Anton J. Cozijnsen

Effector diversification within compartments of the Leptosphaeria maculans genome affected by repeat induced point mutations

Fungi are of primary ecological, biotechnological and economic importance. Many fundamental biological processes that are shared by animals and fungi are studied in fungi due to their experimental tractability. Many fungi are pathogens or mutualists and are model systems to analyse effector genes and their mechanisms of diversification. In this study, we report the genome sequence of the phytopathogenic ascomycete Leptosphaeria maculans and characterize its repertoire of protein effectors. The L. maculans genome has an unusual bipartite structure with alternating distinct guanine and cytosine-equilibrated and adenine and thymine (AT)-rich blocks of homogenous nucleotide composition. The AT-rich blocks comprise one-third of the genome and contain effector genes and families of transposable elements, both of which are affected by repeat-induced point mutation, a fungal-specific genome defence mechanism. This genomic environment for effectors promotes rapid sequence diversification and underpins the evolutionary potential of the fungus to adapt rapidly to novel host-derived constraints.

The sirodesmin biosynthetic gene cluster of the plant pathogenic fungus Leptosphaeria maculans

Sirodesmin PL is a phytotoxin produced by the fungus Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). This phytotoxin belongs to the epipolythiodioxopiperazine (ETP) class of toxins produced by fungi including mammalian and plant pathogens. We report the cloning of a cluster of genes with predicted roles in the biosynthesis of sirodesmin PL and show via gene disruption that one of these genes (encoding a two‐module non‐ribosomal peptide synthetase) is essential for sirodesmin PL biosynthesis. Of the nine genes in the cluster tested, all are co‐regulated with the production of sirodesmin PL in culture. A similar cluster is present in the genome of the opportunistic human pathogen Aspergillus fumigatus and is most likely responsible for the production of gliotoxin, which is also an ETP. Homologues of the genes in the cluster were also identified in expressed sequence tags of the ETP producing fungus Chaetomium globosum. Two other fungi with publicly available genome sequences, Magnaporthe grisea and Fusarium graminearum, had similar gene clusters. A comparative analysis of all four clusters is presented. This is the first report of the genes responsible for the biosynthesis of an ETP.

Nitrogen limitation induces expression of the avirulence gene avr9 in the tomato pathogen Cladosporium fulvum

The avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum encodes a race-specific peptide elicitor that induces the hypersensitive response in tomato plants carrying the complementary resistance gene Cf9. The avr9 gene is not expressed under optimal growth conditions in vitro, but is highly expressed when the fungus grows inside the tomato leaf. In this paper we present evidence for the induction of avr9 gene expression in C. fulvum grown in vitro under conditions of nitrogen limitation. Only growth medium with very low amounts of nitrogen (nitrate, ammonium, glutamate or glutamine) induced the expression of avr9. Limitation of other macronutrients or the addition of plant factors did not induce the expression of avr9. The induced expression of avr9 is possibly mediated by a positive-acting nitrogen regulatory protein, homologous to the Neurospora crassa NIT2 protein, which induces the expression of many genes under conditions of nitrogen limitation. The avr9 promoter contains several putative NIT2 binding sites. The expression of avr9 during the infection process was explored cytologically using transformants of C. fulvum carrying an avr9 promoter-β-glucuronidase reporter gene fusion. The possibility that expression of avr9 in C. fulvum growing in planta is caused by nitrogen limitation in the apoplast of the tomato leaf is discussed.

Origin and distribution of epipolythiodioxopiperazine (ETP) gene clusters in filamentous ascomycetes

BackgroundGenes responsible for biosynthesis of fungal secondary metabolites are usually tightly clustered in the genome and co-regulated with metabolite production. Epipolythiodioxopiperazines (ETPs) are a class of secondary metabolite toxins produced by disparate ascomycete fungi and implicated in several animal and plant diseases. Gene clusters responsible for their production have previously been defined in only two fungi. Fungal genome sequence data have been surveyed for the presence of putative ETP clusters and cluster data have been generated from several fungal taxa where genome sequences are not available. Phylogenetic analysis of cluster genes has been used to investigate the assembly and heredity of these gene clusters.ResultsPutative ETP gene clusters are present in 14 ascomycete taxa, but absent in numerous other ascomycetes examined. These clusters are discontinuously distributed in ascomycete lineages. Gene content is not absolutely fixed, however, common genes are identified and phylogenies of six of these are separately inferred. In each phylogeny almost all cluster genes form monophyletic clades with non-cluster fungal paralogues being the nearest outgroups. This relatedness of cluster genes suggests that a progenitor ETP gene cluster assembled within an ancestral taxon. Within each of the cluster clades, the cluster genes group together in consistent subclades, however, these relationships do not always reflect the phylogeny of ascomycetes. Micro-synteny of several of the genes within the clusters provides further support for these subclades.ConclusionETP gene clusters appear to have a single origin and have been inherited relatively intact rather than assembling independently in the different ascomycete lineages. This progenitor cluster has given rise to a small number of distinct phylogenetic classes of clusters that are represented in a discontinuous pattern throughout ascomycetes. The disjunct heredity of these clusters is discussed with consideration to multiple instances of independent cluster loss and lateral transfer of gene clusters between lineages.

