The bZIP transcription factor BIP1 of the rice blast fungus is essential for infection and regulates a specific set of appressorium genes.

The rice blast fungus Magnaporthe oryzae differentiates specialized cells called appressoria that are required for fungal penetration into host leaves. In this study, we identified the novel basic leucine zipper (bZIP) transcription factor BIP1 (B-ZIP Involved in Pathogenesis-1) that is essential for pathogenicity. BIP1 is required for the infection of plant leaves, even if they are wounded, but not for appressorium-mediated penetration of artificial cellophane membranes. This phenotype suggests that BIP1 is not implicated in the differentiation of the penetration peg but is necessary for the initial establishment of the fungus within plant cells. BIP1 expression was restricted to the appressorium by both transcriptional and post-transcriptional control. Genome-wide transcriptome analysis showed that 40 genes were down regulated in a BIP1 deletion mutant. Most of these genes were specifically expressed in the appressorium. They encode proteins with pathogenesis-related functions such as enzymes involved in secondary metabolism including those encoded by the ACE1 gene cluster, small secreted proteins such as SLP2, BAS2, BAS3, and AVR-Pi9 effectors, as well as plant cuticle and cell wall degrading enzymes. Interestingly, this BIP1 network is different from other known infection-related regulatory networks, highlighting the complexity of gene expression control during plant-fungal interactions. Promoters of BIP1-regulated genes shared a GCN4/bZIP-binding DNA motif (TGACTC) binding in vitro to BIP1. Mutation of this motif in the promoter of MGG_08381.7 from the ACE1 gene cluster abolished its appressorium-specific expression, showing that BIP1 behaves as a transcriptional activator. In summary, our findings demonstrate that BIP1 is critical for the expression of early invasion-related genes in appressoria. These genes are likely needed for biotrophic invasion of the first infected host cell, but not for the penetration process itself. Through these mechanisms, the blast fungus strategically anticipates the host plant environment and responses during appressorium-mediated penetration.

Chitin synthases from Saprolegnia are involved in tip growth and represent a potential target for anti-oomycete drugs.

Oomycetes represent some of the most devastating plant and animal pathogens. Typical examples are Phytophthora infestans, which causes potato and tomato late blight, and Saprolegnia parasitica, responsible for fish diseases. Despite the economical and environmental importance of oomycete diseases, their control is difficult, particularly in the aquaculture industry. Carbohydrate synthases are vital for hyphal growth and represent interesting targets for tackling the pathogens. The existence of 2 different chitin synthase genes (SmChs1 and SmChs2) in Saprolegnia monoica was demonstrated using bioinformatics and molecular biology approaches. The function of SmCHS2 was unequivocally demonstrated by showing its catalytic activity in vitro after expression in Pichia pastoris. The recombinant SmCHS1 protein did not exhibit any activity in vitro, suggesting that it requires other partners or effectors to be active, or that it is involved in a different process than chitin biosynthesis. Both proteins contained N-terminal Microtubule Interacting and Trafficking domains, which have never been reported in any other known carbohydrate synthases. These domains are involved in protein recycling by endocytosis. Enzyme kinetics revealed that Saprolegnia chitin synthases are competitively inhibited by nikkomycin Z and quantitative PCR showed that their expression is higher in presence of the inhibitor. The use of nikkomycin Z combined with microscopy showed that chitin synthases are active essentially at the hyphal tips, which burst in the presence of the inhibitor, leading to cell death. S. parasitica was more sensitive to nikkomycin Z than S. monoica. In conclusion, chitin synthases with species-specific characteristics are involved in tip growth in Saprolegnia species and chitin is vital for the micro-organisms despite its very low abundance in the cell walls. Chitin is most likely synthesized transiently at the apex of the cells before cellulose, the major cell wall component in oomycetes. Our results provide important fundamental information on cell wall biogenesis in economically important species, and demonstrate the potential of targeting oomycete chitin synthases for disease control.

The corky root rot pathogen Pyrenochaeta lycopersici secretes a proteinaceous inducer of cell death affecting host plants differentially.

