Supplementary Material: File 2
Figure 1A. Pairwise sequence alignment of AdDR-13 and AdDR-60 partial cDNA nucleotide sequences.
Figure 1B. Pairwise sequence alignment of deduced amino acid sequence of AdDR-13 and AdDR-60 partial cDNA clones
Figure 2Nucleotide and deduced amino acid sequences of AdCyp. The nucleotides and amino acids are numbered. Asterisk (*) indicates the stop codon.
1 aaa acc cta aaa ttc tca tcc ttc ttc gtt gca ata cga atc gat 4546 cga ttt ctt ttc tct aca gca aaa ATG GCT AAC CCT AAG GTT TAC 90
M A N P K V Y 7
91 TTC GAC ATG TCC ATC GGA GGA CAA CCA GCC GGA AGA GTC GTC TTC 135
8 F D M S I G G Q P A G R V V F 22
136 GAG CTC TTC GCC GAC ACG GTT CCC CGC ACC GCC GAG AAT TTT CGG 180
23 E L F A D T V P R T A E N F R 37
181 GCC CTC TGC ACC GGT GAG AAA GGC GTC GGT CGC GGC GGC AAG CCT 225
38 A L C T G E K G V G R G G K P 52
226 CTC CAC TAC AAG GGA TCA TCC TTC CAC CGT GTG ATC CCT AAC TTC 270
53 L H Y K G S S F H R V I P N F 67
271 ATG TGT CAG GGA GGT GAC TTC ACC GCT GGA AAC GGC ACC GGA GGT 315
68 M C Q G G D F T A G N G T G G 82
316 GAG TCG ATC TAC GGC TCC AAG TTT GCC GAT GAG AAC TTT ATC AAG 360
83 E S I Y G S K F A D E N F I K 97
361 AAG CAC ACC GGT CCT GGG ATC CTT TCA ATG GCG AAT GCA GGA CCT 405
98 K H T G P G I L S M A N A G P 112
406 GGG ACG AAC GGA TCT CAG TTC TTC ATC TGC ACC TCG AAG ACG GAG 450
113 G T N G S Q F F I C T S K T E 127
451 TGG CTC GAC GGA AAG CAC GTG GTG TTC GGC CAA GTT GTT GAA GGA 495
128 W L D G K H V V F G Q V V E G 142
496 ATG GAC GTC GTT AAG GCG GTC GAG AAG GTT GGA TCT AGC TCC GGC 540
143 M D V V K A V E K V G S S S G 157
541 AAG ACC ACC AAA CCT GTT GTG ATC GCC GAT TGC GGT CAA CTC TCT 585
158 K T T K P V V I A D C G Q L S 172
586 TAG aac tat tgc gtt gat cgg aag ctc agt ctt ttt ccc gtg gtg 630
173 *
631 gtg tct ccc tct ttc tct ctg att ctc tct ctg aaa agt tga tgt 675
676 atg gta cta agc ggt gtc gtt ttg gtg ttt tca aat gat aat ctc 720
721 ttt tct gag tcg tgt aag ggt ttc gtg gtg atg tta ctc tct gcg 765
766 tag agt tga acc cgt ttc cca ttc gtc att taa cta gtg tct ttg 810
811 tga tga tga taa aca taa aac tga ttg agc tac ctt caa taa aat 855
856 tag gat tta cat caa aaa aaa aaa aaa aaa 886
Figure 3
5' upstream promoter region of AdCyp. Transcription start site was desiganated as (+1). Putative cis-regulatery elements were shaded and named. The arrows indicate the presence of cis-element on the positive or negative strand.
