Search

Strategies for plant resistance gene deployment aim to preserve their durability to highly adaptable fungal pathogens. While the pyramiding of resistance genes is often proposed as an effective way to increase their durability, molecular mechanisms by which the pathogen can overcome the resistance also are important aspects to take into account. Here, we report a counterexample where pyramiding of two resistance genes of Brassica napus, Rlm3 and Rlm7, matching the Leptosphaeria maculans avirulence genes AvrLm3 and AvrLm4-7, respectively, favored the selection of double-virulent isolates. We previously demonstrated that the presence of a functional AvrLm4-7 gene in an isolate masks the Rlm3-AvrLm3 recognition. Rlm7 was massively deployed in France since 2004. L. maculans populations were surveyed on a large scale (>7,600 isolates) over a period of 20 years, and resistance gene deployment at the regional scale was determined. Mutations in isolates overcoming both resistance genes were analyzed. All data indicated that the simultaneous success of Rlm7, the deployment of varieties pyramiding Rlm3 and Rlm7, along with the decrease in areas cultivated with Rlm3 only, contributed to the success of virulent isolates toward Rlm7, and more recently to both Rlm3 and Rlm7. Experimental field assays proved that resistance gene alternation was a better strategy compared with pyramiding in this context. Our study also illustrated an unusually high sequence diversification of AvrLm3 and AvrLm4-7 under such a selection pressure, and identified a few regions of the AvrLm4-7 protein involved in both its recognition by Rlm7 and in its AvrLm3-Rlm3 masking ability. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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.

The fungal phytopathogen Leptosphaeria maculans, causing stem canker (blackleg) of rapeseed (Brassica napus), is mainly controlled worldwide by genetic resistance including major resistance genes (Rlm). This model is one of those for which the highest number of avirulence genes (AvrLm) has been cloned. In many systems, including L. maculans-B. napus interaction, intense use of resistance genes exerts strong selection pressure on the corresponding avirulent isolates, and the fungi may rapidly escape the resistance by various molecular events modifying the avirulence genes. In the literature, the study of polymorphism at avirulence loci is often focused on single genes under selection pressure. In this study, we investigate allelic polymorphism at eleven avirulence loci in a French population of 89 L. maculans isolates collected on a trap cultivar in four geographic locations in the 2017-2018 cropping season. The corresponding Rlm genes have been (i) used for a long time, (ii) recently used, or (iii) unused yet in agricultural practice. The sequence data generated indicate an extreme diversity of situations. For example, genes submitted to an ancient selection may have either been deleted in populations (AvrLm1) or replaced by a single-nucleotide mutated virulent version (AvrLm2, AvrLm5-9). Genes that have never been under selection may either be nearly invariant (AvrLm6, AvrLm10A, AvrLm10B), exhibit rare deletions (AvrLm11, AvrLm14), or display a high diversity of alleles and isoforms (AvrLmS-Lep2). These data suggest that the evolutionary trajectory of avirulence/virulence alleles is gene-dependent and selection pressure-independent in L. maculans.

Strategies for plant resistance gene deployment aim to preserve their durability to highly adaptable fungal pathogens. While the pyramiding of resistance genes is often proposed as an effective way to increase their durability, molecular mechanisms by which the pathogen can overcome the resistance also are important aspects to take into account. Here, we report a counterexample where pyramiding of two resistance genes of

With only a few exceptions, fungal effectors (small secreted proteins) have long been considered as species- or even isolate-specific. With the increasing availability of high-quality fungal genomes and annotations, trans-species or trans-genera families of effectors are being uncovered. Two avirulence effectors, AvrLm10A and AvrLm10B, of Leptosphaeria maculans, the fungus responsible for stem canker of oilseed rape, are members of such a large family of effectors. AvrLm10A and AvrLm10B are neighboring genes, organized in divergent transcriptional orientation. Sequence searches within the L. maculans genome show 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 Lmb_jn3_08094/Lmb_jn3_08095 and Lmb_jn3_09745 / Lmb_jn3_09746, one (Lmb_jn3_09745 / Lmb_jn3_09746) had the ability to physically interact, similarly to what was previously described for the AvrLm10A/AvrLm10B pair. AvrLm10A homologues are present in more than 30 Dothideomycete and Sordariomycete plant-pathogenic fungi whereas fewer AvrLm10B homologues were identified. One of the AvrLm10A homologues, SIX5, is an effector from Fusarium oxysporum f.sp. lycopersici physically interacting with the avirulence effector Avr2. We found that AvrLm10A homologues were associated with at least eight distinct putative effector families, suggesting an ability of AvrLm10A/SIX5 to cooperate with diverse effectors. These results point to a general role of the AvrLm10A/SIX5 protein as a ‘cooperator protein’, able to interact with diverse families of effectors whose encoding gene is co-regulated with the neighboring AvrLm10A homologue.

