Evolution of virulence in fungal plant pathogens: exploiting fungal genomics to control plant disease.

The propensity of a fungal pathogen to evolve virulence depends on features of its biology (e.g. mode of reproduction) and of its genome (e.g. amount of repetitive DNA). Populations of Leptosphaeria maculans, a pathogen of Brassica napus (canola), can evolve and overcome disease resistance bred into canola within three years of commercial release of a cultivar. Avirulence effector genes are key fungal genes that are complementary to resistance genes. In L. maculans these genes are embedded within inactivated transposable elements in genomic regions where they are readily mutated or deleted. The risk of resistance breakdown in the field can be minimised by monitoring disease severity of canola cultivars and virulence of fungal populations using high throughput molecular assays and by sowing canola cultivars with different resistance genes in subsequent years. This strategy has been exploited to avert yield losses due to blackleg disease in Australia.

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

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

Mitochondrial microsatellite markers for the Australian ectomycorrhizal fungus Laccaria sp. A (Hydnangiaceae).

PREMISE OF THE STUDY: Microsatellite loci were developed for the ectomycorrhizal fungus Laccaria sp. A to investigate the population genetic structure of this fungal symbiont across its fragmented distribution in southeastern Australia. • METHODS AND RESULTS: A partial genome sequence from an individual collection of Laccaria sp. A was obtained by 454 genome sequencing. Eight microsatellite markers were selected from 66 loci identified in the genome. The selected markers were highly polymorphic (4-19 alleles per locus, average 13 alleles) and amplified reproducibly from collections made across the distribution of this species. Five of these markers also amplified reproducibly in the sister species Laccaria sp. E (1). All eight of the selected microsatellite loci were from the mitochondrial genome. • CONCLUSIONS: The highly polymorphic markers described here will enable population structure of Laccaria sp. A to be determined, contributing to research on mycorrhizal fungi from a novel distribution.

Genomes and transcriptomes of partners in plant-fungal-interactions between canola (Brassica napus) and two Leptosphaeria species.

Leptosphaeria maculans 'brassicae' is a damaging fungal pathogen of canola (Brassica napus), causing lesions on cotyledons and leaves, and cankers on the lower stem. A related species, L. biglobosa 'canadensis', colonises cotyledons but causes few stem cankers. We describe the complement of genes encoding carbohydrate-active enzymes (CAZys) and peptidases of these fungi, as well as of four related plant pathogens. We also report dual-organism RNA-seq transcriptomes of these two Leptosphaeria species and B. napus during disease. During the first seven days of infection L. biglobosa 'canadensis', a necrotroph, expressed more cell wall degrading genes than L. maculans 'brassicae', a hemi-biotroph. L. maculans 'brassicae' expressed many genes in the Carbohydrate Binding Module class of CAZy, particularly CBM50 genes, with potential roles in the evasion of basal innate immunity in the host plant. At this time, three avirulence genes were amongst the top 20 most highly upregulated L. maculans 'brassicae' genes in planta. The two fungi had a similar number of peptidase genes, and trypsin was transcribed at high levels by both fungi early in infection. L. biglobosa 'canadensis' infection activated the jasmonic acid and salicylic acid defence pathways in B. napus, consistent with defence against necrotrophs. L. maculans 'brassicae' triggered a high level of expression of isochorismate synthase 1, a reporter for salicylic acid signalling. L. biglobosa 'canadensis' infection triggered coordinated shutdown of photosynthesis genes, and a concomitant increase in transcription of cell wall remodelling genes of the host plant. Expression of particular classes of CAZy genes and the triggering of host defence and particular metabolic pathways are consistent with the necrotrophic lifestyle of L. biglobosa 'canadensis', and the hemibiotrophic life style of L. maculans 'brassicae'.

An avirulence gene, AvrLmJ1, from the blackleg fungus, Leptosphaeria maculans, confers avirulence to Brassica juncea cultivars.

The fungus Leptosphaeria maculans causes blackleg of Brassica species. Here, we report the mapping and subsequent cloning of an avirulence gene from L. maculans. This gene, termed AvrLmJ1, confers avirulence towards all three Brassica juncea cultivars tested. Analysis of RNA-seq data showed that AvrLmJ1 is housed in a region of the L. maculans genome which contains only one gene that is highly expressed in planta. The closest genes are 57 and 33 kb away and, like other avirulence genes of L. maculans, AvrLmJ1 is located within an AT-rich, gene-poor region of the genome. The encoded protein is 141 amino acids, has a predicted signal peptide and is cysteine rich. Two virulent isolates contain a premature stop codon in AvrLmJ1. Complementation of an isolate that forms cotyledonary lesions on B. juncea with the wild-type allele of AvrLmJ1 confers avirulence towards all three B. juncea cultivars tested, suggesting that the gene may confer species-specific avirulence activity.

