Angela P. Van de Wouw

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.

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

BackgroundMany 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.ResultsL. 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.ConclusionsInvasion 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.

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.

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.

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.

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’.

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.

Identification of Leptosphaeria biglobosa ‘canadensis’ on Brassica juncea stubble from northern New South Wales, Australia

Leptosphaeria biglobosa ‘canadensis’ is reported for the first time in Australia. All 88 Leptosphaeria isolates cultured from Brassica juncea stubble from northern NSW were L. biglobosa ‘canadensis’ whilst all 55 isolates cultured from Victorian stubble of the same B. juncea lines were L. maculans. Both L. biglobosa ‘canadensis’ and L. maculans formed similar sized lesions on B. juncea cotyledons after 14 days. However, L. biglobosa ‘canadensis’ isolates colonised stems less effectively than L. maculans and consequently caused less crown cankering.

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.

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.