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

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

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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

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Oral Presentation CRDC 5 22/07/54 1

Re gulator y c ontr

  • l of he xose tr

anspor te r ge ne e xpr e ssion in the ye ast Sacchar

  • myce s ce r

e visiae

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

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Plant Pathogens Plant Pathogens

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Oral Presentation CRDC 5 22/07/54 2

Co lle to tric hum Co lle to tric hum g raminic o la g raminic o la

  • Caused anthracnose of

i d h t maize and wheat

  • After 48-72 hours;

necrotrophic development breaching the plant

l b plasma membrane

gaining access to cell wall-

derived

Absorb carbon source

Co lle to tric hum g raminic o la

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Role of Role of melibiose melibiose transporter gene ( transporter gene (MBT MBT 1 1) )

(Lingner et al., 2011)

Δmbt1 mutants are unable to grow on melibiose.

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Oral Presentation CRDC 5 22/07/54 3

Role of Role of melibiose melibiose transporter gene ( transporter gene (MBT MBT 1 1) )

(Lingner et al., 2011)

Δmbt1 mutants do not show virulence defects on maize.

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S ac c haro myc e s S ac c haro myc e s c e re visiae c e re visiae

d l i

  • model organism
  • utilizes glucose as the main

fermentable carbon source

  • can

adapts to glucose p g exhaustion through various mechanisms.

S ac c haro myc e s c e re visiae (MacPherson et al., 2006)

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Oral Presentation CRDC 5 22/07/54 4

Zinc cluster Zinc cluster proteins proteins

  • transcription factors that

found exclusively in fungi

  • consensus sequence

Cys-X2-Cys-X6-Cys-X5–12- Cys X Cys X Cys Cys-X2-Cys-X6–8-Cys

  • over 50 members in S.

cerevisiae

Gal4p

(MacPherson et al., 2006)

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Rds Rds2 2 regulator regulator

  • Regulator of drug sensitivity
  • Direct

binding to a key gluconeogenic pathway called PCK1 in glucose

  • When the carbon source was shifted from

glucose to alternative carbon sources the glucose to alternative carbon sources, the binding of Rds2 on many promoters are dramatically altered.

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(Soontorngun et. at, 2007)

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Oral Presentation CRDC 5 22/07/54 5

Hexose Transporter Hexose Transporter genes genes

  • The hexose transporters are large integral

membrane proteins.

  • S

ac c haro myc e s c e re visiae has 20 genes that encode proteins similar to glucose (hexose) transporters (HXT 1 to HXT 17, GAL 2, S NF 3, and RGT 2)

  • 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 Induction of HXT HXT 2 2/ HXT / HXT 4 4 genes transcription genes transcription by different levels of glucose by different levels of glucose

  • Induction of Transcription by Low

Levels of Glucose

  • In the absence of glucose;

are repressed by Rgt1

  • On high glucose concentrations;

are repressed by Mig1

(Özcan and Johnston, 1999)

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Oral Presentation CRDC 5 22/07/54 6

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

  • HXT

9 and HXT 11, which are nearly identical

  • HXT

9 and HXT 11, which are nearly identical

  • may play a central role in pleiotropic drug

resistance

  • deletion
  • f

HXT 9 and/or HXT 11 causes resistance to several drugs resistance to several drugs

  • HXT

11 and HXT 9 might be directly involved in uptake of drugs

(Özcan and Johnston, 1999)

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Oral Presentation CRDC 5 22/07/54 7

Research Research Ploblems Ploblems

It is unknown regarding the involvement

  • f Rds2 in the regulation of HXT

9 and HXT 11 during growth on non-fermentable carbon.

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Objectives Objectives

T i th i t f Rd 2 To examine the importance

  • f

Rds2 regulator on the expression of HXT 9 and HXT 11 genes

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Oral Presentation CRDC 5 22/07/54 8

Strains and Strains and media media

B 4741

1% Ye ast e xtr ac t

Sacchar

  • myce s ce r

e visiae

By4741

Δr

ds2

2% Pe ptone 2% Car bon sour c e Gluc ose E thanol

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Gene induction Gene induction

3 hours

  • vernight

5 hours

YP Glucose YP Glucose YP Ethanol

RNA extraction (Hot phenol method ) RNA purification Purified RNA

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Oral Presentation CRDC 5 22/07/54 9

RT RT-

  • PCR analysis

PCR analysis

RNA primer 17

Result from RT Result from RT-

  • PCR equipment

PCR equipment

Table 1 The expression of HXTgenes under different growth diti

Gr

  • wth c ondition

Gluc ose Ge ne s

HXT 9 HXT 11

T

  • tal Ave r

age F

  • ld Change

0.8 0.7 conditions

(Wild-type ve r sus Δr

ds2 str

ain)

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Oral Presentation CRDC 5 22/07/54 10

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 = Δrds2 ACT 1 gene Lane 6-7 = Wild-type HXT 9 gene Lane 8-9 = Δrds2 HXT 9 gene Lane 10-11 = Wild-type HXT 11 gene Lane 12-13 = Δrds2 HXT 11 gene

F igur e 1 The PCR products obtained from RT-PCR analysis during

growth in glucose

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Result from RT Result from RT-

  • PCR equipment

PCR equipment

Table 2 The expression of HXTgenes under different growth diti

Gr

  • wth c ondition

E thanol Ge ne s

HXT 9 HXT 11

T

  • tal Ave r

age F

  • ld Change

(Wild t Δ d 2 t i )

1.5 1.6 conditions

(Wild-type ve r sus Δr

ds2 str

ain)

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Oral Presentation CRDC 5 22/07/54 11

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 = Δrds2 ACT 1 gene Lane 6-7 = Wild-type HXT 9 gene Lane 8-9 = Δrds2 HXT 9 gene Lane 10-11 = Wild-type HXT 11 gene Lane 12-13 = Δrds2 HXT 11 gene

F igur e 2 The PCR products obtained from RT-PCR analysis during

growth in ethanol

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Discussion Discussion

  • Rds2 is not required for expression of HXT9 and HXT11 in

glucose

  • Rds2 regulator acts as positive regulator of both HXT9

and HXT11 genes in ethanol.

  • This agrees well with increased binding of Rds2 regulator

at these HXT promoters in ethanol (Soontorngun et. at, 2007)

  • The expression of HXT9 and HXT11 genes are required to

transport glucose into cells

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Oral Presentation CRDC 5 22/07/54 12

Conclusion Conclusion

The transcriptional

HXT 9/ HXT 11

regulator Rds2 is important for induction of these two hexose transporter genes in ethanol.

High glucose Hxt Low glucose

?

Hxk2 High signal Low signal Reg1 Sak1/T

  • s3/Elm1

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Snf1 Mig1 Rds2 HXT9/11

Expected benefit Expected benefit

The role

  • f

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.

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Oral Presentation CRDC 5 22/07/54 13

  • Dr. Bernard Turcotte -McGill
  • Dr. Khanok Ratanakhanokchai (Assoc. Prof.) -KMUTT
  • Dr. Nitnipa Soontorngun -KMUTT

CRDC 5 Organizing committee

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