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.

Modulation of innate immune signaling by a Coxiella burnetii eukaryotic-like effector protein.

The Q fever agent Coxiella burnetii uses a defect in organelle trafficking/intracellular multiplication (Dot/Icm) type 4b secretion system (T4SS) to silence the host innate immune response during infection. By investigating C. burnetii effector proteins containing eukaryotic-like domains, here we identify NopA (nucleolar protein A), which displays four regulator of chromosome condensation (RCC) repeats, homologous to those found in the eukaryotic Ras-related nuclear protein (Ran) guanine nucleotide exchange factor (GEF) RCC1. Accordingly, NopA is found associated with the chromatin nuclear fraction of cells and uses the RCC-like domain to interact with Ran. Interestingly, NopA triggers an accumulation of Ran-GTP, which accumulates at nucleoli of transfected or infected cells, thus perturbing the nuclear import of transcription factors of the innate immune signaling pathway. Accordingly, qRT-PCR analysis on a panel of cytokines shows that cells exposed to the C. burnetii nopA::Tn or a Dot/Icm-defective dotA::Tn mutant strain present a functional innate immune response, as opposed to cells exposed to wild-type C. burnetii or the corresponding nopA complemented strain. Thus, NopA is an important regulator of the innate immune response allowing Coxiella to behave as a stealth pathogen.

Pathway of glycine betaine biosynthesis in Aspergillus fumigatus.

The choline oxidase (CHOA) and betaine aldehyde dehydrogenase (BADH) genes identified in Aspergillus fumigatus are present as a cluster specific for fungal genomes. Biochemical and molecular analyses of this cluster showed that it has very specific biochemical and functional features that make it unique and different from its plant and bacterial homologs. A. fumigatus ChoAp catalyzed the oxidation of choline to glycine betaine with betaine aldehyde as an intermediate and reduced molecular oxygen to hydrogen peroxide using FAD as a cofactor. A. fumigatus Badhp oxidized betaine aldehyde to glycine betaine with reduction of NAD(+) to NADH. Analysis of the AfchoAΔ::HPH and AfbadAΔ::HPH single mutants and the AfchoAΔAfbadAΔ::HPH double mutant showed that AfChoAp is essential for the use of choline as the sole nitrogen, carbon, or carbon and nitrogen source during the germination process. AfChoAp and AfBadAp were localized in the cytosol of germinating conidia and mycelia but were absent from resting conidia. Characterization of the mutant phenotypes showed that glycine betaine in A. fumigatus functions exclusively as a metabolic intermediate in the catabolism of choline and not as a stress protectant. This study in A. fumigatus is the first molecular, cellular, and biochemical characterization of the glycine betaine biosynthetic pathway in the fungal kingdom.

Pyricularia oryzae: Lab star and field scourge.

Pyricularia oryzae (syn. Magnaporthe oryzae), is a filamentous ascomycete that causes a major disease called blast on cereal crops, as well as on a wide variety of wild and cultivated grasses. Blast diseases have a tremendous impact worldwide particularly on rice and on wheat, where the disease emerged in South America in the 1980s, before spreading to Asia and Africa. Its economic importance, coupled with its amenability to molecular and genetic manipulation, have inspired extensive research efforts aiming at understanding its biology and evolution. In the past 40 years, this plant-pathogenic fungus has emerged as a major model in molecular plant-microbe interactions. In this review, we focus on the clarification of the taxonomy and genetic structure of the species and its host range determinants. We also discuss recent molecular studies deciphering its lifecycle. TAXONOMY: Kingdom: Fungi, phylum: Ascomycota, sub-phylum: Pezizomycotina, class: Sordariomycetes, order: Magnaporthales, family: Pyriculariaceae, genus: Pyricularia. HOST RANGE: P. oryzae has the ability to infect a wide range of Poaceae. It is structured into different host-specialized lineages that are each associated with a few host plant genera. The fungus is best known to cause tremendous damage to rice crops, but it can also attack other economically important crops such as wheat, maize, barley, and finger millet. DISEASE SYMPTOMS: P. oryzae can cause necrotic lesions or bleaching on all aerial parts of its host plants, including leaf blades, sheaths, and inflorescences (panicles, spikes, and seeds). Characteristic symptoms on leaves are diamond-shaped silver lesions that often have a brown margin and whose appearance is influenced by numerous factors such as the plant genotype and environmental conditions. USEFUL WEBSITES Resources URL Genomic data repositories http://genome.jouy.inra.fr/gemo/ Genomic data repositories http://openriceblast.org/ Genomic data repositories http://openwheatblast.net/ Genome browser for fungi (including P. oryzae) http://fungi.ensembl.org/index.html Comparative genomics database https://mycocosm.jgi.doe.gov/mycocosm/home T-DNA mutant database http://atmt.snu.kr/ T-DNA mutant database http://www.phi-base.org/ SNP and expression data https://fungidb.org/fungidb/app/.