-841 ATTAATTTTTTTTGGCACAGGGGCCTCGTTGTCCTTTCGAGGTAATTGTCAGGGGCCGGG
-781 TTGGGTTTTTTTTTTTTGTCAGGGGCCGAGTTGGGATTTTTTTTTGTCAGGGGCCTCGTT
-721 GTCTTTCGAGGTAATTGTCAGGGCTGATTTGGGTTTTTCTCGAAAAGGGAATATCACTAC
-661 AGTTGTCTGTGCCATCTATAATCTTATACTATTTCACCATAAATCATTACTTTTGCTATT
-601 TATTTTTCAGTACTATGCCCGTTTTTTCGACGATTTTTGCCATAAAAAAATAACAAATCC
-541 CCTGACATATACCAGAGTTTCTAACGAATCACCACCGTTAGATTAAGCTCTCCATGTTGG
-481 TCCCTTCAAAATAAATCAACCACTGTTCACATTCTGGCACCACATGAGCTCCCGTACAAT
-421 CTTTCTGATACATCAAGCAACTCTCGCGCAACGGATATATCATGATCAATTTTCCTGGAT
-361 TATTTAAAAGTAGAAGAAAAATTTTCCTTTCATTTTTTTAAATCCAAAAGCATATATTAT
-301 ATATCTCTAGCTAAATAACATTAAAAAAGAAAAACAAATTAAACTATGCCTCGAAGCGGC
-241 GTCGTTTTCTGGGTGTCAAAGGGAAAAAAGGGCAAGTTGGGGCATAAAGAGTTAAAGACA
-181 AGAGCATATCCGTGTCCTCGGATCCGCTCCGCTAACCCTGGTCAATCCCGAACCCAACCA
-121 CAGTTACTTCACCTCACTCAAAACGTGCCACGTGTCCAAATCCAACGGCGACTTCTGAAG
-61 ATTTTCAGAACCACGAGCTACTATAAATATCAGTTACACTCCCCTCTGCACTCTCATAGT
-1 CAAAACCCTAAAATTCTCATCCTTCTTCGTTGCAATACGAATCGATCGATTTCTTTTCTC
+60 TACAGCAAAAATGGCTAACCCTAAGGTTACTTCGACATGT
Isolation of upstream promoter region
Upstream promoter region of AdCyp was cloned using universal genome walking kit (Clontech,USA) following manufacturer’s instructions. In brief,A. diogoi genomic DNA was digested with different blunt cutters like DraI, EcoRV, StuI and SspI. Genome walker adaptors were ligated to the DNA fragments. Primary PCR was performed using adaptor primer 1(AP1) and gene specific primer-1(GSP1). Diluted primary PCR products were amplified using adaptor primer-2(AP2) and gene specific primer 2(GSP2). The amplified products were run on 1.5% agarose gel, eluted, cloned into pTZ57R and sequenced. GSP1 and GSP2 primers were designed based on the AdCyp cDNA sequence (Supplementary File 2 Table 1). The upstream promoter region isolated was analyzed for potential cis-regulatory elements using PLACE database ( promoter sequence was submitted to the NCBI GenBank under the accession number HQ874666.
In silico anlysis of 5' upstream promoter region of AdCyp
Based on the sequence of the 5' RACE products, the putative transcription start site was identified and designated as +1(Supplementary Fig. 3). The transcription start site is present 69bp upstream of translation initiation codonATG. The sequence of AdCyp promoter region was analyzed for potential cis-acting regulatory elements using the PLACE database ( (Higo et al. 1999). A putative TATA box was found at –34 bp position upstream to the transcription start site. In silico analysis showed that several biotic and abiotic stress related cis-acting elements were overrepresented (Supplementary Fig. 3). Two W-BOX elements and several WRKY71 elements were found in the identified promoter region. W-BOX elements were recognizedspecifically by salicylic acid (SA)-induced WRKY DNA binding proteins.WRKY71 element is the core of TGAC-containing W-BOX. Several GT1GMSCAM4 elements were also present in the promoter region. Apart from these, there are ABRE elements known to be present in the ABA regulated genes and MYB and MYC transcription factor binding sites as well.
Construction of pRD400:AdCyp-GFP
The AdCyp ORF was amplified using primers ORF-F and ORF-R3 primers harboringNcoI and SmaI sites respectively and cloned into pTZ57R vector. Similarly GFP was amplified and cloned from the pEGAD vector using primers GFP-F and GFP-R with SmaI and NheI sites. A polyalanine linker sequence was inserted between AdCyp and GFP by incorporating the sequence in the GFP-F primer. AdCyp and GFPORFswere digested with corresponding restriction enzymes and cloned into the pRT100 vector digested with NcoI and XbaI. The resulting 35S:AdCyp:GFP cassette from pRT100 was digested with HindIII and subcloned into pRD400 digested with the same restriction enzyme to create the recombinant vector pRD400:AdCyp-GFP.
