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

Tracing the Origin of the Fungal α1 Domain Places Its Ancestor in the HMG-Box Superfamily: Implication for Fungal Mating-Type Evolution

BACKGROUND: Fungal mating types in self-incompatible Pezizomycotina are specified by one of two alternate sequences occupying the same locus on corresponding chromosomes. One sequence is characterized by a gene encoding an HMG protein, while the hallmark of the other is a gene encoding a protein with an α1 domain showing similarity to the Matα1p protein of Saccharomyces cerevisiae. DNA-binding HMG proteins are ubiquitous and well characterized. In contrast, α1 domain proteins have limited distribution and their evolutionary origin is obscure, precluding a complete understanding of mating-type evolution in Ascomycota. Although much work has focused on the role of the S. cerevisiae Matα1p protein as a transcription factor, it has not yet been placed in any of the large families of sequence-specific DNA-binding proteins. METHODOLOGY/PRINCIPAL FINDINGS: We present sequence comparisons, phylogenetic analyses, and in silico predictions of secondary and tertiary structures, which support our hypothesis that the α1 domain is related to the HMG domain. We have also characterized a new conserved motif in α1 proteins of Pezizomycotina. This motif is immediately adjacent to and downstream of the α1 domain and consists of a core sequence Y-[LMIF]-x(3)-G-[WL] embedded in a larger conserved motif. CONCLUSIONS/SIGNIFICANCE: Our data suggest that extant α1-box genes originated from an ancestral HMG gene, which confirms the current model of mating-type evolution within the fungal kingdom. We propose to incorporate α1 proteins in a new subclass of HMG proteins termed MATα_HMG

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

The dispensable chromosome of Leptosphaeria maculans shelters an effector gene conferring avirulence towards Brassica rapa.

International audiencePhytopathogenic fungi frequently contain dispensable chromosomes, some of which contribute to host range or pathogenicity. In Leptosphaeria maculans, the stem canker agent of oilseed rape (Brassica napus), the minichromosome was previously suggested to be dispensable, without evidence for any role in pathogenicity. Using genetic and genomic approaches, we investigated the inheritance and molecular determinant of an L. maculans-Brassica rapa incompatible interaction.Single gene control of the resistance was found, while all markers located on the L. maculans minichromosome, absent in the virulent parental isolate, co-segregated with the avirulent phenotype. Only one candidate avirulence gene was identified on the minichromosome, validated by complementation experiments and termed AvrLm11. The minichromosome was frequently lost following meiosis, but the frequency of isolates lacking it remained stable in field populations sampled at a 10-yr time interval, despite a yearly sexual stage in the L. maculans life cycle.This work led to the cloning of a new 'lost in the middle of nowhere' avirulence gene of L. maculans, interacting with a B. rapa resistance gene termed Rlm11 and introgressed into B. napus. It demonstrated the dispensability of the L. maculans minichromosome and suggested that its loss generates a fitness deficit