The unconventional resistance protein PTR recognizes the Magnaporthe oryzae effector AVR-Pita in an allele-specific manner.

Blast disease caused by the fungus Magnaporthe oryzae is one of the most devastating rice diseases. Disease resistance genes such as Pi-ta or Pi-ta2 are critical in protecting rice production from blast. Published work reports that Pi-ta codes for a nucleotide-binding and leucine-rich repeat domain protein (NLR) that recognizes the fungal protease-like effector AVR-Pita by direct binding. However, this model was challenged by the recent discovery that Pi-ta2 resistance, which also relies on AVR-Pita detection, is conferred by the unconventional resistance gene Ptr, which codes for a membrane protein with a cytoplasmic armadillo repeat domain. Here, using NLR Pi-ta and Ptr RNAi knockdown and CRISPR/Cas9 knockout mutant rice lines, we found that AVR-Pita recognition relies solely on Ptr and that the NLR Pi-ta has no role in it, indicating that it is not the Pi-ta resistance gene. Different alleles of Ptr confer different recognition specificities. The A allele of Ptr (PtrA) detects all natural sequence variants of the effector and confers Pi-ta2 resistance, while the B allele of Ptr (PtrB) recognizes a restricted set of AVR-Pita alleles and, thereby, confers Pi-ta resistance. Analysis of the natural diversity in AVR-Pita and of mutant and transgenic strains identified one specific polymorphism in the effector sequence that controls escape from PtrB-mediated resistance. Taken together, our work establishes that the M. oryzae effector AVR-Pita is detected in an allele-specific manner by the unconventional rice resistance protein Ptr and that the NLR Pi-ta has no function in Pi-ta resistance and the recognition of AVR-Pita.

Functional analysis of the superoxide dismutase family in Aspergillus fumigatus.

Reactive oxidant species produced by phagocytes have been reported as being involved in the killing of Aspergillus fumigatus. Fungal superoxide dismutases (SODs) that detoxify superoxide anions could be putative virulence factors for this opportunistic pathogen. Four genes encoding putative Sods have been identified in the A. fumigatus genome: a cytoplasmic Cu/ZnSOD (AfSod1p), a mitochondrial MnSOD (AfSod2p), a cytoplasmic MnSOD (AfSod3p) and AfSod4 displaying a MnSOD C-terminal domain. During growth, AfSOD1 and AfSOD2 were highly expressed in conidia whereas AfSOD3 was only strongly expressed in mycelium. AfSOD4 was weakly expressed compared with other SODs. The deletion of AfSOD4 was lethal. Delta sod1 and Delta sod2 mutants showed a growth inhibition at high temperature and a hypersensitivity to menadione whereas the sod3 mutant had only a slight growth delay at high temperature. Multiple mutations had only an additive effect on the phenotype. The triple sod1/sod2/sod3 mutant was characterized by a delay in conidial germination, a reduced conidial survival during storage overtime, the highest sensitivity to menadione and an increased sensitivity to killing by alveolar macrophage of immunocompetent mice. In spite of these phenotypes, no significant virulence difference was observed between the triple mutant and parental strain in experimental murine aspergillosis models in immunocompromised animals.

Comparative functional analysis of the OCH1 mannosyltransferase families in Aspergillus fumigatus and Saccharomyces cerevisiae.

Alpha1,6-linked mannans are an essential component of the Aspergillus fumigatus galactomannan, which is either GPI-anchored to the plasma membrane or covalently bound to the polysaccharide core of the cell wall. In Saccharomyces cerevisiae, the OCH1 gene encodes an alpha1,6-mannosyltransferase that initiates the synthesis of the alpha1,6 linked-mannan. In the A. fumigatus genome, four orthologous genes of Saccharomyces cerevisiae ScOCH1 gene were identified. Single deletion and the quadruple deletion mutants (Afoch1-4Delta) were constructed in A. fumigatus. No phenotype different from the wild-type strain was observed in all single and the quadruple mutants. The corresponding cDNAs of the AfOCH1-4 A. fumigatus orthologues were expressed in the S. cerevisiae Scoch1Delta mutant. Among the four orthologues, only AfOCH1 complemented the aggregation phenotype, the growth inhibition, the hypersensitivity to hygromycin and the protein glycosylation defects observed in the Scoch1Deltamutant.