Figure 4 Molecularanalyses of T0AdCyp transgenic tobacco plants.
A. Southern analysis of T0trangenic plants. Genomic DNA of wild type (WT) and T0 transgenic plants were digested with Eco RI and electrophoresed, blotted and hybridized with [α-32P]–labelled nptII gene as probe
B. Semi-quantitative RT-PCR analysis of AdCyp expression in WT and transgenic plants. cDNA was synthesized from total RNA of WT and transgenic plants and amplified with AdCyp ORF-F and ORF-R primers. Actin served as internal control.
Figure 5
Leaf area damaged by the R. solani infection. Detached leaves of two month old WT and transgenic plants were used in the bioassay. R. solani was grown on PDA and a mycelial plug of 0.5 cm was placed on leaves after making a cut with a razor blade to facilitate fungal infection and maintained in growth room as described in materials and methods (main text). Seven days post inoculation, the diameter of the disease lesion/ leaf damage was measured. Data represent the mean values ± SE of three experimental replications. Asterisk indicates significant difference between WT and transgenic plants (*P<0.05)
Figure 6
Semi-quantitative RT-PCR analysis of AdCyp and actin in mock treatments.
Supplementary Table 1
Name of the Primer / Primer Sequence (5’-3’)Cyp262R / CTT AAC GAC GTC CAT TCC TTC
Cyp141R / ATT GAA AGG ATC CCA GGA CC
ORF-F / GCCATGGCTAACCCTAAGGTTTAC
ORF-R / GTCTAGACTAAGAGAGTTGACCGCAATC
GSP1 / GGAAGGATGATCCCTTGTAGTGGAGA
GSP2 / ACATGTCGAAGTAAACCTTAGGGTTA
AdCyp -841F / GGAATTCATTAATTTTTTTTGGCACAGGG
ORF-F2 / GCCCGGGATGGCTAACCCTAAGGTTTAC
ORF-R2 / CAAGCTTCTAAGAGAGTTGACCGCAATC
ORF-R3 / GCCCGGGAGAGAGTTGACCGCAATCGG
GFP-F / GCCCGGGGCTGCGGCCGCTGCCGCTGCGGCAGCGGCC ATGGTGAGCAAGGGCGAGG
GFP-R / CGCTAGCCTATCCGGACTTGTACAGCTCG
AdDR13-F / CAAGAAGCACACCGGTCCT
AdDR13-R / ATCATCATCACAAAGACACTAG
Actin F / TGGCATCACACTTTCTACAA
Actin R / CAACGGAATCTCTCAGCTCC
Oligo Name / Forward / Reverse
PI-I / ATGGTGAAGTT TGCTCACGT / AATCCCTTAGCCAACCTGG
PR1a / CTTCTTGTCTCTACACTTCTC / GCAAGAGACAACATATCCTC
PR1b / TCTTAACCCTCACAATGCAG / AGGGTTGCTCCTCAAGATC
LOX1 / CACTTCCTACTGATCTCATC / CTCATCGACATTCATCTGCA
LOX3 / aatgacagagaactccaagc / tagaacgcttcgacaatctc
Glucanase / ATGGCTTTATGCATTAAAAATGGC / AGCATTGAAGACATTTGTTTCTGG
Chitinase / CTGAAGAATAGGAACGACGGTAG / ATACCTCCTGTAGTATCCAATTCG
Osmotin / ACTTGAGATCTTCTTTTGTTTTCTTCC / ACTTCCAGGCATTTCCAAGGGAAAATTT
Defensin / GAGGCAAGAACTTGTGAGTC / AAGCCGAAACCATTATTCATAAC
PR1a (X12737), PR1b (X14065), PI-I (Z12623), LOX1 (X84040), LOX3 (AY254349), Glucanase (X69794), Chitinase (X51425), Osmotin (S44889), Defensin (X99403)
References
Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database. Nucleic Acids Res 27:297-300
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