Many people with disabilities, especially those with physical disabilities, could work, draw or produce art. However, very few workstations and tools are developed to meet their needs and conditions. The objective of this study is to evaluate the needs of an artist with a disability for an ergonomic workstation and to propose a prototype that increases the comfort and capabilities of people with physical disabilities to perform their tasks. First, we analyzed the tasks/activities, postures, and needs of an artist with a high level of disability while performing his drawing tasks in a real-life situation. Second, we simulated an art studio with the participation of six non-artist subjects with motor and mental disabilities to evaluate the user's experience and difficulties when painting in a similar real situation. Based on these studies, we proposed a specification for the design and prototyping of a painting workstation adapted for people with disabilities.Our analysis showed that very few workstations are adapted for people with disabilities and they must work on the floor with awkward postures, insufficient lighting and limited space. They have to make excessive efforts to perform most of their activities because the tools and workstations do not meet their needs. Based on the specifications extracted from our study, we propose a new design of painting workstation dedicated to disabled people to improve their comfort and performance.

The Dothideomycete

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. Supplementary Information The online version contains supplementary material available at 10.1007/s10577-021-09658-1.

International audience; Phoma stem canker, a highly damaging disease of oilseed rape (Brassica napus), is caused by fungi of the Leptosphaeria maculans/Leptosphaeria biglobosa species complex. On B. napus, at least in Europe, L. maculans ‘brassicae’ (LMB) and L. biglobosa ‘brassicae’ (LBB) are often found together, in the same fields. The biology and epidemiology of LMB have been intensively studied, but few studies have investigated the infection and colonization processes of LBB, and unanswered questions remain concerning the relative contributions of these two species to phoma stem canker disease. We addressed this question by developing two species-specific robust quantitative PCR (qPCR) assays, one for each species, for detecting and quantifying these species in field samples of oilseed rape. The assays were highly specific, reproducible, and sensitive. We used them to obtain an overview of stem canker epidemiology in a two-year field study of a B. napus genotype susceptible to LMB, and a near-isogenic line harbouring Rlm11, a gene conferring effective resistance to LMB. Inoculum pressure differed considerably between the two years of the experiment, and we found that LMB leaf spotting intensity at limited observation time points was insufficient for predicting final disease severity. We also found that LBB was more prevalent than previously thought, with epidemiological and colonization biology characteristics very similar to those of LMB, but a necrotrophic lifestyle. The general presence of these two species together on leaves and in the stem tissues of the same individual plant raises questions about their relative incidence and biological interactions within plant tissues.

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.

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.Author summaryAn efficient strategy to control fungal diseases in the field is genetic control using resistant crop cultivars. Crop resistance mainly relies on gene-for-gene relationships between plant resistance (R) genes and pathogen avirulence (AVR) genes, as defined by Flor in the 1940s. However, such gene-for-gene relationships can increase in complexity over the course of plant-pathogen co-evolution. Resistance against the plant-pathogenic fungus Leptosphaeria maculans by Brassica napus and other Brassica species relies on the recognition of effector (AVR) proteins by R proteins; however, L. maculans produces an effector that suppresses a subset of these specific resistances. Using a protein structure approach, we revealed structural analogy between several of the resistance-triggering effectors, the resistance-suppressing effector, and effectors from other plant-pathogenic species in the Dothideomycetes and Sordariomycetes classes, defining a new family of effectors called LARS. Notably, cross-species expression of one LARS effector from Fulvia fulva, a pathogen of tomato, in L. maculans resulted in recognition by several resistant cultivars of oilseed rape. These results highlight the need to integrate knowledge on effector structures to improve resistance management and to develop broad-spectrum resistances for multi-pathogen control of diseases.

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 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. 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 located in regions enriched in heterochromatin, either constitutive or facultative. These findings provide support for the hypothesis that fungal genes involved in niche adaptation are located in heterochromatic regions of the genome, conferring an extreme plasticity of expression. 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.Author SummaryFungi are extremely important organisms in the global ecosystem. Some are damaging plant pathogens that threaten global food security. A knowledge of their biology and pathogenic cycle is vital for the design of environmentally-friendly control strategies. Unfortunately, many parts of their life cycle remain unknown, due to the complexity of their life-cycles and technical limitations. Here, we use a rapeseed pathogen, Leptosphaeria maculans, which has a particularly complex life-cycle, to show that large-scale RNA-Seq analyses of fungal gene expression can decipher all stages of the fungal cycle over two years of interaction with living or dead hosts, in laboratory and agricultural conditions. We found that the fungus uses about 9% of the genes of its genome specifically during interactions with the plant, and observed waves of extremely tight, complex regulation during the colonization of specific tissues and specific parts of the life-cycle. Our findings highlight the importance of genes encoding effectors, small secreted proteins manipulating the host. This work opens up new avenues for plant disease control through the identification of stage-specific effectors leading to the discovery of novel durable disease resistance genes, or the analysis of epigenetic regulation, which could be manipulated to interfere with effector gene expression.

SummaryLeptosphaeria maculans, the causal agent of blackleg disease, interacts with Brassica napus (oilseed rape, canola) in a gene-for-gene manner. The avirulence genes AvrLmS and AvrLep2 were described to be perceived by the resistance genes RlmS and LepR2, respectively, present in the cultivar 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) to rapidly identify one candidate avirulence gene present in the effector repertoire of L. maculans. AvrLep2 was cloned using a bi-parental 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 and Topas-LepR2. The gene renamed AvrLmS-Lep2 encodes for a small cysteine-rich protein of unknown function with an N-terminal secretory signal peptide, which are common features 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 to intermediate resistance sometimes at the edge of, or evolving toward, susceptibility depending on the inoculation conditions. AvrLmS-Lep2 was nevertheless sufficient to significantly reduce the stem lesion size on plant genotypes with LepR2, indicating the potential efficiency of this resistance to control the disease in the field.

