mRNA vaccine against malaria tailored for liver-resident memory T cells.

Malaria is caused by Plasmodium species transmitted by Anopheles mosquitoes. Following a mosquito bite, Plasmodium sporozoites migrate from skin to liver, where extensive replication occurs, emerging later as merozoites that can infect red blood cells and cause symptoms of disease. As liver tissue-resident memory T cells (Trm cells) have recently been shown to control liver-stage infections, we embarked on a messenger RNA (mRNA)-based vaccine strategy to induce liver Trm cells to prevent malaria. Although a standard mRNA vaccine was unable to generate liver Trm or protect against challenge with Plasmodium berghei sporozoites in mice, addition of an agonist that recruits T cell help from type I natural killer T cells under mRNA-vaccination conditions resulted in significant generation of liver Trm cells and effective protection. Moreover, whereas previous exposure of mice to blood-stage infection impaired traditional vaccines based on attenuated sporozoites, mRNA vaccination was unaffected, underlining the potential for such a rational mRNA-based strategy in malaria-endemic regions.

Liver-Resident Memory CD8+ T Cells Form a Front-Line Defense against Malaria Liver-Stage Infection.

In recent years, various intervention strategies have reduced malaria morbidity and mortality, but further improvements probably depend upon development of a broadly protective vaccine. To better understand immune requirement for protection, we examined liver-stage immunity after vaccination with irradiated sporozoites, an effective though logistically difficult vaccine. We identified a population of memory CD8+ T cells that expressed the gene signature of tissue-resident memory T (Trm) cells and remained permanently within the liver, where they patrolled the sinusoids. Exploring the requirements for liver Trm cell induction, we showed that by combining dendritic cell-targeted priming with liver inflammation and antigen recognition on hepatocytes, high frequencies of Trm cells could be induced and these cells were essential for protection against malaria sporozoite challenge. Our study highlights the immune potential of liver Trm cells and provides approaches for their selective transfer, expansion, or depletion, which may be harnessed to control liver infections or autoimmunity.

Development of Plasmodium-specific liver-resident memory CD8+ T cells after heat-killed sporozoite immunization in mice.

Malaria remains a major cause of mortality in the world and an efficient vaccine is the best chance of reducing the disease burden. Vaccination strategies for the liver stage of disease that utilise injection of live radiation-attenuated sporozoites (RAS) confer sterile immunity, which is mediated by CD8+ memory T cells, with liver-resident memory T cells (TRM ) being particularly important. We have previously described a TCR transgenic mouse, termed PbT-I, where all CD8+ T cells recognize a specific peptide from Plasmodium. PbT-I form liver TRM cells upon RAS injection and are capable of protecting mice against challenge infection. Here, we utilize this transgenic system to examine whether nonliving sporozoites, killed by heat treatment (HKS), could trigger the development of Plasmodium-specific liver TRM cells. We found that HKS vaccination induced the formation of memory CD8+ T cells in the spleen and liver, and importantly, liver TRM cells were fewer in number than that induced by RAS. Crucially, we showed the number of TRM cells was significantly higher when HKS were combined with the glycolipid α-galactosylceramide as an adjuvant. In the future, this work could lead to development of an antimalaria vaccination strategy that does not require live sporozoites, providing greater utility.

Development of a Novel CD4+ TCR Transgenic Line That Reveals a Dominant Role for CD8+ Dendritic Cells and CD40 Signaling in the Generation of Helper and CTL Responses to Blood-Stage Malaria.

We describe an MHC class II (I-Ab)-restricted TCR transgenic mouse line that produces CD4+ T cells specific for Plasmodium species. This line, termed PbT-II, was derived from a CD4+ T cell hybridoma generated to blood-stage Plasmodium berghei ANKA (PbA). PbT-II cells responded to all Plasmodium species and stages tested so far, including rodent (PbA, P. berghei NK65, Plasmodium chabaudi AS, and Plasmodium yoelii 17XNL) and human (Plasmodium falciparum) blood-stage parasites as well as irradiated PbA sporozoites. PbT-II cells can provide help for generation of Ab to P. chabaudi infection and can control this otherwise lethal infection in CD40L-deficient mice. PbT-II cells can also provide help for development of CD8+ T cell-mediated experimental cerebral malaria (ECM) during PbA infection. Using PbT-II CD4+ T cells and the previously described PbT-I CD8+ T cells, we determined the dendritic cell (DC) subsets responsible for immunity to PbA blood-stage infection. CD8+ DC (a subset of XCR1+ DC) were the major APC responsible for activation of both T cell subsets, although other DC also contributed to CD4+ T cell responses. Depletion of CD8+ DC at the beginning of infection prevented ECM development and impaired both Th1 and follicular Th cell responses; in contrast, late depletion did not affect ECM. This study describes a novel and versatile tool for examining CD4+ T cell immunity during malaria and provides evidence that CD4+ T cell help, acting via CD40L signaling, can promote immunity or pathology to blood-stage malaria largely through Ag presentation by CD8+ DC.