CD8+ T Cells from a Novel T Cell Receptor Transgenic Mouse Induce Liver-Stage Immunity That Can Be Boosted by Blood-Stage Infection in Rodent Malaria

To follow the fate of CD8+ T cells responsive to Plasmodium berghei ANKA (PbA) infection, we generated an MHC I-restricted TCR transgenic mouse line against this pathogen. T cells from this line, termed PbT-I T cells, were able to respond to blood-stage infection by PbA and two other rodent malaria species, P. yoelii XNL and P. chabaudi AS. These PbT-I T cells were also able to respond to sporozoites and to protect mice from liver-stage infection. Examination of the requirements for priming after intravenous administration of irradiated sporozoites, an effective vaccination approach, showed that the spleen rather than the liver was the main site of priming and that responses depended on CD8α+ dendritic cells. Importantly, sequential exposure to irradiated sporozoites followed two days later by blood-stage infection led to augmented PbT-I T cell expansion. These findings indicate that PbT-I T cells are a highly versatile tool for studying multiple stages and species of rodent malaria and suggest that cross-stage reactive CD8+ T cells may be utilized in liver-stage vaccine design to enable boosting by blood-stage infections.

Evolution of Linked Avirulence Effectors in Leptosphaeria maculans Is Affected by Genomic Environment and Exposure to Resistance Genes in Host Plants

Brassica napus (canola) cultivars and isolates of the blackleg fungus, Leptosphaeria maculans interact in a ‘gene for gene’ manner whereby plant resistance (R) genes are complementary to pathogen avirulence (Avr) genes. Avirulence genes encode proteins that belong to a class of pathogen molecules known as effectors, which includes small secreted proteins that play a role in disease. In Australia in 2003 canola cultivars with the Rlm1 resistance gene suffered a breakdown of disease resistance, resulting in severe yield losses. This was associated with a large increase in the frequency of virulence alleles of the complementary avirulence gene, AvrLm1, in fungal populations. Surprisingly, the frequency of virulence alleles of AvrLm6 (complementary to Rlm6) also increased dramatically, even though the cultivars did not contain Rlm6. In the L. maculans genome, AvrLm1 and AvrLm6 are linked along with five other genes in a region interspersed with transposable elements that have been degenerated by Repeat-Induced Point (RIP) mutations. Analyses of 295 Australian isolates showed deletions, RIP mutations and/or non-RIP derived amino acid substitutions in the predicted proteins encoded by these seven genes. The degree of RIP mutations within single copy sequences in this region was proportional to their proximity to the degenerated transposable elements. The RIP alleles were monophyletic and were present only in isolates collected after resistance conferred by Rlm1 broke down, whereas deletion alleles belonged to several polyphyletic lineages and were present before and after the resistance breakdown. Thus, genomic environment and exposure to resistance genes in B. napus has affected the evolution of these linked avirulence genes in L. maculans.

Genetic diversity of isolates of the Leptosphaeria maculans species complex from Australia, Europe and North America using amplified fragment length polymorphism analysis

Amplified Fragment Length Polymorphism (AFLP) analysis has been used to analyse 100 Australian, European and North American isolates of Leptosphaeria maculans. All isolates had distinct AFLP profiles. They could be classified into five types, which had very few AFLP bands in common and corresponded to classifications made previously on the basis of ability to cause stem cankers on Brassica napus (A group), or inability to do so (B group), and on host range. Four isolates had AFLP profiles completely dissimilar to these groups and to each other. Genetic diversity and geographic differentiation were analysed separately within AFLP types 1 and 2. UPGMA analysis of the 66 AFLP type 1 (A group) isolates using 50 polymorphic bands did not provide evidence for clustering according to geographic origin. Non-metric multidimensional scaling (NMDS) analysis suggested that the Australian and European populations were separate adjacent clusters, while the North American population partially overlapped both the others. This geographic differentiation was supported by Wrights fixation index (Fst) analysis. Three measures of genetic variability between isolates within regions (effective number of alleles, gene diversity, and Shannon index) showed that the North American A group isolates were less diverse than those from Australia and Europe. The 21 AFLP type 2 (B group; NA1 sub-group) isolates did not cluster based on geographic region, which was confirmed by NMDS and Fst analysis. There was a similar degree of genetic diversity within A group and the NA1 sub-group of B group isolates. Unlike other techniques, AFLP analysis can readily discriminate between group B isolates of the L. maculans complex that were previously difficult to classify and also provides individual fingerprints for isolates. Isolates of the A group and of the NA1 sub-group of B group could be also distinguished readily by electrophoretic karyotyping, as the latter isolates had more bands smaller than 1.4 Mb than the A group isolates.