In plant-associated fungi, the role of the epigenome is increasingly recognized as an important regulator of genome structure and of the expression of genes involved in interaction(s) with the host plant. Two closely-related phytopathogenic species, Leptosphaeria maculans "brassicae" (Lmb) and L. maculans "lepidii" (Lml) exhibit a large conservation of genome synteny but contrasting genome structure. Lmb has undergone massive invasion of its genome by transposable elements amounting to one third of its genome and clustered in large TE-rich regions on chromosomal arms, while Lml genome has only a small amount of repeats (3% of the genome). Previous studies showed that the TE-rich regions of Lmb harbour a few species-specific effector genes, expressed during plant infection. The distinct genome structures shown by Lmb and Lml thus provides an excellent model for comparing the organization of pathogenicity/effector genes in relation to the chromatin landscape in two closely related phytopathogenic fungi. Here, we performed chromatin immunoprecipitation during axenic culture, targeting either histone modifications typical for heterochromatin or euchromatin, combined with transcriptomic analysis to analyse the influence of chromatin organisation on gene expression. In both species, we found that facultative heterochromatin landscapes associated with H3K27me3-domains are enriched with genes lacking functional annotation, including numerous candidate effector and species-specific genes. Notably, orthologous genes located in H3K27me3-domains in both species are enriched with genes encoding putative proteinaceous and metabolic effectors. These genes are mostly silenced in axenic growth conditions and are likely to be involved in interaction with the host. Compared to other fungal species, including Lml, Lmb is distinct in having large H3K9me3-domains associated with TE-rich regions that contain numerous species-specific effector-encoding 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.

Leptosphaeria maculans, the causal agent of blackleg disease, interacts with Brassica napus (oilseed rape, canola) and other Brassica hosts in a gene-for-gene manner. The avirulence gene AvrLmJ1 has been cloned previously and shown to interact with an unidentified Brassica juncea resistance gene. In this study, we show that the AvrLmJ1 gene maps to the same position as the AvrLm5 locus. Furthermore, isolates complemented with the AvrLmJ1 locus confer avirulence towards B. juncea genotypes harbouring Rlm5. These findings demonstrate that AvrLmJ1 is AvrLm5 and highlight the need for shared resources to characterize accurately avirulence and/or resistance genes.

Contents 526 I. 526 II. 527 III. 527 IV. 529 V. 529 VI. 530 VII. 530 531 References 531 SUMMARY: In agricultural systems, major (R) genes for resistance in plants exert strong selection pressure on cognate/corresponding avirulence effector genes of phytopathogens. However, a complex interplay often exists between trade-offs linked to effector function and the need to escape R gene recognition. Here, using the Leptosphaeria maculans-oilseed rape pathosystem we review evolution of effectors submitted to multiple resistance gene selection. Characteristics of this pathosystem include a crop in which resistance genes have been deployed intensively resulting in 'boom and bust' cycles; a fungal pathogen with a high adaptive potential in which seven avirulence genes are cloned and for which population surveys have been coupled with molecular analysis of events responsible for virulence. The mode of evolution of avirulence genes, all located in dispensable parts of the 'two-speed' genome, is a highly dynamic gene-specific process. In some instances, avirulence genes are readily deleted under selection. However, others, even when located in the most plastic genome regions, undergo only limited point mutations or their avirulence phenotype is 'camouflaged' by another avirulence gene. Thus, while hundreds of effector genes are present, some effectors are likely to have an important and nonredundant function, suggesting functional redundancy and dispensability of effectors might not be the rule.

International audience

International audience; 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.

Leptosphaeria maculans, the causal agent of stem canker disease, colonizes oilseed rape (Brassica napus) in two stages: a short and early colonization stage corresponding to cotyledon or leaf colonization, and a late colonization stage during which the fungus colonizes systemically and symptomlessly the plant during several months before stem canker appears. To date, the determinants of the late colonization stage are poorly understood; L. maculans may either successfully escape plant defences, leading to stem canker development, or the plant may develop an 'adult-stage' resistance reducing canker incidence. To obtain an insight into these determinants, we performed an RNA-sequencing (RNA-seq) pilot project comparing fungal gene expression in infected cotyledons and in symptomless or necrotic stems. Despite the low fraction of fungal material in infected stems, sufficient fungal transcripts were detected and a large number of fungal genes were expressed, thus validating the feasibility of the approach. Our analysis showed that all avirulence genes previously identified are under-expressed during stem colonization compared with cotyledon colonization. A validation RNA-seq experiment was then performed to investigate the expression of candidate effector genes during systemic colonization. Three hundred and seven 'late' effector candidates, under-expressed in the early colonization stage and over-expressed in the infected stems, were identified. Finally, our analysis revealed a link between the regulation of expression of effectors and their genomic location: the 'late' effector candidates, putatively involved in systemic colonization, are located in gene-rich genomic regions, whereas the 'early' effector genes, over-expressed in the early colonization stage, are located in gene-poor regions of the genome.

