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Leptosphaeria maculans
A phytopathological fungus displaying complex parasitic strategies |
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Introduction
A severe attack of Leptosphaeria maculans causing intensive lodging in an oilseed rape field (credits: CETIOM). |
Fungi are organisms of primary ecological, biotechnological and
economic importance. Many fundamental biological processes (cell
cycle, circadian rhythms, ageing, cytoskeleton dynamics, regulation of
gene expression, cell differentiation) are shared by human and fungi
and are currently studied in fungi as model systems, due to their
experimental tractability and their small genome size. Fungi are
important for food production (edible mushrooms, fermentable products)
and for the industrial production of enzymes and metabolites. These
microbial eukaryotes cause important diseases in humans, particularly
in immunocompromised patients, and are emerging as a major threat
since they are very difficult to control with current
fungicides. Fungi are also responsible for many diseases of plants,
causing large losses in crop production and contaminating harvested
crops with mycotoxins dangerous for health. As such they are the
target of large-scale fungicide use, with the corresponding
environmental and health hazards.
Of the ca. 250 000 fungal species known to date, ca. 75% are
Ascomycetes, of which at least three classes include specialized plant
pathogens. Of these three classes, the main one is
Dothideomycetes. This class encompasses genera Stagonospora,
Ascochyta, Venturia, Mycosphaerella, Alternaria, Cochliobolus and
Leptosphaeria, representing the most important pathogens of wheat,
legumes, barley, apples, bananas, brassicas, etc.
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The complex parasitic strategies of Leptosphaeria maculans
A Leptosphaeria maculans ascus containing 6-celled ascospores (credits: D. Zickler, IGM, Orsay). |
Leptosphaeria maculans is the cause of "stem canker" (also
termed "Blackleg") of oilseed rape (or canola) (Brassica
napus). In France, the disease is responsible for 5-20% average
yield losses due to drastic lodging of the crop (see the picture on
the effect in the field). In addition, L. maculans is a model
fungus, since it exemplifies Dothideomycete infection strategies, but
nevertheless shows specificities in its life traits. During its life
cycle L. maculans is firstly:
- saprophyte on stem residues. It can survive as a saprobe for many
years on the debris where sexual mating takes place resulting in the
generation of ascospores, the primary inoculum (see the ascii
picture);
- necrotrophic for a very short time period, causing leaf spots
where asexual multiplication takes place (see picture).
- This leaf spot stage is followed by a lengthy endophytic systemic
colonisation of leaf tissues, which may last up to 9 months for winter
oilseed rape, e.g., in Europe. This phase of the disease is fully
symptomless, and the fungus is growing in intercellular spaces towards
the crown at the basis of the stem, and the upper root. These plant
tissues are a specific ecological niche where numerous L. maculans
individuals are living quiescent;
- Finally, at the end of the growing season, the fungus suddenly
turns necrotrophic, and destroys the crown tissues, causing the
devastating crown canker responsible for lodging of the plants (see
picture).
The primary symptom of the disease : the leaf spots occurring on oilseed rape leaves in Autumn (credits: T. Rouxel, INRA) |
As compared to more specialized fungal pathogens, for which parasitism
is linked with the loss of genes corresponding to unnecessary
functions, such as sexual reproduction or competition with other
microbes in the environment, Leptosphaeria maculans is unique
in its ability to develop contrasting life modes. As a consequence, it
probably maintains numerous genes required for saprophytic life
(nutrient acquisition, competition with the soil flora, etc.),
necrotrophic parasitism (toxins, degradation enzymes, etc.) and
intercellular cryptic growth (suppression of recognition by the plant
and/or suppression of plant defence responses). These diverse life
traits also suggest a high level of plasticity in terms of nutrient
acquisition and requirements. Such a versatile behaviour and the
successive setting of all these biological or phytopathological
programmes suggest Leptosphaeria maculans is an adequate and
complete model for genome-wide based functional studies of
pathogenicity, and to identify signalling and regulation processes
responsible for shifts in life style. The importance of sexual
recombination in the wild also renders it a very efficient model for
the study of molecular evolution of genes submitted to selection
pressure.
