DSpace Angular :: Browsing by Author "Larrondo, Luis F."

Browsing by Author "Larrondo, Luis F."

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A comprehensive transcription factor and DNA-binding motif resource for the construction of gene regulatory networks in Botrytis cinerea and Trichoderma atroviride
(2021) Olivares-Yanez, Consuelo; Sanchez, Evelyn; Perez-Lara, Gabriel; Seguel, Aldo; Camejo, Pamela Y.; Larrondo, Luis F.; Vidal, Elena A.; Canessa, Paulo
Botrytis cinerea and Trichoderma atroviride are two relevant fungi in agricultural systems. To gain insights into these organisms' transcriptional gene regulatory networks (GRNs), we generated a manually curated transcription factor (TF) dataset for each of them, followed by a GRN inference utilizing available sequence motifs describing DNA-binding specificity and global gene expression data. As a proof of concept of the usefulness of this resource to pinpoint key transcriptional regulators, we employed publicly available transcriptomics data and a newly generated dual RNA-seq dataset to build context-specific Botrytis and Trichoderma GRNs under two different biological paradigms: exposure to continuous light and Botrytis-Trichoderma confrontation assays. Network analysis of fungal responses to constant light revealed striking differences in the transcriptional landscape of both fungi. On the other hand, we found that the confrontation of both microorganisms elicited a distinct set of differentially expressed genes with changes in T. atroviride exceeding those in B. cinerea. Using our regulatory network data, we were able to determine, in both fungi, central TFs involved in this interaction response, including TFs controlling a large set of extracellular peptidases in the biocontrol agent T. atroviride. In summary, our work provides a comprehensive catalog of transcription factors and regulatory interactions for both organisms. This catalog can now serve as a basis for generating novel hypotheses on transcriptional regulatory circuits in different experimental contexts. (C) 2021 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.
A global search for novel transcription factors impacting the Neurospora crassa circadian clock
(2021) Munoz-Guzman, Felipe; Caballero, Valeria; Larrondo, Luis F.
Eukaryotic circadian oscillators share a common circuit architecture, a negative feedback loop in which a positive element activates the transcription of a negative one that then represses the action of the former, inhibiting its own expression. While studies in mammals and insects have revealed additional transcriptional inputs modulating the expression of core clock components, this has been less characterized in the model Neurospora crassa, where the participation of other transcriptional components impacting circadian clock dynamics remains rather unexplored. Thus, we sought to identify additional transcriptional regulators modulating the N. crassa clock, following a reverse genetic screen based on luminescent circadian reporters and a collection of transcription factors (TFs) knockouts, successfully covering close to 60% of them. Besides the canonical core clock components WC-1 and -2, none of the tested transcriptional regulators proved to be essential for rhythmicity. Nevertheless, we identified a set of 23 TFs that when absent lead to discrete, but significant, changes in circadian period. While the current level of analysis does not provide mechanistic information about how these new players modulate circadian parameters, the results of this screen reveal that an important number of light and clock-regulated TFs, involved in a plethora of processes, are capable of modulating the clockworks. This partial reverse genetic clock screen also exemplifies how the N. crassa knockout collection continues to serve as an expedite platform to address broad biological questions.
A global search for novel transcription factors impacting the Neurospora crassa circadian clock
(2021) Munoz-Guzman, Felipe; Caballero, Valeria; Larrondo, Luis F.
In order to identify new players modulating the circadian clock in the model Neurospora crassa, we adopted a reverse genetics strategy. Thus, we focused on transcription factors knockouts and crossed them to strains containing circadian luciferase reporters. Our screen covered close to 60% de the 302 genes encoding for such proteins in Neurospora, identifying that 23 of them appear to modulate period, while none of the tested ones (besides the classic core-clock components) are essential for daily rhythms.
