regulation of arabidopsis tga transcription factors by cysteine ...

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Sd is the modified standard deviation that incorporates size and standard deviations of both of the sample groups. In this study, the 10 degrees of freedom and a 95% confidence level (p = 0.05) was used to determine if the activity observed in the yeast cells was significantly different. 36
CHAPTER 4: RESULTS 4.1 Role of cysteine residues in controlling transactivation by TGA1/TGA4 and the interaction of TGA factors with NPR1 4.1.1 Role of TGA1 cysteines within the 30 amino acid domain Previous research identified a 30 amino acid region of TGA2 that, when used to replace the corresponding sequences from TGA1, enabled the resulting chimeric protein to interact with NPR1 while abolishing transactivation in yeast (Després et al., 2003; see also Figure 4.1 and section 2.6.1). To identify the residues within the 30 amino acid region that are important for these functions, a rational site-directed mutagenesis approach was taken based on a multiple alignment of seven Arabidopsis TGA factors between the region corresponding to amino acids 236 to 266 of TGA1 (Figure 4.2). The selection criteria for targeting an amino acid were that the residue needed to be conserved between TGA1 and TGA4 but had to differ from that of the conserved residues in the remaining TGA factors analyzed. Cysteine 266 (Cys-266) is the only residue that conforms to these criteria (Figure 4.2). It is conserved between TGA1 and TGA4, while in all other TGA factors a conserved serine (Ser) residue is present at the corresponding position. Since Cys residues possess the unique ability to form a bond between their sulfhydryl side chains, called a disulfide bond, the other Cys residue found within the 30 amino acid region, at residue 260 (Cys-260), was also targeted for mutagenesis. Cys-260 conforms to the first criterion as it is conserved between TGA1 37
Page 1 and 2: REGULATION OF ARABIDOPSIS TGA TRANS
Page 3 and 4: ACKNOWLEDGEMENTS I would like to th
Page 5 and 6: argument that oxidoreduction of thi
Page 7 and 8: 3.2.5.2 Electroporation of electro-
Page 9 and 10: LIST OF FIGURES CHAPTER 2: LITERATU
Page 11 and 12: GSSG oxidized glutathione GUS β-gl
Page 13 and 14: CHAPTER 1: INTRODUCTION All plants
Page 15 and 16: 2.1 Induced disease resistance CHAP
Page 17 and 18: PR genes like acd2 (accelerated cel
Page 19 and 20: localization of NPR1, which is medi
Page 21 and 22: expressed under abiotic stress and
Page 23 and 24: TGA2 TGA6 TGA5 TGA1 TGA4 TGA3 TGA7
Page 25 and 26: overexpression of a single member o
Page 27 and 28: closely related tobacco gene TGA2.1
Page 29 and 30: maintaining the reducing environmen
Page 31 and 32: the cell wall, HR, as well as act a
Page 33 and 34: consisted of the N-terminus of TGA2
Page 35 and 36: 3.2.2 Bacterial media Liquid and so
Page 37 and 38: culture volume of cold sterile 10%
Page 39 and 40: 3.3.2 Yeast Plasmids The yeast plas
Page 41 and 42: 20 µl. The ligation mixture was in
Page 43 and 44: Table 3.3 Oligonucleotides used for
Page 45 and 46: oligonucleotides to amplify from th
Page 47: fluorescence by a Perkin Elmer LS50
Page 51 and 52: TGA1 M-----NSTSTHFVPPRRVGIYEPVHQFGM
Page 53 and 54: Description Transactivation Wild ty
Page 55 and 56: of the best characterized targets o
Page 57 and 58: To ensure that GUS activity observe
Page 59 and 60: The codon for Cys at position 256 o
Page 61 and 62: construct showed a slight decrease
Page 63 and 64: not significantly different to that
Page 65 and 66: 4.2 Attempts to alter transactivati
Page 67 and 68: pBI881 and transformed them into ye
Page 69 and 70: Description Transactivation Wild ty
Page 71 and 72: CHAPTER 5: DISCUSSION Following the
Page 73 and 74: protein, interacted with NPR1 in ye
Page 75 and 76: TGA1/TGA4. These results indicate t
Page 77 and 78: Pathogen S S HS HS TGA1 TGACG NPR1
Page 79 and 80: TGA1 C260S SDMs display a great dec
Page 81 and 82: protein to interact with NPR1. The
Page 83 and 84: preferred partner of Cys-260 in dis
Page 85 and 86: incorporated the corresponding Cys
Page 87 and 88: CHAPTER 6: REFERENCES Alvarez,M.E.,
Page 89 and 90: Friedrich,L., Lawton,K., Dietrich,R
Page 91 and 92: Mahoney,C.W., Pak,J.H., and Huang,K
Page 93 and 94: Sticher,L., Mauch-Mani,B., and Metr
Page 95: Zimmerli,L., Jakab,G., Metraux,J.P.

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