RESUMO
Volvocine green algae are a model for understanding the evolution of mating types and sexes. They are facultatively sexual, with gametic differentiation occurring in response to nitrogen starvation (-N) in most genera and to sex inducer hormone in Volvox. The conserved RWP-RK family transcription factor (TF) MID is encoded by the minus mating-type locus or male sex-determining region of heterothallic volvocine species and dominantly determines minus or male gametic differentiation. However, the factor(s) responsible for establishing the default plus or female differentiation programs have remained elusive. We performed a phylo-transcriptomic screen for autosomal RWP-RK TFs induced during gametogenesis in unicellular isogamous Chlamydomonas reinhardtii (Chlamydomonas) and in multicellular oogamous Volvox carteri (Volvox) and identified a single conserved ortho-group we named Volvocine Sex Regulator 1 (VSR1). Chlamydomonas vsr1 mutants of either mating type failed to mate and could not induce expression of key mating-type-specific genes. Similarly, Volvox vsr1 mutants in either sex could initiate sexual embryogenesis, but the presumptive eggs or androgonidia (sperm packet precursors) were infertile and unable to express key sex-specific genes. Yeast two-hybrid assays identified a conserved domain in VSR1 capable of self-interaction or interaction with the conserved N terminal domain of MID. In vivo coimmunoprecipitation experiments demonstrated association of VSR1 and MID in both Chlamydomonas and Volvox. These data support a new model for volvocine sexual differentiation where VSR1 homodimers activate expression of plus/female gamete-specific-genes, but when MID is present, MID-VSR1 heterodimers are preferentially formed and activate minus/male gamete-specific-genes.
Assuntos
Chlamydomonas , Sementes , Sexo , Reprodução , Células Germinativas , Espermatozoides , BiotinaRESUMO
BACKGROUND: Cell type specialization is a hallmark of complex multicellular organisms and is usually established through implementation of cell-type-specific gene expression programs. The multicellular green alga Volvox carteri has just two cell types, germ and soma, that have previously been shown to have very different transcriptome compositions which match their specialized roles. Here we interrogated another potential mechanism for differentiation in V. carteri, cell type specific alternative transcript isoforms (CTSAI). METHODS: We used pre-existing predictions of alternative transcripts and de novo transcript assembly with HISAT2 and Ballgown software to compile a list of loci with two or more transcript isoforms, identified a small subset that were candidates for CTSAI, and manually curated this subset of genes to remove false positives. We experimentally verified three candidates using semi-quantitative RT-PCR to assess relative isoform abundance in each cell type. RESULTS: Of the 1978 loci with two or more predicted transcript isoforms 67 of these also showed cell type isoform expression biases. After curation 15 strong candidates for CTSAI were identified, three of which were experimentally verified, and their predicted gene product functions were evaluated in light of potential cell type specific roles. A comparison of genes with predicted alternative splicing from Chlamydomonas reinhardtii, a unicellular relative of V. carteri, identified little overlap between ortholog pairs with alternative splicing in both species. Finally, we interrogated cell type expression patterns of 126 V. carteri predicted RNA binding protein (RBP) encoding genes and found 40 that showed either somatic or germ cell expression bias. These RBPs are potential mediators of CTSAI in V. carteri and suggest possible pre-adaptation for cell type specific RNA processing and a potential path for generating CTSAI in the early ancestors of metazoans and plants. CONCLUSIONS: We predicted numerous instances of alternative transcript isoforms in Volvox, only a small subset of which showed cell type specific isoform expression bias. However, the validated examples of CTSAI supported existing hypotheses about cell type specialization in V. carteri, and also suggested new hypotheses about mechanisms of functional specialization for their gene products. Our data imply that CTSAI operates as a minor but important component of V. carteri cellular differentiation and could be used as a model for how alternative isoforms emerge and co-evolve with cell type specialization.
