Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSor host colonization. In the oomycete Phytophthora infestans, the potato late blight pathogen, we identified major shifts in mRNA profiles duringdevelopmental transitions using microarrays. We then used those data with three search algorithms to discover more than 100 motifs that are overrepresentedin promoters of genes up-regulated in hyphae, sporangia, sporangia undergoing zoosporogenesis, swimming zoospores, or germinated cystsforming appressoria. Most of the putative stage-specific transcription factor binding sites (TFBSs) thus identified had features typical of TFBSs such asposition or orientation bias, palindromy, and conservation in related species. Each of six motifs tested in P. infestans transformants using the GUS reportergene conferred the expected stage-specific expression pattern, and were shown to bind nuclear proteins in gel-shift assays. Several motifs linked to theappressoria-forming stage were over-represented in promoters of genes encoding effectors and other pathogenesis-related proteins. To understand howpromoter and genome architecture influence expression, we also mapped transcription patterns to the P. infestans genome assembly. Adjacent geneswere not typically induced in the same stage, including genes transcribed from a small shared promoter region. Analyses of global expression, however,demonstrated that co-regulated gene pairs occurred more than expected by random chance. These data help illuminate the processes regulatingdevelopment and pathogenesis, and will enable future attempts to purify the cognate transcription factors. Our approach should be applicable to bothoomycetes and fungi.463. Cooperative regulation of Aspergillus nidulans cellulase genes by transcription factors McmA and ManR/ClrB. Tetsuo Kobayashi 1 , Nuo Li 1 , MikiAoyama 1 , Yohei Yamakawa 1 , Masahiro Ogawa 2 , Yasuji Koyama 2 . 1) Grad Sch of Bioagricultural Sci, Nagoya Univ, Nagoya, Aichi, Japan; 2) Kikkoman Corp,Noda, Chiba, Japan.Expression of the endoglucanase A gene (eglA) in A. nidulans is inducible by cellobiose. Previously, the cis-element responsible for the inductiveexpression, designated CeRE (Cellulose Responsive Element), was identified based on mutational analysis of the eglA promoter. CeRE contained thebinding consensus of SRF-MADS proteins, suggesting involvement of McmA, the sole SRF-MADS protein in A. nidulans. While two Zn 2Cys 6 transcriptionfactors in Aspergillus were recently reported to be essential to cellulase induction. One is ManR in A. oryzae, which regulates both mannanase andcellulase genes, and the other is ClrB in A. nidulans, a homolog of the cellulase regulator CLR-2 in N. crassa. Since these factors were orthologous sharing63% identity, we use the name ManR/ClrB. In this presentation, we provide evidences that McmA and ManR/ClrB cooperatively regulate induction ofcellulase genes. Effects of mcmA mutation and manR/clrB deletion on expression of cellulase genes were examined by qRT-PCR. Expression of eglA, eglB,and cbhA was highly induced by cellobiose in the wild type strain. The induction was significantly impaired by the mcmA mutation and abolished by themanR/clrB deletion, indicating that the cellulase genes under control of McmA and ManR/ClrB are overlapped. Binding of His-tagged McmA and FlagtaggedManR/ClrB-DBD (DNA Binding Domain), which were produced in E. coli and purified, to the CeRE containing region of the eglA promoter wasexamined by EMSA. McmA gave two shifted bands corresponding to single and double occupation of the binding sites, which lay within and just upstreamof CeRE. ManR/ClrB-DBD alone showed very weak binding to the region. When both McmA and ManR/ClrB-DBD were added, a slower-migrating and moreabundant shifted band was appeared, which suggested cooperative binding of McmA and ManR/ClrB-DBD. Presence of McmA and ManR/ClrB-DBD in theshifted band was confirmed by supershift assay with the anti-his-tag and anti-flag-tag antibodies. These results indicated that inductive expression of eglAis regulated by cooperative binding of McmA and ManR/ClrB to its promoter, and suggests that regulation of eglB and cbhA would be similar. This workwas supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry.464. Transcription factor shuttling during cellulase induction in Trichoderma reesei. Alex Lichius, Christian P. Kubicek, Verena Seidl-Seiboth. Institute ofChemical Engineering, Vienna University of Technology, Vienna, Austria.For economically