Next-generation genome sequencing can be used to rapidly characterise sequences flanking T-DNA insertions in random insertional mutants of Leptosphaeria maculans.

BACKGROUND: Banks of mutants with random insertions of T-DNA from Agrobacterium tumefaciens are often used in forward genetics approaches to identify phenotypes of interest. Upon identification of mutants of interest, the flanking sequences of the inserted T-DNA must be identified so that the mutated gene can be characterised. However, for many fungi, this task is not trivial as widely used PCR-based methods such as thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) are not successful. FINDINGS: Next-generation Illumina sequencing was used to locate T-DNA insertion sites in four mutants of Leptosphaeria maculans, a fungal plant pathogen. Sequence reads of up to 150 bp and coverage ranging from 6 to 24 times, were sufficient for identification of insertion sites in all mutants. All T-DNA border sequences were truncated to different extents. Additionally, next-generation sequencing revealed chromosomal rearrangements associated with the insertion in one of the mutants. CONCLUSIONS: Next-generation sequencing is a cost-effective and rapid method of identifying sites of T-DNA insertions, and associated genomic rearrangements in Leptosphaeria maculans and potentially in other fungal species.

Multigene sequence data reveal morphologically cryptic phylogenetic species within the genus Laccaria in southern Australia.

Laccaria (Hydnangiaceae, Agaricales, Basidiomycota) is one of the more intensively studied ectomycorrhizal genera; however, species boundaries within Laccaria and the closely related Hydnangium and Podohydnangium in Australia have not yet been examined with molecular sequence data. Based on morphological characters, eight native species of Laccaria are currently recognized in Australia, as well as three Hydnangium species and the monotypic Podohydnangium australe. Sequences of the internal transcribed spacer region of nuclear rDNA (ITS), RNA polymerase beta subunit II (rpb2) and translation elongation factor 1 alpha (tef-1α) were generated from 77 collections of Laccaria, Hydnangium and Podohydnangium from Australia. Ten phylogenetic species and a further 11 potential species (represented by singletons) of Laccaria in Australia are delimited from sequence analyses. Most of the morphological species contained cryptic phylogenetic species, but these species were always nested entirely within a given morphological species, although not always as sister taxa. The rpb2 locus performed best as a species barcode with pairwise and patristic distance measures. The ITS sequence region returned the least resolved gene tree of the three regions examined and was the least useful as a barcode region. Based on the phylogenetic topology, there appears to have been multiple gains and/or losses of the ectomycorrhizal association of some species with the myrtle beech, Nothofagus cunninghamii as well as of sequestrate basidiocarps and two-spored basidia.

A gene cluster responsible for biosynthesis of phomenoic acid in the plant pathogenic fungus, Leptosphaeria maculans.

Phomenoic acid, a long chain aliphatic carboxylic acid is a major metabolite produced by Leptosphaeria maculans, a fungal pathogen of Brassica napus (canola). This fungus has 15 predicted polyketide synthases (PKS) and seven of them have the appropriate domains for the biosynthesis of phomenoic acid. The most highly expressed PKS gene after 7 days growth in 10% V8 juice, PKS2, was silenced and the resultant mutant produced very low levels of phomenoic acid, indicating that this PKS is involved in phomenoic acid biosynthesis. This gene is part of a co-regulated cluster of genes. Reduced expression of an adjacent gene encoding the transcriptional regulator C6TF, led to reduced expression of genes for PKS2, P450, a cytochrome P450 monoxygenase, YogA, an alcohol dehydrogenase/quinone reductase, RTA1, a lipid transport exporter superfamily member and MFS, a Major Facilitator Superfamily transporter, as well as a marked reduction in phomenoic acid production. Phomenoic acid is toxic towards another canola pathogen Leptosphaeria biglobosa 'canadensis', but not towards L. maculans and only moderately toxic towards the wheat pathogen Stagonospora nodorum. This molecule is detected in infected stems and stubble of B. napus, but biosynthesis of it does not appear to be essential for pathogenicity of L. maculans. Phomenoic acid may play a role in allowing L. maculans to outcompete other fungi in its environmental niche.

