Plant Pathogens Plant Pathogens 2 1 22/07/54 Oral Presentation - PDF document

22/07/54 Oral Presentation CRDC 5 Re gulator y c ontr ol of he xose tr anspor te r ge ne e ssion in the ye ast Sacchar omyce s ce r e visiae e xpr Bar ame e , S. and Soontor ngun, N. Bioc he mic al T e c hnology, Sc hool of Bior e sour c e s and T e c hnology King Mongkut’s Unive r sity of T e c hnology T honbur i CRDC 5: July, 22, 2011 1 Plant Pathogens Plant Pathogens 2 1

22/07/54 Oral Presentation CRDC 5 Co lle to tric hum Co lle to tric hum g raminic o la g raminic o la o Caused anthracnose of maize and wheat i d h t o After 48-72 hours; � necrotrophic development � breaching the plant plasma membrane l b � gaining access to cell wall- derived Co lle to tric hum g raminic o la � Absorb carbon source 3 Role of Role of melibiose melibiose transporter gene ( transporter gene ( MBT 1 ) ) MBT 1 Δ mbt1 mutants are unable to grow on melibiose. (Lingner et al., 2011) 4 2

22/07/54 Oral Presentation CRDC 5 Role of Role of melibiose melibiose transporter gene ( transporter gene ( MBT 1 ) ) MBT 1 Δ mbt1 mutants do not show virulence defects on maize. (Lingner et al., 2011) 5 S S ac c haro myc e s c e re visiae ac c haro myc e s c e re visiae o model organism d l i o utilizes glucose as the main fermentable carbon source o can adapts p to glucose g exhaustion through various mechanisms. S ac c haro myc e s c e re visiae (MacPherson et al., 2006) 6 3

22/07/54 Oral Presentation CRDC 5 Zinc cluster Zinc cluster proteins proteins o transcription factors that found exclusively in fungi o consensus sequence Cys-X 2 -Cys-X 6 -Cys-X 5–12 - Cys X Cys X Cys-X 2 -Cys-X 6–8 -Cys Cys Gal4p o over 50 members in S. cerevisiae (MacPherson et al., 2006) 7 Rds2 Rds 2 regulator regulator o Regulator of drug sensitivity o Direct binding to a key gluconeogenic pathway called PCK1 in glucose o When the carbon source was shifted from glucose to alternative carbon sources glucose to alternative carbon sources, the the binding of Rds2 on many promoters are dramatically altered. (Soontorngun et. at, 2007) 8 4

22/07/54 Oral Presentation CRDC 5 Hexose Transporter Hexose Transporter genes genes o The hexose transporters are large integral membrane proteins. ac c haro myc e s c e re visiae has 20 genes that o S encode proteins similar to glucose (hexose) transporters ( HXT 1 to HXT 17 , GAL 2 , S 3 , and RGT 2 ) NF o The multiple pathways regulate the transcription of several members of the HXT gene family in response to different levels of extracellular glucose (Özcan and Johnston, 1999) 9 Induction of HXT Induction of 4 genes transcription genes transcription HXT 2 2/ / HXT HXT 4 by different levels of glucose by different levels of glucose o Induction of Transcription by Low Levels of Glucose o In the absence of glucose; are repressed by Rgt1 o On high glucose concentrations; are repressed by Mig1 (Özcan and Johnston, 1999) 10 5

22/07/54 Oral Presentation CRDC 5 Regulatory network of Regulatory network of nonfermentable nonfermentable carbon utilization carbon utilization (Turcotte et al, 2010) 11 Role of HXT Role of HXT9 9/HXT /HXT11 11 genes genes 9 and HXT 9 and HXT 11 , which are nearly identical 11 , which are nearly identical o HXT o HXT o may play a central role in pleiotropic drug resistance o deletion of and/or causes HXT 9 HXT 11 resistance to several drugs resistance to several drugs 11 and HXT 9 might be directly involved o HXT in uptake of drugs (Özcan and Johnston, 1999) 12 6