Analysis of the Phlebiopsis gigantea Genome, Transcriptome and Secretome Provides Insight into Its Pioneer Colonization Strategies of Wood
(2014) Hori, Chiaki; Ishida, Takuya; Igarashi, Kiyohiko; Samejima, Masahiro; Suzuki, Hitoshi; Master, Emma; Ferreira, Patricia; Ruiz-Duenas, Francisco J.; Held, Benjamin; Canessa, Paulo; Larrondo, Luis F.; Schmoll, Monika; Druzhinina, Irina S.; Kubicek, Christian P.; Gaskell, Jill A.; Kersten, Phil; St John, Franz; Glasner, Jeremy; Sabat, Grzegorz; BonDurant, Sandra Splinter; Syed, Khajamohiddin; Yadav, Jagjit; Mgbeahuruike, Anthony C.; Kovalchuk, Andriy; Asiegbu, Fred O.; Lackner, Gerald; Hoffmeister, Dirk; Rencoret, Jorge; Gutierrez, Ana; Sun, Hui; Lindquist, Erika; Barry, Kerrie; Riley, Robert; Grigoriev, Igor V.; Henrissat, Bernard; Kuees, Ursula; Berka, Randy M.; Martinez, Angel T.; Covert, Sarah F.; Blanchette, Robert A.; Cullen, Daniel
Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on fresh-cut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea's extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.
Assessing the Effects of Light on Differentiation and Virulence of the Plant Pathogen Botrytis cinerea: Characterization of the White Collar Complex
(2013) Canessa, Paulo; Schumacher, Julia; Hevia, Montserrat A.; Tudzynski, Paul; Larrondo, Luis F.
Organisms are exposed to a tough environment, where acute daily challenges, like light, can strongly affect several aspects of an individual's physiology, including pathogenesis. While several fungal models have been widely employed to understand the physiological and molecular events associated with light perception, various other agricultural-relevant fungi still remain, in terms of their responsiveness to light, in the dark. The fungus Botrytis cinerea is an aggressive pathogen able to cause disease on a wide range of plant species. Natural B. cinerea isolates exhibit a high degree of diversity in their predominant mode of reproduction. Thus, the majority of naturally occurring strains are known to reproduce asexually via conidia and sclerotia, and sexually via apothecia. Studies from the 1970's reported on specific developmental responses to treatments with near-UV, blue, red and far-red light. To unravel the signaling machinery triggering development - and possibly also connected with virulence - we initiated the functional characterization of the transcription factor/photoreceptor BcWCL1 and its partner BcWCL2, that form the White Collar Complex (WCC) in B. cinerea. Using mutants either abolished in or exhibiting enhanced WCC signaling (overexpression of both bcwcl1 and bcwcl2), we demonstrate that the WCC is an integral part of the mentioned machinery by mediating transcriptional responses to white light and the inhibition of conidiation in response to this stimulus. Furthermore, the WCC is required for coping with excessive light, oxidative stress and also to achieve full virulence. Although several transcriptional responses are abolished in the absence of bcwcl1, the expression of some genes is still light induced and a distinct conidiation pattern in response to daily light oscillations is enhanced, revealing a complex underlying photobiology. Though overlaps with well-studied fungal systems exist, the light-associated machinery of B. cinerea appears more complex than those of Neurospora crassa and Aspergillus nidulans.
Circadian oscillations in Trichoderma atroviride and the role of core clock components in secondary metabolism, development, and mycoparasitism against the phytopathogen Botrytis cinerea
(2022) Henriquez-Urrutia, Marlene; Spanner, Rebecca; Olivares-Yanez, Consuelo; Seguel-Avello, Aldo; Perez-Lara, Rodrigo; Guillen-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 modulated 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 (delta frq) 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.
Cloning and functional characterization of the gene encoding the transcription factor Ace1 in the basidiomycete Phanerochaete chrysosporium
(2006) Polanco, Ruben; Canessa, Paulo; Rivas, Alexis; Larrondo, Luis F.; Lobos, Sergio; Vicuna, Rafael
In this report we describe the isolation and characterization of a gene encoding the transcription factor Ace 1 (Activation protein of cup 1 Expression) in the white rot fungus Phanerochaete chrysosporium. Pc-ace 1 encodes a predicted protein of 633 amino acids containing the copper-fist DNA binding domain typically found in fungal transcription factors such as Ace 1 Mac 1 and Haa 1 from Saccharomyces cerevisiae. The Pc-ace 1 gene is localized in Scaffold 5, between coordinates 220841 and 222983. A S. cerevisiae ace 1 null mutant strain unable to grow in high-copper medium was fully complemented by transformation with the cDNA of Pc-ace 1. Moreover, Northern blot hybridization studies indicated that Pc-ace 1 cDNA restores copper inducibility of the yeast cup 1 gene, which encodes the metal-binding protein metallothionein implicated in copper resistance. To our knowledge, this is first report describing an Ace 1 transcription factor in basidiomycetes.