Stem canker observed on oilseed rape plants in the field (original painting by M. Huau, 1950, INRA-PMDV). |
Superimposed on this behaviour versatility, Leptosphaeria
maculans, as other Dothideomycetes, develops an extreme host
specialization termed the gene-for-gene relationship, where the
outcome of the infection (resistance or susceptibility) will depend on
the presence of one major gene for resistance in the plant (R) and one
corresponding "avirulence" (AVR) gene in the pathogen. This
specific recognition lead to the rapid set-up of plant defence
response at the onset of leaf penetration, and fully protects the
plant from the disease.
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The lab tractability of Leptosphaeria maculans
- L. maculans is a haploid fungus, with a small genome size,
of about 34 Mb, and predicted to encode ca. 10 000 genes. Its 15-16
chromosomes, including a non-mendelian-transmitted minichromosome
range between 0.7 and 3.5 Mb and are easily separated by CHEF
electrophoresis, therefore allowing to allocate linkage groups or
contigs to given chromosomes.
- The fungus is easily grown in vitro on a range of media. Culture
conditions have been optimised for enhanced asexual sporulation. It
can be maintained for years in collections; for example, some isolates
of the early 1970s have retained sporulation and pathogenicity and can
be compared to current populations.
- The fungus is strictly heterothallic and, in contrast to most
fungal phytopathogens, it can be crossed easily on synthetic media,
with an excellent level of fertility, whatever the cross undertaken,
and giving rise to an abundant progeny within two months. Tetrad
analysis, though being tedious, is feasible.
- Phenotyping is easy, mainly with regards to phytopathological
traits (aggressiveness, i.e., the amount of disease, and virulence,
i.e., the ability to cause or not disease on a given
host). Miniaturized assays on cotyledons of plantlets can allow the
phenotyping of up to 500 interactions per week in INRA-PMDV conditions
(with 10-12 repeats per interaction). Such high-throughput screening
is suitable to evaluate mutant libraries or progenies.
- L. maculans is amenable to genetic transformation, and
highly efficient agrotransformation protocols have been developed. In
this respect GFP and DsRed reporter gene vectors have been developed,
along with a vector designed to enhance the recovery of fungal
transformants having undergone homologous recombination events
(thymidine kinase negative selection). Also BiBAC vectors for
transformation of large pieces of DNA via agrotransformation are
constructed. Preliminary findings in B. Howlett’s
lab are that RNAi protocols to knock out gene expression are
effective.
Available resources and tools include extensive collections of natural
isolates or progeny, extensive collection of Agrobacterium-mediated
tranformants, genetic maps, BAC libraries, including one 24
genome-equivalent BAC library maintained at INRA-Versailles, etc. In
addition the whole genome sequence of two related species, the wheat
pathogen Stagonospora nodorum (Richard Oliver team,
Murdoch University, Australia) and the Brassica pathogen Alternaria
brassicicola (Christopher
Lawrence, Virginia Bioinformatics Institute, USA), will be
completed in the coming year and made available to the scientific
community for future comparative genomics of pathogenicity, and genome
evolution approaches.
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Biological and phytopathological process studied in Leptosphaeria maculans
Among the main research topics analysed in Leptosphaeria
maculans as a model, regardless of researches performed on the
plant side, one can stress:
- Host specificity linked with avirulence genes ; evolution of AVR
genes and their genome environment under selection pressure (INRA-Versailles);
- Intrinsic function of avirulence genes, loss of function linked
with gain of "virulence" and consequences for the fungal fitness
(INRA-Versailles) ; consequences for the durable management of
resistance sources in oilseed rape (main objective of the EU-funded project
SECURE);
- Structure and evolution of Leptosphaeria maculans
centromeres; consequences for the chromosome size polymorphism
generated by meiosis (INRA-Versailles);
- Identification of fungal pathogenicity genes by forward genetics
(Melbourne
University, INRA-Versailles);
- Transcriptomics and proteomics of the interaction with Brassicas
and Arabidopsis thaliana;
- Organisation and gene function of clusters of secondary metabolite
genes involved in toxic metabolite production (univ. Melbourne ;
univ. Saskatchewan);
- Detoxification of plant defense compounds (i.e., phytoalexins) and
consequences on pathogenicity (University
Saskatchewan).
