Supporting Information
Downregulation of Cap Binding Protein 80 gene expression as a strategy to engineer a droughttolerant potato
Marcin Pieczynski1, Waldemar Marczewski2, Jacek Hennig3, Jakub Dolata1, Dawid Bielewicz1,
Paulina Piontek1, Anna Wyrzykowska1, Dominika Krusiewicz2, Danuta Strzelczyk-Zyta2, Dorota
Konopka-Postupolska3, Magdalena Krzeslowska4, Artur Jarmolowski1 and Zofia SzweykowskaKulinska1*
1
Department of Gene Expression, Faculty of Biology, Adam Mickiewicz University, Umultowska 89,
61-614 Poznan, Poland; 2 Plant Breeding and Acclimatization Institute, National Research Institute,
Department of Potato Genetics and Parental Lines, Laboratory of Biotechnology, Platanowa 19,
05-831 Mlochow, Poland; 3 Institute of Biochemistry and Biophysics, Polish Academy of Sciences,
Laboratory of Plant Pathogenesis, Pawinskiego 5a, 02-106 Warszawa, Poland; 4 Department of
General Botany, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznan.
* Author for correspondence:
Zofia Szweykowska-Kulinska, Tel: +4861-829-5950, Fax: +4861-829-5949,
Email: zofszwey@amu.edu.pl
Table S1. The oligonucleotide primers and probes used.
Primers used for cDNA of CBP20 gene amplification from A. thaliana
SEN203
SEN205
SEN803
SEN805
5’- GCTTCTTTGTTCAAGGAGCAAGC - 3’
5’- GCGATCATCGTCTCTTTGTCG - 3’
5’- GTTTTGTCGTGGAGGAGTATTTGC - 3’
5’- GCGGTTTGTTTTTCCTTGACCTTCC - 3’
Primers used for cDNA of CBP80/ABH1 gene amplification and sequencing from S. tuberosum var. Desiree
F80ST
R80ST
cbp80-1R
cbp80-1F
cbp80-2F
cbp80-2R
F-actin
R-actin
5’-GGAATGAGTAGTTGGCGG- 3’
5’-TAAATCATTCCTCCAGAGGTC- 3’
5’-CAGTGAAAGAAACAAAGCCTC- 3’
5’-GTGCCTTCCTTGGGGTGGTGC- 3’
5’-CTGCCTGTACCGTTCAGATATG- 3’
5’-GGGCAGTTTTCCTACCTTTCAC- 3’
5’-GGAAACATTGTGCTCAGTGGTGG- 3’
5’-CTCTGCCTTTGCAATCCACATC- 3’
Primers used during expression construct preparation generating artificial microRNA
A-RS300
B-RS300
I-amiRNA80.1
II-amiRNA80.1
III-amiRNA80.1
5’-CTGCAAGGCGATTAAGTTGGGTAAC- 3’
5’-GCGGATAACAATTTCACACAGGAAACAG- 3’
5’-GATAACGGTACAGGCAGGCCGACTCTCTCTTTTGTATTCC- 3’
5’-GAGTCGGCCTGCCTGTACCGTTATCAAAGAGAATCAATGA- 3’
5’-GAGCCGGCCTGCCTCTACCGTTATCACAGGTCGTGATATG- 3’
5’-GATAACGGTAGAGGCAGGCCGGCTCTACATATATATTCCT- 3’
IV-amiRNA80.1
5’-GATAACGGTACAGGCAGCCGGACTCTCTCTTTTGTATTCC- 3’
I-amiRNA80.2
5’-GAGTCCGGCTGCCTGTACCGTTATCAAAGAGAATCAATGA- 3’
II-amiRNA80.2
5’-GAGTACGGCTGCCTGAACCGTTTTCACAGGTCGTGATATG- 3’
III-amiRNA80.2
5’-GAAAACGGTTCAGGCAGCCGTACTCTACATATATATTCCT- 3’
IV-amiRNA80.2
Primers used for cDNA fragments amplification of CBP80/ABH1 and cyclofilin genes from S. tuberosum
during real-time PCR reactions
5’-TCCTTCAAATAAAACTGAGGATC- 3’
Fcbp80
5’-CCTGGCAGAGCCTTGC- 3’
Rcbp80
5’-CTCTTCGCCGATACCACTCC- 3’
F_cyclofilin
5’-TCACACGGTGGAAGGTTGAG- 3’
R_cyclofilin
5’-GCCCGGGTAATCTTTGAAAT- 3’
F-18S rRNA
5’-GTACAAAGGGCAGGGACGTA- 3’
R-18S rRNA
Oligonucleotides used as probes during northern hybridization
probe-80.1
probe-80.2
microRNA 159
U6 snRNA
5’-GTCGGCCTGCCTGTACCGTTA5’-GTCCGGCTGCCTGTACCGTTA5’- AAATGCTCCCTTTAATCCAAA
5’- TCATCCTTGCGCAGGGGCCAG
3’
3’
-3’
-3’
Primers used for cDNA fragment amplification of RAB18 gene (At5g66400)
5’- GAAACCCGATCCAGCAGCAG -3’
RAB-F
5’- TCTTGTCCATCATCCCCTTCT -3’
RAB-R
Primers used for cDNA fragments amplification of different genes from A.thaliana and S.tuberosum during
real-time PCR reactions
5’- AGCATGGTGAGGGTAACTGG- 3’
AtMYB33-F
5’- TTGGCCTCAGATGATTAGCC- 3’
AtMYB33-R
5’- GGGAGAGGGTTGAAGAAAGG- 3’
AtMYB101-F
5’- CACCGTAGACGGCAACTTTT- 3’
AtMYB101-R
5’- CTCTTCCTCTGGGGTGAATG- 3’
