Embed Size (px)
Citation preview
Supporting InformationBrioudes et al. 10.1073/pnas.1007926107SI Materials and MethodsConstructions and Plant Lines. AtTCTP knockout lines. The two in-dependent A. thaliana (both in Col-0 accession background)T-DNA-insertion knockout lines, tctp-1 and tctp-2 (SAIL_28_C03and GABI_901E08, respectively), were obtained from the Not-tingham Arabidopsis Stock Centre. The presence of T-DNA in-sertionwithin theAtTCTP genewas confirmed by PCRanalysis. InSAIL_28_C03, the T-DNA was inserted into the fourth exon ofAtTCTP (948-bp downstream from the start codon in the genomicsequence), whereas in GABI_901E08 the T-DNA was insertedinto the first exon ofAtTCTP (34-bp downstream the start codon).35S::AtTCTP, 35S::dTCTP, and 35S::YFP-AtTCTP overexpression lines.DNAfragments corresponding to theORF ofAtTCTP (At3g16640) or theDrosophila melanogaster TCTP (dTCTP, Dmel_CG4800) werecloned into pK2GW7 or pK7WGY2 vectors (1) using Gatewaytechnology (Invitrogen).The resulting constructs, p35S::AtTCTPandp35S::dTCTP, express either theAtTCTPor the dTCTP, respectively,under the control of the cauliflower mosaic virus (CaMV) 35S con-stitutive promoter. The p35S::YFP-AtTCTP construct harbors theAtTCTP fused to the YFP under the control of the 35S promoter.These constructs were then used to transform A. thaliana Col-0plants. These overexpression lines are referred to as 35S::AtTCTP,35S::dTCTP, and 35::YFP-AtTCTP.Anti-AtTCTP RNAi-expressing plants. The DNA fragment corre-sponding to the ORF of AtTCTP was cloned into the vectorpB7GWIWG2 (II) (1) after the CaMV 35S constitutive promoter.The resulting construct, referred to as p35S::RNAi-AtTCTP, wasthen used to transformA. thalianaCol-0 plants. Lines are referredto as RNAi-AtTCTP.AtTCTPg-GFP line. The 3,179-bp genomic DNA sequence containingthe promoter region, exons, and introns and excluding the stop co-don was PCR amplified and cloned into the pDONRP4-P1r. The 3′UTR was PCR amplified and cloned into the pDONR P2r-P3. TheeGFPDNA sequence was inserted into the pDONR207. The threepDONR vectors above were then subjected to recombination alongwith the pK7m34GW vector (1) using the Multisite Gateway tech-nology (Invitrogen). The resulting construct (referred to as pTCT-P2K::AtTCTPg-GFP-3UTR) harbors theAtTCTP genomic sequenceincluding the promoter region, exons, introns, and the 3′ UTR re-gion in which the GFP was inserted in frame with AtTCTP at the Cterminus. This construct was then used to transform A. thalianaCol-0 plants, and the resulting line is referred to as AtTCTPg-GFP.The presence of the different transgenes was confirmed by PCR,
and their expression was checked by semiquantitative RT-PCRexperiments and, when indicated, by Western blotting using anti-AtTCTP antibody.Arabidopsis plants were grown in soil in growthchambers under continuous long-day condition (16 h/8 h day/nightat 22 °C/19 °C and 100 μEm−2 s−1 or 70 μEm−2 s−1) or plants werefirst grown under short-day condition (8 h/16 h day/night at 22 °C/19 °C and 100 μE m−2 s−1 or 70 μE m−2 s−1) and then transferredunder long-day condition to promote flowering.Embryos homozygous for tctp mutation (white seeds) were
dissected from heterozygous tctp-1 and tctp-2 siliques at stage 17B(2). At least 20 siliques from three independent heterozygousmutant plants for both tctp-1 and tctp-2 lines were analyzed.RNAi-NtTCTP BY-2 cell line. The DNA fragment corresponding to theORF of NtTCTP (AF107842.1) was cloned as an inverted tandemrepeat into the vector pK7GWIWG2D(II) (1). The resulting con-struct (p35S::RNAi-NtTCTP) expresses an anti-NtTCTP RNAi un-der the control of the CaMV 35S constitutive promoter. p35S::RNAi-NtTCTP was then used to transform tobacco BY-2 (N. ta-bacum L. cv. Bright Yellow-2) cell suspension as previously de-
scribed (3). Lines are referred to asRNAi-NtTCTP. BY2 cells werecultured as previously described (4).