To achieve host colonization, successful pathogens need to overcome plant basal defences. For this, (hemi)biotrophic pathogens secrete effectors that interfere with a range of physiological processes of the host plant. AvrLm4-7 is one of the cloned effectors from the hemibiotrophic fungus Leptosphaeria maculans 'brassicaceae' infecting mainly oilseed rape (Brassica napus). Although its mode of action is still unknown, AvrLm4-7 is strongly involved in L. maculans virulence. Here, we investigated the effect of AvrLm4-7 on plant defence responses in a susceptible cultivar of B. napus. Using two isogenic L. maculans isolates differing in the presence of a functional AvrLm4-7 allele [absence ('a4a7') and presence ('A4A7') of the allele], the plant hormone concentrations, defence-related gene transcription and reactive oxygen species (ROS) accumulation were analysed in infected B. napus cotyledons. Various components of the plant immune system were affected. Infection with the 'A4A7' isolate caused suppression of salicylic acid- and ethylene-dependent signalling, the pathways regulating an effective defence against L. maculans infection. Furthermore, ROS accumulation was decreased in cotyledons infected with the 'A4A7' isolate. Treatment with an antioxidant agent, ascorbic acid, increased the aggressiveness of the 'a4a7' L. maculans isolate, but not that of the 'A4A7' isolate. Together, our results suggest that the increased aggressiveness of the 'A4A7' L. maculans isolate could be caused by defects in ROS-dependent defence and/or linked to suppressed SA and ET signalling. This is the first study to provide insights into the manipulation of B. napus defence responses by an effector of L. maculans.

International audience; The recent finding of a novel fungal strategy to manipulate salicylic acid (SA) in a nonmodel plant pathogen interaction not only establishes the universality of the strategy to ensure the success of biotrophs and hemibiotrophs, but also illustrates current limitations and challenges to identify targets of fungal effector in crop plants.

International audience

International audience

International audience; Plant-associated fungi often present in their genome areas enriched in repeat sequences and effector genes, the latter being specifically induced in planta. The location of effector genes in regions enriched in repeats has been shown to have an impact on adaptability of fungi but could also provide for tight control of effector gene expression through chromatin-based regulation. The distribution of two repressive histone marks was shown to be an important regulatory layer in two fungal species with different lifestyles. Chromatin-based control of effector gene expression is likely to provide an evolutionary advantage by preventing the expression of genes not needed during vegetative growth and allow for a massive concerted expression at particular time-points of plant infection.

Summary Leptosphaeria maculans causes stem canker of oilseed rape (Brassica napus). The APSES transcription factor StuA is a key developmental regulator of fungi, involved in morphogenesis, conidia production and also more recently described as required for secondary metabolite production and for effector gene expression in phytopathogenic fungi. We investigated the involvement of the orthologue of StuA in L. maculans, LmStuA, in morphogenesis, pathogenicity and effector gene expression. LmStuA is induced during mycelial growth and at 14 days after infection, corresponding to the development of pycnidia on oilseed rape leaves, consistent with the function of StuA described so far. We set up the functional characterization of LmStuA using an RNA interference approach. Silenced LmStuA transformants showed typical phenotypic defects of StuA mutants with altered growth in axenic culture and impaired conidia production and perithecia formation. Silencing of LmStuA abolished the pathogenicity of L. maculans on oilseed rape leaves and also resulted in a drastic decrease in expression of at least three effector genes during in planta infection, suggesting either that LmStuA regulates, directly or indirectly, the expression of several effector genes in L. maculans or that the infection stage in which effectors are expressed is not reached when LmStuA expression is silenced.

Summary Five avirulence genes from Leptosphaeria maculans, the causal agent of blackleg of canola (Brassica napus), have been identified previously through map-based cloning. In this study, a comparative genomic approach was used to clone the previously mapped AvrLm2. Given the lack of a presence–absence gene polymorphism coincident with the AvrLm2 phenotype, 36 L. maculans isolates were resequenced and analysed for single-nucleotide polymorphisms (SNPs) in predicted small secreted protein-encoding genes present within the map interval. Three SNPs coincident with the AvrLm2 phenotype were identified within LmCys1, previously identified as a putative effector-coding gene. Complementation of a virulent isolate with LmCys1, as the candidate AvrLm2 allele, restored the avirulent phenotype on Rlm2-containing B. napus lines. AvrLm2 encodes a small cysteine-rich protein with low similarity to other proteins in the public databases. Unlike other avirulence genes, AvrLm2 resides in a small GC island within an AT-rich isochore of the genome, and was never found to be deleted completely in virulent isolates.

