Examinando por Autor "Larrondo, Luis F."
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Ítem Circadian oscillations in Trichoderma atroviride and the role of core clock components in secondary metabolism, development, and mycoparasitism against the phytopathogen Botrytis cinerea(eLife Sciences Publications Ltd, 2022) Henríquez-Urrutia, Marlene; Spanner, Rebecca; Olivares-Yánez, Consuelo; Seguel-Avello, Aldo; Pérez-Lara, Rodrigo; Guillén-Alonso, Hector; Winkler, Robert; Herrera-Estrella, Alfredo; Canessa, Paulo; Larrondo, Luis F.Circadian clocks are important for an individual’s fitness, and recent studies have underlined their role in the outcome of biological interactions. However, the relevance of circadian clocks in fungal–fungal interactions remains largely unexplored. We sought to characterize a functional clock in the biocontrol agent Trichoderma atroviride to assess its importance in the mycoparasitic interaction against the phytopathogen Botrytis cinerea. Thus, we confirmed the existence of circadian rhythms in T. atroviride, which are temperature-compensated and modu-lated by environmental cues such as light and temperature. Nevertheless, the presence of such molecular rhythms appears to be highly dependent on the nutritional composition of the media. Complementation of a clock null (Δfrq) Neurospora crassa strain with the T. atroviride-negative clock component (tafrq) restored core clock function, with the same period observed in the latter fungus, confirming the role of tafrq as a bona fide core clock component. Confrontation assays between wild-type and clock mutant strains of T. atroviride and B. cinerea, in constant light or darkness, revealed an inhibitory effect of light on T. atroviride’s mycoparasitic capabilities. Interestingly, when confrontation assays were performed under light/dark cycles, T. atroviride’s overgrowth capacity was enhanced when inoculations were at dawn compared to dusk. Deleting the core clock-negative element FRQ in B. cinerea, but not in T. atroviride, was vital for the daily differential phenotype, suggesting that the B. cinerea clock has a more significant influence on the result of this interaction. Additionally, we observed that T. atroviride clock components largely modulate development and secondary metabolism in this fungus, including the rhythmic production of distinct volatile organic compounds (VOCs). Thus, this study provides evidence on how clock components impact diverse aspects of T. atroviride lifestyle and how daily changes modulate fungal interactions and dynamics. © Henríquez-Urrutia et al.Ítem Comprehensive re-analysis of hairpin small RNAs in fungi reveals loci with conserved links(eLife Sciences Publications Ltd, 2022-12) Johnson, Nathan R.; Larrondo, Luis F.; Álvarez, José M.; Vidal, Elena A.RNA interference is an ancient mechanism with many regulatory roles in eukaryotic genomes, with small RNAs acting as their functional element. While there is a wide array of classes of small-RNA-producing loci, those resulting from stem-loop structures (hairpins) have received profuse attention. Such is the case of microRNAs (miRNAs), which have distinct roles in plants and animals. Fungi also produce small RNAs, and several publications have identified miRNAs and miRNA-like (mi/milRNA) hairpin RNAs in diverse fungal species using deep sequencing technolo-gies. Despite this relevant source of information, relatively little is known about mi/milRNA features in fungi, mostly due to a lack of established criteria for their annotation. To systematically assess mi/milRNA characteristics and annotation confidence, we searched for publications describing mi/ milRNA loci and re-assessed the annotations for 41 fungal species. We extracted and normalized the annotation data for 1727 reported mi/milRNA loci and determined their abundance profiles, concluding that less than half of the reported loci passed basic standards used for hairpin RNA discovery. We found that fungal mi/milRNA are generally more similar in size to animal miRNAs and were frequently associated with protein-coding genes. The compiled genomic analyses identified 25 mi/milRNA loci conserved in multiple species. Our pipeline allowed us to build a general hierarchy of locus quality, identifying more than 150 loci with high-quality annotations. We provide a centralized annotation of identified mi/milRNA hairpin RNAs in fungi which will serve as a resource for future research and advance in understanding the characteristics and functions of mi/milRNAs in fungal organisms. © Johnson et al.Ítem Genome-Wide Characterization of Light-Regulated Gene Expression in Botrytis cinerea Reveals Underlying Complex Photobiology(Multidisciplinary Digital Publishing Institute (MDPI), 2023-05) Pérez-Lara, Gabriel; Olivares-Yañez, Consuelo; van Bakel, Harm; Larrondo, Luis F.; Canessa, PauloBotrytis cinerea is a necrotrophic fungus characterized mainly by its wide host range of infected plants. The deletion of the white-collar-1 gene (bcwcl1), which encodes for a blue-light receptor/transcription factor, causes a decrease in virulence, particularly when assays are conducted in the presence of light or photocycles. However, despite ample characterization, the extent of the light-modulated transcriptional responses regulated by BcWCL1 remains unknown. In this study, pathogen and pathogen:host RNA-seq analyses, conducted during non-infective in vitro plate growth and when infecting Arabidopsis thaliana leaves, respectively, informed on the global gene expression patterns after a 60 min light pulse on the wild-type B05.10 or ∆bcwcl1 B. cinerea strains. The results revealed a complex fungal photobiology, where