Longitudinal Sections and Staining of Arabidopsis Stems. Arabidopsisstem segments cut from secondary inflorescence stems were fixedin 4% paraformaldehyde, 1% glutharaldehyde in phosphate buf-fer, pH 6.8, overnight at 4 °C. Stem segments were dehydratedthrough a series of ethanol solution and embedded in Technovit7100 (Heraeus Kulzer) according to the manufacturer’s in-structions. Sections (5 μm) were stained for 5 min with 0.5% to-luidine blue.
Kinematic Analysis of Organ Growth. Kinematic analysis of leafgrowth was performed as previously described (5) on the two firstinitiated-leaves of WT and RNAi-AtTCTP plants grown in vitro.Leaf size and number and size of abaxial epidermal cells were de-termined startingat day4until day 18after sowing.Theaverage celldivision rates were determined by calculating the slope of the Ne-perian Logarithmic-transformed number of cells per leaf, whichwas done using five-point differentiation formulas (6).
BY-2 Cell Cycle Progression, DNA Content, and Cell Cycle GenesExpression Analyses. BY-2 cells were synchronized using aphi-dicholin essentially as previously described by Kumagai-Sano et al.(7). Briefly, stationary cultures of BY-2 cells were 1/10 diluted inculture medium supplemented with 5 mg/L aphidicholin for 24 h.Aphidicholinwas then removedbyextensivewashing and cellswereresuspended in fresh medium. The mitotic index was measuredeveryhour under afluorescencemicroscope after staining cellswith2 μg/mL Hoechst 33258 in 1% Triton X 100.For flow cytometric analysis, BY-2 cell nuclei were extracted
in Galbraith buffer (8) by repetitive pipetting. Nuclei were filteredthrough a 20-μm nylon filter and then stained with 2 μg/mLHoechst 33258. Fluorescence intensity of 10,000 stained nucleiwasmeasuredwith aBDLSRII FlowCytometer (BDBiosciences).Background noise was eliminated by gating, and DNA contentanalysis was performed using ModFit LT software.TotalRNAwasextracted fromsynchronizedBY-2cellsusing the
Spectrum Plant Total RNA kit (Sigma). mRNA were reversetranscribed, and target cDNAswere quantified by qRT-PCRusingFast SYBR green master mix (Applied Biosystems) on a Step-OnePlus Real-Time PCR System (Applied Biosystems). Expres-sion levels were normalized with Tubulin. DNA primers used forqRT-PCRs are listed in Table S1.
GST Pull-Down Experiments. DNA fragments corresponding to theDrosophila dRheb and the Arabidopsis AtRABA4a were PCR ampli-fied and then cloned into the bacterial expression vector pDEST15.The resulting clone expressed dRheb andAtRABA4a fused toGST.Bacterial protein extracts containing GST-dRheb or GST-AtRA-BA4a proteins were incubated with Gluthatione Sepharose (Amer-sham) in PBS in the presence of 1mMDTT for 1 h at 4 °C. The resinwas washed three times with PBS and then incubated with extractsfrom bacteria harboring His-tagged proteins His-AtTCTP or His-dTCTP. After washing with PBS, proteins were eluted with 10 mMreduced gluthatione and analyzed by Western blot using mousemonoclonal anti-polyhistidine antibody (1/3,000 dilution, cloneHIS1; Sigma). HRP-conjugated sheep anti-mouse IgG (1/5,000 di-lution, Amersham) was used as secondary antibody.