Genome analysis of the plant pathogenic ascomycete Leptosphaeria maculans; mapping mating type and host specificity loci

Abstract A genetic and physical map has been developed for the loculoascomycete Leptosphaeria maculans, a pathogen of oilseed Brassicas. The genetic map was constructed from 58 F(1) progeny and comprises 155 amplified fragment length polymorphic (AFLP) markers, three random amplified polymorphic DNA (RAPD) markers, the mating type locus and a host specificity locus conferring the ability to form lesions on Brassica juncea. Twenty-one linkage groups, 5 pairs, and 18 unlinked markers were assigned, and the genome size was 1520 cM. Pulsed field gel electrophoresis experiments showed that the parental isolates each had 16 chromosomes and a genome size of about 33.5 Mb. Attempts to anchor a large number of markers to chromosomes were hampered by difficulties in converting AFLPs into RFLP markers, and because many markers bound to every chromosome, indicating that L. maculans has a high level of dispersed repetitive sequences. This fungus displays chromosomal length polymorphisms, but in the cross examined, the linkage and physical maps were essentially congruent and there was no evidence of translocations. The host specificity locus is 18 cM from the nearest AFLP marker and is located on a chromosome sized 1.85 Mb in the virulent parent. The mating type locus is on a chromosome sized 2.6 Mb and coincident on an AFLP marker amplified from the virulent parent. The derived amino acid sequence of part of this marker has some conserved amino acids present in the High Mobility Group DNA binding domain of MAT-2 mating type genes of other ascomycetes.

Production of the toxin sirodesmin PL by Leptosphaeria maculans during infection of Brassica napus

SUMMARY Sirodesmin PL is a non-host-selective phytotoxin produced by Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). Previous studies have shown that sirodesmin PL biosynthesis involves a cluster of 18 co-regulated genes and that disruption of the two-module non-ribosomal peptide synthetase gene (sirP) in this cluster prevents the production of sirodesmin PL. Loss of sirodesmin PL did not affect the growth or fertility of the sirP mutant in vitro, but this mutant had less antibacterial and antifungal activity than the wild-type. When the sirP mutant was inoculated on to cotyledons of B. napus, it caused similar-sized lesions on cotyledons as the wild-type isolate, but subsequently caused fewer lesions and was half as effective as the wild-type in colonizing stems, as shown by quantitative PCR analyses. However, no significant difference was observed in size of lesions when either wild-type or mutant isolates were injected directly into the stem. The expression of two cluster genes, sirP and an ABC transporter, sirA, was studied in planta. Fungal isolates containing fusions of the green fluorescent protein gene with the promoters of these genes fluoresced after 10 days post-inoculation (dpi). Transcripts of sirP and sirA were detected after 11 dpi in cotyledons by reverse transcriptase PCR, and expression of both genes increased dramatically in stem tissue. This expression pattern was consistent with the distribution of sirodesmin PL in planta as revealed by mass spectrometry experiments.

Identifying resistance genes to Leptosphaeria maculans in Australian Brassica napus cultivars based on reactions to isolates with known avirulence genotypes

Abstract. Blackleg disease, caused by the fungus Leptosphaeria maculans, is the major disease of canola (Brassica napus) worldwide. A set of 12 Australian L. maculans isolates was developed and used to characterise seedling resistance in 127 Australian cultivars and advanced breeding lines. Plant mortality data used to assess the effectiveness of seedling resistance in canola growing regions of Australia showed that Rlm3 and Rlm4 resistance genes were less effective than other seedling resistance genes. This finding was consistent with regional surveys of the pathogen, which showed the frequency of Rlm4-attacking isolates was >70% in fungal populations over a 10-year period. Differences in adult plant resistance were identified in a subset of Australian cultivars, indicating that some adult gene resistance is isolate-specific.