regulation of arabidopsis tga transcription factors by cysteine ...

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GAL4 DB:TGA1 GAL4 DB:TGA2 GAL4 DB:TGA1/ TGA2 30aa/TGA1 GAL4 DB:TGA1 C260N GAL4 DB:TGA1 C266S Transactivation Interaction with GAL4 DB:NPR1 GAL4 TA:TGA1 GAL4 TA:TGA2 GAL4 TA:TGA1/ TGA2 30aa/TGA1 GAL4 TA:TGA1 C260N GAL4 TA:TGA1 C266S Figure 4.1 Transactivation and NPR1-interaction properties of TGA1 containing mutations within the 30 amino acid region. β-galactosidase assays were performed on yeast cells transformed with TGA constructs fused to the domains indicated. Labels at top indicate the property tested. Each spot represents an independent transformation event. A blue colour observed with GAL4:DB fusions to TGA factors indicates autonomous transactivation of the construct. Blue colour observed with GAL4:TA fusions of TGA factors in cells also containing DB:NPR1 indicates interaction between the test protein pair. 38
TGA1 M-----NSTSTHFVPPRRVGIYEPVHQFGMWGE-----SFKSNISNGTMNTPNHII--IPNNQKLDNNVSED----TSHGTAGTPHMFDQEASTSRHPDK 84 TGA4 M-----NTTSTHFVPPRRFEVYEPLNQIGMWEE-----SFKNN---GDMYTPGSII--IPTNEK-PDSLSED----TSHGTEGTPHKFDQEASTSRHPDK 80 TGA2 MA------------------------------------------------------DTSPRTDVSTDDDTDHPDLGSEGALVNTAASDSSDRSKGKMDQK 46 TGA6 MH------------------------------------------------------SLN--ETVIPDVDYMQSDRGH----MHAAASDSSDRSKDKLDQK 40 TGA5 ------------------------------------------------------------RTSVSTDGDTDHNNLMFDEGHLGIGASDSSDRSKSKMDQK 40 TGA3 MEMMSSSSSTTQVVSFRDMGMYEPFQQLSGWESPFKSDINNITSNQNNNQSSSTTLEVDARPEADDNN-RVNYTSVYNNSLEAEPSSNNDQDED-RINDK 98 TGA7 MM---SSSSPTQLASLRDMGIYEPFQQIVGWGNVFKSDIND----HSPNTATSSIIQVDPRIDDHNNNIKINYDSSHNQIEAEQPSSNDNQDDDGRIHDK 93 TGA1 IQRRLAQNREAARKSRLRKKAYVQQLETSRLKLIQLEQELDRARQQGFYVGNGIDTNSLGFSETMNPGIAAFEMEYGHWVEEQNRQICELRTVLHGHIND 184 TGA4 IQRRLAQNREAARKSRLRKKAYVQQLETSRLKLIHLEQELDRARQQGFYVGNGVDTNALSFSDNMSSGIVAFEMEYGHWVEEQNRQICELRTVLHGQVSD 180 TGA2 TLRRLAQNREAARKSRLRKKAYVQQLENSRLKLTQLEQELQRARQQGVFISGTGDQAH---STGGNG-ALAFDAEHSRWLEEKNKQMNELRSALNAHAGD 142 TGA6 TLRRLAQNREAARKSRLRKKAYVQQLEDSRLKLTQVEQELQRARQQGVFISSSGDQAH---STGGNGGALAFDAEHSRWLEEKNRQMNELRSALNAHAGD 137 TGA5 TLRRLAQNREAARKSRLRKKAYVQQLENSRLKLTQLEQELQRARQQGVFISSSGDQAH---STAGDG-AMAFDVEYRRWQEDKNRQMKELSSAIDSHATD 136 TGA3 MKRRLAQNREAARKSRLRKKAHVQQLEESRLKLSQLEQELVRARQQGLCVRNSSDTSYLGPAGNMNSGIAAFEMEYTHWLEEQNRRVSEIRTALQAHIGD 198 TGA7 MKRRLAQNREAARKSRLRKKAYVQQLEESRLKLSQLEQELEKVKQQG----------HLGPSGSINTGIASFEMEYSHWLQEQSRRVSELRTALQSHISD 183 TGA1 IELRSLVENAMKHYFELFRMKSSAAKADVFFVMSGMWRTSAERFFLWIGGFRPSDLLKVLLPHFDVLTDQQLLDVCNLKQSCQQAEDALTQGMEKLQHTL 