BiFC Experiments. DNA fragments corresponding to the codingsequences ofAtTCTP, dTCTP, dRheb (Dmel_CG1081),AtRABA4a(At5g65270),AtRABA4b (At4g39990),AtRABF1 (At3g54840), and
Brioudes et al. www.pnas.org/cgi/content/short/1007926107 1 of 11
ATRABF2b (At4g19640) were PCR amplified and then cloned intothe pBiFP1, pBiFP2, pBiFP3, and pBiFP4 vectors (9) using Gate-way technology. Resulting constructs were used to infiltrate leafepidermal cells of Nicotiana benthamiana plants as previously de-scribed (10). Bimolecular fluorescence complementation usingYFPNterm and YFPCterm fusion proteins was checked 4 d post-infiltration using a Leica TCS SP5 Spectral Confocal microscope.
Transgene Constructions and Drosophila Lines. DNA fragmentscontaining the ORF of theWT plant TCTP (AtTCTP) or mutatedat amino acid Glu12 (AtTCTPE12V) were amplified using the pri-mers P-AtTCTP-F and P-AtTCTP-R or P-AtTCTP-FE12V and P-
AtTCTP-R, respectively (Table S1). PCR products were digestedwithEcoRI andXbaI and then cloned into the pUAST vector (11)to place the cDNA under the control of UAS sequence. The re-sulting constructs referred to as pUAST::AtTCTP and pUAST::AtTCTPE12V, respectively, were then used to generate transgenicDrosophila via embryo injections (BestGene Inc.). These lines arereferred to asUAS::AtTCTP andUAS::AtTCTPE12V, respectively.Three independent lines were analyzed for the expression of eachtransgene with similar results. Flies were grown at 29 °C to en-hance GAL4 expression in eyes and wings. For all experiments, atleast 20 organs and at least 10 flies were used.
1. Karimi M, Inzé D, Depicker A (2002) GATEWAY vectors for Agrobacterium-mediatedplant transformation. Trends Plant Sci 7:193–195.
2. Roeder AH, Yanofsky MF (2009) Fruit development in Arabidopsis. The ArabidopsisBook (The American Society of Plant Biologists, Rockville, MD), pp 1–50.
3. Mayo KJ, Gonzales BJ, Mason HS (2006) Genetic transformation of tobacco NT1 cellswith Agrobacterium tumefaciens. Nat Protoc 1:1105–1111.
4. Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the “HeLa” cell inthe cell biology of higher plants. Int Rev Cytol 132:1–30.
5. De Veylder L, et al. (2001) Functional analysis of cyclin-dependent kinase inhibitors ofArabidopsis. Plant Cell 13:1653–1668.
6. Erickson RO (1976) Modeling of plant growth. Annu Rev Plant Physiol 27:407–434.
7. Kumagai-Sano F, Hayashi T, Sano T, Hasezawa S (2006) Cell cycle synchronization oftobacco BY-2 cells. Nat Protoc 1:2621–2627.
8. Galbraith DW, et al. (1983) Rapid flow cytometric analysis of the cell cycle in intactplant tissues. Science 220:1049–1051.
9. Desprez T, et al. (2007) Organization of cellulose synthase complexes involved in primarycell wall synthesis in Arabidopsis thaliana. Proc Natl Acad Sci USA 104:15572–15577.
10. Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expressionsystem in plants based on suppression of gene silencing by the p19 protein of tomatobushy stunt virus. Plant J 33:949–956.
11. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cellfates and generating dominant phenotypes. Development 118:401–415.
wt tctp- 2-/- A
wt B
Pollinated
tctp - 2-/-
Non
pollinated tctp - 2-/-
wt
Pollinated
tctp - 2-/-
Non
pollinated tctp - 2-/-
C
Wt embryo
E D
tctp /TCTP embryo
in tctp - 2-/- genetic background
Fig. S1. (A) Homozygous tctp-2 plants exhibit anther dehiscence defects: Dehiscing anthers from mature flowers of WT and tctp-2−/− are shown. Note that nopollen grains are released by tctp-2 anthers. (B–E) The development of heterozygous tctp/TCTP embryos is delayed in tctp mutant genetic background. (B)Siliques produced by WT and tctp-2−/− flowers pollinated at the same time with WT pollen. A silique produced by nonpollinated tctp-2−/− flower is shown ascontrol. (C) The same siliques as in B with open valves. Note that few seeds in tctp-2−/− siliques pollinated with WT pollen could develop (white arrows). Thetctp/TCTP heterozygous embryos that developed in tctp-2−/− genetic background (E) exhibited delayed development compared with WT embryos of the sameage (D). [Scale bars, 200 μm (A), 2 mm (B and C), and 50 μm (D and E).]
Brioudes et al. www.pnas.org/cgi/content/short/1007926107 2 of 11
tctp -2-/-
35S::AtTCTP tctp -2-/-
AtTCTPg-GFP tctp -2-/-
35S::YFP- AtTCTP
wt
Fig. S2. The embryonic lethality of homozygous tctp-2 mutant is rescued by expression of AtTCTP ORF (35S::AtTCTP), YFP-AtTCTP fusion (35S::YFP-AtTCTP), orAtTCTP genomic construct (AtTCTPg-GFP).
Fig. S3. RNAi-AtTCTP plants show retarded growth, whereas overexpression of AtTCTP results in accelerated growth. (A, Left) Rosette maximal diameter wasmeasured during the development of WT, 35S::AtTCTP and RNAi-AtTCTP plants up to 48 dpg. RNAi-AtTCTP line exhibits a significant (pStudent < 0.001) re-duction of rosette size ranging from 21% at 30 dpg to 18% at 48 dpg. The 35S::AtTCTP line exhibits a significant (pStudent < 0.001) accelerated rosette growthranging from 1 d at 30 dpg to 2 d at 48 dpg. Thirty plants were used for each line. Bars indicate SE. (Right) Semiquantitative RT-PCR (top) shows that no or verylittle expression of AtTCTP is observed in RNAi-AtTCTP leaves and high level of AtTCTP mRNA accumulation is observed in the 35S::AtTCTP overexpressing line.Western blot analysis (bottom) of AtTCTP accumulation using an anti-AtTCTP antibody (1/500 dilution). An HRP-conjugated donkey antirabbit IgG (1/4,000dilution, Amersham) was used as secondary antibody. (B) Petal surface area and petal conical cell density measurements show no significant difference be-tween 35S::AtTCTP and WT.
Brioudes et al. www.pnas.org/cgi/content/short/1007926107 3 of 11
wt RNAi-AtTCTP
B
wt
RNAi-AtTCTP
D
A
wt RNAi-AtTCTP
C
wt RNAi-AtTCTP
0
20
40
60
80
100
120 %
of W
T p
eta
l cell
density
* *
wt
tctp -1
tctp -2
E
Fig. S4. AtTCTP loss of function affects cell division. (A) Image of longitudinal sections of inflorescence stems colored by toluidine blue showing no significantdifference in medullar parenchyma cell length between WT and RNAi-AtTCTP. (Scale bar, 200 μm.) Cell length quantification is provided in Fig. 3B. (B and C)Leaf epidermal cells (C) and petal conical cells (D) of WT and RNAi-AtTCTP observed with Nomarski optics show a significant decrease of cell density in bothleaves and petals of RNAi-AtTCTP plants compared with WT. [Scale bars, 50 μm (B) and 20 μm (C).] Quantifications of cell density are shown in Fig. 3 C and D. (D)Phenotypic appearances of petals in WT and RNAi-AtTCTP plants. No significant change of petal size could be detected. Petal surface area measurement isprovided in Fig. 3E. (E) Petal conical cells in tctp-1 and tctp-2 are bigger in size but exhibit a lower cell density compared with WT. Asterisk indicates significantstatistical difference with PStudent < 0.001. (Scale bars, 20 μm.)