Mitochondrial ATP synthase is dispensable in blood-stage Plasmodium berghei rodent malaria but essential in the mosquito phase.

Mitochondrial ATP synthase is driven by chemiosmotic oxidation of pyruvate derived from glycolysis. Blood-stage malaria parasites eschew chemiosmosis, instead relying almost solely on glycolysis for their ATP generation, which begs the question of whether mitochondrial ATP synthase is necessary during the blood stage of the parasite life cycle. We knocked out the mitochondrial ATP synthase ß subunit gene in the rodent malaria parasite, Plasmodium berghei, ablating the protein that converts ADP to ATP. Disruption of the ß subunit gene of the ATP synthase only marginally reduced asexual blood-stage parasite growth but completely blocked mouse-to-mouse transmission via Anopheles stephensi mosquitoes. Parasites lacking the ß subunit gene of the ATP synthase generated viable gametes that fuse and form ookinetes but cannot progress beyond this stage. Ookinetes lacking the ß subunit gene of the ATP synthase had normal motility but were not viable in the mosquito midgut and never made oocysts or sporozoites, thereby abrogating transmission to naive mice via mosquito bite. We crossed the self-infertile ATP synthase ß subunit knockout parasites with a male-deficient, self-infertile strain of P. berghei, which restored fertility and production of oocysts and sporozoites, which demonstrates that mitochondrial ATP synthase is essential for ongoing viability through the female, mitochondrion-carrying line of sexual reproduction in P. berghei malaria. Perturbation of ATP synthase completely blocks transmission to the mosquito vector and could potentially be targeted for disease control.

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.

Quantitative analysis of Plasmodium ookinete motion in three dimensions suggests a critical role for cell shape in the biomechanics of malaria parasite gliding motility.

Motility is a fundamental part of cellular life and survival, including for Plasmodium parasites--single-celled protozoan pathogens responsible for human malaria. The motile life cycle forms achieve motility, called gliding, via the activity of an internal actomyosin motor. Although gliding is based on the well-studied system of actin and myosin, its core biomechanics are not completely understood. Currently accepted models suggest it results from a specifically organized cellular motor that produces a rearward directional force. When linked to surface-bound adhesins, this force is passaged to the cell posterior, propelling the parasite forwards. Gliding motility is observed in all three life cycle stages of Plasmodium: sporozoites, merozoites and ookinetes. However, it is only the ookinetes--formed inside the midgut of infected mosquitoes--that display continuous gliding without the necessity of host cell entry. This makes them ideal candidates for invasion-free biomechanical analysis. Here we apply a plate-based imaging approach to study ookinete motion in three-dimensional (3D) space to understand Plasmodium cell motility and how movement facilitates midgut colonization. Using single-cell tracking and numerical analysis of parasite motion in 3D, our analysis demonstrates that ookinetes move with a conserved left-handed helical trajectory. Investigation of cell morphology suggests this trajectory may be based on the ookinete subpellicular cytoskeleton, with complementary whole and subcellular electron microscopy showing that, like their motion paths, ookinetes share a conserved left-handed corkscrew shape and underlying twisted microtubular architecture. Through comparisons of 3D movement between wild-type ookinetes and a cytoskeleton-knockout mutant we demonstrate that perturbation of cell shape changes motion from helical to broadly linear. Therefore, while the precise linkages between cellular architecture and actomyosin motor organization remain unknown, our analysis suggests that the molecular basis of cell shape may, in addition to motor force, be a key adaptive strategy for malaria parasite dissemination and, as such, transmission.

Complexing CpG adjuvants with cationic liposomes enhances vaccine-induced formation of liver TRM cells.