In phytopathogenic fungi, the expression of hundreds of small secreted protein (SSP)-encoding genes is induced upon primary infection of plants while no or a low level of expression is observed during vegetative growth. In some species such as Leptosphaeria maculans, this coordinated in-planta upregulation of SSP-encoding genes expression relies on an epigenetic control but the signals triggering gene expression in-planta are unknown. In the present study, biotic and abiotic factors that may relieve suppression of SSP-encoding gene expression during axenic growth of L. maculans were investigated. Some abiotic factors (temperature, pH) could have a limited effect on SSP gene expression. In contrast, two types of cellular stresses induced by antibiotics (cycloheximide, phleomycin) activated strongly the transcription of SSP genes. A transcriptomic analysis to cycloheximide exposure revealed that biological processes such as ribosome biosynthesis and rRNA processing were induced whereas important metabolic pathways such as glycogen and nitrogen metabolism, glycolysis and tricarboxylic acid cycle activity were down regulated. A quantitatively different expression of SSP-encoding genes compared to plant infection was also detected. Interestingly, the same physico-chemical parameters as those identified here for L. maculans effectors were identified to regulate positively or negatively the expression of bacterial effectors. This suggests that apoplastic phytopathogens may react to similar physiological parameters for regulation of their effector genes. (C) 2017 Elsevier Inc. All rights reserved.

Summary Leptosphaeria maculans is the fungus responsible for the stem canker disease of oilseed rape (Brassica napus). AvrLm3 and AvrLm4-7, two avirulence effector genes of L. maculans, are involved in an unusual relationship: AvrLm4-7 suppresses the Rlm3-mediated resistance. Here, we assessed AvrLm3 polymorphism in a collection of 235 L. maculans isolates. No field isolates exhibited deletion or inactivating mutations in AvrLm3, as observed for other L. maculans avirulence genes. Eleven isoforms of the AvrLm3 protein were found. In isolates virulent towards both Rlm3 and Rlm7 (a3a7), the loss of the Rlm3-mediated resistance response was due to two distinct mechanisms. First, when AvrLm4-7 was inactivated (deletion or inactivating mutations), amino acid substitutions in AvrLm3 generated virulent isoforms of the protein. Secondly, when only point mutations were observed in AvrLm4-7, a3a7 isolates still contained an avirulent allele of AvrLm3. Directed mutagenesis confirmed that some point mutations in AvrLm4-7 were sufficient for the fungus to escape Rlm7-mediated resistance while maintaining the suppression of the AvrLm3 phenotype. Signatures of positive selection were also identified in AvrLm3. The complex evolutionary mechanisms enabling L. maculans to escape Rlm3-mediated resistance while preserving AvrLm3 integrity, along with observed reduced aggressiveness of isolates silenced for AvrLm3, serves to emphasize the importance of this effector in pathogenicity towards B. napus. While the common response to resistance gene pressure is local selection of isolates depleted in the cognate avirulence gene, this example contributes to complexify the gene-for-gene concept of plant-pathogen evolution with a “camouflaged” model allowing retention of non-dispensable avirulence effectors. This article is protected by copyright. All rights reserved.

Les champignons phytopathogenes, menace majeure pour la securite alimentaire mondiale, presentent une plasticite de modes de vie remarquable et une extreme capacitae` s’adapter aux methodes de luttes qui leur sont opposees par l’homme dans les agro-systemes. La genomique et la genomique comparative sont utilisees ici pour evaluer le lien entre plasticite genomique et potentiel evolutif et adaptatif. Une serie evolutive de champignons phytopathogenes infeodes aux cruciferes, le complexe d’especes Leptosphaeria maculans-Leptosphaeria biglobosa , a ete choisi ici comme modele puisqu’il regroupe cinq entites dont le statut con- ou heterospecifique est peu clair mais presente des differences de gamme d’hote ou de pouvoir pathogene. En particulier, l’espece la mieux adaptee sur le colza (et la plus dommageable pour l’agriculture), L. maculans « brassicae », possede, par rapport aux autres membres du complexe d’especes, un genome caracterise par l’expansion recente, mais massive, de quelques familles d’elements transposables (ET). Celle-ci a sans doute eu un effet encore peu clair sur la speciation, mais a surtout contribuae` la diversification de molecules de type « effecteurs », donc a l’acquisition de nouvelles specificites parasitaires. La localisation des genes codant pour des effecteurs dans des regions genomiques enrichies en ET a par ailleurs un effet direct sur l’adaptation aux resistances varietales en favorisant une diversite d’evenements mutationnels. Ces donnees sont confrontees a d’autres exemples de la litterature qui tendent a generaliser l’idee que les champignons phytopathogenes ont, au cours de l’evolution, developpe des « genomes a deux vitesses » incluant un compartiment plastique enrichi en ET et en genes impliques dans le pouvoir pathogene et l’adaptation a l’hote.

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).

International audience; 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.

The race structure and genotypic diversity of four Leptosphaeria maculans populations isolated from Brassica oleracea (broccoli, cauliflower, cabbage, etc.) in central Mexico (Aguascalientes, Guanajuato and Zacatecas states) were analysed. Race structure was characterized by an unusually low diversity at three locations out of four. Fourteen minisatellite markers revealed a high proportion of repeated multilocus genotypes in these populations, combined with a significant linkage disequilibrium and strong clonal fraction (65‐87%). The occurrence of the mating-type idiomorphs always significantly departed from the 1:1 proportion expected in the case of random mating. Each population thus consists of a few (four to nine) multilocus genotypes which are specific to each location. These data strongly support the hypothesis of exclusive, or a high rate of, clonal multiplication. Comparison of cropping practices between B. oleracea and B. napus indicate that the shift in reproductive behaviour of the fungus is chiefly man-mediated.

