Protocols

Maize two-component trans-activation system (LhG4-pOp)

I. Background

In the maize two-component trans-activation system LhG4-pOp, a tissue specific promoter drives expression of the LhG4 transcription factor, which in turns binds to the pOp regulatory sequences and activates expression of a fluorescent tag (e.g. NLS-RFP) and the GUS reporter in the same cells (cis). This reporter line can also be crossed to another pOp-carrying line, to activate a different target gene (trans). This system can be used for tissue specific gene expression, knockouts or other genomic applications.



II. Construct development

First, to generate the driver construct the native promoter from the gene of interest, the LhG4 transcription factor, and the terminator sequence from the gene of interest are individually PCR-amplified and subcloned into the pDONR vector. Second, in the responder construct the pOp regulatory sequence is used to drive the expression of a fluorescent tag (e.g. NLS-RFP) and the GUS reporter gene in a Gateway destination vector. Third, the three pDONR subclones carrying parts of the driver contruct are transferred into the pOp Gateway destination vector through the Gateway LR reaction system. Finally, the expression clone will contain two cassettes: the driver and responder. The driver cassette carries the LhG4 transcription factor driven by the native promoter from the gene of interest. The responder cassette carries a fluorescent tag and the GUS reporter gene both under the control of the pOp promoter in response to LhG4.



Fluorescent Tagging of Full-Length Maize Genes

I. Fluorescent tags: Citrine-YFP and CFP

We routinely tag proteins with the Citrine variant of Yellow Fluorescent Protein (YFP) (Griesbeck et al. 2001), which can be used not only to visualize a single protein but also to study protein-protein interactions in vivo as an energy acceptor in BRET (Xu et al. 1999) and FRET assays (Tsien et al. 1998; Pollok et al. 1999). Moreover, Citrine-YFP has enhanced photostability and is much less sensitive to pH and anions, such as chloride, compared to other YFP variants (Griesbeck et al. 2001). The reduced sensitivity to pH allows detection of proteins targeted to the extracellular matrix or to other relatively acidic subcellular compartments, thus making this reporter more suitable for tagging proteins with a wide range of targeting specificities. Some proteins are also tagged with Cyan Fluorescent Protein (CFP) (ECFP, Clontech) for comparison of localization patterns obtained with different tags and for future colocalization and interaction studies.

For tagging, Citrine-YFP/CFP coding sequences, which lack start and stop codons, are flanked by linker peptides that function as flexible tethers, minimizing potential folding interference between Citrine-YFP/CFP and the tagged protein (Doyle et al. 1996). To avoid placing identical nucleotide sequences on each side of the tag, we use two different linkers: the N-terminus of the tag is linked to a glycine-rich linker peptide (Gly)5Ala, and the C-terminus is linked to an alanine-rich linker peptide AlaGly(Ala)5GlyAla.

The Citrine-YFP tag is amplified from the pRSETB-Citrine plasmid (Griesbeck et al. 2001) and the CFP tag from the pECFP-C1 plasmid (Clontech) using the ExTaq DNA polymerase (TaKaRa) and two primers shown in Figure 1. The products are cloned into the pTOPO TA vector (Invitrogen). The resulting cDNAs encoding the fluorescent tags contain recognition sequences for FseI and SfiI restriction endonucleases at their 3'- and 3'-ends, respectively. Plasmid with Citrine-YFP is designated as pCitrine-3 and plasmid with CFP as pCFP-3 (Figure 2).

Next, the fluorescent tag cDNA sequences are amplified from the pCitrine-3 and pCFP-3 plasmids using Pfu DNA polymerase (Invitrogen) and the forward and reverse Citrine-YFP/CFP primers (Figure 3) to produce the "TT-Citrine" and "TT-CFP" fragments.

PCR reaction mixture

PCR cycles

100 ng dna template
1x pfu-turbo reaction buffer
0.2 mm 4 x dntp
0.2 µm of each primer
0.025 u/µl pfu-turbo (invitrogen)
total volume: 25 µl

1 cycle:
94°C for 3 min
30 cycles:
94°C for 30 sec
70°C for 2 min
1 cycle:
70°C for 2 min

The PCR products are gel-purified using the GFX PCR purification kit (Amersham) or PCR Purification Kit (Qiagen) to remove dNTPs, primers and enzyme, and used in TT-PCR (see below).

II. Gene tagging

The entire protocol is summarized in Figure 4 and described in detail below.