Tissue resident memory T cells (TRM cells) can provide effective tissue surveillance and can respond rapidly to infection. Vaccination strategies aimed at generating TRM cells have shown promise against a range of pathogens. We have previously shown that the choice of adjuvant critically influences CD8+ TRM cell formation in the liver. However, the range of adjuvants tested was limited. Here, we assessed the ability of a broad range of adjuvants stimulating membrane (TLR4), endosomal (TLR3, TLR7 and TLR9) and cytosolic (cGAS, RIG-I) pathogen recognition receptors for their capacity to induce CD8+ TRM formation in a subunit vaccination model. We show that CpG oligodeoxynucleotides (ODN) remain the most efficient inducers of liver TRM cells among all adjuvants tested. Moreover, their combination with the cationic liposome DOTAP further enhances the potency, particularly of the class B ODN CpG 1668 and the human TLR9 ligand CpG 2006 (CpG 7909). This study informs the design of efficient liver TRM-based vaccines for their potential translation.

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.

6″-Modifed α-GalCer-peptide conjugate vaccine candidates protect against liver-stage malaria.

Self-adjuvanting vaccines consisting of peptide epitopes conjugated to immune adjuvants are a powerful way of generating antigen-specific immune responses. We previously showed that a Plasmodium-derived peptide conjugated to a rearranged form of α-galactosylceramide (α-GalCer) could stimulate liver-resident memory T (TRM) cells that were effective killers of liver-stage Plasmodium berghei ANKA (Pba)-infected cells. To investigate if similar or even superior TRM responses can be induced by modifying the α-GalCer adjuvant, we created new conjugate vaccine cadidates by attaching an immunogenic Plasmodium-derived peptide antigen to 6″-substituted α-GalCer analogues. Vaccine synthesis involved developing an efficient route to α-galactosylphytosphingosine (α-GalPhs), from which the prototypical iNKT cell agonist, α-GalCer, and its 6″-deoxy-6″-thio and -amino analogues were derived. Attaching a cathepsin B-cleavable linker to the 6″-modified α-GalCer created pro-adjuvants bearing a pendant ketone group available for peptide conjugation. Optimized reaction conditions were developed that allow for the efficient conjugation of peptide antigens to the pro-adjuvants via oxime ligation to create new glycolipid-peptide (GLP) conjugate vaccines. A single dose of the vaccine candidates induced acute NKT and Plasmodium-specific CD8+ T cell responses that generated potent hepatic TRM responses in mice. Our findings demonstrate that attaching antigenic peptides to 6″-modifed α-GalCer generates powerful self-adjuvanting conjugate vaccine candidates that could potentially control hepatotropic infections such as liver-stage malaria.

Plasmodium berghei Hsp90 contains a natural immunogenic I-Ab-restricted antigen common to rodent and human Plasmodium species.

Thorough understanding of the role of CD4 T cells in immunity can be greatly assisted by the study of responses to defined specificities. This requires knowledge of Plasmodium-derived immunogenic epitopes, of which only a few have been identified, especially for the mouse C57BL/6 background. We recently developed a TCR transgenic mouse line, termed PbT-II, that produces CD4+ T cells specific for an MHC class II (I-Ab)-restricted Plasmodium epitope and is responsive to both sporozoites and blood-stage P. berghei. Here, we identify a peptide within the P. berghei heat shock protein 90 as the cognate epitope recognised by PbT-II cells. We show that C57BL/6 mice infected with P. berghei blood-stage induce an endogenous CD4 T cell response specific for this epitope, indicating cells of similar specificity to PbT-II cells are present in the naïve repertoire. Adoptive transfer of in vitro activated TH1-, or particularly TH2-polarised PbT-II cells improved control of P. berghei parasitemia in C57BL/6 mice and drastically reduced the onset of experimental cerebral malaria. Our results identify a versatile, potentially protective MHC-II restricted epitope useful for exploration of CD4 T cell-mediated immunity and vaccination strategies against malaria.

Mechanisms and targets of Fcγ-receptor mediated immunity to malaria sporozoites.