Assuntos
Volvox , Volvox/genética , Transcriptoma , Isoformas de Proteínas/genéticaRESUMO
Sarocladium zeae is a fungal endophyte of maize and can be found co-inhabiting a single seed with Fusarium verticillioides, a major mycotoxigenic food safety threat. S. zeae produces pyrrocidines A and B that inhibit the growth of F. verticillioides and may limit its spread within the seed to locations lacking S. zeae. Although coinhabiting single seeds, the fungi are generally segregated in separate tissues. To understand F. verticillioides' protective physiological response to pyrrocidines we sequenced the F. verticillioides transcriptome upon exposure to purified pyrrocidine A or B at sub-inhibitory concentrations. Through this work we identified a F. verticillioides locus FvABC3 (FVEG_11089) encoding a transporter critical for resistance to pyrrocidine. We also identified FvZBD1 (FVEG_00314), a gene directly adjacent to the fumonisin biosynthetic gene cluster that was induced several thousand-fold in response to pyrrocidines. FvZBD1 is postulated to act as a genetic repressor of fumonisin production since deletion of the gene resulted in orders of magnitude increase in fumonisin. Further, pyrrocidine acts, likely through FvZBD1, to shut off fumonisin biosynthesis. This suggests that S. zeae is able to hack the secondary metabolic program of a competitor fungus, perhaps as preemptive self-protection, in this case impacting a mycotoxin of central concern for food safety.
Assuntos
Acremonium , Fumonisinas/metabolismo , Fusarium/genética , Micoses/microbiologia , Doenças das Plantas/microbiologia , Zea mays/microbiologia , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Coinfecção , Resistência à Doença/genética , Genes Fúngicos , Micoses/metabolismo , Pirrolidinonas/metabolismo , Pirrolidinonas/farmacologiaRESUMO
The importance of understanding the biology of the mycotoxigenic fungus Fusarium verticillioides and its various microbial and plant host interactions is critical given its threat to maize, one of the world's most valuable food crops. Disease outbreaks and mycotoxin contamination of grain threaten economic returns and have grave implications for human and animal health and food security. Furthermore, F. verticillioides is a member of a genus of significant phytopathogens and, thus, data regarding its host association, biosynthesis of secondary metabolites, and other metabolic (degradative) capabilities are consequential to both basic and applied research efforts across multiple pathosystems. Notorious among its secondary metabolites are the fumonisin mycotoxins, which cause severe animal diseases and are implicated in human disease. Additionally, studies of these mycotoxins have led to new understandings of F. verticillioides plant pathogenicity and provide tools for research into cellular processes and host-pathogen interaction strategies. This review presents current knowledge regarding several significant lines of F. verticillioides research, including facets of toxin production, virulence, and novel fitness strategies exhibited by this fungus across rhizosphere and plant environments.
Assuntos
Fusarium/fisiologia , Fusarium/patogenicidade , Micotoxinas/metabolismo , Zea mays/microbiologia , Animais , Contaminação de Alimentos , Humanos , Micotoxinas/toxicidade , Doenças das Plantas/microbiologia , VirulênciaRESUMO
The important cereal crops of maize, rye, and wheat constitutively produce precursors to 2-benzoxazolinone, a phytochemical having antifungal effects towards many Fusarium species. However, Fusarium verticillioides can tolerate 2-benzoxazolinone by converting it into non-toxic metabolites through the synergism of two previously identified gene clusters, FDB1 and FDB2. Inspired by the induction of these two clusters upon exposure to 2-benzoxazolinone, RNA sequencing experiments were carried out by challenging F. verticillioides individually with 2-benzoxazolinone and three related chemical compounds, 2-oxindole, 2-coumaranone, and chlorzoxazone. These compounds all contain lactam and/or lactone moieties, and transcriptional analysis provided inferences regarding the degradation of such lactams and lactones. Besides induction of FDB1 and FDB2 gene clusters, four additional clusters were identified as induced by 2-benzoxazolinone exposure, including a cluster thought to be responsible for biosynthesis of pyridoxine (vitamin B6), a known antioxidant providing tolerance to reactive oxygen species. Three putative gene clusters were identified as induced by challenging F. verticillioides with 2-oxindole, two with 2-coumaranone, and two with chlorzoxazone. Interestingly, 2-benzoxazolinone and 2-oxindole each induced two specific gene clusters with similar composition of enzymatic functions. Exposure to 2-coumranone elicited the expression of the fusaric acid biosynthetic gene cluster. Another gene cluster that may encode enzymes responsible for degrading intermediate catabolic metabolites with carboxylic ester bonds was induced by 2-benzoxazolinone, 2-oxindole, and chlorzoxazone. Also, the induction of a dehalogenase encoding gene during chlorzoxazone exposure suggested its role in the removal of the chlorine atom. Together, this work identifies genes and putative gene clusters responsive to the 2-benzoxazolinone-like compounds with metabolic inferences. Potential targets for future functional analyses are discussed.