The ever-increasing generation of sequence data is accompanied by unsatisfactory functional annotation, and complex genomes, such as those of plants and filamentous fungi, show a large number of genes with no predicted or known function. For functional annotation of unknown or hypothetical genes, the production of collections of mutants using Agrobacterium tumefaciens–mediated transformation (ATMT) associated with genotyping and phenotyping has gained wide acceptance. ATMT is also widely used to identify pathogenicity determinants in pathogenic fungi. A systematic analysis of T-DNA borders was performed in an ATMT-mutagenized collection of the phytopathogenic fungus Leptosphaeria maculans to evaluate the features of T-DNA integration in its particular transposable element-rich compartmentalized genome. A total of 318 T-DNA tags were recovered and analyzed for biases in chromosome and genic compartments, existence of CG/AT skews at the insertion site, and occurrence of microhomologies between the T-DNA left border (LB) and the target sequence. Functional annotation of targeted genes was done using the Gene Ontology annotation. The T-DNA integration mainly targeted gene-rich, transcriptionally active regions, and it favored biological processes consistent with the physiological status of a germinating spore. T-DNA integration was strongly biased toward regulatory regions, and mainly promoters. Consistent with the T-DNA intranuclear-targeting model, the density of T-DNA insertion correlated with CG skew near the transcription initiation site. The existence of microhomologies between promoter sequences and the T-DNA LB flanking sequence was also consistent with T-DNA integration to host DNA mediated by homologous recombination based on the microhomology-mediated end-joining pathway.

Pathogen introductions into novel areas can lead to the emergence of new fungal diseases of plants. Understanding the origin, introduction pathways, possible changes in reproductive system and population size of fungal pathogens is essential in devising an integrated strategy for the control of these diseases. We used minisatellite markers to infer the worldwide invasion history of the fungal plant pathogen Leptosphaeria maculans, which causes stem canker (blackleg) of oilseed and vegetable brassicas. Clustering analyses partitioned genotypes into distinct populations corresponding to major geographic regions, along with two differentiated populations in Western Canada. Comparison of invasion scenarios using Approximate Bayesian Computation suggested an origin of the pathogen in the USA, the region where epidemics were first recorded, and independent introductions from there over the last few decades into Eastern Canada (Ontario), Europe and Australia. The population in Western Canada appeared to be founded from a source in Ontario and the population in Chile resulted from an admixture between multiple sources. A bottleneck was inferred for the introduction into Western Canada but not into Europe, Ontario or Australia. Clonality appeared high in Western Canada, possibly because environmental conditions there were less conducive to sexual reproduction. Leptosphaeria maculans is a model invasive pathogen with contrasting features in different regions: shallow population structure, high genetic variability and regular sexual recombination in some regions, by comparison with reduced genetic variability, high rates of asexual multiplication, strong population structure or admixture in others.

International audience; The secreted proteins (secretome) of fungi play a key role in interactions of pathogenic and symbiotic fungi with plants. Using the plant pathogenic fungus Leptosphaeria maculans and symbiont Laccaria bicolor grown in culture, we have established a proteomic protocol for extraction, concentration and resolution of the fungal secretome. As no proteomic data were available on mycelium tissues from both L. maculans and L. bicolor, mycelial proteins were studied; they also helped verifying the purity of secretome samples. The quality of protein extracts was initially assessed by both 1-DE and 2-DE using first a broad pH range for IEF, and then narrower acidic and basic pH ranges, prior to 2-DE. Compared with the previously published protocols for which only dozens of 2-D spots were recovered from fungal secretome samples, up to approximately 2000 2-D spots were resolved by our method. MS identification of proteins along several pH gradients confirmed this high resolution, as well as the presence of major secretome markers such as endopolygalacturonases, -glucanosyltransferases, pectate lyases and endoglucanases. Shotgun proteomic experiments evidenced the enrichment of secreted protein within the liquid medium. This is the first description of the proteome of L. maculans and L. bicolor, and the first application of liquid-phase IEF to any fungal extracts.

International audience; Stem canker of oilseed rape (canola, Brassica napus) is associated with a species complex of two closely related fungal species, Leptosphaeria maculans and L. biglobosa. Of these, L. maculans is the most damaging and develops gene-for-gene relationships with the host. Here, a wide scale analysis of the L. maculans - L. biglobosa species complex was performed throughout the American continent (23 locations from Chile to Canada) plus several locations inWestern Australia for comparison purposes, based on a collection of 1132 isolates from infected tissues of a susceptible cultivar. Fungal species were discriminated on the basis of morphological, phytopathological and molecular criteria and showed that L. biglobosa was closely associated with L. maculans in most of the locations. Multiple gene phylogeny using sequences of ITS, actin and b-tubulin confirmed the prevalence of the L. biglobosa ‘canadensis’ sub-clade in Canada, whereas up to three different sub-clades of L. biglobosa were found in Georgia (USA). Race structure of L. maculans was investigated using a combination of pathogenicity tests and PCR amplification of avirulence alleles AvrLm1, AvrLm4 and AvrLm6. Three contrasting situations were observed: (i) race structure in Ontario, Chile and Georgia was related to that of European and Western Australian populations, with a low race diversity; (ii) only one race was found in Mexico, and not found outside of this country; (iii) a large diversity of races was observed in central Canada (Manitoba, Alberta and Saskatchewan) with very specific features including maintenance of avirulence alleles absent from Europe, absence of the AvrLm7 allele common in Europe (or eastern Canada) and wide location-to-location variability.