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Objectives of the sequencing project
The gene-for-gene paradigm, now established for numerous
plant-pathogen interactions, has major economic and scientific
consequences. Such strict host specificities led breeders to widely
breed for, and deploy, the corresponding resistance, i.e., major gene
resistance in crops. The plants harbouring these genes are fully
immune to the disease. The unwanted counterpart is that such
resistance genes exert extremely strong selection pressure on pathogen
populations that usually can easily develop single mutations in their
avirulence genes, which are enough to overwhelm the corresponding
major resistance. For example, the large scale deployment of major
resistance gene Rlm1(recognizing avirulence gene AvrLm1)
in France since 1995 led, in three years only, to the surge and
prevalence of populations of Leptosphaeria maculans, having
lost the functional allele of AvrLm1. Consequently, the
efficiency of Rlm1-harbouring varieties to control the disease
was lost in three years only, whereas more that 10 years were needed
for breeders to introduce this resistance source in commercial
varieties.
An example of the gene-for-gene interaction as expressed on cotyledons of oilseed rape, the AvrLm1-Rlm1 incompatibility. |
On a fundamental point of view, there is a definite lack of data on
fungal avirulence genes, which are broadly defined by the
resistance phenotype they induce on resistant plants. In this respect
their intrinsic function, their role in fungal biology or
pathogenicity, the recognition mechanism between the R and AVR genes,
and the mode of evolution under selection pressure are widely unknown,
with only 10 avirulence genes in only 3 three ascomycete species
currently identified at the molecular level, and a definite lack of
common traits between these genes.
The INRA-PMDV group at Versailles develops map-based cloning
strategies to identify such avirulence genes in L. maculans
(termed AvrLm). Eight AvrLm genes are currently mapped
on the fungal genetic map, and six of them are organized as two
unlinked genetic clusters comprising three genes each. The main target
of the Rouxel’s team at INRA-Versailles currently is the
AvrLm1-AvrLm2-AvrLm6 cluster, due to (i) the wide use of the
Rlm1 resistance gene these last years, and the concomitant
availability of large collections of isolates having been submitted to
the selection pressure; (ii) the very different crop history of the
three resistance gene corresponding to avirulence genes of this
cluster, with Rlm2 having been used in the most ancient oilseed
rape genotypes, whereas Rlm6 has never been used in crops in
Europe; and (iii) the important size polymorphism of the chromosome
harbouring this gene cluster, ranging between 1.90 Mb and 2.80 Mb
depending on the isolates.
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History of the sequencing project
Scanning electron microscopy of ascospores germinating on oilseed rape leaves (credits: AM Jaunet & MH Balesdent, INRA). |
A formal genetic approach allowed us to genetically delineate the
AvrLm1-AvrLm2-AvrLm6 cluster on both sides with two markers of
the genetic map separated by 9.8 cM. These markers were used to screen
the 24-X BAC library in order to physically border the gene cluster
with two BAC contigs.