Comprehensive re-analysis of hairpin small RNAs in fungi reveals loci with conserved links
(2022) Johnson, Nathan R.; Larrondo, Luis F.; Alvarez, Jose 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 technologies. 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.
Developing a Temperature-Inducible Transcriptional Rheostat in Neurospora crassa
(2023) Tabilo-Agurto, Cyndi; Del Rio-Pinilla, Veronica; Eltit-Villarroel, Valeria; Goity, Alejandra; Munoz-Guzman, Felipe; Larrondo, Luis F.
Heat shock protein (HSP)-encoding genes (hsp), part of the highly conserved heat shock response (HSR), are known to be induced by thermal stress in several organisms. In Neurospora crassa, three hsp genes, hsp30, hsp70, and hsp80, have been characterized; however, the role of defined cis elements in their responses to discrete changes in temperature remains largely unexplored. To fill this gap, while also aiming to obtain a reliable fungal heat shock-inducible system, we analyzed different sections of each hsp promoter by assessing the expression of real-time transcriptional reporters. Whereas all three promoters and their resected versions were acutely induced by high temperatures, only hsp30 displayed a broad range of expression and high tunability, amply exceeding other inducible promoter systems existing in Neurospora, such as quinic acid- or light-inducible ones. As proof of concept, we employed one of these promoters to control the expression of clr-2, which encodes the master regulator of Neurospora cellulolytic capabilities. The resulting strain fails to grow on cellulose at 25 degrees C, whereas it grows robustly if heat shock pulses are delivered daily. Additionally, we designed two hsp30 synthetic promoters and characterized them, as well as the native promoters, using a gradient of high temperatures, yielding a wide range of responses to thermal stimuli. Thus, Neurospora hsp30-based promoters represent a new set of modular elements that can be used as transcriptional rheostats to adjust the expression of a gene of interest or for the implementation of regulated circuitries for synthetic biology and biotechnological strategies.
Functional analysis of the endoxylanase B (xynB) promoter from Penicillium purpurogenum
(2008) Diaz, Jheimmy; Chavez, Renato; Larrondo, Luis F.; Eyzaguirre, Jaime; Bull, Paulina
In Penicillium purpurogenum, the gene encoding endoxylanase B (xynB) is highly expressed by xylan and repressed by glucose at the transcriptional level. The promoter of this gene has a modular structure, with eight putative XlnR binding sites in tandem (XlnR module), and upstream from them, four putative CreA binding sites (CreA module). Promoter fragments containing different modules were inserted into a plasmid, pAN49-1, which contains a basal fungal promoter linked to a reporter gene (lacZ) and its expression was studied in vivo in Aspergillus nidulans. The XlnR module is able to trigger high beta-galactosidase activity in the presence of xylan, but the lack of most XlnR sites notoriously reduces this enzymatic activity. No enzyme induction is observed if the orientation of the promoter fragment is inverted. The presence of the CreA module is necessary for glucose repression when beta-galactosidase activity is previously induced by xylan. However, when transformant strains containing the XlnR module but lacking all CreA sites were grown in glucose without pre-induction in xylan, a low beta-galactosidase activity was observed compared with the same transformants grown in xylan. These results agree with a double-lock regulatory mechanism for both direct and indirect repression of xylanolytic genes by glucose.
