Transcriptome profiling identified multiple jasmonate ZIM-domain proteins involved in the regulation of alkaloid biosynthesis in tobacco BY-2 cells
Yuping Yang, Jing Guo, Pengcheng Yan, Ping Gao, Heping Zhao, Yubao Chen, Yingdian Wang, Michael P. Timko and Shengcheng Han
Table S1. Primer sequences used for cloning the NtJAZ genes from tobacco BY-2 cells.
Gene name / Primer sequence (5’→3’)NtJAZ1 FR / CACCATGGGGTCATCGGAGATTGTAGA
NtJAZ1 RR / CTAAAAGAACTGCTCAGTTTTCAC
NtJAZ2b FR / CACCATGGAGAGAGATTTCATGGG
NtJAZ2b RR / GGTCTCCTTACCTGCTATCA
NtJAZ3 FR / CACCATGGCATCATCGGAGATTGTGGA
NtJAZ3 RR / CTAGAATTGCTCAGCTTTCACTGG
NtJAZ3b FR / CACCATGGTGGATTCCGGTAGATTCGC
NtJAZ3b RR / CTAGAATTGCTCAGTTTTCACTGG
NtJAZ5 FR / CACCATGATGCTTGAAAAGCAAGG
NtJAZ5 RR / ATTGGTAGCAGGAAGAACAG
NtJAZ7a FR / CACCATGCAGTGGTCATTCTCGAACAA
NtJAZ7a RR / TCACGTCTCCTTGACCAAATTG
NtJAZ10 FR / CACCATGTCAAATTCGCAAAATTCTTTT
NtJAZ10 RR / CTATAACTTGAAATTGAGATCGAGT
NtJAZ11b FR / CACCATGAAGCACAGAATTGGCCT
NtJAZ11b RR / GTATGGATTAGCTGCTTGAATC
NtJAZ12b FR / CACCATGAGAAGAAACTGTAACTT
NtJAZ12b RR / GTGATGATAAGGAGAAGTTGCT
Table S2. Primer sequences used in RT-PCR and qRT-PCR experiments.
Gene name / Primer sequence (5’→3’)NtJAZ1 FR / CGAACTCACGTGCCAATATC
NtJAZ1 RR / CACCTAATCCAAGCCATGC
NtJAZ2b.2 FR / GGCGGGTATACCTCATTTCA
NtJAZ2b.2 RR / TGCATGCACATTCTGGATTT
NtJAZ3 FR / GGTTCCTTTGGAGATCTCAGC
NtJAZ3 RR / GACCGCCGTAGAATATCGTC
NtJAZ3b FR / ACGATATTCTACGGCGGTCA
NtJAZ3b RR / TAGGCTGAGGCACCAAATCT
NtJAZ5 FR / CATGGTCGTCAGCCCTTAAT
NtJAZ5 RR / TCAGTAGTGCCCACAACACC
NtJAZ7a FR / GCTGTCGACTCAAGTCATCG
NtJAZ7a RR / GGAAACTGGGAGCACTCTGA
NtJAZ10 FR / GCACCACAACAACAAAAGCA
NtJAZ10 RR / GCGACGCAGAAGAAGTTGAT
NtJAZ11b FR / GGCCTTCAACTTTTCCCTCT
NtJAZ11b RR / TGCATGGCATAAGATGGCTA
NtJAZ12b FR / GAGAAGCAACAACAGCTAACCA
NtJAZ12b RR / GACGAAGGCTCTGAAATTGG
ACTIN FR / AGCACCTCTTAACCCGAAGG
ACTIN RR / GGACAGTGTGGCTAACACCA
NtPMT1a FR / TATGCACACAGGCTGAAAGC
NtPMT1a RR / AGTCAACTTCTGGCCCTTCA
NtADC FR / GGTGGCCTTGGAATTGACTA
NtADC RR / ACGTTCTTCCGGTCACAAAC
NtODC FR** / ATTGGACCCAGCAGCTTTAG
NtODC RR** / GTTTCCGACGACTGTGTTTG
NtMPO1 FR / GGCACCTTCTCCTTCTCCTT
NtMPO1 RR / CCGTCTTTTTCTGCTTCTCG
A622 FR* / CATAGCGACATACACTATCG
A622 RR* / GGCATATGGCCAAATTAGTC
NtQPT2 FR / AGAGGTGAAACCACCAGCAC
NtQPT2 RR / TCCCGTCTTCCTTTGCTAGA
NtERF189 FR* / GCAGCTTCGACTGCAGCTTCCT
NtERF189 RR* / CTCCTCGGACTCGGAGCACTTC
NtMYC2a FR** / TCACCCATTTCTCTCTCTCTCTC
NtMYC2a RR** / GAGGTAACAGCAGCAGTAGTAG
NtPI-II FR*** / GGGGAGCCTCAAAGTGCTGC
NtPI-II RR*** / CAGAGTTTAGCATGACAGTGC
* Primers have been described (Shoji, Kajikawa & Hashimoto, 2010).
