Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSAcuM binding to sites in the 5’ upstream region. Studies with an acuF-lacZ gene fusion indicate positive control by AcuK and AcuM but a loss of the glucoserepression observed in Northerns suggesting negative regulation acting via 3’ sequences in response to growth on glycolytic carbon sources. Support forthis is provided by transcription studies. Modulation of the balance between the opposing activities of these two gene products is proposed to result fromtranscriptional interference involving collision of RNA polymerase molecules.424. ClbR and its paralog, ClbR2, regulate gene expression of cellulase genes in response to cellobiose in Aspergillus aculeatus. E. Kunitake, S. Tani, J.Sumitani, T. Kawaguchi. Life & Environmental Science, Osaka Prefecture University, Sakai, Osaka, Japan.The cellobiose- and cellulose-responsive induction of the cellobiohydrolase I (cbhI) and carboxymethylcellulase 2 (cmc2) genes is not regulated by XlnR, aZn(II) 2Cys 6 transcriptional activator, in Aspergillus aculeatus. We have identified a novel activator containing a Zn(II) 2Cys 6 binuclear cluster motif designatedas cellobiose-response regulator (ClbR), and which is not homologous to Clr-2/ClrB, a transcriptional activator controlling cellobiose-responsive inductionin Neurospora crassa and Aspergillus nidulans. Interestingly ClbR regulates not only the expression of cbhI and cmc2 but also genes regulated by XlnRunder the cellulose-inducing condition. However, the clbR overexpression did not increase the expression of all genes under control of ClbR. Therefore, wepredict that ClbR functions cooperatively with other factor(s). This study reports the ClbR function and the functional relationships among ClbR, ClbRinteractingfactor, and ClrB homolog on cellobiose-responsive induction in A. aculeatus. ClbR-interacting proteins were screened from a prey librarycomposed of ClbR paralogs and transcription factors controlling the expression of glycoside hydrolase genes by yeast two-hybrid method. ClbR2, 42%identity to ClbR, was so far isolated as a ClbR interacting protein. To investigate the correlation between ClbR and ClbR2 function, gene expression profilesunder control of ClbR were assessed in the clbR amd clbR2 single disruption mutants, and clbR/clbR2 double disruption mutant. The expression of cbhI andcmc2 decreased to the same level in all three mutants under the cellobiose-inducing condition. Furthermore, transcripts of the cbhI and cmc2 genesdrastically decreased in the clrB disruption mutant. This result suggests that ClrB regulates gene expression controlled via the XlnR-independent signalingpathway in this fungus. Because the clrB gene is induced by cellulosic compounds, we investigated if clrB expression is regulated by ClbR and ClbR2. In theclbR, clbR2, and clbR/clbR2 mutants, clrB transcripts under the inducing condition reduced to almost the same level as those under the uninducingcondition. Taken together, these data demonstrate that ClbR and ClbR2 regulate the cellulase gene expression in response to cellobiose by regulating theclrB gene expression.425. Expression of a bacterial xylanase in Trichoderma reesei under the egl2 and cbh2 glycosyl hydrolase gene promoters. Helena Nevalainen 1,2 , ShingoMiyauchi 1,2 , Junior Te'o 1,2 , Peter Bergquist 1,2,3 . 1) Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia; 2)Biomolecular Frontiers Research Centre, Macquarie University, NSW 2109, Australia; 3) Department of Molecular Medicine & Pathology, University ofAuckland Medical School, Auckland 1142, New Zealand.Our main aim in this study was to isolate a suite of promoters, other than cbh1, to establish a gene expression platform in which they could be usedsynergistically for the expression of recombinant gene products, using a bacterial thermophilic xylanase (XynB) as an example. The egl2 and cbh2promoters were selected on the basis of their relative strength and functionality under the same cultivation conditions that induce cbh1 so that thispromoter, widely used for recombinant expression, could be included in the ‘promoter mix’ when desired in the future. Together, these promoterspossess considerable expression capacity that can be harnessed for the synthesis of recombinant gene products in T. reesei. We also explored the mode ofglycosylation of the recombinant bacterial xylanase