaded genome (32.5%) compared to the other members genomes (<4%). These TEs had an impact at the structural level by creating large TE-rich regions and are suspected to have been instrumental in chromosomal rearrangements possibly leading to speciation. TEs, associated with species-specific genes involved in disease process, also possibly had an incidence on evolution of pathogenicity by promoting translocations of effector genes to highly dynamic regions and thus tuning the regulation of effector gene expression in planta. CONCLUSIONS: Invasion of L. maculans 'brassicae' genome by TEs followed by bursts of TE activity allowed this species to evolve and to better adapt to its host, making this genome species a peculiarity within its own species complex as well as in the Pleosporales lineage.

Genome structure and reproductive behaviour influence the evolutionary potential of a fungal phytopathogen.

Modern agriculture favours the selection and spread of novel plant diseases. Furthermore, crop genetic resistance against pathogens is often rendered ineffective within a few years of its commercial deployment. Leptosphaeria maculans, the cause of phoma stem canker of oilseed rape, develops gene-for-gene interactions with its host plant, and has a high evolutionary potential to render ineffective novel sources of resistance in crops. Here, we established a four-year field experiment to monitor the evolution of populations confronted with the newly released Rlm7 resistance and to investigate the nature of the mutations responsible for virulence against Rlm7. A total of 2551 fungal isolates were collected from experimental crops of a Rlm7 cultivar or a cultivar without Rlm7. All isolates were phenotyped for virulence and a subset was genotyped with neutral genetic markers. Virulent isolates were investigated for molecular events at the AvrLm4-7 locus. Whilst virulent isolates were not found in neighbouring crops, their frequency had reached 36% in the experimental field after four years. An extreme diversity of independent molecular events leading to virulence was identified in populations, with large-scale Repeat Induced Point mutations or complete deletion of AvrLm4-7 being the most frequent. Our data suggest that increased mutability of fungal genes involved in the interactions with plants is directly related to their genomic environment and reproductive system. Thus, rapid allelic diversification of avirulence genes can be generated in L. maculans populations in a single field provided that large population sizes and sexual reproduction are favoured by agricultural practices.

Microbe-independent entry of oomycete RxLR effectors and fungal RxLR-like effectors into plant and animal cells is specific and reproducible.

A wide diversity of pathogens and mutualists of plant and animal hosts, including oomycetes and fungi, produce effector proteins that enter the cytoplasm of host cells. A major question has been whether or not entry by these effectors can occur independently of the microbe or requires machinery provided by the microbe. Numerous publications have documented that oomycete RxLR effectors and fungal RxLR-like effectors can enter plant and animal cells independent of the microbe. A recent reexamination of whether the RxLR domain of oomycete RxLR effectors is sufficient for microbe-independent entry into host cells concluded that the RxLR domains of Phytophthora infestans Avr3a and of P. sojae Avr1b alone are NOT sufficient to enable microbe-independent entry of proteins into host and nonhost plant and animal cells. Here, we present new, more detailed data that unambiguously demonstrate that the RxLR domain of Avr1b does show efficient and specific entry into soybean root cells and also into wheat leaf cells, at levels well above background nonspecific entry. We also summarize host cell entry experiments with a wide diversity of oomycete and fungal effectors with RxLR or RxLR-like motifs that have been independently carried out by the seven different labs that coauthored this letter. Finally we discuss possible technical reasons why specific cell entry may have been not detected by Wawra et al. (2013).

The dispensable chromosome of Leptosphaeria maculans shelters an effector gene conferring avirulence towards Brassica rapa.

Phytopathogenic fungi frequently contain dispensable chromosomes, some of which contribute to host range or pathogenicity. In Leptosphaeria maculans, the stem canker agent of oilseed rape (Brassica napus), the minichromosome was previously suggested to be dispensable, without evidence for any role in pathogenicity. Using genetic and genomic approaches, we investigated the inheritance and molecular determinant of an L. maculans-Brassica rapa incompatible interaction. Single gene control of the resistance was found, while all markers located on the L. maculans minichromosome, absent in the virulent parental isolate, co-segregated with the avirulent phenotype. Only one candidate avirulence gene was identified on the minichromosome, validated by complementation experiments and termed AvrLm11. The minichromosome was frequently lost following meiosis, but the frequency of isolates lacking it remained stable in field populations sampled at a 10-yr time interval, despite a yearly sexual stage in the L. maculans life cycle. This work led to the cloning of a new 'lost in the middle of nowhere' avirulence gene of L. maculans, interacting with a B. rapa resistance gene termed Rlm11 and introgressed into B. napus. It demonstrated the dispensability of the L. maculans minichromosome and suggested that its loss generates a fitness deficit.

The neighbouring genes AvrLm10A and AvrLm10B are part of a large multigene family of cooperating effector genes conserved in Dothideomycetes and Sordariomycetes.