Eplt4 proteinaceous elicitor produced in Pichia pastoris has a protective effect against Cercosporidium sofinum infections of soybean leaves.

A complementary DNA library was constructed from the mycelium of Trichoderma asperellum T4, and a highly expressed gene fragment named EplT4 was found. In order to find a more efficient and cost-effective way of obtaining EplT4, this study attempted to produce EplT4 using a Pichia pastoris expression system. The gene encoding EplT4, with an additional 6-His tag at the C-terminus, was cloned into the yeast vector pPIC9K and expressed in the P. pastoris strain GS115 to obtaining more protein for the further research. Transformants of P. pastoris were selected by PCR analysis, and the ability to secrete high levels of the EplT4 protein was determined. The optimal conditions for induction were assayed using the shake flask method and an enzyme-linked immunosorbent assay. The yield of purified EplT4 was approximately 20 mg/L by nickel affinity chromatography and gel-filtration chromatography. Western blot and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis revealed that the recombinant EplT4 was expressed in both its monomers and dimers. Soybean leaves treated with the EplT4 monomer demonstrated the induction of glucanase, chitinase III-A, cysteine proteinase inhibitor, and peroxidase genes. Early cellular events in plant defense response were also observed after incubation with EplT4. Soybean leaves protected by EplT4 against the pathogen Cercosporidium sofinum (Hara) indicated that EplT4 produced in P. pastoris was biologically active and would be potentially useful for improving food security.

Spot Form of Net Blotch Resistance in a Diverse Set of Barley Lines in Australia and Canada.

The responses of 95 barley lines and cultivars to spot form of net blotch (SFNB) caused by Pyrenophora teres f. maculata were analyzed as seedlings and adults in Australia and Canada. Cluster analyses revealed complex reaction responses. Only 2 lines (Esperance Orge 289 and TR3189) were resistant to all isolates at the seedling stage, whereas 15 lines and cultivars (81-82/033, Arimont, BYDV-018, CBSS97M00855T-B2-M1-Y1-M2-Y-1M-0Y, CI9776, Keel, Sloop, Torrens, TR326, VB0111, Yarra, VB0229, WI-2477, WI2553, and Wisconsin Pedigree) were resistant toward the two Canadian isolates and mixture of Australian isolates at the adult stages. In Australian field experiments, the effectiveness of SFNB resistance in three barley cultivars (Barque, Cowabbie, and Schooner) and one breeding line (VB9104) with a different source of resistance was tested. Barque, which possessed a resistance gene that provided complete resistance to SFNB, was the most effective and showed no effect on grain yield or quality in the presence of inoculum. Generally, cultivars with seedling or adult resistance had less disease and better grain quality than the susceptible control, Dash, but they were not as effective as Barque. A preliminary differential set of 19 barley lines and cultivars for P. teres f. maculata is proposed.

Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea.

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.

The cross-pathway control system regulates production of the secondary metabolite toxin, sirodesmin PL, in the ascomycete, Leptosphaeria maculans.

BACKGROUND: Sirodesmin PL is a secondary metabolite toxin made by the ascomycetous plant pathogen, Leptosphaeria maculans. The sirodesmin biosynthetic genes are clustered in the genome. The key genes are a non-ribosomal peptide synthetase, sirP, and a pathway-specific transcription factor, sirZ. Little is known about regulation of sirodesmin production. RESULTS: Genes involved in regulation of sirodesmin PL in L. maculans have been identified. Two hundred random insertional T-DNA mutants were screened with an antibacterial assay for ones producing low levels of sirodesmin PL. Three such mutants were isolated and each transcribed sirZ at very low levels. One of the affected genes had high sequence similarity to Aspergillus fumigatus cpcA, which regulates the cross-pathway control system in response to amino acid availability. This gene was silenced in L. maculans and the resultant mutant characterised. When amino acid starvation was artificially-induced by addition of 3-aminotriazole for 5 h, transcript levels of sirP and sirZ did not change in the wild type. In contrast, levels of sirP and sirZ transcripts increased in the silenced cpcA mutant. After prolonged amino acid starvation the silenced cpcA mutant produced much higher amounts of sirodesmin PL than the wild type. CONCLUSIONS: Production of sirodesmin PL in L. maculans is regulated by the cross pathway control gene, cpcA, either directly or indirectly via the pathway-specific transcription factor, sirZ.

