Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSShedding light on Botrytis biology: characterization of the WC1 photoreceptor and FRQ homologues in the necrotrophic plant pathogen Botrytiscinerea. Paulo Canessa 1 , Montserrat Hevia 1 , Julia Schumacher 2 , Paul Tudzynski 3 , Luis F Larrondo 1 . 1) Depto. Genética Molecular y Microbiologia, PontificiaUniversidad Catolica de Chile, Santiago, Chile; 2) Purdue University, USA; 3) West F Wilhelms Univ Muenster Germany.The non-pathogenic fungus Neurospora crassa has been a premier model for the study of circadian clocks, but it is the only fungus where clocks havebeen molecularly characterized. In Neurospora the circadian oscillator is based on the transcriptional control of frq expression by the White Collar complex(WCC) composed of WC-1, a blue-light photoreceptor and WC-2, its functional partner. The WCC activates frq expression until FRQ promotes thephosphorylation of the WCC, shutting down its transcriptional activity. Then FRQ is progressively phosphorylated and degraded setting the bases ofcircadian rhythmicity. While circadian clocks modulate defense in plants, little is known about its participation in fungal pathogenicity. In an effort tocharacterize light responses and the circadian clock in B. cinerea, we have generated KO of homologues of wc-1 and frq (bcwcl1 and bcfrql). Our resultsindicate that light can modulate B. cinerea growth and activates transcriptional responses. Most -but not all of them- are mediated by BcWCL1. Moreover,the obtained bcwcl1 KO mutant strains exhibit enhanced conidiation patterns and susceptibility to hydrogen peroxide. Virulence assays show significantlyreduced lesion formation under constant light conditions (LL) and light:dark cycles (LD), but not under constant darkness (DD). Further analysis of thebcwcl1 KO strains indicates non-altered ROS patterns in planta but delayed penetration on onion epidermis. RT-qPCR experiments have confirmed dailyoscillations in bcfrql levels under LD and DD conditions. Using a BcFRQL-luciferase translational reporter, we have further observed oscillatory levels of theBcFRQL protein under both temperature cycles and constant culture conditions. Both BcFRQL and bcfrql respond to light pulses, while under oscillatoryculture conditions, BcFRQL anticipates cyclical-environmental changes, a key characteristic of circadian behavior. Finally, bcfrql KO mutants also exhibitimpaired pathogenicity, with a drastic reduction in spore production. To our knowledge, these results provide the first evidence of functionallight/circadian machineries in B. cinerea, showing that in this necrotroph, homologues of circadian clock components modulate the plant pathogeninteraction from a fungal perspective. Fondecyt 1090513, postdoc 3110127, AT24121100, ICGEB CRPCHI0902.The transcription factor FL is phosphorylated and interacts with a trehalose related protein in Neurospora crassa. Carmen Ruger-Herreros 1 , GencerSancar 2 , Michael Brunner 2 , Luis M. Corrochano 1 . 1) Departamento de Genetica, Universidad de Sevilla, Spain; 2) BZH, Universität Heidelberg, Germany.Several environmental cues, including light, promote a developmental transition in Neurospora crassa that leads to the formation of conidia. Conidiationis controlled by FLUFFY (FL), a zinc finger transcription factor. Light activates the transcription of fl through the transient binding of the WC complex to thefl promoter. Light also activates the transcription of several conidiation genes in Aspergillus nidulans, and their Neurospora homologs have been identifiedin the Neurospora genome. We have assayed the activation by light of the Neurospora homologs of A. nidulans conidiation genes (flbA, flbC, flbD, medAand stuA), and the Neurospora conidiation gene con-10 as a control. Unlike con-10, none of the Neurospora homologs of the A. nidulans conidiation geneswere induced by light in vegetative mycelia. However, we found that deletion fl resulted in light-dependent mRNA accumulation for all the conidiationgenes. This result indicated that the absence of FL allows the binding of the WC complex to the promoter of these genes to activate transcription in a lightdependentmanner. We have assayed the amount of WC proteins in the Dfl and wild type strains but we did not find any difference between the twostrains. We expect to identify additional genes deregulated by the absence of FL after massive sequencing of total RNA (RNAseq) using a Dfl strain andwild-type strain in dark and light conditions. We have investigated the role of FL during conidiation in Neurospora using a tagged version of FL. FL ispresent in vegetative mycelia but the amount increses after light exposure. We observed several forms of FL due to phosphorylation, and and we havedetermined by mass spectrometry that FL is phosphorylated in several residues. We have immunoprecipitated FL to identify proteins that may interactwith FL. We have found a protein that interacts with FL in different growth conditions. This protein has been described in other organisms and plays a rolein the ability to grow in the presence of trehalose. Since FL is a transcription factor, we have use FL::3XFLAG strain to do ChIPseq in order to identify theputative binding sites of FL to the DNA. We expect that the results from these experiments will help us to understand in more detail the role of FL in theactivation of gene transcription during development.Regulation of gene expression in response to light in Trichoderma atroviride. Jose Cetz 1 , Nohemi Carreras-Villaseñor 1 , Monica Garcia-Esquivel 1 , UlisesEsquivel-Naranjo 2 , Jose M. Villalobos-Escobedo 1 , Cei Abreu-Goodger 1 , Alfredo H. Herrera-Estrella 1 . 1) National Laboratory of Genomics for Biodiversity,CINVESTAV-IPN, Irapuato, Irapuato, Mexico; 2) Facultad de Ciencias Naturales. Universidad Autónoma de Queretaro. Queretaro, Mexico.Trichoderma is used as a photomorphogenetic model due to its ability to conidiate upon exposure to light. In total darkness, T. atroviride growsindefinitely as a mycelium provided that nutrients are not limiting. However, a brief pulse of blue light given to a radially growing colony inducessynchronous sporulation. Photoconidiation in Trichoderma is controlled by the blr1 and blr2, orthologs of the N. crassa white-collar genes. We appliedhigh-throughput sequencing technology to RNA samples from the wild type strain grown in the dark or after exposure to a pulse of white or blue-light, aswell as from a photoreceptor mutant (Dblr-1) exposed to white light. We identified 331 transcripts regulated by white light and 204 responsive specificallyto blue light, both induced and repressed, the majority of them blr1 dependent. Among the genes identified there is a set of transcription factors. We haveobtained gene disruption mutants of several of the transcription factors, and all potential light receptors. Using this strategy we have obtained mutantsthat do not conidiate in response to light, as well as mutants that do not require this stimulus to conidiate, and a conection between light and carbonmetabolism. Further, transcriptional analysis in RNAi machinery mutants indicated that light induced conidiation is defective in the Ddcr2, Ddcr1Ddcr2,Drdr3 and Dago2 mutants, and significant differences were found in the set of light responsive genes between the dicer mutants and the wild type. Thesedata indicate that in T. atroviride, the RNAi machinery plays an important role in controlling development.27th Fungal Genetics Conference | 91