Brioudes et al. www.pnas.org/cgi/content/short/1007926107 4 of 11
RNAi-AtTCTP
102 103 104 105
0
C
ount
16888
Fluorescence intensity
0
10
20
30
40
50
60 2C
4C
8C
16C
32C
Fre
quency (
%)
wt RNAi-AtTCTP
wt
0
Count
1
0642
Fluorescence intensity102 103 104 105
Fig. S5. Nuclear DNA content analysis by flow cytometry reveals no endoreduplication in RNAi-AtTCTP leaves compared with the WT. To measure endor-eduplication levels, whole rosette leaves were chopped in Galbraith’s buffer (1). Extracts were passed through two nylon filters (pore size, 50 and 20 μm) andreleased nuclei were stained with Hoechst 33258 (2 μg/mL). DNA content was analyzed on a BD LSRII flow cytometer. Background noise was eliminated bygating. These analyses show no significant modifications in RNAi-AtTCTP compared with that of the WT, demonstrating that the cell size increase observed inleaves of the RNAi-AtTCTP line was not correlated with endoreduplication, a common landmark of cell expansion.
1. Galbraith DW, et al. (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220:1049–1051.
Brioudes et al. www.pnas.org/cgi/content/short/1007926107 5 of 11
wt
RNAi-AtTCTP
35S::AtTCTP
% o
f W
T h
ypocoty
l le
ngth
0
20
40
60
80
100
120
Fig. S6. Cell elongation is not affected in RNAi-AtTCTP and 35::AtTCTP lines etiolated plants compared with the WT. Hypocotyl length of corresponding plantshas been quantified 4 d after germination and does not show any significant difference (n > 20).
Brioudes et al. www.pnas.org/cgi/content/short/1007926107 6 of 11
Fig. S7. 5′ UTR and/or 3′ UTR are required for regulation of translation of AtTCTP. (A and B) GFP fluorescence in Arabidopsis plantlets that harbor the GFP-tagged genomic construct of AtTCTP (containing the promoter and 5′ and the 3′ UTRs) shows that AtTCTP-GFP fusion protein accumulates mainly in shootapical meristem (red arrow) and in primary and secondary root meristems (white arrows). (B) Detail of secondary root meristem. (C and D) GFP fluorescence inplantlets that harbor 35S::YFP-AtTCTP construct (without 5′ and 3′ UTRs). Note that AtTCTP-YFP fluorescence is observed in all tissues. (D) Detail of secondaryroot meristem. (E and F) The 5′ (E) and 3′ UTR (F) sequences of human, mouse, zebrafish, Drosophila, and Arabidopsis TCTP mRNAs. The 5′ TOP in the 5′ UTRsequence (E) and the instability AUUUA motifs in the 3′ UTR sequence (F) are highlighted by black boxes. 5′ TOP containing mRNAs, such as ribosomal proteinsmRNAs, are known to be translationally controlled in response to mitogenic and nutritional stimuli (1). AUUUA motifs have been shown to be required for theMos (Moloney sarcoma oncogene) mRNA translation arrest upon fertilization of Xenopus eggs (2). Polyadenylation signatures are underlined in the 3′UTR sequences(F). Asterisk indicates an alternative 5′ end of AtTCTP mRNA (E), according to Castelli et al. (3). The GC and AT percentages in 5′ UTR and 3′ UTR are indicated.
1. Meyuhas O (2000) Synthesis of the translational apparatus is regulated at the translational level. Eur J Biochem 267:6321–6330.2. Ueno S, Sagata N (2002) Requirement for both EDEN and AUUUA motifs in translational arrest of Mos mRNA upon fertilization of Xenopus eggs. Dev Biol 250:156–167.3. Castelli V, et al. (2004) Whole genome sequence comparisons and “full-length” cDNA sequences: A combined approach to evaluate and improve Arabidopsis genome annotation.
