1
SUPPORTING METHODS
2
General molecular and bioinformatic methods
3
cDNA sequence were analyzed using BLAST (http://www.ncbi.nlm.nih.gov/blast/) and
4
ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Protein sequences were
5
analyzed using ProtParam (http://www.expasy.ch/tools/protparam.html) for primary
6
structure. Conserved domains were deduced using NCBI (http://www.ncbi.nlm.nih.gov/
7
Structure/cdd/wrpsb.cgi). PSORT (http://psort.nibb.ac.jp/form.html) was used for
8
predicting subcellular localization. ClustalX (Thompson et al., 1994) and DNAMAN (ver.
9
5.2.2, Lynnon Biosoft, Quebec, Canada) were used for the sequence alignment analyses.
10
MEGA4 was used for phylogenetic analyses (Tamura et al., 2007).
11
Chromosomal assignment of TaADF7
12
Conserved primers capable of amplifying all three replications of TaADF7 in genomic
13
PCR (Table S1) were designed. Intron length polymorphisms were employed to
14
distinguish the fragments from different replications (Yu et al., 2010). The templates for
15
the PCR amplifications were genomic DNA samples prepared from Triticum aestivum cv.
16
Suwon 11, cv. Chinese Spring (CS) or the associated nulli-tetrasomic lines. Water was
17
used as the negative control. The amplified products were separated using 3% sepharose
18
gel. The designed length of fragment on chromosome 1A, 1B, and 1D were 288, 423, and
19
366 bp, respectively. The amplified products are marked with blue underline in Figure S2.
20
The three amplified fragments obtained by genomic PCR using Suwon 11 or CS genomic
21
DNA as templates were sequenced.
22
1
23
Supplementary details for VIGS
24
Selection of silencing fragments and detection of silencing efficiency
25
The first silenced fragment (303 bp) was located at the 3'-UTR (Figure S2, blue underline)
26
and the second (177 bp) was located at the partial open reading frame and the 5'-UTR
27
(Figure S2, black underline). The two fragments did not show any similarity with any
28
other wheat gene in BLAST analyses (http://blast.ncbi.nlm.nih.gov/Blast/). Three copies
29
shared 94.46% and 89.83% nucleotide identity with each other for two VIGS fragments,
30
respectively. A pair of primers located at 5'-UTR (Figure S2, red underline) was designed
31
to test the silencing efficiency of the first silenced fragment for every copy, and another
32
pair of primers located at 3'-UTR (Figure S2, green underline) was designed to test the
33
silencing efficiency of the second silenced fragment for every copy. The productions of
34
six pair of primers (Table S1) were sequenced to assure the specificity of production.
35
Virus inoculation
36
The two fragments were used to replace the TaPDS (wheat phytoene desaturase) coding
37
sequence in BSMV:TaPDS. Recombinant plasmids were linearized. Capped in vitro
38
transcripts were produced from linearized plasmids with RiboMAX Large Scale RNA
39
Production System-T7 (Promega, Madison, WI, USA). Transcripts of each of the BSMV
40
genomes (wild type or genetically modified) were mixed in a 1:1:1 ratio. A 7.5- ml
41
aliquot of the transcription mix was combined with 45 μl of 1× Fes buffer (Pogue et al.,
42
1998) and directly applied (Holzberg et al., 2002). Four BSMV viruses (BSMV:γ,
43
BSMV:TaPDS, BSMV:TaADF7-1 and BSMV:TaADF7-2) were individually inoculated
44
on the second leaf of two-leaf wheat seedlings by gently rubbing the leaves with gloved
2
45
fingers (Hein et al., 2005). After they were stored for 24 h in the dark, the seedlings were
46
placed in a growth chamber at 25 °C and examined for symptoms.
47
Control design and reproducibility of the data for VIGS
48
To confirm the VIGS system functioned correctly, BSMV:TaPDS was used as a negative
49
control to induce photo-bleaching on inoculated plants (Holzberg et al., 2002). Control
50
plants were treated with 1× Fes buffer to replace virus. Expression level of TaADF2,
51
another wheat ADF gene that shares 49.35% nucleotide identity with TaADF7, was tested
52
as the systems control. At least three plants from different pots were treated as one
53
sample, and each time point has three samples. Wheat elongation factor TaEF-1a was
54
used as internal reference for qRT-PCR analysis. The whole experiment was repeated
55
three times. One-way analysis of variance was performed by the SPSS software program
56
(ver. 16.0, SPSS Inc., Chicago, IL, U.S.A.).
57
Accession numbers
58
The nucleotide sequences used in this study had the following GenBank accession
59
numbers: TaADF7 (JX486723), OsADF7 (Q0DLA3), BdADF7 (XP_003569002), TaEF-
60
1a (Q03033), AtFim1 (NM_118804), Rattus norvegicus BAX (U32098), TaPDS
61
(FJ517553) TaPR1 (AAK60565), TaPR2 (DQ090946), TaPR5 (FG618781), TaSOD
62
(CB307850), TaCAT (X94352), and TaNOX (AY561153).
63
References
64
65
66
67
68
69
Hein, I., Barciszewska-Pacak, M., Hrubikova, K., Williamson, S., Dinesen, M., Soenderby, I.E.,
Sundar, S., Jarmolowski, A., Shirasu, K. and Lacomme, C. (2005) Virus–induced gene
silencing-based functional characterization of genes associated with powdery mildew resistance in
barley. Plant Physiol., 138, 2155-2164.
Holzberg, S., Brosio, P., Gross, C. and Pogue, G.P. (2002) Barley stripe mosaic virus‐induced gene
silencing in a monocot plant. Plant J., 30, 315-327.
3
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
Pogue, G., Lindbo, J., Dawson, W. and Turpen, T. (1998) Tobamovirus transient expression vectors:
tools for plant biology and high-level expression of foreign proteins in plants. In Plant Molecular
Biology Manual (Gelvin, S.B. and Schilperoort, R.A. eds). Berlin: Springer, pp. 63-97.
Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4: molecular evolutionary genetics
analysis (MEGA) software version 4.0. Mol. Biol. Evol., 24, 1596-1599.
Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of
progressive multiple sequence alignment through sequence weighting, position-specific gap
penalties and weight matrix choice. Nucleic Acids Res., 22, 4673-4680.
Tolvanen, M.E., Ortutay, C., Barker, H.R., Aspatwar, A., Patrikainen, M. and Parkkila, S. (2013)
Analysis of evolution of carbonic anhydrases IV and XV reveals a rich history of gene
duplications and a new group of isozymes. Biorg. Med. Chem., 21, 1303-1310.
Yu, C., Li, Y., Li, B., Liu, X., Hao, L., Chen, J., Qian, W., Li, S., Wang, G. and Bai, S. (2010)
Molecular analysis of phosphomannomutase (PMM) genes reveals a unique PMM duplication
event in diverse Triticeae species and the main PMM isozymes in bread wheat tissues. BMC Plant
Biol., 10, 214.
4