Fungal Endophytes Enhance the Photoprotective Mechanisms and Photochemical Efficiency in the Antarctic Colobanthus quitensis (Kunth) Bartl. Exposed to UV-B Radiation
(FRONTIERS MEDIA SA, 2020) Barrera, Andrea; Hereme, Rasme; Ruiz Lara, Simon; Larrondo, Luis F.; Gundel, Pedro E.; Pollmann, Stephan; Molina Montenegro, Marco A.; Ramos, Patricio
Antarctic plants have developed mechanisms to deal with one or more adverse factors which allow them to successfully survive such extreme environment. Certain effective mechanisms to face adverse stress factors can arise from the establishment of functional symbiosis with endophytic fungi. In this work, we explored the role of fungal endophytes on host plant performance under high level of UV-B radiation, a harmful factor known to damage structure and function of cell components. In order to unveil the underlying mechanisms, we characterized the expression of genes associated to UV-B photoreception, accumulation of key flavonoids, and physiological responses of Colobanthus quitensis plants with (E+) and without (E-) fungal endophytes, under contrasting levels of UV-B radiation. The deduced proteins of CqUVR8, CqHY5, and CqFLS share the characteristic domains and display high degrees of similarity with other corresponding proteins in plants. Endophyte symbiotic plants showed lower lipid peroxidation and higher photosynthesis efficiency under high UV-B radiation. In comparison with E-, E+ plants showed lower CqUVR8, CqHY5, and CqFLS transcript levels. The content of quercetin, a ROS-scavenger flavonoid, in leaves of E- plants exposed to high UV-B was almost 8-fold higher than that in E+ plants 48 h after treatment. Our results suggest that endophyte fungi minimize cell damage and boost physiological performance in the Antarctic plants increasing the tolerance to UV-B radiation. Fungal endophytes appear as fundamental biological partners for plants to cope with the highly damaging UV-B radiation of Antarctica.
Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina
(2008) Martinez, Diego; Berka, Randy M.; Henrissat, Bernard; Saloheimo, Markku; Arvas, Mikko; Baker, Scott E.; Chapman, Jarod; Chertkov, Olga; Coutinho, Pedro M.; Cullen, Dan; Danchin, Etienne G. J.; Grigoriev, Igor V.; Harris, Paul; Jackson, Melissa; Kubicek, Christian P.; Han, Cliff S.; Ho, Isaac; Larrondo, Luis F.; de Leon, Alfredo Lopez; Magnuson, Jon K.; Merino, Sandy; Misra, Monica; Nelson, Beth; Putnam, Nicholas; Robbertse, Barbara; Salamov, Asaf A.; Schmoll, Monika; Terry, Astrid; Thayer, Nina; Westerholm-Parvinen, Ann; Schoch, Conrad L.; Yao, Jian; Barbote, Ravi; Nelson, Mary Anne; Detter, Chris; Bruce, David; Kuske, Cheryl R.; Xie, Gary; Richardson, Paul; Rokhsar, Daniel S.; Lucas, Susan M.; Rubin, Edward M.; Dunn-Coleman, Nigel; Ward, Michael; Brettin, Thomas S.
Trichoderma reesei is the main industrial source of cellulases and hemicellulases used to depolymerize biomass to simple sugars that are converted to chemical intermediates and biofuels, such as ethanol. We assembled 89 scaffolds (sets of ordered and oriented contigs) to generate 34 Mbp of nearly contiguous T. reesei genome sequence comprising 9,129 predicted gene models. Unexpectedly, considering the industrial utility and effectiveness of the carbohydrate-active enzymes of T. reesei, its genome encodes fewer cellulases and hemicellulases than any other sequenced fungus able to hydrolyze plant cell wall polysaccharides. Many T. reesei genes encoding carbohydrate-active enzymes are distributed nonrandomly in clusters that lie between regions of synteny with other Sordariomycetes. Numerous genes encoding biosynthetic pathways for secondary metabolites may promote survival of T. reesei in its competitive soil habitat, but genome analysis provided little mechanistic insight into its extraordinary capacity for protein secretion. Our analysis, coupled with the genome sequence data, provides a roadmap for constructing enhanced T. reesei strains for industrial applications such as biofuel production.
Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion
(2009) Martinez, Diego; Challacombe, Jean; Morgenstern, Ingo; Hibbett, David; Schmoll, Monika; Kubicek, Christian P.; Ferreira, Patricia; Ruiz-Duenas, Francisco J.; Martinez, Angel T.; Kersten, Phil; Hammel, Kenneth E.; Wymelenberg, Amber Vanden; Gaskell, Jill; Lindquist, Erika; Sabat, Grzegorz; BonDurant, Sandra Splinter; Larrondo, Luis F.; Canessa, Paulo; Vicuna, Rafael; Yadav, Jagjit; Doddapaneni, Harshavardhan; Subramanian, Venkataramanan; Pisabarro, Antonio G.; Lavin, Jose L.; Oguiza, Jose A.; Master, Emma; Henrissat, Bernard; Coutinho, Pedro M.; Harris, Paul; Magnuson, Jon Karl; Baker, Scott E.; Bruno, Kenneth; Kenealy, William; Hoegger, Patrik J.; Kuees, Ursula; Ramaiya, Preethi; Lucash, Susan; Salamov, Asaf; Shapiro, Harris; Tu, Hank; Chee, Christine L.; Misra, Monica; Xie, Gary; Teter, Sarah; Yaver, Debbie; James, Tim; Mokrejs, Martin; Pospisek, Martin; Grigoriev, Igor V.; Brettin, Thomas; Rokhsar, Dan; Berka, Randy; Cullen, Dan
Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exo-cellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative beta-1-4 endoglucanase were expressed at high levels relative to glucose-grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also up-regulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H2O2. These observations are consistent with a biodegradative role for Fenton chemistry in which Fe( II) and H2O2 react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons with the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost.