Genome Res 14:406–413.
Brioudes et al. www.pnas.org/cgi/content/short/1007926107 7 of 11
Fig. S8. AtTCTP expression is translationally controlled. (A) Semiquantitative RT-PCR shows that AtTCTP is expressed in all organs of WT A. thaliana plants. Ct:control RT-PCR performed in absence of cDNA. (B) Western blot analysis of AtTCTP accumulation in different plant organs using an anti-AtTCTP antibody (1/500dilution) generated by immunizing rabbits (CovalAb) with a HIS-tagged AtTCTP protein produced in E. coli. An HRP-conjugated donkey anti-rabbit IgG (1/4,000dilution, Amersham) was used as secondary antibody. Note that AtTCTP protein accumulation does not always correlate with AtTCTP mRNA accumulation andthat there is little AtTCTP protein in stems. Red Ponceau staining of total proteins is shown as control.
ptc >GFP (ptc:GAL4; UAS:GFP)
nub >GFP (nub:GAL4; UAS:GFP)
ey >GFP (ey:GAL4; UAS:GFP) A
C
B
Fig. S9. Drosophila line harboring GFP reporter under the control of UAS was crossed with lines expressing GAL4-drivers. (A) Antenna and eye imaginal discsof Drosophila harboring eyless promoter driving GAL4 (ey-GAL4). (B) Wing imaginal disk of Drosophila harboring nubbin promoter driving GAL4 (nub-GAL4).(C) Wing imaginal disk of Drosophila harboring patch promoter driving GAL4 (ptc-GAL4). Transmission (Left) and fluorescence (Right) images are shown. Thefigures show that these promoters are highly active and also demonstrate their organ specificity.
Brioudes et al. www.pnas.org/cgi/content/short/1007926107 8 of 11
Fig. S10. Conservation of key amino acids and domains in TCTP protein sequences. Protein sequences of TCTP from human (NP_003286), mouse (NP_033455),rat (NP_446319), zebrafish (NP_937783), Drosophila (NP_650048), Caenorhabditis elegans (NP_492767), fission yeast (NP_594328), and A. thaliana (NP_188286)were aligned using Clustalw algorithm. Asterisks indicate absolutely conserved amino acids, colons indicate strongly similar amino acids, and dots indicateweakly similar amino acids. Colored boxes indicate TCTP interacting domains with Mcl-1/Bcl-xL (1, 2), microtubules (3), Na+/K+ ATPase (4), or Polo-like kinase (5).The green box indicates the putative homodimerization domain of TCTP (6) (Leu122 and Cys168 according to AtTCTP numbering are indicated with arrows).Black boxes indicate TCTP signatures (PROSITE documentation PDOC00768). Amino acids highlighted in yellow correspond to serine residues phosphorylatedby the Polo-like kinase in mammals (5), those in gray correspond to the putative amino acids involved in calcium binding (7), and those in black correspond tothe conserved amino acids involved in Ras GTPases binding (8).
1. Yang Y, et al. (2005) An N-terminal region of translationally controlled tumor protein is required for its antiapoptotic activity. Oncogene 24:4778–4788.2. Zhang D, Li F, Weidner D, Mnjoyan ZH, Fujise K (2002) Physical and functional interaction between myeloid cell leukemia 1 protein (MCL1) and Fortilin. The potential role of MCL1 as
a fortilin chaperone. J Biol Chem 277:37430–37438.3. Gachet Y, et al. (1999) The growth-related, translationally controlled protein P23 has properties of a tubulin binding protein and associates transiently with microtubules during the cell
cycle. J Cell Sci 112:1257–1271.4. Jung J, et al. (2004) Translationally controlled tumor protein interacts with the third cytoplasmic domain of Na,K-ATPase alpha subunit and inhibits the pump activity in HeLa cells. J Biol
Chem 279:49868–49875.5. Yarm FR (2002) Plk phosphorylation regulates the microtubule-stabilizing protein TCTP. Mol Cell Biol 22:6209–6221.6. Yoon T, et al. (2000) Identification of the self-interaction of rat TCTP/IgE-dependent histamine-releasing factor using yeast two-hybrid system. Arch Biochem Biophys 384:379–382.7. Feng Y, Liu D, Yao H, Wang J (2007) Solution structure and mapping of a very weak calcium-binding site of human translationally controlled tumor protein by NMR. Arch Biochem
Biophys 467:48–57.8. Thaw P, et al. (2001) Structure of TCTP reveals unexpected relationship with guanine nucleotide-free chaperones. Nat Struct Biol 8:701–704.
