RESUMO
Pandora formicae is an obligate entomopathogenic fungus from the phylum Entomophthoromycota, known to infect only ants from the genus Formica. In the final stages of infection, the fungus induces the so-called summit disease syndrome, manipulating the host to climb up vegetation prior to death and fixing the dead cadaver to the surface, all to increase efficient spore dispersal. To investigate this fascinating pathogen-host interaction, we constructed interaction transcriptome libraries from two final infection stages from the material sampled in the field: (1) when the cadavers were fixed, but the fungus had not grown out through the cuticle and (2) when the fungus was growing out from host cadaver and producing spores. These phases mark the switch from within-host growth to reproduction on the host surface, after fungus outgrowth through host integument. In this first de novo transcriptome of an entomophthoralean fungus, we detected expression of many pathogenicity-related genes, including secreted hydrolytic enzymes and genes related to morphological reorganization and nutrition uptake. Differences in expression of genes in these two infection phases were compared and showed a switch in enzyme expression related to either cuticle breakdown or cell proliferation and cell wall remodeling, particularly in subtilisin-like serine protease and trypsin-like protease transcripts.
Assuntos
Formigas/parasitologia , Entomophthorales/genética , Entomophthorales/patogenicidade , Interações Hospedeiro-Patógeno/genética , Animais , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/fisiologia , Filogenia , Reação em Cadeia da Polimerase , TranscriptomaRESUMO
BACKGROUND: The fungus gardens of leaf-cutting ants are natural biomass conversion systems that turn fresh plant forage into fungal biomass to feed the farming ants. However, the decomposition potential of the symbiont Leucocoprinus gongylophorus for processing polysaccharides has remained controversial. We therefore used quantifiable DeepSAGE technology to obtain mRNA expression patterns of genes coding for secreted enzymes from top, middle, and bottom sections of a laboratory fungus-garden of Acromyrmex echinatior leaf-cutting ants. RESULTS: A broad spectrum of biomass-conversion-relevant enzyme genes was found to be expressed in situ: cellulases (GH3, GH5, GH6, GH7, AA9 [formerly GH61]), hemicellulases (GH5, GH10, CE1, GH12, GH74), pectinolytic enzymes (CE8, GH28, GH43, PL1, PL3, PL4), glucoamylase (GH15), α-galactosidase (GH27), and various cutinases, esterases, and lipases. In general, expression of these genes reached maximal values in the bottom section of the garden, particularly for an AA9 lytic polysaccharide monooxygenase and for a GH5 (endocellulase), a GH7 (reducing end-acting cellobiohydrolase), and a GH10 (xylanase), all containing a carbohydrate binding module that specifically binds cellulose (CBM1). Although we did not directly quantify enzyme abundance, the profile of expressed cellulase genes indicates that both hydrolytic and oxidative degradation is taking place. CONCLUSIONS: The fungal symbiont of Acromyrmex leaf-cutting ants can degrade a large range of plant polymers, but the conversion of cellulose, hemicellulose, and part of the pectin occurs primarily towards the end of the decomposition process, i.e. in the bottom section of the fungus garden. These conversions are likely to provide nutrients for the fungus itself rather than for the ants, whose colony growth and reproductive success are limited by proteins obtained from ingesting fungal gongylidia. These specialized hyphal tips are hardly produced in the bottom section of fungus gardens, consistent with the ants discarding old fungal biomass from this part of the garden. The transcripts that we found suggest that actively growing mycelium in the bottom of gardens helps to maintain an optimal water balance to avoid hyphal disintegration, so the ants can ultimately discard healthy rather than decaying and diseased garden material, and to buffer negative effects of varying availability and quality of substrate across the seasons.
Assuntos
Agaricales/genética , Formigas/microbiologia , Parede Celular/química , Celulose/metabolismo , Agaricales/enzimologia , Animais , Biomassa , Celulases/metabolismo , Etiquetas de Sequências Expressas , Regulação Fúngica da Expressão Gênica , Biblioteca Gênica , Genes Fúngicos , Polissacarídeos/metabolismo , SimbioseRESUMO
The vision of the European common research programme for 2014-2020, called Horizon 2020, is to create a smarter, more sustainable and more inclusive society. However, this is a global endeavor, which is important for mycologists all over the world because it includes a special role for fungi and fungal products. After ten years of research on industrial scale conversion of biowaste, the conclusion is that the most efficient and gentle way of converting recalcitrant lignocellulosic materials into high value products for industrial purposes, is through the use of fungal enzymes. Moreover, fungi and fungal products are also instrumental in producing fermented foods, to give storage stability and improved health. Climate change will lead to increasingly severe stress on agricultural production and productivity, and here the solution may very well be that fungi will be brought into use as a new generation of agricultural inoculants to provide more robust, more nutrient efficient, and more drought tolerant crop plants. However, much more knowledge is required in order to be able to fully exploit the potentials of fungi, to deliver what is needed and to address the major global challenges through new biological processes, products, and solutions. This knowledge can be obtained by studying the fungal proteome and metabolome; the biology of fungal RNA and epigenetics; protein expression, homologous as well as heterologous; fungal host/substrate relations; physiology, especially of extremophiles; and, not the least, the extent of global fungal biodiversity. We also need much more knowledge and understanding of how fungi degrade biomass in nature.The projects in our group in Aalborg University are examples of the basic and applied research going on to increase the understanding of the biology of the fungal secretome and to discover new enzymes and new molecular/bioinformatics tools.However, we need to put Mycology higher up on global agendas, e.g. by positioning Mycology as a candidate for an OECD Excellency Program. This could pave the way for increased funding of international collaboration, increased global visibility, and higher priority among decision makers all over the world.