Genome-Wide Characterization of Light-Regulated Gene Expression in Botrytis cinerea Reveals Underlying Complex Photobiology
(2023) Perez-Lara, Gabriel; Olivares-Yanez, Consuelo; van Bakel, Harm; Larrondo, Luis F.; Canessa, Paulo
Botrytis 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 increment 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 increment 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.
High-resolution spatiotemporal analysis of gene expression in real time: In vivo analysis of circadian rhythms in Neurospora crassa using a FREQUENCY-luciferase translational reporter
(2012) Larrondo, Luis F.; Loros, Jennifer J.; Dunlap, Jay C.
The pacemaker of the Neurospora circadian clock is composed of a transcriptional-translational feedback loop that has been intensively studied during the last two decades. Invaluable information has been derived from measuring the expression of the central clock component frequency (frq) under liquid culture conditions. Direct analyses of frq mRNA and protein levels on solid media - where overt circadian rhythms are normally visualized - have not been trivial due to technical issues. Nevertheless, a frq promoter-luciferase reporter has recently allowed the study of frq transcription under these conditions. It is known that FRQ undergoes extensive posttranslational modifications, and changes in its levels provide important information regarding the clockworks. Here we describe a FRQ-luciferase translational fusion reporter that directly tracks FRQ levels, granting access to a better understanding and analysis of FRQ dynamics in vivo. More generally the method, which allows the investigator to follow continuous gene expression in real time in a spatially and temporally unrestricted manner, should be widely applicable to analyses of environmentally and developmentally regulated gene expression in ascomycete filamentous fungi as well as in basidiomycetes. (C) 2012 Elsevier Inc. All rights reserved.
bcpmr1 encodes a P-type Ca²⁺/Mn²⁺-ATPase mediating cell-wall integrity and virulence in the phytopathogen Botrytis cinerea
(2015) Plaza, Veronica; Laguees, Yanssuy; Carvajal, Mauro; Perez-Garcia, Luis A.; Mora-Montes, Hector M.; Canessa, Paulo; Larrondo, Luis F.; Castillo, Luis
The cell wall of fungi is generally composed of an inner skeletal layer consisting of various polysaccharides surrounded by a layer of glycoproteins. These usually contain both N- and O-linked oligosaccharides, coupled to the proteins by stepwise addition of mannose residues by mannosyltransferases in the endoplasmic reticulum and the Golgi apparatus. In yeast, an essential luminal cofactor for these mannosyltransferases is Mn2+ provided by the Ca2+/Mn2+-ATPase known as Pmr1. In this study, we have identified and characterized the Botrytis cinerea pmr1 gene, the closest homolog of yeast PMR1. We hypothesized that bcpmr1 also encodes a Ca2+/Mn2+-ATPase that plays an important role in the protein glycosylation pathway. Phenotypic analysis showed that bcpmr1 null mutants displayed a significant reduction in conidial production, radial growth and diameter of sclerotia. Significant alterations in hyphal cell wall composition were observed including a 83% decrease of mannan levels and an increase in the amount of chitin and glucan. These changes were accompanied by a hypersensitivity to cell wall-perturbing agents such as Calcofluor white, Congo red and zymolyase. Importantly, the Delta bcpmr1 mutant showed reduced virulence in tomato (leafs and fruits) and apple (fruits) and reduced biofilm formation. Together, our results highlight the importance of bcpmr1 for protein glycosylation, cell wall structure and virulence of B. cinerea. (C) 2015 Elsevier Inc. All rights reserved.
Identification of a common secondary mutation in the Neurospora crassa knockout collection conferring a cell fusion-defective phenotype
(2023) 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 Delta ada-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 similar to 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.
