Oral Presentation CRDC 5 22/07/54 CRDC-5 (22 July 2011) Phenotypic analysis of transcriptional regulators associated transcriptional regulators associated with environmental stress tolerance in the yeast Saccharomyces cerevisiae Miss Chalinee Tangsombatvichit Division of Biochemical Technology School of Bioresources and Technology King Mongkut’s University of Technology Thonburi Introduction Plant disease Susceptible plant Pathogen can develop and induce diseases Unsuitable environment for plant growth 2 http://www.sustland.umn.edu/maint/images/disease.gif 1
Oral Presentation CRDC 5 22/07/54 Cell division and fungal development http://www.phschool.com/science/biology_place/biocoach/images/transcription/euovrvw.gif 3 http://scienceblogs.com/pharyngula/upload/2006/07/transcriptionfactor.jpg Zinc cluster proteins Gal4p Hap1p Leu3p � Sub-classes of eukaryotic zinc finger proteins � Major class of transcriptional regulator � Consensus amino acid sequence as shown Cys-X 2 -Cys-X 6 -Cys-X 5-12 -Cys-X 2 -Cys-X 6-8 -Cys � Identified exclusively in fungi 4 MacPherson et al ., 2006. Microbiology and Molecular Biology Review , Vol. 70, pp. 583-604. 2
Oral Presentation CRDC 5 22/07/54 Fungal organism Non Pathogenic fungi Saccharomyces Saccharomyces Kluyveromyces Kluyveromyces Schizosaccharomyces Schizosaccharomyces cerevisiae lactis pombe 5 MacPherson et al ., 2006. Microbiology and Molecular Biology Review , Vol. 70, pp. 583-604. Pathogenic fungi Candida Candida albicans albicans Aspergillus Aspergillus nidulans nidulans Fusarium oxysporum Fusarium oxysporum 6 MacPherson et al ., 2006. Microbiology and Molecular Biology Review , Vol. 70, pp. 583-604. 3
Oral Presentation CRDC 5 22/07/54 Fungal pathogen in plant Fusarium oxysporum Fusarium oxysporum : widespread in economic plants such as tomato, pepper, maize or banana etc. : Soil borne : Filamentous fungi : Mycotoxin : Vascular wilt 7 Roles of zinc cluster proteins in Fusarium sp. Imazaki et al ., 2007. Fow2 , a Zn(II) 2 Cys 6 -type transcription regulator, controls plant infection of the vascular wilt fungus Fusarium oxysporum. Molec. Microbiol. 63: 737-753. 737 753 Fig. 1 Pathogenicity of Fow2 mutants. g g y Melon plants with a single true leaf were inoculated with bud cell suspension by the root dip method. WT Fow2 WT Fow2 mutant mutant 8 4
Oral Presentation CRDC 5 22/07/54 WT Fow2 mutant Fig. 2 Invasive hyphae of wild-type and Fow2 mutant strains within hypocotyls. Cross-sections of hypocotyls were observed with a fluorescence microscope. DIC, differential interference contrast images; GFP, GFP fluorescence images 9 Ramos et al ., 2007. The gene coding for a new transcription factor ( ftf1 ) of Fusarium oxysporum is only expressed during infection of common bean . Fungal Gent. Biol. 44: 864-876. Isolated virulent genes from highly virulent strains of Fusarium oxysporum during infection of common bean Found virulent gene FTF1 (Fusarium transcription factor 1) which encodes zinc cluster protein of common bean FTF1 is presented and involved in plant disease. 10 5
Oral Presentation CRDC 5 22/07/54 The yeast S. cerevisiae genetic model organism Genome sequencing of yeast Saccharomyces cerevisiae identified a total of 55 putative zinc p cluster proteins and many have unknown roles ??? 11 Schjerling, P. and Holmberg, S., 1996. Nucleic Acids Research , Vol. 24, pp. 4599-4607. Roles of zinc cluster proteins in S. cerevisiae Sugar metabolism Gene Name Characteristic GAL4 Activator of genes involved in galactose metabolism MAL33 Activator of genes involved in maltose metabolism Stress response Gene Name Characteristic ASG1 ASG1 Activator of stress response genes, involved in respiration, Activator of stress response genes, involved in respiration, or antifungal drug resistance HAL9 Involved in salt tolerance STB5 Activator of genes involved in multidrug resistance and oxidative stress response 12 MacPherson et al ., 2006. Microbiology and Molecular Biology Review , Vol. 70, pp. 583-604. 6