Pathogenic isolates of Pyrenochaeta lycopersici, the causal agent of corky root rot of tomato, secrete cell death in tomato 1 (CDiT1), a homodimeric protein of 35 kDa inducing cell death after infiltration into the leaf apoplast of tomato. CDiT1 was purified by fast protein liquid chromatography, characterized by mass spectrometry and cDNA cloning. Its activity was confirmed after infiltration of an affinity-purified recombinant fusion of the protein with a C-terminal polyhistidine tag. CDiT1 is highly expressed during tomato root infection compared with axenic culture, and has a putative ortholog in other pathogenic Pleosporales species producing proteinaceous toxins that contribute to virulence. Infiltration of CDiT1 into leaves of other plants susceptible to P. lycopersici revealed that the protein affects them differentially. All varieties of cultivated tomato (Solanum lycopersicum) tested were more sensitive to CDiT1 than those of currant tomato (S. pimpinellifolium). Root infection assays showed that varieties of currant tomato are also significantly less prone to intracellular colonization of their root cells by hyphae of P. lycopersici than varieties of cultivated tomato. Therefore, secretion of this novel type of inducer of cell death during penetration of the fungus inside root cells might favor infection of host species that are highly sensitive to this molecule.

Mitonuclear Genetic Interactions in the Basidiomycete Heterobasidion parviporum Involve a Non-conserved Mitochondrial Open Reading Frame.

The mitochondrial and nuclear genomes of Eukaryotes are inherited separately and consequently follow distinct evolutionary paths. Nevertheless, the encoding of many mitochondrial proteins by the nuclear genome shows the high level of integration they have reached, which makes mitonuclear genetic interactions all the more conceivable. For each species, natural selection has fostered the evolution of coadapted alleles in both genomes, but a population-wise divergence of such alleles could lead to important phenotypic variation, and, ultimately, to speciation. In this study in the Basidiomycete Heterobasidion parviporum, we have investigated the genetic basis of phenotypic variation among laboratory-designed heterokaryons carrying the same pair of haploid nuclei, but a different mitochondrial genome. Radial growth rate data of thirteen unrelated homokaryotic parents and of their heterokaryotic offspring were combined with SNP data extracted from parental genome sequences to identify nuclear and mitochondrial loci involved in mitonuclear interactions. Two nuclear loci encoding mitochondrial proteins appeared as best candidates to engage in a genetic interaction affecting radial growth rate with a non-conserved mitochondrial open reading frame of unknown function and not reported apart from the Russulales order of Basidiomycete fungi. We believe our approach could be useful to investigate several important traits of fungal biology where mitonuclear interactions play a role, including virulence of fungal pathogens.

Estimating the Fitness Effect of Deleterious Mutations During the Two Phases of the Life Cycle: A New Method Applied to the Root-Rot Fungus Heterobasidion parviporum.

Many eukaryote species, including taxa such as fungi or algae, have a lifecycle with substantial haploid and diploid phases. A recent theoretical model predicts that such haploid-diploid lifecycles are stable over long evolutionary time scales when segregating deleterious mutations have stronger effects in homozygous diploids than in haploids and when they are partially recessive in heterozygous diploids. The model predicts that effective dominance-a measure that accounts for these two effects-should be close to 0.5 in these species. It also predicts that diploids should have higher fitness than haploids on average. However, an appropriate statistical framework to conjointly investigate these predictions is currently lacking. In this study, we derive a new quantitative genetic model to test these predictions using fitness data of two haploid parents and their diploid offspring, and genome-wide genetic distance between haploid parents. We apply this model to the root-rot basidiomycete fungus Heterobasidion parviporum-a species where the heterokaryotic (equivalent to the diploid) phase is longer than the homokaryotic (haploid) phase. We measured two fitness-related traits (mycelium growth rate and the ability to degrade wood) in both homokaryons and heterokaryons, and we used whole-genome sequencing to estimate nuclear genetic distance between parents. Possibly due to a lack of power, we did not find that deleterious mutations were recessive or more deleterious when expressed during the heterokaryotic phase. Using this model to compare effective dominance among haploid-diploid species where the relative importance of the two phases varies should help better understand the evolution of haploid-diploid life cycles.

Nitrogen controls in planta expression of Cladosporium fulvum Avr9 but no other effector genes.

SUMMARY During growth on its host tomato, the apoplast-colonizing fungal pathogen Cladosporium fulvum secretes several effector proteins. The expression of the Avr9 gene encoding one of these effector proteins has previously been shown to be strongly induced in vitro during nitrogen deprivation. This led to the hypothesis that expression of additional effector genes in C. fulvum could be triggered by nitrogen starvation conditions that are encountered in the host. We now show that expression of most effectors is not affected by varying levels of nitrogen supplementation in vitro. In addition, we demonstrate that the nitrogen response regulator Nrf1 only regulates Avr9 expression during infection of the host, whereas none of the other known effectors is significantly controlled by this transcription factor in planta. Deletion of Nrf1, but not of Avr9, significantly reduces C. fulvum virulence. Therefore, it is concluded that Nrf1 controls, in addition to Avr9, unidentified effector genes that are required for full virulence of C. fulvum.