Interactions between Core Elements of the Botrytis cinerea Circadian Clock Are Modulated by Light and Different Protein Domains
(2022) Rojas, Vicente; Salinas, Francisco; Romero, Andres; Larrondo, Luis F.; Canessa, Paulo
Botrytis 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 (BcWCL1(PAS increment )) or BcWCL2 (BcWCL2(PAS increment )) severely impairs the interaction between these proteins. Interestingly, the BcWCL1(PAS increment ) protein shows a blue-light response and interacts with BcWCL2 or BcWCL2(PAS increment ) upon light stimulation. Finally, we demonstrate that BcWCL1 and BcWCL1(PAS increment ) 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.
Methylxanthines Modulate Circadian Period Length Independently of the Action of Phosphodiesterase
(2023) Olivares-Yanez, Consuelo; Alessandri, Maria P.; Salas, Loreto; Larrondo, Luis F.
In Neurospora crassa, caffeine and other methylxanthines are known to inhibit phosphodiesterase (PDE) activity, leading to augmented cAMP levels. In this organism, it has also been shown that the addition of these drugs significantly lengthens the circadian period, as seen by conidiation rhythms. Utilizing in vivo bioluminescence reporters, pharmacological inhibitors, and cAMP analogs, we revisited the effect of methylxanthines and the role of cAMP signaling in the Neurospora clockworks. We observed that caffeine, like all tested methylxanthines, led to significant period lengthening, visualized with both core-clock transcriptional and translational reporters. Remarkably, this phenotype is still observed when phosphodiesterase (PDE) activity is genetically or chemically (via 3-isobutyl-1-methylxanthine) abrogated. Likewise, methylxanthines still exert a period effect in several cAMP signaling pathway mutants, including adenylate cyclase (cr-1) and protein kinase A (PKA) (Delta pkac-1) mutants, suggesting that these drugs lead to circadian phenotypes through mechanisms different from the canonical PDE-cAMP-PKA signaling axis. Thus, this study highlights the strong impact of methylxanthines on circadian period in Neurospora, albeit the exact mechanisms somehow remain elusive.IMPORTANCE Evidence from diverse organisms show that caffeine causes changes in the circadian clock, causing period lengthening. The fungus Neurospora crassa is no exception; here, several methylxanthines such as caffeine, theophylline, and aminophylline cause period lengthening in a concentration-dependent manner. Although methylxanthines are expected to inhibit phosphodiesterase activity, we were able to show by genetic and pharmacological means that these drugs exert their effects through a different mechanism. Moreover, our results indicate that increases in cAMP levels and changes in PKA activity do not impact the circadian period and therefore are not part of underlying effects of methylxanthine. These results set the stage for future analyses dissecting the molecular mechanisms by which these drugs dramatically modify the circadian period.
Modular and Molecular Optimization of a LOV (Light-Oxygen-Voltage)-Based Optogenetic Switch in Yeast
(2021) Romero, Andres; Rojas, Vicente; Delgado, Veronica; Salinas, Francisco; Larrondo, Luis F.
Optogenetic switches allow light-controlled gene expression with reversible and spatiotemporal resolution. In Saccharomyces cerevisiae, optogenetic tools hold great potential for a variety of metabolic engineering and biotechnology applications. In this work, we report on the modular optimization of the fungal light-oxygen-voltage (FUN-LOV) system, an optogenetic switch based on photoreceptors from the fungus Neurospora crassa. We also describe new switch variants obtained by replacing the Gal4 DNA-binding domain (DBD) of FUN-LOV with nine different DBDs from yeast transcription factors of the zinc cluster family. Among the tested modules, the variant carrying the Hap1p DBD, which we call "HAP-LOV", displayed higher levels of luciferase expression upon induction compared to FUN-LOV. Further, the combination of the Hap1p DBD with either p65 or VP16 activation domains also resulted in higher levels of reporter expression compared to the original switch. Finally, we assessed the effects of the plasmid copy number and promoter strength controlling the expression of the FUN-LOV and HAP-LOV components, and observed that when low-copy plasmids and strong promoters were used, a stronger response was achieved in both systems. Altogether, we describe a new set of blue-light optogenetic switches carrying different protein modules, which expands the available suite of optogenetic tools in yeast and can additionally be applied to other systems.

Browsing by Author "Larrondo, Luis F."

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