22/07/54 Oral Presentation CRDC 5 Research Research Ploblems Ploblems It is unknown regarding the involvement of Rds2 in the regulation of HXT 9 and HXT 11 during growth on non-fermentable carbon. 13 Objectives Objectives To T examine i the th i importance t of f Rd 2 Rds2 regulator on the expression of HXT 9 and HXT 11 genes 14 7

22/07/54 Oral Presentation CRDC 5 Strains and Strains and media media B 4741 By4741 Sacchar omyce s ce r e visiae Δ r ds2 1% Ye ast e xtr ac t 2% Pe ptone Gluc ose 2% Car bon sour c e E thanol 15 Gene induction Gene induction overnight 5 hours 3 hours YP YP YP Glucose Glucose Ethanol Purified RNA RNA purification RNA extraction (Hot phenol method ) 16 8

22/07/54 Oral Presentation CRDC 5 RT RT- -PCR analysis PCR analysis RNA primer 17 Result from RT Result from RT- -PCR equipment PCR equipment Table 1 The expression of HXT genes under different growth conditions diti Gr owth c ondition Gluc ose HXT 9 HXT 11 Ge ne s T otal Ave r age F old Change 0.8 0.7 sus Δ r ds2 str (Wild-type ve r ain) 18 9

22/07/54 Oral Presentation CRDC 5 Result Result from from PCR product gel PCR product gel 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 10 11 12 13 Lane 1 = 1 kb Ladder marker Lane 2-3 = Wild-type ACT 1 gene Lane 4-5 = Δ rds2 ACT 1 gene Lane 6-7 = Wild-type HXT 9 gene Lane 8-9 = Δ rds2 HXT 9 gene Lane 10-11 = Wild-type HXT 11 gene Lane 12-13 = Δ rds2 HXT 11 gene e 1 The PCR products obtained from RT-PCR analysis during F igur growth in glucose 19 Result from RT Result from RT- -PCR equipment PCR equipment Table 2 The expression of HXT genes under different growth conditions diti Gr owth c ondition E thanol HXT 9 HXT 11 Ge ne s T otal Ave r age F old Change 1.5 1.6 sus Δ r Δ d 2 t (Wild t (Wild-type ve r ds2 str ain) i ) 20 10

22/07/54 Oral Presentation CRDC 5 Result from PCR product gel Result from PCR product gel 1 2 3 4 5 6 7 8 9 10 11 12 13 Lane 1 = 1 kb Ladder marker Lane 2-3 = Wild-type ACT 1 gene Lane 4-5 = Δ rds2 ACT 1 gene Lane 6-7 = Wild-type HXT 9 gene Lane 8-9 = Δ rds2 HXT 9 gene Lane 10-11 = Wild-type HXT 11 gene Lane 12-13 = Δ rds2 HXT 11 gene e 2 The PCR products obtained from RT-PCR analysis during F igur growth in ethanol 21 Discussion Discussion o Rds2 is not required for expression of HXT9 and HXT11 in glucose o Rds2 regulator acts as positive regulator of both HXT9 and HXT11 genes in ethanol. o This agrees well with increased binding of Rds2 regulator at these HXT promoters in ethanol (Soontorngun et. at, 2007) o The expression of HXT9 and HXT11 genes are required to transport glucose into cells 22 11

22/07/54 Oral Presentation CRDC 5 Conclusion Conclusion HXT 9/ HXT 11 The transcriptional High glucose Low glucose regulator Rds2 is important for induction of these two ? Hxt Hxk2 hexose transporter genes in High signal Low signal ethanol. Reg1 Sak1/T os3/Elm1 Snf1 Mig1 Rds2 HXT9/11 23 Expected benefit Expected benefit The role of Rds2 homolog in hexose transport and virulence in C. g raminic o la is highly interesting and may be useful for improvement of crop quality and yield. 24 12

22/07/54 Oral Presentation CRDC 5 Dr. Bernard Turcotte -McGill Dr. Khanok Ratanakhanokchai (Assoc. Prof.) -KMUTT Dr. Nitnipa Soontorngun -KMUTT CRDC 5 Organizing committee 25 26 13