RESUMO
Fungi are absorptive feeders and thus must colonize and ramify through their substrate to survive. In so doing they are in competition, particularly in the soil, with myriad microbes. These microbes use xenobiotic compounds as offensive weapons to compete for nutrition, and fungi must be sufficiently resistant to these xenobiotics. One prominent mechanism of xenobiotic resistance is through production of corresponding degrading enzymes. As typical examples, bacterial ß-lactamases are well known for their ability to degrade and consequently confer resistance to ß-lactam antibiotics, a serious emerging problem in health care. We have identified many fungal genes that putatively encode proteins exhibiting a high degree of similarity to ß-lactamases. However, fungal cell walls are structurally different from the bacterial peptidoglycan target of ß-lactams. This raises the question, why do fungi have lactamases and what are their functions? Previously, we identified and characterized one Fusarium verticillioides lactamase encoding gene (FVEG_08291) that confers resistance to the benzoxazinoid phytoanticipins produced by maize, wheat, and rye. Since benzoxazinoids are γ-lactams with five-membered rings rather than the four-membered ß-lactams, we refer to the predicted enzymes simply as lactamases, rather than ß-lactamases. An overview of fungal genomes suggests a strong positive correlation between environmental niche complexity and the number of fungal lactamase encoding genes, with soil-borne fungi showing dramatic amplification of lactamase encoding genes compared to those fungi found in less biologically complex environments. Remarkably, Fusarium species frequently possess large (>40) numbers of these genes. We hypothesize that many fungal hydrolytic lactamases are responsible for the degradation of plant or microbial xenobiotic lactam compounds. Alignment of protein sequences revealed two conserved patterns resembling bacterial ß-lactamases, specifically those possessing PFAM domains PF00753 or PF00144. Structural predictions of F. verticillioides lactamases also suggested similar catalytic mechanisms to those of their bacterial counterparts. Overall, we present the first in-depth analysis of lactamases in fungi, and discuss their potential relevance to fitness and resistance to antimicrobials in the environment.
RESUMO
Microbes encounter a broad spectrum of antimicrobial compounds in their environments and often possess metabolic strategies to detoxify such xenobiotics. We have previously shown that Fusarium verticillioides, a fungal pathogen of maize known for its production of fumonisin mycotoxins, possesses two unlinked loci, FDB1 and FDB2, necessary for detoxification of antimicrobial compounds produced by maize, including the γ-lactam 2-benzoxazolinone (BOA). In support of these earlier studies, microarray analysis of F. verticillioides exposed to BOA identified the induction of multiple genes at FDB1 and FDB2, indicating the loci consist of gene clusters. One of the FDB1 cluster genes encoded a protein having domain homology to the metallo-ß-lactamase (MBL) superfamily. Deletion of this gene (MBL1) rendered F. verticillioides incapable of metabolizing BOA and thus unable to grow on BOA-amended media. Deletion of other FDB1 cluster genes, in particular AMD1 and DLH1, did not affect BOA degradation. Phylogenetic analyses and topology testing of the FDB1 and FDB2 cluster genes suggested two horizontal transfer events among fungi, one being transfer of FDB1 from Fusarium to Colletotrichum, and the second being transfer of the FDB2 cluster from Fusarium to Aspergillus. Together, the results suggest that plant-derived xenobiotics have exerted evolutionary pressure on these fungi, leading to horizontal transfer of genes that enhance fitness or virulence.