A preliminary collaboration between Genoscope and INRA, in 2000,
provided us with the whole sequence of the 5' side bordering BAC
contig in isolate JN3 (whose genotype at the three avirulence loci is
AvrLm1-avrLm2-AvrLm6). This 184-kb genomic region has peculiar
features in that it is extremely A+T-rich, and that it mainly consists
of mosaic of a few families of retrotransposons, degenerated following
RIP (Repeat Induced Point mutations) and usually severely
truncated. The 5' contig nevertheless contains a 32-kb G+C
equilibrated region, containing functional ORFs. The contig contains
numerous genetic markers co-segregating with
AvrLm1. Unfortunately, the very strong recombination-deficient
nature of the whole genome environment was misleading in targeting
AvrLm1 within the 32-kb ORF-rich region, and recent data
suggested AvrLm1 was actually located 3' of the 184-kb
contig. For subsequent steps, the availability of the 184-kb contig
precise sequence was instrumental to design PCR markers based on
Single Nucleotide Polymorphism (SNPs) between degenerated repeats,
which could be used to screen 3-D BAC pools in order to expand the BAC
contig in the 3' part. This allowed to delineate a 1 Mb region (out of
the 2.80 Mb of the chromosome), containing AvrLm1,
AvrLm6, and most probably AvrLm2. The whole region
structure seems to be very similar to that of the 5' 184 kb contig,
since it appears to be also A+T-rich, to contain small ORF islands
drowned within oceans of degenerated retroelements, to correspond to a
recombination-deficient region, and since it is under-represented in
the BAC library. These characteristics are reminiscent of those of
regional centromeres with a central nucleus bordered by
heterochromatic regions that may expand several hundred of
kb. Regional centromeres are specific of upper Eukaryotes, and remain
largely unexplored, whichever the species, due to difficulties in
cloning, sequencing, assembling and annotation.
Additional characterization of natural populations of
L. maculans, encompassing virulent and avirulent isolates at
the AvrLm1 locus, along with genetic studies furthermore
pointed out:
- one 210-kb region within which recombination events are strongly
suppressed, therefore corresponding to a genetic distance of less than
1 cM, whereas the average correspondence between physical and genetic
distances is 8 kb per cM in the Versailles genetic map
- one 530-kb region surrounding AvrLm1 which is submitted to
drastic deletions when the isolates are losing the avirulent
phenotype. Such deletions can reach 210 kb and fully explain the
chromosome size polymorphism observed (see above).
These data strongly suggest that this pericentromeric,
recombination-deficient region, may constitute a "genomic refuge"
for genes involved in pathogenicity or avirulence.
In 2003, Genoscope undertook the sequencing of the whole
pericentromeric region encompassing AvrLm1, AvrLm6, and
probably AvrLm2, and corresponding to a 1-Mb BAC contig of
isolate JN3 (AvrLm1 avrLm2 AvrLm6). In parallel, BAC contigs
originating from two other isolates with different avirulent allele
combinations (isolate NZ-T : avrLm1 avrLm2 AvrLm6, which is a
field isolate from New Zealand and isolate v29 : avrLm1 AvrLm2
avrLm6, which is a lab progeny from a cross with one Australian
isolate as a grand-father) will be partially sequenced with a special
focus on G+C-rich regions, likely to contain target genes. In contrast
to JN3, these two isolates are virulent on Rlm1-harbouring plant
genotypes, and their sequencing will provide a precise comparison of
the avirulent and virulent loci.
Once the sequence is assembled, a precise inventory of the genes
present in the region will be performed in order to identify both
putative avirulence genes and pathogenicity genes clustered in this
region. Structural features of the region will be thoroughly analysed
with special focus on the degree of conservation of the mosaic of
repeats, in order to better define the deletion extent and borders,
and possibly to identify deletion "hot spots". In addition to the
immediate deliverables of this sequencing project, the data thus
obtained will be the basis for :
- The cloning of avirulence genes AvrLm1 and AvrLm6,
including functional validation and accurate analysis of their
function, kinetics of expression, regulation and cell localisation of
the gene product.
- A study of the molecular evolution of avirulence genes and of the
events leading to virulence in natural populations of the pathogen (in
collaboration with INAPG). The Genoscope sequencing will be completed
by a large-scale sequencing of virulent alleles in natural population
to analyse the different events leading to virulence, as a function of
the intensity and duration of the selection pressure, and will allow
to infer a sequential evolution towards virulence and between virulent
alleles.
- Analysis of structural features specific of L. maculans
genes based on the sequence of ca. 30 genes (and promoter region)
present in the contig sequenced here by comparison with cDNAs analysed
in parallel. This will be the base for refined annotation of the
L. maculans genome when whole-genome sequencing is
performed.
- The design of improved race markers for the PCR-based
identification and monitoring of virulent races avrLm1 and
avrLm6.
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