The crucial role of the Pls1 tetraspanin during ascospore germination in Podospora anserina provides an example of the convergent evolution of morphogenetic processes in fungal plant pathogens and saprobes.

Pls1 tetraspanins were shown for some pathogenic fungi to be essential for appressorium-mediated penetration into their host plants. We show here that Podospora anserina, a saprobic fungus lacking appressorium, contains PaPls1, a gene orthologous to known PLS1 genes. Inactivation of PaPls1 demonstrates that this gene is specifically required for the germination of ascospores in P. anserina. These ascospores are heavily melanized cells that germinate under inducing conditions through a specific pore. On the contrary, MgPLS1, which fully complements a DeltaPaPls1 ascospore germination defect, has no role in the germination of Magnaporthe grisea nonmelanized ascospores but is required for the formation of the penetration peg at the pore of its melanized appressorium. P. anserina mutants with mutation of PaNox2, which encodes the NADPH oxidase of the NOX2 family, display the same ascospore-specific germination defect as the DeltaPaPls1 mutant. Both mutant phenotypes are suppressed by the inhibition of melanin biosynthesis, suggesting that they are involved in the same cellular process required for the germination of P. anserina melanized ascospores. The analysis of the distribution of PLS1 and NOX2 genes in fungal genomes shows that they are either both present or both absent. These results indicate that the germination of P. anserina ascospores and the formation of the M. grisea appressorium penetration peg use the same molecular machinery that includes Pls1 and Nox2. This machinery is specifically required for the emergence of polarized hyphae from reinforced structures such as appressoria and ascospores. Its recurrent recruitment during fungal evolution may account for some of the morphogenetic convergence observed in fungi.

Fungi have three tetraspanin families with distinct functions.

BACKGROUND: Tetraspanins are small membrane proteins that belong to a superfamily encompassing 33 members in human and mouse. These proteins act as organizers of membrane-signalling complexes. So far only two tetraspanin families have been identified in fungi. These are Pls1, which is required for pathogenicity of the plant pathogenic ascomycetes, Magnaporthe grisea, Botrytis cinerea and Colletotrichum lindemuthianum, and Tsp2, whose function is unknown. In this report, we describe a third family of tetraspanins (Tsp3) and a new family of tetraspanin-like proteins (Tpl1) in fungi. We also describe expression of some of these genes in M. grisea and a basidiomycete, Laccaria bicolor, and also their functional analysis in M. grisea. RESULTS: The exhaustive search for tetraspanins in fungal genomes reveals that higher fungi (basidiomycetes and ascomycetes) contain three families of tetraspanins (Pls1, Tsp2 and Tsp3) with different distribution amongst phyla. Pls1 is found in ascomycetes and basidiomycetes, whereas Tsp2 is restricted to basidiomycetes and Tsp3 to ascomycetes. A unique copy of each of PLS1 and TSP3 was found in ascomycetes in contrast to TSP2, which has several paralogs in the basidiomycetes, Coprinus cinereus and Laccaria bicolor. A tetraspanin-like family (Tpl1) was also identified in ascomycetes. Transcriptional analyses in various tissues of L. bicolor and M. grisea showed that PLS1 and TSP2 are expressed in all tissues in L. bicolor and that TSP3 and TPL1 are overexpressed in the sexual fruiting bodies (perithecia) and mycelia of M. grisea, suggesting that these genes are not pseudogenes. Phenotypic analysis of gene replacementmutants Deltatsp3 and Deltatpl1 of M. grisea revealed a reduction of the pathogenicity only on rice, in contrast to Deltapls1 mutants, which are completely non-pathogenic on barley and rice. CONCLUSION: A new tetraspanin family (Tsp3) and a tetraspanin-like protein family (Tpl1) have been identified in fungi. Functional analysis by gene replacement showed that these proteins, as well as Pls1, are involved in the infection process of the plant pathogenic fungus M. grisea. The next challenge will be to decipher the role(s) of tetraspanins in a range of symbiotic, saprophytic and human pathogenic fungi.