Brioudes et al. www.pnas.org/cgi/content/short/1007926107 9 of 11
GS
T-A
tRA
BA
4a
GS
T
GS
T-d
Rheb
6H
is-d
TC
TP
Input
6His-dTCTP
C
GS
T
GS
T-d
Rheb
GS
T-A
tRA
BA
4a
6H
is-A
tTC
TP
Input
6His-AtTCTP
D
A GFP
YF
PC
Te
r -A
tTC
TP
+ YFPNTer-AtTCTP
YF
PC
Te
r -dT
CT
P
+ YFPNTer-dTCTP
YF
PC
Te
r -dT
CT
P
+ YFPNTer-AtTCTP
a b
c d
B + YFPNTer-dRheb
YF
PC
Te
r -A
tTC
TP
+ YFPNTer-AtRABA4a + YFPNTer-AtRABA4b
YF
PC
Te
r -dT
CT
P
+ YFPNTer-AtRABF1 + YFPNTer-AtRABF2b
a b c d
f g h
e
i j
Fig. S11. AtTCTP and dTCTP homodimerize and interact with RAB GTPases. (A) We tested the ability of AtTCTP or dTCTP to homodimerize in vivo using BiFC(1). AtTCTP and dTCTP fused to split YFP were expressed in tobacco leaf epidermal cells. The confocal images of tobacco leaf epidermal cells expressing BiFCconstructions show in vivo homodimerization of AtTCTP (b) and of dTCTP (c). (d) AtTCTP and dTCTP are able to homodimerize with each other. GFP wasexpressed as a control (a). (Scale bars, 50 μm.) (B-D). Plant and animal TCTPs interact with Drosophila dRheb and with Arabidopsis AtRABA4a (At5g65270),AtRABA4b (At4g39990), AtRABF1 (ARA6; At3g54840), and AtRABF2b (ARA7; At4g19640) Rab GTPases. (B) BiFC assays in infiltrated tobacco leaves showedinteractions between Arabidopsis or Drosophila TCTP and Rab GTPases in vivo. The confocal images of tobacco epidermal leaf cells expressing BiFC con-structions show that AtTCTP can interact in vivo with the Drosophila dRheb (a) and with the plant GTPases AtRABA4a (b), AtRABA4b (c), AtRABF1 (d), andAtRABF2b (e). dTCTP also interacts with dRheb (f), AtRABA4a (g), AtRABA4b (h), AtRABF1 (i), and AtRABF2b (j). (Scale bars, 50 μm.) (C and D) The GST pull-down experiment further confirmed that dTCTP interacts in vitro with dRheb, in agreement with Hsu et al. (2), and also with the Arabidopsis Rab GTPaseAtRABA4a (C). Similarly, the plant AtTCTP could interact with the Drosophila dRheb and with AtRABA4a (D).
1. Hu CD, Kerppola TK (2003) Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat Biotechnol 21:539–545.
2. Hsu YC, Chern JJ, Cai Y, Liu M, Choi KW (2007) Drosophila TCTP is essential for growth and proliferation through regulation of dRheb GTPase. Nature 445:785–788.