Fungal effectors (small-secreted proteins) have long been considered as species or even subpopulation-specific. The increasing availability of high-quality fungal genomes and annotations has allowed the identification of trans-species or trans-genera families of effectors. Two avirulence effectors, AvrLm10A and AvrLm10B, of Leptosphaeria maculans, the fungus causing stem canker of oilseed rape, are members of such a large family of effectors. AvrLm10A and AvrLm10B are neighbouring genes, organized in divergent transcriptional orientation. Sequence searches within the L. maculans genome showed that AvrLm10A/AvrLm10B belong to a multigene family comprising five pairs of genes with a similar tail-to-tail organization. The two genes, in a pair, always had the same expression pattern and two expression profiles were distinguished, associated with the biotrophic colonization of cotyledons and/or petioles and stems. Of the two protein pairs further investigated, AvrLm10A_like1/AvrLm10B_like1 and AvrLm10A_like2/AvrLm10B_like2, the second one had the ability to physically interact, similarly to what was previously described for the AvrLm10A/AvrLm10B pair, and cross-interactions were also detected for two pairs. AvrLm10A homologues were identified in more than 30 Dothideomycete and Sordariomycete plant-pathogenic fungi. One of them, SIX5, is an effector from Fusarium oxysporum f. sp. lycopersici physically interacting with the avirulence effector Avr2. We found that AvrLm10A/SIX5 homologues were associated with at least eight distinct putative effector families, suggesting that AvrLm10A/SIX5 is able to cooperate with different effectors. These results point to a general role of the AvrLm10A/SIX5 proteins as "cooperating proteins", able to interact with diverse families of effectors whose encoding gene is co-regulated with the neighbouring AvrLm10A homologue.

A new avirulence gene of Leptosphaeria maculans, AvrLm14, identifies a resistance source in American broccoli (Brassica oleracea) genotypes.

In many cultivated crops, sources of resistance to diseases are sparse and rely on introgression from wild relatives. Agricultural crops often are allopolyploids resulting from interspecific crosses between related species, which are sources of diversity for resistance genes. This is the case for Brassica napus (oilseed rape, canola), an interspecific hybrid between Brassica rapa (turnip) and Brassica oleracea (cabbage). B. napus has a narrow genetic basis and few effective resistance genes against stem canker (blackleg) disease, caused by the fungus Leptosphaeria maculans, are currently available. B. rapa diversity has proven to be a valuable source of resistance (Rlm, LepR) genes, while B. oleracea genotypes were mostly considered susceptible. Here we identified a new resistance source in B. oleracea genotypes from America, potentially effective against French L. maculans isolates under both controlled and field conditions. Genetic analysis of fungal avirulence and subsequent cloning and validation identified a new avirulence gene termed AvrLm14 and suggested a typical gene-for-gene interaction between AvrLm14 and the postulated Rlm14 gene. AvrLm14 shares all the usual characteristics of L. maculans avirulence genes: it is hosted in a genomic region enriched in transposable elements and heterochromatin marks H3K9me3, its expression is repressed during vegetative growth but shows a strong overexpression 5-9 days following cotyledon infection, and it encodes a small secreted protein enriched in cysteine residues with few matches in databases. Similar to the previously cloned AvrLm10-A, AvrLm14 contributes to reduce lesion size on susceptible cotyledons, pointing to a complex interplay between effectors promoting or reducing lesion development.

Leptosphaeria maculans avirulence gene AvrLm4-7 confers a dual recognition specificity by the Rlm4 and Rlm7 resistance genes of oilseed rape, and circumvents Rlm4-mediated recognition through a single amino acid change.

Leptosphaeria maculans is the ascomycete responsible for one of the most damaging diseases of oilseed rape (Brassica napus), stem canker of crucifers. Both avirulence (AvrLm) genes in the fungus and resistance (Rlm) genes in the plant are genetically clustered. Using a map-based cloning strategy, we delineated a 238 kb region containing the AvrLm7 locus. Structural features of the region were reminiscent of those previously found on another chromosome for genomic regions encompassing AvrLm1 and AvrLm6, i.e. GC-equilibrated, gene-rich isochores alternating with AT-rich, recombination-deficient, gene-poor isochores. These latter corresponded to mosaics of degenerated and truncated transposable elements. AvrLm7 is the only gene located within a 60 kb AT-rich isochore. It induced resistance responses in plants harbouring either Rlm7 or Rlm4, and was thus renamed AvrLm4-7. It encodes a 143-amino-acid cysteine-rich protein, predicted to be secreted, and strongly induced during early stages of plant infection. Sequencing and restriction analyses of AvrLm4-AvrLm7 or avrLm4-AvrLm7 alleles in L. maculans field isolates, and targeted point mutagenesis strongly suggested that one single base mutation, leading to the change of a glycine to an arginine residue, was responsible for the loss of AvrLm4 specificity whereas AvrLm7 recognition was unaltered.