StMYB33-F
5’- TTCAGGACTTGCTCGTTGTG- 3’
StMYB33-R
5’- TACTGGATCAATGCCCATCC- 3’
StMYB101-F
5’- GCTTGCCTGGAAGAACAGAC- 3’
StMYB101-R
5’-GCCCGGGTAATCTTTGAAAT- 3’
F-18S rRNA
5’-GTACAAAGGGCAGGGACGTA- 3’
R-18S rRNA
Figure S1. A comparison of the A.thaliana T-DNA insertion and posttranscriptionally silenced cbp20
and cbp80 mutants. (a) The same minor morphological changes are present in both the
posttranscriptionally silenced cbp20 and cbp80 mutants and T-DNA knockouts. (b) Both classes of
mutants exhibited hypersensitivity to ABA during germination. (c) The phenotype that shows a
reduction in wilting during drought stress was obtained in cbp20 and cbp80 mutants using insertion
mutagenesis and posttranscriptional gene silencing. Plants are shown after 6 days without watering.
Plants were grown in a growth chamber for five weeks under normal conditions before completely
ceasing irrigation.
Figure S2. The potato CBP80 full-length cDNA from the Desiree cultivar
(line 3) was
analyzed on a 1% agarose gel. Line 2 – actin (Accession number: DQ252512.1), lines 1 and 4 – 100
bp and 1 kbp DNA ladder, respectively.
Figure S3. A comparison of the cDNA sequences of the CBP80 gene alleles from the potato cultivar
Desiree. The black color depicts the nucleotide differences between the sequences. The red and violet
colors depict the START and STOP codons, respectively. The green color depicts the sequences
chosen as targets for the artificial micro RNA.
Figure S4. An analysis of the CBP80 amino acid (aa) sequence from the Desiree plants. When
compared with the A.thaliana CBP80 protein using the ClustalW2 program,
a 67% identity
and 83% similarity of the aa sequences were found. Desiree-1, Desiree-2, and Desiree-3 represent the
aa sequences of individual alleles of the CBP80 protein found in the cultivar Desiree. The green, blue,
and red frames depict domains from MIF4G, and amino acid letters with a red background represent
the aa substitutions found in the three CBP80 alleles of the cultivar Desiree. The amino acid letters
with a black background represent the aa substitutions found in the A.thaliana and potato CBP80
proteins.
Figure S5. The level of silencing of CBP80 gene expression using artificial miRNAs in selected
potato plants. Individual plants from the two potato transgenic lines amiR80.1-8 and amiR80.2-14
were tested for the silencing of CBP80 gene expression during the third year of vegetative
reproduction via tubers. Real-time measurements of the level of CBP80 mRNA in selected transgenic
plants were calculated as a percentage of the expression of CBP80 mRNA in the Desiree plants. As a
comparison, the level of silencing from one individual plant in 2010 is shown.
Levels of
transcript were normalized against cyclophilin. Values are shown as the mean ±SD (n=3) from three
independent experiments. Asterisk - P<0.02, Mann-Whitney U-Test.
Figure S6. Quantitative real-time PCR of the Arabidopsis pri-mir319a in wild-type and cbp80 mutant
plants. The levels of transcript were normalized against GAPDH (At1g13440). The calculation shows
the mean ±SD from three biological replicates.
Figure S7. Real-time measurements of the levels of the StMYB1R-1 mRNA using the ΔΔCT method
in the Desiree and amiRNA80.2-14 plants after 14 days without watering.
The
mRNA level is presented as a percentage of the StMYB1R-1 mRNA level in the control plants at day 0
of drought. Levels of transcript were normalized against cyclophilin.