International audience; Blackleg disease (phoma stem canker) of Brassica napus (canola, oilseed rape) is caused by the fungus Leptosphaeria maculans. In some regions of Australia, resistance in oilseed rape cultivars derived from B. rapa subs. sylvestris (e.g. cv. Surpass 400) became ineffective within three years of commercial release. The genetic control of avirulence in L. maculans towards cv. Surpass 400 is described. When Australian field isolates were screened on this cultivar, three phenotypic classes were observed; virulent, intermediate and avirulent. Analysis of crosses between fungal isolates varying in their ability to infect cv. Surpass 400 demonstrated the presence of two unlinked avirulence genes, AvrLm1 and AvrLmS. Complementation of isolates (genotype avrLm1) with a functional copy of AvrLm1, and genotyping of field isolates using a molecular marker for AvrLm1 showed that virulence towards Rlm1 is necessary, but not sufficient, for expression of a virulent phenotype on cv. Surpass 400. Taken together, these data strongly suggest that cv. Surpass 400, with ‘sylvestris-derived’ resistance, contains at least two resistance genes, one of which is Rlm1.

International audience; Following Agrobacterium tumefaciens-mediated mutagenesis in Leptosphaeria maculans, we identified the mutant 210, displaying total loss of pathogenicity towards its host plant (Brassica napus). Microscopic observations showed that m210 is unable to germinate on the host leaf surface and is thus blocked at the pre-penetration stage. The pathogenicity phenotype is linked with a single T-DNA insertion into the promoter region of a typical plasma membrane H(+)-ATPase-encoding gene, termed Lmpma1, thus leading to a twofold reduction in Lmpma1 expression. Since LmPMA1 is involved in intracellular pH homeostasis, we postulate that reduction in LmPMA1 activity disturbs the electrochemical transmembrane gradient in m210, thus leading to conidia defective in turgor pressure generation on leaf surface. Whole genome survey showed that L. maculans possesses a second plasma membrane H(+)-ATPase-encoding gene, termed Lmpma2. Silencing experiments, expression analyses and phylogenetic studies allowed us to highlight the essential role assumed by the Lmpma1 isoform in L.maculans pathogenicity.

International audience; Leptosphaeria maculans, a dothideomycete fungus causing stem canker on oilseed rape, develops gene-for-gene interactions with its host plants. It has the ability to rapidly adapt to selection pressure exerted by cultivars harbouring novel resistance genes as exemplified recently by the 3-year evolution towards virulence at the AvrLm1 locus in French populations. The AvrLm1 avirulence gene was recently cloned and shown to be a solo gene within a 269 kb non-coding, heterochromatin-like region. Here we describe the sequencing of the AvrLm1 genomic region in one avirulent and two virulent isolates to investigate the molecular basis of evolution towards virulence at the AvrLm1 locus. For these virulent isolates, the gain of virulence was linked to a 260 kb deletion of a chromosomal segment spanning AvrLm1 and deletion breakpoints were identical or similar. Among the 460 isolates analysed from France, Australia and Mexico, a similar large deletion was apparent in > 90% of the virulent isolates. Deletion breakpoints were also strongly conserved in most of the virulent isolates, which led to the hypothesis that a unique deletion event leading to the avrLm1 virulence has diffused in pathogen populations. These data finally suggest that retrotransposons are key drivers in genome evolution and adaptation to novel selection pressure in L. maculans.

International audience; 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.

Agrobacterium species are soilborne gram-negative bacteria exhibiting predominantly a saprophytic lifestyle. Only a few of these species are capable of parasitic growth on plants, causing either hairy root or crown gall diseases. The core of the infection strategy of pathogenic Agrobacteria is a genetic transformation of the host cell, via stable integration into the host genome of a DNA fragment called T-DNA. This genetic transformation results in oncogenic reprogramming of the host to the benefit of the pathogen. This unique ability of interkingdom DNA transfer was largely used as a tool for genetic engineering. Thus, the artificial host range of Agrobacterium is continuously expanding and includes plant and nonplant organisms. The increasing availability of genomic tools encouraged genome-wide surveys of T-DNA tagged libraries, and the pattern of T-DNA integration in eukaryotic genomes was studied. Therefore, data have been collected in numerous laboratories to attain a better understanding of T-DNA integration mechanisms and potential biases. This review focuses on the intranuclear mechanisms necessary for proper targeting and stable expression of Agrobacterium oncogenic T-DNA in the host cell. More specifically, the role of genome features and the putative involvement of host’s transcriptional machinery in relation to the T-DNA integration and effects on gene expression are discussed. Also, the mechanisms underlying T-DNA integration into specific genome compartments is reviewed, and a theoretical model for T-DNA intranuclear targeting is presented.