RESUMO
The discovery of novel secretome proteins can add to our understanding of host-pathogen interactions. Here we report a rich diversity of secreted proteins from the interaction between grain aphids (host, insect order Hemiptera) and fungi of the order Entomophthorales (insect pathogens). The proteins were identified using a unique method unbiased by known sequences or functions to screen a cDNA library constructed directly from field-sampled material. We show for the first time that fungi from the genera Pandora and Entomophthora are armed with a battery of hydrolytic enzymes for penetrating the host cuticle. This enables both access to the hemolymph and exit for sporulation. Further, they secrete enzymes, most notably a number of lipases, for digestion of easily accessible high-energy compounds in the hemolymph. In contrast, we identified only few host genes potentially involved in the interaction, indicating that aphids respond only weakly to the pathogens. These results support recent findings that aphids have a reduced immune repertoire.
Assuntos
Entomophthorales/patogenicidade , Proteínas Fúngicas/metabolismo , Hemípteros/microbiologia , Interações Hospedeiro-Patógeno , Proteínas de Insetos/metabolismo , Animais , Proteínas Fúngicas/genética , Biblioteca Gênica , Proteínas de Insetos/genética , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The red pigmentation of Fusarium graminearum and related species that cause stem and head blight of cereals is due to the deposition of aurofusarin in the cell walls. To determine the importance of this polyketide for fungal physiology and pathogenicity, aurofusarin deficient mutants were produced by random and targeted mutagenesis of F. pseudograminearum and F. graminearum. We show that a gene cluster, including the F. graminearum PKS12 gene, is responsible for the biosynthesis of aurofusarin. Three F. pseudograminearum aurofusarin deficient mutants were disrupted in a region upstream from a gene with sequence homology to the aflatoxin regulatory gene aflR. Comparative PCR analyses of the aurofusarin gene cluster in F. graminearum, F. culmorum, and F. pseudograminearum show conserved organization and expression analyses detected no PKS12 transcripts in any of the mutants. To confirm that PKS12 encodes the precursor for aurofusarin, targeted mutagenesis was carried out in F. graminearum. All disruptants showed an albino phenotype. The DeltaPKS12 mutants have higher growth rate and a 10-fold increase in conidia production compared to the wild type. Aurofusarin does not appear to aid in radiation protection and all the mutants are fully pathogenic on wheat and barley. HPLC analyses of aurofusarin deficient mutants confirm the absence of aurofusarin and show an increase in the level of the mycotoxin zearalenone.
Assuntos
Fusarium/genética , Genes Fúngicos , Naftoquinonas/metabolismo , Pigmentos Biológicos/biossíntese , Análise Mutacional de DNA , DNA Fúngico/química , Fusarium/metabolismo , Fusarium/patogenicidade , Hordeum/microbiologia , Dados de Sequência Molecular , Família Multigênica , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Mutação , Doenças das Plantas/microbiologia , Raízes de Plantas/microbiologia , RNA Fúngico/análise , RNA Mensageiro/análise , Análise de Sequência de DNA , Virulência/genética , Zearalenona/biossínteseRESUMO
In a study aimed at characterising, at the molecular level, the obligate biotrophic fungus Blumeria graminis f. sp. hordei (Bgh), we have identified a novel group of genes, the Egh16H genes, and shown that two of these are up-regulated during primary infection of barley leaves. The genes have partial homology to a previously characterised Bgh gene family, Egh16. Egh16 and Egh16H are subfamilies of a larger multigene family with presently about 15 members identified in Bgh. Egh16H has about ten members, and we show that five of these are expressed as highly conserved mRNAs that are predicted to encode proteins with a C-terminal variable region. Egh16H has high homology to sequences in Magnaporthe grisea and other plant pathogenic fungi, as well as sequences of both the insect pathogen Metarhizium anisopliae and the human pathogen Aspergillus fumigatus. No close homologues of Egh16H were found in the non-pathogenic fungi Neurospora crassa and Aspergillus nidulans. We predict that Egh16H plays a general role in the interaction between pathogenic fungi and their hosts. At present, the large number of gene family members with C-terminal variation appears to be unique for Bgh, and the Egh16/Egh16H gene family is to our knowledge the largest gene family so far characterised in this fungus.