the mutant did not react to the light pulse during its interaction with the plant. Indeed, when infecting Arabidopsis, no photoreceptor-encoding genes were upregulated upon the light pulse in the ∆bcwcl1 mutant. Differentially expressed genes (DEGs) in B. cinerea under non-infecting conditions were predominantly related to decreased energy production in response to the light pulse. In contrast, DEGs during infection significantly differ in the B05.10 strain and the ∆bcwcl1 mutant. Upon illumination at 24 h post-infection in planta, a decrease in the B. cinerea virulence-associated transcripts was observed. Accordingly, after a light pulse, biological functions associated with plant defense appear enriched among light-repressed genes in fungus-infected plants. Taken together, our results show the main transcriptomic differences between wild-type B. cinerea B05.10 and ∆bcwcl1 after a 60 min light pulse when growing saprophytically on a Petri dish and necrotrophically over A. thaliana. © 2023 by the authors.Ítem Identification of a common secondary mutation in the Neurospora crassa knockout collection conferring a cell fusion-defective phenotype(American Society for Microbiology, 2023-10) Montenegro-Montero, Alejandro; Goity, Alejandra; Canessa, Paulo F.; Larrondo, Luis F.Gene-deletion mutants represent a powerful tool to study gene function. The filamentous fungus Neurospora crassa is a well-established model organism, and features a comprehensive gene knockout strain collection. While these mutant strains have been used in numerous studies, resulting in the functional annotation of many Neurospora genes, direct confirmation of gene-phenotype relationships is often lacking, which is particularly relevant given the possibility of background mutations, sample contamination, and/or strain mislabeling. Indeed, spontaneous mutations resulting in phenotypes resembling many cell fusion mutants have long been known to occur at relatively high frequency in N. crassa, and these secondary mutations are common in the Neurospora deletion collection. The identity of these mutations, however, is largely unknown. Here, we report that the Δada-3 strain from the N. crassa knockout collection, which exhibits a cell fusion defect, harbors a secondary mutation responsible for this phenotype. Through whole-genome sequencing and genetic analyses, we found a ~30-Kb deletion in this strain affecting a known cell fusion-related gene, so/ham-1, and show that it is the absence of this gene—and not of ada-3—that underlies its cell fusion defect. We additionally found three other knockout strains harboring the same deletion, suggesting that this mutation may be common in the collection and could have impacted previous studies. Our findings provide a cautionary note and highlight the importance of proper functional validation of strains from mutant collections. We discuss our results in the context of the spread of cell fusion-defective cheater variants in N. crassa cultures. IMPORTANCE This study emphasizes the need for careful and detailed characterization of strains from mutant collections. Specifically, we found a common deletion in various strains from the Neurospora crassa gene knockout collection that results in a cell fusion-defective phenotype. This is noteworthy because this collection is known to contain background mutations—of a largely unclear nature—that produce cell fusion-defective phenotypes. Our results describe an example of such mutations, and highlight how this common genetic defect could have impacted previous studies that have used the affected strains. Furthermore, they provide a cautionary note about the use of Neurospora strains with similar phenotypes. Lastly, these findings offer additional details relevant to our understanding of the origin and spread of cell fusion-defective cheater variants in N. crassa cultures. Copyright © 2023 Montenegro-Montero et al.Ítem Interactions between Core Elements of the Botrytis cinerea Circadian Clock Are Modulated by Light and Different Protein Domains(MDPI, 2022-05) Rojas, Vicente; Salinas, Francisco; Romero, Andrés; Larrondo, Luis F.; Canessa, PauloBotrytis cinerea possesses a complex light-sensing system composed of eleven photoreceptors. In B. cinerea, bcwcl1 encodes for the BcWCL1 protein, the orthologue of the blue-light photoreceptor WC-1 from Neurospora crassa. The functional partner of BcWCL1 is the BcWCL2 protein, both interacting in the nucleus and forming the B. cinerea white collar complex (BcWCC). This complex is required for photomorphogenesis and circadian regulation. However, no molecular evidence shows a light-dependent interaction between the BcWCC components or light-sensing capabilities in BcWCL1. In this work, by employing a yeast two-hybrid system that allows for the in vivo analysis of protein–protein interactions, we confirm that BcWCL1 and BcWCL2 interact in the absence of light as well as upon blue-light stimulation, primarily through their PAS (Per-Arnt-Sim) domains. Deletion of the PAS domains present in BcWCL1 (BcWCL1PAS∆) or BcWCL2 (BcWCL2PAS∆) severely impairs the interaction between these proteins. Interestingly, the BcWCL1PAS∆ protein shows a blue-light response and interacts with BcWCL2 or BcWCL2PAS∆ upon light stimulation. Finally, we demonstrate that BcWCL1 and BcWCL1PAS∆ respond to blue light by introducing a point mutation in the photoactive cysteine, confirming that both proteins are capable of light sensing. Altogether, the results revealed the complexity of protein–protein interactions occurring between the core elements of the B. cinerea circadian clock. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.