Identification of cryptic products of the gliotoxin gene cluster using NMR-based comparative metabolomics and a model for gliotoxin biosynthesis.

Gliotoxin, a major product of the gli non-ribosomal peptide synthetase gene cluster, is strongly associated with virulence of the opportunistic human pathogen Aspergillus fumigatus. Despite identification of the gli cluster, the pathway of gliotoxin biosynthesis has remained elusive, in part because few potential intermediates have been identified. In addition, previous studies suggest that knowledge of gli-dependent metabolites is incomplete. Here we use differential analysis by 2D NMR spectroscopy (DANS) of metabolite extracts derived from gli knock-out and wild-type (WT) strains to obtain a detailed inventory of gli-dependent metabolites. DANS-based comparison of the WT metabolome with that of ΔgliZ, a knock-out strain devoid of the gene encoding the transcriptional regulator of the gli cluster, revealed nine novel gliZ-dependent metabolites including unexpected structural motifs. Their identification provides insight into gliotoxin biosynthesis and may benefit studies of the role of the gli cluster in A. fumigatus virulence. Our study demonstrates the utility of DANS for correlating gene expression and metabolite biosynthesis in microorganisms.

A novel mode of chromosomal evolution peculiar to filamentous Ascomycete fungi.

BACKGROUND: Gene loss, inversions, translocations, and other chromosomal rearrangements vary among species, resulting in different rates of structural genome evolution. Major chromosomal rearrangements are rare in most eukaryotes, giving large regions with the same genes in the same order and orientation across species. These regions of macrosynteny have been very useful for locating homologous genes in different species and to guide the assembly of genome sequences. Previous analyses in the fungi have indicated that macrosynteny is rare; instead, comparisons across species show no synteny or only microsyntenic regions encompassing usually five or fewer genes. To test the hypothesis that chromosomal evolution is different in the fungi compared to other eukaryotes, synteny was compared between species of the major fungal taxa. RESULTS: These analyses identified a novel form of evolution in which genes are conserved within homologous chromosomes, but with randomized orders and orientations. This mode of evolution is designated mesosynteny, to differentiate it from micro- and macrosynteny seen in other organisms. Mesosynteny is an alternative evolutionary pathway very different from macrosyntenic conservation. Surprisingly, mesosynteny was not found in all fungal groups. Instead, mesosynteny appears to be restricted to filamentous Ascomycetes and was most striking between species in the Dothideomycetes. CONCLUSIONS: The existence of mesosynteny between relatively distantly related Ascomycetes could be explained by a high frequency of chromosomal inversions, but translocations must be extremely rare. The mechanism for this phenomenon is not known, but presumably involves generation of frequent inversions during meiosis.

Fungal pathogenicity genes in the age of 'omics'.

The identification of the fungal genes essential for disease underpins the development of disease control strategies. Improved technologies for gene identification and functional analyses, as well as a plethora of sequenced fungal genomes, have led to the characterization of hundreds of genes, denoted as pathogenicity genes, which are required by fungi to cause disease. We describe recent technologies applied to characterize the fungal genes involved in disease and focus on some genes that are likely to attract continuing research activity.

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.

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.

Hunting down fungal secretomes using liquid-phase IEF prior to high resolution 2-DE.

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, beta-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.

Mutations to LmIFRD affect cell wall integrity, development and pathogenicity of the ascomycete Leptosphaeria maculans.

Maintaining cell wall integrity is essential for fungal growth and development. We describe two mutants with altered expression of a gene, LmIFRD, from the ascomycete Leptosphaeria maculans. Truncation of the LmIFRD transcript in a T-DNA insertional mutant led to slower germination, less sporulation and loss-of-pathogenicity towards Brassica napus, whereas silencing of the LmIFRD transcript led to increased germination, sporulation and earlier infection. The increased tolerance to cell wall lysing enzymes and cell wall-disrupting compounds of the T-DNA mutant contrasts with decreased tolerance of the silenced mutant and suggests altered cell wall integrity and accessibility to 1,3-linked glucan and chitin. Lectin binding experiments and monosaccharide analysis revealed altered polysaccharide content and structure within the cell wall of the LmIFRD mutants, notably increased 1,3-linked galactose and chitin within the cell wall of the T-DNA mutant. This is the first analysis of monosaccharide linkage composition of cell walls of spores and mycelia for any dothideomycete.