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IMPACT OF FUNGAL SECONDARY METABOLITES ON TRANSCRIPT REGULATION OF FOLSOMIA CANDIDA

ABSTRACT: Genetic and molecular evidence to support the hypothesis that fungal secondary metabolites play a significant role in protecting the fungi against fungivory is scarce. We investigated the impact of fungal secondary metabolites on transcript regulation of stress related expressed sequence tags (ESTs) of the Collembola Folsomia candida feeding on mixed vs. single diets. Aspergillus nidulans wildtype (WT; Ascomycota) able to produce secondary metabolites including sterigmatocystin (ST) and a knockout mutant with reduced secondary metabolism (A. nidulans ΔLaeA) were combined with the high quality fungus Cladosporium cladosporioides as mixed diets or offered as single diets. We hypothesized that (i) A. nidulans WT triggers more genes associated with stress responses compared to the A. nidulans ΔlaeA strain with suppressed secondary metabolism, (ii) C. cladosporioides causes significantly different transcript regulation than the A. nidulans strains ΔlaeA and WT, and (iii) mixed diets will cause significantly different transcript expression levels than single diets. All three hypotheses are generally supported despite the fact that many functions of the affected ESTs are unknown. The results bring molecular evidence for the existence of a link between fungal secondary metabolites and responses in springtails supporting the hypothesis that fungal secondary metabolites act as a shield against fungivory.

ORGANISM(S): Folsomia candida

PROVIDER: GSE19930 | GEO | 2010/02/03

SECONDARY ACCESSION(S): PRJNA122007

REPOSITORIES: GEO

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Project description:Genetic and molecular evidence to support the hypothesis that fungal secondary metabolites play a significant role in protecting the fungi against fungivory is scarce. We investigated the impact of fungal secondary metabolites on transcript regulation of stress related expressed sequence tags (ESTs) of the Collembola Folsomia candida feeding on mixed vs. single diets. Aspergillus nidulans wildtype (WT; Ascomycota) able to produce secondary metabolites including sterigmatocystin (ST) and a knockout mutant with reduced secondary metabolism (A. nidulans ?LaeA) were combined with the high quality fungus Cladosporium cladosporioides as mixed diets or offered as single diets. We hypothesized that (i) A. nidulans WT triggers more genes associated with stress responses compared to the A. nidulans ?laeA strain with suppressed secondary metabolism, (ii) C. cladosporioides causes significantly different transcript regulation than the A. nidulans strains ?laeA and WT, and (iii) mixed diets will cause significantly different transcript expression levels than single diets. All three hypotheses are generally supported despite the fact that many functions of the affected ESTs are unknown. The results bring molecular evidence for the existence of a link between fungal secondary metabolites and responses in springtails supporting the hypothesis that fungal secondary metabolites act as a shield against fungivory. Twenty-three day old Folsomia candida were fed ad libitum for five days to fungal cuts respectively Cladosporium cladosporoides, Aspergillus nidulans WT, Aspergillus nidulans ?LaeA and two mixed diets of C.cladosporoides/A. nidulans WT (mix 1) and C. cladosporoides/A. nudlans ?LaeA (mix2) respectively. Four biological replicates were used for every treatment and a dye swap was used with the Cy3/Cy5 labels. This resulted in 20 samples which were analysed in 10 hybridisations executed in an interwoven loop design. The C. cladosporoides diet was used as the reference in the data analysis.

Project description:Gaining new knowledge through fungal monoculture responses to lignocellulose is a widely used approach that can lead to better cocktails for lignocellulose saccharification (the enzymatic release of sugars which are subsequently used to make biofuels). However, responses in lignocellulose mixed cultures are rarely studied in the same detail even though in nature fungi often degrade lignocellulose as mixed communities. Using a dual RNA-seq approach, we describe the first study of the transcriptional responses of wild-type strains of Aspergillus niger, Trichoderma reesei and Penicillium chrysogenum in two and three mixed species shake-flask cultures with wheat straw. Based on quantification of species-specific rRNA, a set of conditions was identified where mixed cultures could be sampled so as to obtain sufficient RNA-seq reads for analysis from each species. The number of differentially-expressed genes varied from a couple of thousand to fewer than one hundred. The proportion of carbohydrate active enzyme (CAZy) encoding transcripts was lower in the majority of the mixed cultures compared to the respective straw monocultures. A small subset of P. chrysogenum CAZy genes showed five to ten-fold significantly increased transcript abundance in a two-species mixed culture with T. reesei. However, a substantial number of T. reesei CAZy transcripts showed reduced abundance in mixed cultures. The highly induced genes in mixed cultures indicated that fungal antagonism was a major part of the mixed cultures. In line with this, secondary metabolite producing gene clusters showed increased transcript abundance in mixed cultures and also mixed cultures with T. reesei led to a decrease in the mycelial biomass of A. niger. Significantly higher monomeric sugar release from straw was only measured using a minority of the mixed culture filtrates and there was no overall improvement. This study demonstrates fungal interaction with changes in transcripts, enzyme activities and biomass in the mixed cultures and whilst there were minor beneficial effects for CAZy transcripts and activities, the competitive interaction between T. reesei and the other fungi was the most prominent feature of this study.

Project description:Neurospora crassa is a reference organism to study carotene biosynthesis and light regulation for decades. However, there is no evidence of its capacity to produce secondary metabolites, a characteristic of many filamentous fungi. In this work, we report the role of the fungal specific velvet regulator complex in development and secondary metabolism in N. crassa. Four velvet genes were found in the genome. Deletion of ve-1 or ve-2 affects asexual and sexual development, secondary metabolism and light-dependent carotene biosynthesis. Deletion of vos-1 did not show significant differences in comparison to wild type. Deletion of lae-1 resulted in reduced protoperithecia formation and affected secondary metabolism. VE-1, VE-2, LAE-1 and VOS-1 showed nucleo-cytoplasmic localization, which was independent of light input. Two distinct velvet complexes were found in vivo: a heterotrimeric VE-1/VE-2/LAE-1 and a heterodimeric VE-2/VOS-1 complex, respectively. The heterotrimer-complex positively regulated sexual development and repressed asexual spore formation. Moreover, it repressed siderophore coprogen production under iron starvation conditions. The VE-1/VE-2 heterodimer controlled the production of carotene pigments. VE-1 regulated the expression of more than 15% of the whole genome, which corresponded mainly to regulatory, developmental and redox proteins. We also studied intergenera functions of the velvet complex through complementation of A. nidulans veA, velB, laeA, vosA mutants with their Neurospora crassa orthologues ve-1, ve-2, lae-1 and vos-1, respectively. Expression of VE-1 and VE-2 in A. nidulans successfully substituted the developmental and secondary metabolite functions of VeA and VelB by forming two functional chimeric velvet complexes in vivo, VelB/VE-1/LaeA and VE-2/VeA/LaeA, respectively. The N. crassa lae-1 and vos-1 genes did not complement respective A. nidulans mutants and failed to form corresponding complexes. Reciprocally, expression of veA restored the phenotypes of the N. crassa ve-1 mutant. All N. crassa velvet proteins heterologously expressed in A. nidulans were predominantly localized to nuclear fraction independent of light signal. These data highlight the conservation of the complex formation potential in N. crassa and A. nidulans. However, they also underline the similarities and differences of the velvet roles across genera according to the different life styles, niches and ontogenetic processes.

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IMPACT OF FUNGAL SECONDARY METABOLITES ON TRANSCRIPT REGULATION OF FOLSOMIA CANDIDA

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