** Primers have been described(Zhang, Bokowiec, Rushton, Han & Timko, 2012)
***. Primers were designed according to the DNA sequence of tobacco proteinase inhibitor II (accession number: Z29537)
Table S3. Primer sequences used for the construction of RNAi constructs.
Gene name / Primer sequence (5’→3’)NtJAZ1 FR / CACCATGGGGTCATCGGAGATTGTAGA
NtJAZ1 RR / AATCAGCAACAGAGGATTGTGA
NtJAZ3 FR / CACCATGGCATCATCGGAGATTGTGGA
NtJAZ3 RR / TTCTTTTCTCTAAAAACCTAGT
NtJAZ7a FR / CACCATGCAGTGGTCATTCTCGAACAA
NtJAZ7a RR / AAGAGTAGGTGTTGTGTTGTGG
NtJAZ10 FR / CACCATGGAGGTGGCCGTAAATCAGCC
NtJAZ10 RR / AGAGCTGCTAGTTCCAGGTGTC
Table S4. Primer sequences used in the cloning of NtMYC2a and NtERF189.
Gene name / Primer sequence (5’→3’)NtERF189 FR / CACCATGGAAATGAATCTAGCTGA
NtERF189 RR / TTAAGCTAATCTCTGTAAATTT
NtMYC2a FR / CACCATGACTGATTACAGCTTACC
NtMYC2a RR / TTAGCGTGTTTCAGCAACTCT
Figure S1.Sequence similarities among the Jas domains of NtJAZs. Partial amino acid sequences were used to generate a sequence alignment of the Jas domains from 17 NtJAZs using the BioEdit software (ver. 7.0.9). The highly conserved Jas motif – SLX2FX2KRX2RX5PY – is boxed.
Figure S2.qRT-PCR analysis of NtJAZ transcript levels in JA-induced BY-2 cells. Treated and untreated BY-2 cells were harvested at the indicated times after MeJA stimulation and analysed for gene expression by qRT-PCR. To determine the effect of CHX on MeJA-induced NtJAZ expression, BY-2 cells were pre-treated with 40 µM CHX for 20 min prior to the addition of MeJA.Error bars indicate the SD of three biological replicates.
Figure S3.Diluted cells detected in the yeast two-hybrid assay. Yeast colonies were grown in liquid selective medium lacking Leu and Trp (-2) to OD600 = 1 and diluted 100-fold to assess the protein interaction in drop-out medium lacking His, Leu, and Trp (-3).
Figure S4.BiFC assay to examine the homomeric interaction of NtJAZ1 and NtJAZ3b proteins in BY-2 cells. The NtJAZs coding sequence without the stop codon were cloned into BIFC vectors. Mixtures of two constructs were then transformed into tobacco BY-2 cell protoplasts. Each of the four possible combinations wasassayed by confocal laser microscopy to examine protein-protein interactions.The homomeric interaction of NtJAZ1 and NtJAZ3b were showed here.
Figure S5.BiFC assay to examine heteromeric interactions between various NtJAZs in BY-2 cells. The coding sequences (without stop codons) of various NtJAZs were cloned into BIFC vectors. The various constructs were pair-wise transformed into BY-2 cell protoplasts. Each of the four possible combinations was assayed to examine their interaction by confocal laser microscopy. Detected heteromeric interactions, including NtJAZ1 and NtJAZ3, NtJAZ3 and NtJAZ3b, and NtJAZ7 and NtJAZ3, are shown.
Figure S6.Interactions between various JA-induced NtJAZs with NtERF189. Yeast cells were co-transformed with one of nine pGBKT7-NtJAZ proteins (bait) and pGADT7-NtERF189 protein (prey). The cells were grown on agar plates containing synthetic medium lacking Leu and Trp (-2) to confirm transformation, or drop-out medium lacking His, Leu, and Trp (3) to test for interactions.
Figure S7. Sequence alignment of the coding sequence of TIFY domains in NtJAZs which were used to construct RNAi vector. About 69 bp cDNA sequence of TIFY domains were used to generate a sequence alignment from 4NtJAZs using the BioEdit software (ver. 7.0.9).
Figure S8.Expression levels of tobacco proteinase inhibitor II (NtPI-II) in NtJAZ7a-RNAi and NtJAZ10-RNAitransformed BY-2 cell lines. Transgenic BY-2 cell lines were cultured in the absence or presence of 50 µM MeJA for 12 h. Transcript levels of NtPI-II in two corresponding transgenic lines are shown relative to empty vector (control values) by qRT-PCR. Error bars represent the SD of three biological replicates. Different letters (a-d) indicate statistically significant differences (P< 0.05) according to Tukey’s multiple range test.
References
Shoji T., Kajikawa M. & Hashimoto T. (2010) Clustered Transcription Factor Genes Regulate Nicotine Biosynthesis in Tobacco. Plant Cell, 22, 3390-3409.
Zhang H.B., Bokowiec M.T., Rushton P.J., Han S. & Timko M.P. (2012) Tobacco Transcription Factors NtMYC2a and NtMYC2b Form Nuclear Complexes with the NtJAZ1 Repressor and Regulate Multiple Jasmonate-Inducible Steps in Nicotine Biosynthesis. Molecular Plant, 5, 73-84.
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