The dothideomycete fungus, Leptosphaeria maculans, is poorly described at the genomic level. Two genomic regions have been analysed - one (55 kb) comprises a cluster of 18 genes encoding the biosynthetic enymes for a phytotoxin, sirodesmin, whilst the other (184 kb) is the pericentromeric region of a 2.80 Megabase chromosome. Transcription of all genes in the sirodesmin gene cluster is co-regulated with the production of sirodesmin in culture. Disruption of one of these genes (encoding a two-module non-ribosomal peptide synthetase) is essential for production of sirodesmin. The 184 kb sequence contains 6.980 kb repetitive element named Pholy bordered by two Long Terminal Repeats (LTRs), five Pholy-related sequences, mostly truncated at their 3′ ends; and five solo-LTRs. This element, Pholy, comprises a previously described element, LMR1. Structural features suggest that Pholy corresponds to an ancient copia-like retrotransposon, as it has high sequence similarity to the ELSA retrotransposon of the closely related fungus, Stagonospora nodorum. Comparative analysis of the structure of the Pholy-like sequences in the 184 kb contig and in other parts of the genome suggests that this family of repetitive elements has undergone extensive Repeat Induced Point (RIP) mutation.

Summary Leptosphaeria maculans, a Dothideomycete causing stem canker on oilseed rape (Brassica napus), develops gene-for-gene interactions with its host plants. To date, nine resistance genes (Rlm1–9) have been identified in Brassica spp. The corresponding nine avirulence genes (AvrLm1–9) in L. maculans have been mapped at four independent loci, thereby revealing two clusters of three and four linked avirulence genes. Here, we report the completion of map-based cloning of AvrLm1. AvrLm1 was genetically delineated within a 7.3 centimorgan interval corresponding to a 439 kb BAC contig. AvrLm1 is a single copy gene isolated within a 269 kb non-coding, heterochromatin-like region. The region comprised a number of degenerated, nested copies of four long-terminal repeat (LTR) retrotransposons, including Pholy and three novel Gypsy-like retrotransposons. AvrLm1 restored the avirulent phenotype on Rlm1 cultivars following functional complementation of virulent isolates. AvrLm1 homologues were not detected in other Leptosphaeria species or in known fungal genomes including the closely related species Stagonospora nodorum. The predicted AvrLm1 protein is composed of 205 amino acids, of which only one is a cysteine residue. It contains a peptide signal suggesting extracellular localization. Unlike most other fungal avirulence genes, AvrLm1 is constitutively expressed, with a probable increased level of expression upon plant infection, suggesting the absence of tight regulation of AvrLm1 expression.

Leptosphaeria maculans is the most ubiquitous fungal pathogen of Brassica crops and causes the devastating stem canker disease of oilseed rape worldwide. We used minisatellite markers to determine the genetic structure of L. maculans in four field populations from France. Isolates were collected at three different spatial scales (leaf, 2-m 2 field plot, and field) enabling the evaluation of spatial distribution of the mating type alleles and of genetic variability within and among field populations. Within each field population, no gametic disequilibrium between the minisatellite loci was detected and the mating type alleles were present at equal frequencies. Both sexual and asexual reproduction occur in the field, but the genetic structure of these populations is consistent with annual cycles of randomly mating sexual reproduction. All L. maculans field populations had a high level of gene diversity ( H = 0.68 to 0.75) and genotypic diversity. Within each field population, the number of genotypes often was very close to the number of isolates. Analysis of molecular variance indicated that >99.5% of the total genetic variability was distributed at a small spatial scale, i.e., within 2-m 2 field plots. Population differentiation among the four field populations was low ( G ST < 0.02), suggesting a high degree of gene exchange between these populations. The high gene flow evidenced here in French populations of L. maculans suggests a rapid countrywide diffusion of novel virulence alleles whenever novel resistance sources are used.

Leptosphaeria maculans, the causal agent of stem canker of oilseed rape, develops gene-for-gene interactions with its hosts. To date, eight L. maculans avirulence (Avr) genes, AvrLm1 to AvrLm8, have been genetically characterized. An additional Avr gene, AvrLm9, that interacts with the resistance gene Rlm9, was genetically characterized here following in vitro crosses of the pathogen. A worldwide collection of 63 isolates, including the International Blackleg of Crucifers Network collection, was genotyped at these nine Avr loci. In a first step, isolates were classified into pathogenicity groups (PGs) using two published differential sets. This analysis revealed geographical disparities as regards the proportion of each PG. Genotyping of isolates at all Avr loci confirmed the disparities between continents, in terms of Avr allele frequencies, particularly for AvrLm2, AvrLm3, AvrLm7, AvrLm8, and AvrLm9, or in terms of race structure, diversity, and complexity. Twenty-six distinct races were identified in the collection. A larger number of races (n = 18) was found in Australia than in Europe (n = 8). Mean number of virulence alleles per isolate was also higher in Australia (5.11 virulence alleles) than in Europe (4.33) and Canada (3.46). Due to the diversity of populations of L. maculans evidenced here at the race level, a new, open terminology is proposed for L. maculans race designation, indicating all Avr loci for which the isolate is avirulent.