Fungal Pls1 tetraspanins as key factors of penetration into host plants: a role in re-establishing polarized growth in the appressorium?

The ability of plant pathogenic fungi to infect their host depends on successful penetration into plant tissues. This process often involves the differentiation of a specialized cell, the appressorium. Signalling pathways required for appressorium formation are conserved among fungi. However, the functions involved in appressorium maturation and penetration peg formation are still poorly understood. Recent studies have shown that Pls1 tetraspanins control an appressorial function required for penetration into host plants and are likely conserved among plant pathogenic fungi. Tetraspanins are small membrane proteins widely distributed among ascomycetes and basidiomycetes defining two distinct families; Pls1 tetraspanins are found in both ascomycetes and basidiomycetes and Tsp2 tetraspanins are specific to basidiomycetes. Both fungal tetraspanins families have similar secondary structures shared with animal tetraspanins. Pls1 tetraspanins are present as single genes in genomes of ascomycetes, allowing a unique opportunity to study their function in appressorium mediated penetration. Experimental evidence suggests that Pls1 tetraspanins are required for the formation of the penetration peg at the base of the appressorium, probably through re-establishing cell polarity.

Characterization of glucuronic acid containing glycolipid in Aspergillus fumigatus mycelium.

A glucuronic acid containing glycerolipid was isolated from the filamentous fungi Aspergillus fumigatus. This acidic glycolipid was extracted from the membrane of mycelium and purified by two successive chromatographic steps on DEAE-Sephadex and Silica columns. Chemical structural analysis was performed using methylation, gas-chromatography, gas-chromatography-mass spectrometry, nano-electrospray mass spectrometry and (1)H/(13)C NMR spectra. The corresponding structure is a 3-(O-alpha-glucuronyl)-1,2-diacyl-sn-glycerol, where acyl chains are mainly C(16:0), C(18:0), C(18:1), and C(18:2). This alpha-GlcA-diacylglycerol is not present in fungal conidia. This acidic glycerolipid is described here for the first time in a fungal species. Two homologs of UDP-glucose dehydrogenase that convert UDP-glucose into UDP-glucuronic acid, are present in A. fumigatus genome, UGD1 and UGD2. Gene deletion showed that only UGD1 is essential for the biosynthesis of GlcA-DG. However, no particular phenotype has been observed in the Ugd1Delta mutant. Biological function of this acidic glycolipid remains unknown in A. fumigatus.

Convergent evolution of morphogenetic processes in fungi: Role of tetraspanins and NADPH oxidases 2 in plant pathogens and saprobes.

Convergent evolution of trophic life style and morphological characters are very common in the fungal kingdom. Recently, we have shown that the same molecular machinery containing a tetraspanin and a NADPH oxidase has been recruited in two different fungal species for the same purpose (exiting from a melanized re-enforced cell at a focal weakened point), but at different stages of their development (ascospore germination and appressorium mediated penetration). Although this molecular machinery is required at these key developmental steps, it is also likely involved in specialized cellular functions at other stages of fungal development, as shown here for nutrient acquisition by Podospora anserina.

Improved gene targeting in Magnaporthe grisea by inactivation of MgKU80 required for non-homologous end joining.

The ascomycete Magnaporthe grisea is a model species for the study of plant fungal interactions. As in many filamentous fungi, targeted gene replacement occurs at low frequency in M. grisea (average 7%). mus52/KU80 is a gene essential for non-homologous end joining (NHEJ) of DNA double-strand breaks. Its deletion increases the frequency of targeted gene replacement in fungi [Ninomiya, Y., Suzuki, K., Ishii, C., Inoue, H., 2004. Highly efficient gene replacements in Neurospora strains deficient for non-homologous end joining. Proc. Natl. Acad. Sci. USA 101(33), 12248-53]. M. grisea KU80 deletion mutants were constructed and displayed wild-type phenotypes regarding pathogenicity, growth, sporulation and mating. MgADE4 targeted gene replacement frequency was increased in Deltaku80 mutants (80% vs 5%) and high frequencies (>80%) were observed at seven other loci. However, the deletion of MgKU80 did not increase the frequency of ACE1 replacement indicating that this locus has an intrinsic reduced ability for gene replacement. These results open the way to large-scale reverse genetics experiments in M. grisea facilitating the study of the infection process.