A highly protective vaccine will greatly facilitate achieving and sustaining malaria elimination. Understanding mechanisms of antibody-mediated immunity is crucial for developing vaccines with high efficacy. Here, we identify key roles in humoral immunity for Fcγ-receptor (FcγR) interactions and opsonic phagocytosis of sporozoites. We identify a major role for neutrophils in mediating phagocytic clearance of sporozoites in peripheral blood, whereas monocytes contribute a minor role. Antibodies also promote natural killer cell activity. Mechanistically, antibody interactions with FcγRIII appear essential, with FcγRIIa also required for maximum activity. All regions of the circumsporozoite protein are targets of functional antibodies against sporozoites, and N-terminal antibodies have more activity in some assays. Functional antibodies are slowly acquired following natural exposure to malaria, being present among some exposed adults, but uncommon among children. Our findings reveal targets and mechanisms of immunity that could be exploited in vaccine design to maximize efficacy.

A Natural Peptide Antigen within the Plasmodium Ribosomal Protein RPL6 Confers Liver TRM Cell-Mediated Immunity against Malaria in Mice.

Liver-resident memory CD8+ T (TRM) cells remain in and constantly patrol the liver to elicit rapid immunity upon antigen encounter and can mediate efficient protection against liver-stage Plasmodium infection. This finding has prompted the development of immunization strategies where T cells are activated in the spleen and then trapped in the liver to form TRM cells. Here, we identify PbRPL6120-127, a H2-Kb-restricted epitope from the putative 60S ribosomal protein L6 (RPL6) of Plasmodium berghei ANKA, as an optimal antigen for endogenous liver TRM cell generation and protection against malaria. A single dose vaccination targeting RPL6 provided effective and prolonged sterilizing immunity against high dose sporozoite challenges. Expressed throughout the parasite life cycle, across Plasmodium species, and highly conserved, RPL6 exhibits strong translation potential as a vaccine candidate. This is further advocated by the identification of a broadly conserved, immunogenic HLA-A∗02:01-restricted epitope in P. falciparum RPL6.

Glycolipid-peptide vaccination induces liver-resident memory CD8+ T cells that protect against rodent malaria.

Liver resident-memory CD8+ T cells (TRM cells) can kill liver-stage Plasmodium-infected cells and prevent malaria, but simple vaccines for generating this important immune population are lacking. Here, we report the development of a fully synthetic self-adjuvanting glycolipid-peptide conjugate vaccine designed to efficiently induce liver TRM cells. Upon cleavage in vivo, the glycolipid-peptide conjugate vaccine releases an MHC I-restricted peptide epitope (to stimulate Plasmodium-specific CD8+ T cells) and an adjuvant component, the NKT cell agonist α-galactosylceramide (α-GalCer). A single dose of this vaccine in mice induced substantial numbers of intrahepatic malaria-specific CD8+ T cells expressing canonical markers of liver TRM cells (CD69, CXCR6, and CD101), and these cells could be further increased in number upon vaccine boosting. We show that modifications to the peptide, such as addition of proteasomal-cleavage sequences or epitope-flanking sequences, or the use of alternative conjugation methods to link the peptide to the glycolipid improved liver TRM cell generation and led to the development of a vaccine able to induce sterile protection in C57BL/6 mice against Plasmodium berghei sporozoite challenge after a single dose. Furthermore, this vaccine induced endogenous liver TRM cells that were long-lived (half-life of ~425 days) and were able to maintain >90% sterile protection to day 200. Our findings describe an ideal synthetic vaccine platform for generating large numbers of liver TRM cells for effective control of liver-stage malaria and, potentially, a variety of other hepatotropic infections.

Cloning, purification and characterisation of brassinin glucosyltransferase, a phytoalexin-detoxifying enzyme from the plant pathogen Sclerotinia sclerotiorum.

The plant-pathogenic fungus Sclerotinia sclerotiorum can detoxify cruciferous phytoalexins such as brassinin via glucosylation. Here we describe a multifaceted approach including genome mining, transcriptional induction, phytoalexin quantification, protein expression and enzyme purification that led to identification of a S. sclerotiorum glucosyltransferase that detoxifies brassinin. Transcription of this gene, denoted as brassinin glucosyltransferase 1 (SsBGT1), was induced significantly in response to the cruciferous phytoalexins camalexin, cyclobrassinin, brassilexin, brassinin and 3-phenylindole, a camalexin analogue. This gene was also up-regulated during infection of Brassica napus leaves. Levels of brassinin decreased significantly between 48 and 72h post-inoculation, with a concomitant increase in levels of 1-beta-d-glucopyranosylbrassinin, the product of the reaction catalysed by SsBGT1. These findings strongly implicate the involvement of this gene during infection of B. napus. This gene was cloned and expressed in Saccharomyces cerevisiae. The purified recombinant enzyme was able to glucosylate brassinin and two other phytoalexins, albeit much less effectively. This is the first report of a fungal gene involved in detoxification of plant defence molecules via glucosylation.

