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1.
Molecules ; 28(11)2023 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-37298936

RESUMO

The search for safe and efficient new antifungal compounds for agriculture has led to more efforts in finding new modes of action. This involves the discovery of new molecular targets, including coding and non-coding RNA. Rarely found in plants and animals but present in fungi, group I introns are of interest as their complex tertiary structure may allow selective targeting using small molecules. In this work, we demonstrate that group I introns present in phytopathogenic fungi have a self-splicing activity in vitro that can be adapted in a high-throughput screening to find new antifungal compounds. Ten candidate introns from different filamentous fungi were tested and one group ID intron found in F. oxysporum showed high self-splicing efficiency in vitro. We designed the Fusarium intron to act as a trans-acting ribozyme and used a fluorescence-based reporter system to monitor its real time splicing activity. Together, these results are opening the way to study the druggability of such introns in crop pathogen and potentially discover small molecules selectively targeting group I introns in future high-throughput screenings.


Assuntos
Ensaios de Triagem em Larga Escala , RNA Catalítico , Animais , Íntrons/genética , Antifúngicos/farmacologia , Trans-Splicing , Splicing de RNA , RNA Catalítico/química
2.
Front Microbiol ; 15: 1466375, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39355424

RESUMO

Using oral swabs to collect the remnants of stomach content regurgitation during rumination in dairy cows can replicate up to 70% of the ruminal bacterial community, offering potential for broad-scale population-based studies on the rumen microbiome. The swabs collected from dairy cows often vary widely with respect to sample quality, likely due to several factors such as time of sample collection and cow rumination behavior, which may limit the ability of a given swab to accurately represent the ruminal microbiome. One such factor is the color of the swab, which can vary significantly across different cows. Here, we hypothesize that darker-colored swabs contain more rumen contents, thereby better representing the ruminal bacterial community than lighter-colored swabs. To address this, we collected oral swabs from 402 dairy cows and rumen samples from 13 cannulated cows on a research farm in Wisconsin, United States and subjected them to 16S rRNA sequencing. In addition, given that little is known about the ability of oral swabs to recapitulate the ruminal fungal community, we also conducted ITS sequencing of these samples. To correlate swab color to the microbiota we developed and utilized a novel imaging approach to colorimetrically quantify each swab from a range of light to dark. We found that swabs with increasing darkness scores were significantly associated with increased bacterial alpha diversity (p < 0.05). Lighter swabs exhibited greater variation in their community structure, with many identified amplicon sequence variants (ASVs) categorized as belonging to known bovine oral and environmental taxa. Our analysis of the fungal microbiome found that swabs with increasing darkness scores were associated with decreased alpha diversity (p < 0.05) and were also significantly associated with the ruminal solids fungal community, but not with the ruminal liquid community. Our study refines the utility of oral swabs as a useful proxy for capturing the ruminal microbiome and demonstrates that swab color is an important factor to consider when using this approach for documenting both the bacterial and fungal communities.

3.
Nat Commun ; 14(1): 1835, 2023 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-37005409

RESUMO

With >7000 species the order of rust fungi has a disproportionately large impact on agriculture, horticulture, forestry and foreign ecosystems. The infectious spores are typically dikaryotic, a feature unique to fungi in which two haploid nuclei reside in the same cell. A key example is Phakopsora pachyrhizi, the causal agent of Asian soybean rust disease, one of the world's most economically damaging agricultural diseases. Despite P. pachyrhizi's impact, the exceptional size and complexity of its genome prevented generation of an accurate genome assembly. Here, we sequence three independent P. pachyrhizi genomes and uncover a genome up to 1.25 Gb comprising two haplotypes with a transposable element (TE) content of ~93%. We study the incursion and dominant impact of these TEs on the genome and show how they have a key impact on various processes such as host range adaptation, stress responses and genetic plasticity.


Assuntos
Basidiomycota , Phakopsora pachyrhizi , Elementos de DNA Transponíveis/genética , Glycine max/genética , Glycine max/microbiologia , Ecossistema , Basidiomycota/genética , Proliferação de Células
4.
Plant Biotechnol J ; 6(1): 46-61, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17944820

RESUMO

Plant chloroplasts are promising vehicles for recombinant protein production, but the process of protein folding in these organelles is not well understood in comparison with that in prokaryotic systems, such as Escherichia coli. This is particularly true for disulphide bond formation which is crucial for the biological activity of many therapeutic proteins. We have investigated the capacity of tobacco (Nicotiana tabacum) chloroplasts to efficiently form disulphide bonds in proteins by expressing in this plant cell organelle a well-known bacterial enzyme, alkaline phosphatase, whose activity and stability strictly depend on the correct formation of two intramolecular disulphide bonds. Plastid transformants have been generated that express either the mature enzyme, localized in the stroma, or the full-length coding region, including its signal peptide. The latter has the potential to direct the recombinant alkaline phosphatase into the lumen of thylakoids, giving access to this even less well-characterized organellar compartment. We show that the chloroplast stroma supports the formation of an active enzyme, unlike a normal bacterial cytosol. Sorting of alkaline phosphatase to the thylakoid lumen occurs in the plastid transformants translating the full-length coding region, and leads to larger amounts and more active enzyme. These results are compared with those obtained in bacteria. The implications of these findings on protein folding properties and competency of chloroplasts for disulphide bond formation are discussed.


Assuntos
Cloroplastos/metabolismo , Dissulfetos/metabolismo , Nicotiana/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Proteínas Recombinantes/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos , Plantas Geneticamente Modificadas/metabolismo , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/genética , Nicotiana/genética , Transformação Genética
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