feasible production of liquid fuels and other value-added compounds from lignocellulosic plant material, strategies are required toboost cellulolytic and hemicellulolytic enzyme production by industrially relevant fungi. One promising approach is to modulate the transcriptional controlmediating release from carbon catabolite repression (CCR) and induction of cellulase, hemicellulase and xylanase gene expression. To better understandthe underlying molecular dynamics during induction, we characterized nucleo-cytoplamic shuttling of the two transcription factors carbon cataboliterepressor 1 (CRE1) and xylanase regulator 1 (XYR1) of Trichoderma reesei by means of live-cell imaging. In submerged cultures, nuclear import and exportof CRE1 upon repression and induction, respectively, occurred within minutes and therefore was generally faster than shuttling of XYR1. Under CCRconditions XYR1 expression levels were very low, and its nuclear signal required up to one hour to significantly increase upon replacement into an inducingcarbon source. Cultured directly under inducing conditions, nuclear accumulation of XYR1 was detectable after about 20h post inoculation, and stronglyincreased within the following 24 hours. CRE1 under the same conditions was localized exclusively to the cytoplasm. In plate cultures, nuclear recruitmentof CRE1 and XYR1 differed within the central area, the subperiphery and the periphery of the colony depending on the provided carbon source. Mostinterestingly, under inducing conditions we found evidence for increased nuclear recruitment of CRE1 in the central area, correlating with strong nuclearimport of XYR1 in the same region. Notably, the cytoplasmic signal of CRE1 was usually elevated in leading hyphae, whereas XYR1 was never significantlyrecruited to the colony periphery. Taken together our data provide the first temporal resolution of transcription factor shuttling during the induction ofcellulase gene expression in Trichoderma reesei, and reveal some interesting differences between the subcellular localization of CRE1 and XYR1 insubmerged and plate cultures, respectively. These differences indicate that the mycelial organization during fungal growth might be another importantregulatory element to consider for the industrial scale production of cellulolytic enzymes.465. Trichophyton rubrum ap-1 gene expression in response to environmental challenges. Nalu TA Peres, Gabriela F Persinoti, Larissa G Silva, Tiago RJacob, Antonio Rossi, Nilce M Martinez-Rossi. School of Medicine, Univiversity of Sao Paulo, Ribeirao Preto, Brazil.Several families of transcription factors (TF) are found in fungal cells, which contribute for the broad range of cellular responses triggered byenvironmental changes in these organisms. These TF regulate the expression of genes involved in different cellular processes allowing cell survival understressful as well as physiological conditions. The AP-1 TF belongs to the bZIP family (basic leucine zipper), and is involved in the conidiation process,response to oxidative stress, multidrug resistance, and pathogenicity of some fungi. In dermatophytes, a group of keratinophilic fungi, little is known aboutthis TF and the processes in which it is required. Trichophyton rubrum is the major etiologic agent isolated from clinical cases of cutaneous mycoses inhumans, and studies of the responses of this fungus to several environmental conditions allow a better understanding of its physiology and pathogenicity,thus providing information of how to decrease its growth and to establish more efficient therapeutic measures. Here, we evaluated the ap-1 geneexpression profile during growth of T. rubrum in several nutrient sources (keratin, ex vivo skin and nail), exposure to antifungal drugs, andoxidative/osmotic stresses. An up-regulation of ap-1 gene expression was observed during ex vivo infection and keratin utilization, compared to growth ina glucose containing medium. In response to different antifungal agents, ap-1 was up-regulated, while osmotic and oxidative stress did not alter itsexpression level. These results provide insights into the regulation of the ap-1 TF gene expression, suggesting its involvement in T. rubrum pathogenicity,possibly regulating several genes that allow the utilization of proteins from the host tissues as nutrient sources, and also protecting the cell against thedamages caused by antifungal drugs. Financial Support: FAPESP, CNPq, CAPES, and FAEPA.27th Fungal Genetics Conference | 235