in T. reesei to confirm that the higher molecular weight bands seen in activity zymograms were indeedpost-translationally modified by glycosylation and to characterize the sugars attached to the protein. The highest XynB production was achieved from atransformant containing 1-2 copies of the EGL2sigpro vector (egl2 promoter). Best xylanase producers did not show any particular pattern in terms of thenumber of gene copies and their mode of integration into the chromosomal DNA. Transformants produced multiple forms of XynB which were decoratedwith various N- and O-glycans. One of the O-glycans was identified as hexuronic acid, whose presence had not been observed previously in theglycosylation patterns of T. reesei.426. A single argonaute gene participates in exogenous and endogenous RNAi and controls different cellular functions in the basal fungus Mucorcircinelloides. F. E. Nicolas-Molina 1,2 , M. Cervantes 1 , A. Vila 1 , S. Moxon 3 , J. P. De Haro 1 , T. Dalmay 3 , S. Torres-Martínez 1 , R. M. Ruiz-Vázquez 1 . 1) Departmentof Genetics and Microbiology, University of Murcia, Murcia, Spain; 2) Regional Campus of International Excellence "Campus Mare Nostrum", Murcia,Spain; 3) School of Biological Sciences, University of East Anglia, Norwich, UK.Regulation by RNAi of diverse cellular functions in metazoans is largely known. However, although different classes of endogenous small RNAs (esRNAs)have been identified in fungi, their biological roles are poorly described. The Argonaute proteins are the core component of all known RNAi pathways.Here we identified three argonaute genes of the basal fungus Mucor circinelloides and investigated their participation in exogenous and endogenous RNAi.Only ago-1 is required for transgene-induced RNA silencing. Ago-1 is also required for the production of distinct classes of esRNAs derived from exons (exsiRNAs),which differ in the silencing proteins required for their biogenesis. Classes I and II ex-siRNAs bind to Ago-1 to control mRNA accumulation of thetarget protein coding genes. Class III ex-siRNAs do not specifically bind to Ago-1, but require this protein for their production, revealing the complexity ofthe biogenesis pathways of ex-siRNAs. We also show that ago-1 gene is involved in the response to environmental signals, since vegetative developmentand autolysis induced by nutritional stress are affected in ago-1 - mutants. Our results highlight the role of ex-siRNAs in the regulation of endogenous genesin basal fungi and expand the range of biological functions modulated by RNAi silencing. This work was funded by MICINN (BFU2009-07220) and MINECO(BFU2012-32246), Spain.427. The transcription factor, AtrR, regulates the expression of ABC transporter genes and ergosterol biosynthesis genes in aspergilli. Ayumi Ohba 1 ,Kiminori Shimizu 2 , Daisuke Hagiwara 2 , Takahiro Shintani 1 , Susumu Kawamoto 2 , Katsuya Gomi 1 . 1) Div. Biosci. Biotechnol. Future Bioind., Grad Sch. Agric.Sci., Tohoku Univ., Japan; 2) MMRC, Chiba Univ., Japan.We previously demonstrated that a novel Zn(II)2Cys6 transcriptional factor, AoAtrR, regulates gene expression of the ABC transporters that wouldfunction as drug efflux pumps and contributes to the azole resistance in Aspergillus oryzae. Moreover, we showed that a deletion mutant of the AoatrRortholog (AfatrR) in Aspergillus fumigatus was similarly hypersensitive to azole drugs. However, little is known about target genes regulated by AfAtrR. Inthis study, we comprehensively examined the target genes regulated by AfAtrR using next-generation DNA sequencing technology (RNA-seq). RNA-seqanalysis indicated that AfAtrR similarly regulated at least one ABC transporter. In addition, surprisingly, AfAtrR also regulated several ergosterolbiosynthetic pathway genes including erg11(cyp51A). It has been known that the basic helix-loop-helix transcription factor, SrbA, has a critical role inergosterol biosynthesis and resistance to the azole drugs in A. fumigatus. Interestingly, the ergosterol biosynthetic pathway genes regulated by AfSrbAwere nearly identical with those regulated by AfAtrR. Therefore, we investigated difference in function between AtrR and SrbA in A. oryzae. The expressionof ergosterol biosynthetic pathway genes such as erg11, erg24, and erg25 etc. and three ABC transporter genes was significantly down-regulated in the27th Fungal Genetics Conference | 225