284 TGA4 IELRSLVENAMKHYFQLFRMKSAAAKIDVFYVMSGMWKTSAERFFLWIGGFRPSELLKVLLPHFDPLTDQQLLDVCNLRQSCQQSEDALSQGMEKLQHTL 280 TGA2 SELRIIVDGVMAHYEELFRIKSNAAKNDVFHLLSGMWKTPAERCFLWLGGFRSSELLKLLANQLEPMTERQLMGINNLQQTSQQAEDALSQGMESLQQSL 242 TGA6 TELRIIVDGVMAHYEELFRIKSNASKNDVFHLLSGMWKTPAERCFLWLGGFPSSELLKLLANQLEPMTERQVMGINSLQQTSQQAEDALSQGMESLQQSL 237 TGA5 SELRIIVDGVIAHYEELYRIKGNAAKSDVFHLLSGMWKTPAERCFLWLGGFRSSELLKLIACQLEPLTEQQSLDINNLQQSTQQAEDALSQGMDNLQQSL S 236 TGA3 IELKMLVDSCLNHYANLFRMKADAAKADVFFLMSGMWRTSTERFFQWIGGFRPSELLNVVMPYVEPLTDQQLLEVRNLQQSSQQAEEALSQGLDKLQQGL 298 TGA7 IELKMLVESCLNHYANLFQMKSDAAKADVFYLISGMWRTSTERFFQWIGGFRPSELLNVVMPYLQPLTDQQILEVRNLQQSSQQAEDALSQGIDKLQQSL 283 TGA1 ADC -RTVAAGQ--LGEGS----YIPQVNSAMDRLEALVSFVNQADHLRHETLQQMYRILTTRQAARGLLALGEYFQRLRALSSSWATRHREPT 367 TGA4 AESVAAGK--LGEGS----YIPQMTCAMERLEALVSFVNQADHLRHETLQQMHRILTTRQAARGLLALGEYFQRLRALSSSWAARQREPT 364 TGA2 ADTLSSGTLGSSSSGNVASYMGQMAMAMGKLGTLEGFIRQADNLRLQTLQQMIRVLTTRQSARALLAIHDYFSRLRALSSLWLARPRE 330 TGA6 ADTLSSGTLGSSSSDNVASYMGQMAMAMGKLGTLEGFIRQADNLRLQTLQQMLRVLTTRQSARALLAIHDYSSRLRALSSLWLARPRE 325 TGA5 ADTLSSGTLGSSSSGNVASYMGQMAMAMGKLGTLEGFIRQADNLRLQTYQQMVRLLTTRQSARALLAVHNYTLRLRALSSLWLARPRE 324 TGA3 VESIAIQIKVVESVN----HGAPMASAMENLQALESFVNQADHLRQQTLQQMSKILTTRQAARGLLALGEYFHRLRALSSLWAARPRE--H 383 TGA7 AESIVIDA-VIESTH----YPTHMAAAIENLQALEGFVNQADHLRQQTLQQMAKILTTRQSARGLLALGEYLHRLRALSSLWAARPQEPT. 369 Figure 4.2 Alignment of seven of the Arabidopsis TGA factors. Dashed and solid blue lines indicate the basic and leucine zipper domains respectively. The dark purple box indicates cysteines of TGA1 and TGA4 at positions 172 and 168 respectively. The blue/green closed box indicates cysteine of TGA2 at position 186. The green closed box indicates residues or TGA1, TGA4, and TGA2 at positions 260, 256, and 218 respectively. The orange closed box indicates residues of TGA1, TGA4, and TGA2 at positions 266, 262, and 224 respectively. The yellow closed boxes indicate residues of TGA1 and TGA2 at positions 287 and 245 respectively. The light purple closed box and solid lines indicate regions used to mark changes for chimeric proteins. The red open box indicates the 30 amino acid span between position 236 and 266 of TGA1 that was replaced with the corresponding region from TGA2. Dashes indicate gaps introduced to facilitate proper alignment. 39
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 and 48: fluorescence by a Perkin Elmer LS50
Page 49: CHAPTER 4: RESULTS 4.1 Role of cyst
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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