Alternative splicing is required for stage differentiation in malaria parasites.

BACKGROUND: In multicellular organisms, alternative splicing is central to tissue differentiation and identity. Unicellular protists lack multicellular tissue but differentiate into variable cell types during their life cycles. The role of alternative splicing in transitions between cell types and establishing cellular identity is currently unknown in any unicellular organism. RESULTS: To test whether alternative splicing in unicellular protists plays a role in cellular differentiation, we conduct RNA-seq to compare splicing in female and male sexual stages to asexual intraerythrocytic stages in the rodent malaria parasite Plasmodium berghei. We find extensive changes in alternative splicing between stages and a role for alternative splicing in sexual differentiation. Previously, general gametocyte differentiation was shown to be modulated by specific transcription factors. Here, we show that alternative splicing establishes a subsequent layer of regulation, controlling genes relating to consequent sex-specific differentiation of gametocytes. CONCLUSIONS: We demonstrate that alternative splicing is reprogrammed during cellular differentiation of a unicellular protist. Disruption of an alternative splicing factor, PbSR-MG, perturbs sex-specific alternative splicing and decreases the ability of the parasites to differentiate into male gametes and oocysts, thereby reducing transmission between vertebrate and insect hosts. Our results reveal alternative splicing as an integral, stage-specific phenomenon in these protists and as a regulator of cellular differentiation that arose early in eukaryotic evolution.

CD8+ T Cell Activation Leads to Constitutive Formation of Liver Tissue-Resident Memory T Cells that Seed a Large and Flexible Niche in the Liver.

Liver tissue-resident memory T (Trm) cells migrate throughout the sinusoids and are capable of protecting against malaria sporozoite challenge. To gain an understanding of liver Trm cell development, we examined various conditions for their formation. Although liver Trm cells were found in naive mice, their presence was dictated by antigen specificity and required IL-15. Liver Trm cells also formed after adoptive transfer of in vitro-activated but not naive CD8+ T cells, indicating that activation was essential but that antigen presentation within the liver was not obligatory. These Trm cells patrolled the liver sinusoids with a half-life of 36 days and occupied a large niche that could be added to sequentially without effect on subsequent Trm cell cohorts. Together, our findings indicate that liver Trm cells form as a normal consequence of CD8+ T cell activation during essentially any infection but that inflammatory and antigenic signals preferentially tailor their development.

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

BACKGROUND: Genes 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. RESULTS: Putative 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. CONCLUSION: ETP 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.

Microsatellite and Minisatellite Analysis of Leptosphaeria maculans in Australia Reveals Regional Genetic Differentiation.

ABSTRACT The population genetic structure of the fungal pathogen Leptosphaeria maculans was determined in Australia using six microsatellite and two minisatellite markers. Ascospores were sampled from Brassica napus stubble in disease nurseries and commercial fields in different sites over 2 years. The 13 subpopulations of L. maculans exhibited high gene (H = 0.393 to 0.563) and genotypic diversity, with 357 haplotypes identified among 513 isolates. Although the majority of genetic variation was distributed within subpopulations (85%), 10% occurred between the regions of eastern and Western Australia, and 5% within regions. F(ST) analysis of subpopulation pairs also showed the east-west genetic differentiation, whereas factorial correspondence analysis separated Western Australian subpopulations from eastern ones. Bayesian model-based population structure analyses of multilocus haplotypes inferred three distinct populations, one in Western Australia and an admixture of two in eastern Australia. These two regions are separated by 1,200 km of arid desert that may act as a natural barrier to gene flow, resulting in differentiation by random genetic drift. The genetic differentiation of L. maculans isolates between eastern and Western Australia means that these regions can be treated as different management units, and reinforces the need for widespread disease nurseries in each region to screen breeding lines against a range of genetic and pathogenic populations of L. maculans.