Oral Presentation CRDC 5 22/07/54 Multidrug resistance Gene Name Characteristic PDR1 Activators and repressors involved in multidrug resistance RDR1 Repressor of genes involved in the control of multidrug resistance RDS2 Activator and repressor involved in gluconeogenesis and glyoxylate cycle genes, or drug sensitivity YRR1 Activator of genes involved in multidrug resistance http://www.yeastgenome.org/ 13 MacPherson et al ., 2006. Microbiology and Molecular Biology Review , Vol. 70, pp. 583-604. Objective To observe the roles of unknown zinc cluster transcriptional T b th l f k i l t t i ti l regulators that responses to environmental stress > heat tolerance > salt tolerance in the wild-type and zinc cluster deletion strains of S. cerevisiae via phenotypic analysis 14 7
Oral Presentation CRDC 5 22/07/54 Methodology Table 1 S. cerevisiae strains used in this study Strain name Deletion* FA aa 24-167 FB aa 25-166 FC aa 23-206 FI aa 27-200 FZI FZI aa 25-122 25 122 FZJ aa 115-319 FZO aa 25-166 FZP aa 25-196 15 Phenotypic analysis Culture yeast strains Measured OD 600nm in YPD overnight start at 0.1 Spot assay under different Serial dilution and environmental conditions spot on plates 16 8
Oral Presentation CRDC 5 22/07/54 Growth conditions Heat Salt stress Salt stress sensitivity No salt 30 O C 0.5M LiCl 37 O C 1 0M NaCl 1.0M NaCl 39 O C 1.5M KCl 17 Results Table 2 Growth assay of wild-type and zinc cluster deletion strains under increased temperature Temperature ( O C) Temperature ( O C) Strain name 30 37 39 Wild-type ++ ++ + FA ++ ++ + FB ++ ++ + FC ++ ++ + FI FI ++ ++ ++ ++ + + FZI ++ ++ + FZJ ++ ++ + FZO ++ ++ + FZP ++ + X ++ normal growth + moderate growth X impaired growth 18 9
Oral Presentation CRDC 5 22/07/54 cell Wild-type 30 O C FZP FZP Wild-type 39 O C FZP Fig. 3 Phenotypic analysis of wild-type and zinc cluster FZP strains were grown at various temperature of 30 O C or 39 O C 19 Table 3 Growth assay of wild-type and zinc cluster deletion strains during growth on media containing salts Salt concentration Strain name No No 0.5M 0.5M 1.0M 1.0M 1.5M 1.5M salt LiCl NaCl KCl Wild-type ++ + + + FA ++ + + + FB ++ + + + FC ++ + + + FI ++ + + + FZI ++ + + + FZJ FZJ ++ + + + FZO ++ + + + FZP ++ + X X ++ normal growth + moderate growth X impaired growth 20 10
Oral Presentation CRDC 5 22/07/54 cell Wild-type No salt FZP Wild-type 1.0M NaCl FZP Wild-type 1.5M KCl FZP FZP Fig. 4 Phenotypic analysis of wild-type and zinc cluster FZP strain were grown on media containing no salt, 1.0M NaCl or 1.5M KCl 21 Fig. 5 Schematic proposed model for transcriptional regulator named FZP is involved in heat tolerance or osmotic stress response in the yeast 22 11
Oral Presentation CRDC 5 22/07/54 Conclusion 1 1 Th The deletion FZP strain showed impaired growth at temperature d l ti FZP t i h d i i d th t t t of 39 O C but not 30 O C when compared with wild-type . 2 In the presence of salt, the deletion FZP strain also revealed impaired growth on media containing high salt concentration of 1 0M impaired growth on media containing high salt concentration of 1.0M NaCl or 1.5M KCl but not 0.5M LiCl 23 3 The zinc cluster transcriptional regulator named FZP may regulate genes encoding for enzymes involved in heat tolerance or salt stress response. 24 12
Oral Presentation CRDC 5 22/07/54 Expected benefit Our phenotypic analysis indicates new functions for zinc cluster transcription factor of S. cerevisiae and may be useful for designing new antifungal agent for control of fungal infection in economic plants. 25 Acknowledgements : Assoc Prof Dr Bernard Turcotte : Assoc.Prof.Dr. Bernard Turcotte : Assoc.Prof.Dr. Kanok Rattanakanokchai : Dr. Nitnipa Soontorngun 13
Oral Presentation CRDC 5 22/07/54 Thank you for your attention! 14
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