Supplementary Fig. S1 Alignment of the deduced protein of GhWRKY3 with other plant WRKY proteins. The amino acid sequences used in the alignment were GhWRKY3 (Gossypium hirsutum, FJ966887), NtWRKY2 (Nicotiana tabacum, AAD16139), AtWRKY3 (Arabidopsis thaliana, NP_178433), CaWRKY2 (Capsicum annuum, ABA56495), VvWRKY2 (Vitis vinifera, XP_002264243). The identical amino acids were shaded in black. The two conserved WRKY domains were underlined and the completely conserved WRKYGQK amino acids were boxed. The cysteine and histidine residues of the putative zinc finger motif were indicated by arrowheads (▼). The putative NLS was marked by asterisks (※).

Supplementary Fig. S2 Phylogenic analysis of WRKY proteins from different plants. The phylogenic tree was generated by MEGA4.1. The amino acid sequences of WRKY proteins used for construction of the phylogenic tree were listed: SPF1 (BAA06278) from Ipomoea batatas; NtWRKY1 (BAA82107), NtWRKY2 (AAD16139), NtWRKKY3 (BAA77358) from Nicotiana tabacum; NaWRKY6 (AAS13440) from Nicotiana attenuate; GhWRKY3 (FJ966887) from Gossypium hirsutum; CaWRKY2 (ABA56495) from Capsicum annuum; VvWRKY2 (XP_002264243) from Vitis vinifera; BnWRKY17 (ACQ76799), BnWRKY11-1 (ACI14384) from Brassica napus; AtWRKY4 (NP_172849), AtWRKY3 (NP_178433), AtWRKY11 (Nt_5677878), AtWRKY67 (NP_564877), AtWRKY41 (NP_192845), AtWRKY2 (NP_200438), AtWRKY7 (NP_194155) from Arabidopsis thaliana, PtWRKY7 (ACV92009), PtWRKY24 (ACV92026), PtWRKY27 (ACV92029), PtWRKY6 (ACV92008) from (Populus tomentosa x P. bolleana) × P. tomentosa; GmWRKY50 (ABS18446), GmWRKY20 (ABS18424) from Glycine max; VaWRKY30 (AAR92477) from Vitis aestivalis; OsWRKY15 (NP_001067197), OsWRKY34 (NP_001066844), OsWRKY74 (DAA05139) from Oryza sativa.

Supplementary Fig. S3 Nucleotide sequence of putative 5′-flanking region of GhWRKY3. The transcription and translation start sites were marked with arrows. The putative core promoter consensus sequences were highlighted as grey, and the putative cis-acting elements were boxed.

Supplementary Table S1. The primers used in the present study

Primer Name Sequence(5′-3′) Description
WP1
WP2
5WP2
5WP2
3WP1
3WP2
B26
B25
AAP
AUAP
WR1
WR2
WG1
WG2
SSU1
SSU2
WRT1
WRT2
WQ1
WQ2
TAQ1
TAQ2
WQF
WQN / GGNTAYAAYTGGMGNAARTAY
RTGYTTNCCYTCRTANGTNGTDAT
CGATTTCCAGCTACAACCAGAG
GACAGGGCAACTCGGATGTG
GGCAGAAAGTTGTCAAAGGG
GCAATGTTCGGAAGCATGTGG
GACTCTAGACGACATCGA(T)18
GACTCTAGACGACATCGA
GGCCACGCGTCGACTAGTAC(G)14
GGCCACGCGTCGACTAGTAC
CAGGTGGAAAAGAAAGAAGACAG
ATTCATTTCATACGCGGGTTAGTC
GGATCCAGGTGGAAAAGAAAGAAGACAG
CTCGAGCACTCTTATTTGCTCCTCTTT
AACTTAAAGGAATTGACGGAAG
CAAATCTGAACTTTCGTCTCTCC
GGGACGAAAGTTGTCAAAGGGAA
ATTCATTTCATACGCGGGTTAGTC
CACAGTGAACCAAGGAGAATTACC
CATGGGATTGTAAGGCAGTTTAG
TTTGCTTAACTCTCCTGGTCTC
GGATTGCTTAAGATTATGCTCTTTC
GTTGTATGTTGGAAAATATAGGC
TTCTTTCTTTTCCACCTGTTTT / Degenerate primer, Forward
Degenerate primer, Reverse
5′RACE reverse primer, outer
5′RACE reverse primer, nested
3′RACE forward primer, outer
3′RACE forward primer, nested
Universal adaptor primer, outer
Universal primer, nested
Abridged Anchor primer
Abridged Universal Amplification Primer
Full-length cDNA and genomic sequence
primer, forward
Full-length cDNA and genomic sequence
primer, reverse
Subcellular localization Primers, forward
Subcellular localization Primers, reverse
Standard control primer, forward
Standard control primer, reverse
RT-PCR primer, forward
RT-PCR primer, reverse
IPCR reverse primer, outer
IPCR reverse primer, nested
IPCR forward primer, outer
IPCR forward primer, nested
Promoter specific primer, forward
Promoter specific primer, reverse