Brioudes et al. www.pnas.org/cgi/content/short/1007926107 10 of 11
Table S1. Sequence of primers used in this study
Primers Gene Primer Sequence Experiment
P-AtTCTP-F AtTCTP 5′ CCGGAATTCATGTTGGTGTACCAAGATCTTC Gene cloningP-AtTCTP-FE12V AtTCTP 5′ CCGGAATTCATGTTGGTGTACCAAGATCTT
CTCACCGGTGATGTGCTTGene cloning
P-AtTCTP-R AtTCTP 5′ GCAGATCTAGATCAGCACTTGACCTCCTTCA Gene cloningAtTCTP-F AtTCTP 5′ CACCATGTTGGTGTACCAAGATCTTC Gene cloningAtTCTP-R AtTCTP 5′ TCAGCACTTGACCTCCTTCA Gene cloningdTCTP-F dTCTP 5′ CACCATGAAGATCTACAAGGATATCATC Gene cloningdTCTP-R dTCTP 5′ CTAGCATTTCTCCTCCTCCAGACC Gene cloningdRheb-F dRheb 5′ CACCATGCCAACCAAGGAGCGCCAC Gene cloningdRheb-R dRheb 5′ CTACGATACAAGACAACCGCTCTTC Gene cloningAtRABA4a-F AtRABA4a 5′ CACCATGACTAGTGGAGGAGGATACG Gene cloningAtRABA4a-R AtRABA4a 5′ CTAAGAGTTACAACACATGTTACTCTTG Gene cloningAtRABA4b-F AtRABA4b 5′ CACCATGGCCGGAGGAGGCGGATAC Gene cloningAtRABA4b-R AtRABA4b 5′ TCAAGAAGAAGTACAACAAGTGCTG Gene cloningNtTCTP-F NtTCTP 5′ CACCATGTTGGTTTACCAGGATCTTC Gene cloningNtTCTP-R NtTCTP 5′ TTAACACTTGACCTCCTTGAGTC Gene cloningAtTCTP-qF AtTCTP 5′ GAGCTTCTGTCTGACTCTTTCCC Semiquantitative RT-PCRAtTCTP-qR AtTCTP 5′ GTTGAACCCTCCTTGTAGTAAGC Semiquantitative RT-PCRGAPDH-qF GAPDH 5′ AGGGTGGTGCCAAGAAGGTTG Semiquantitative RT-PCRGAPDH-qR GAPDH 5′ GGAACTGTAACCCCATTCGTTGTC Semiquantitative RT-PCRNtTCTP-qF NtTCTP 5′ GCATGGCTGATGATACTGGA Quantitative RT-PCR (BY-2
cells experiments)NtTCTP-qR NtTCTP 5′ ATAACCGGTCAAGCCCCTAC Quantitative RT-PCR (BY-2
cells experiments)TUB-qF tubulin 5′ CCAGACGGCTCATAGGGTTA Quantitative RT-PCR (BY-2
cells experiments)TUB-qR tubulin 5′ GCAAACGTTGGATGATTCCT Quantitative RT-PCR (BY-2
cells experiments)CYCA1.1-qF cyclin A1.1 5′ TGCCCTCCAACAATACCTGT Quantitative RT-PCR (BY-2
cells experiments)CYCA1.1-qR cyclin A1.1 5′ CGAAGCATCGTTGAAATGAA Quantitative RT-PCR (BY-2
cells experiments)CYCB1.2-qF cyclin B1.2 5′ GGCTGCATCATCATCAAGTG Quantitative RT-PCR (BY-2
cells experiments)CYCB1.2-qR cyclin B1.2 5′ CATGTGGCACCTTTGACAAC Quantitative RT-PCR (BY-2
cells experiments)PCNA-qF PCNA 5′ TCTTTGACTTTTGCCCTGAGA Quantitative RT-PCR (BY-2
cells experiments)PCNA-qR PCNA 5′ GCCCATCTCAGCAATCTTGT Quantitative RT-PCR (BY-2
cells experiments)
Brioudes et al. www.pnas.org/cgi/content/short/1007926107 11 of 11