1. First PCR reaction

a. Genomic DNA template

Genomic DNA is extracted from leaf material of maize plants using any protocol that yields relatively high molecular weight DNA. Genomic DNA isolated by QIAGEN kit works well for PCR.

b. Primers

Two sets of primers (P1/P2, P3/P4) for each gene are designed for the amplification of two genomic fragments using the Primer3 software. Our PCR design program considers a series of criteria including the position of each primer within the genomic sequence, annealing temperature, length, and hairpin structures in an iterative fashion to determine the most suitable sets of P1/P2 and P3/P4 for each gene.

The first set of primers amplifies a fragment (P1-P2) that extends from up to 3 kb upstream of the transcription start of the gene to the tag insertion site within the coding sequence. We assume that most maize promoters should be contained within 3 kb. However, if intergenic regions are <3 kbit can be shorter; we defined a minimal size for the 5' UTR and promoter region as 1 kb, extending P1 into the upstream ORF if the intergenic region is very small.

The second set of primers amplifies a fragment (P3-P4) from the tag insertion site to 0.5-1 kb downstream of the gene to include 3' UTR and regulatory sequences. The default position for the start of the gene-specific region of P2 and P3 primers is at the 30th nucleotide (i.e. 10 amino acids) upstream of the stop codon. However, if a functional domain is predicted at this position, or it does not generate a suitable primer sequence, the positions of P2 and P3 are reiteratively shifted from the initial site until suitable priming sites are determined.

P1 and P4 contain, in addition to gene-specific sequences, sequences partially overlapping the attB1 and attB2 Gateway forward and reverse primers, respectively (used for TT-PCR, see below). P2 and P3 contain sequences partially overlapping the Citrine-YFP/CFP primers (Figure 5).

c. PCR conditions

Because all primers in the TT-PCR reaction (P1-P4) have gene-specific sequences, it is impossible to calculate a standard annealing temperature for all genes to be tagged. Instead, we use touch-down PCR conditions to include a range of temperatures as shown below.

PCR reaction mixture

PCR cycles

100 ng DNA template
1 x KOD reaction buffer
1 x dNTP
0.2 µM of each primer
1U KOD (TaKaRa)
total volume 25 µl

1 cycle:
94°C for 3 min
7 cycles touch-down:
94°C for 30 sec
61°C for 30 sec; reduce by 1°C per cycle
68°C for 1 min per kb
24 cycles:
94°C for 30 sec
52-54°C for 30 sec
68°C for 1 min per kb
1 cycle:
68°C for 10-15min

The PCR products are gel-purified using the GFX PCR purification kit (Amersham).

2. Triple template PCR (TT-PCR)

All three amplified fragments, i.e., TT-Citrine or TT-CFP, P1-P2, and P3-P4, are combined together to serve as three overlapping templates for Long Flanking Homology (LFH) PCR (Wach 1996). This second PCR reaction, designated triple-template PCR (TT-PCR), utilizes two primers containing the complete attB1 and attB2 Gateway sequences (Walhout et al. 2000) and partially overlapping the P1 and P4 primers (Figure 4). Thus, TT-PCR introduces the fluorescent tag into the selected site within the target gene without the need for conventional cloning and results in an internally-tagged full-length gene sequence flanked by attB1 and attB2 sites ready for Gateway recombination cloning.

a. Three templates

P1-P2 fragment, TT-Citrine or TT-CFP fragment, and P3-P4 fragment.

b. Primers

Universal, gene-non-specific primers carrying the Gateway attB1 and attB2 sequences that overlap with the gene-non-specific sequences of P1 and P4 primers (Figure 6).

TT-PCR reaction mixture

TT-PCR cycles

100 ng P1-P2 fragment+50ng P3-P4 fragment+50ng TT-Citrine or TT-CFP
1x KOD reaction buffer
0.2 mM 4 x dNTP
0.2 µM of each primer
1 U KOD (TaKaRa)
total volume: 25 µl

1 cycle:
94°C for 3 min 30 sec
7 cycles touch-down:
94°C for 30 sec
61°C for 30 sec; reduce by 1°C per cycle
68°C for 1 min per kb
23 cycles:
94°C for 30 sec
52-54°C for 30 sec;
68°C for 1 min per kb
1 cycle:
68°C for 10-15 min

The PCR products are gel-purified using the GFX PCR purification kit (Amersham).

3. Gateway cloning of TT-PCR products into pDONR207

The Gateway system (Invitrogen) is based on bacteriophage l site-specific recombination (Landy 1989). Gateway cloning introduces the amplified TT-PCR product into the donor vector, pDONR207 (Invitrogen), by in vitro recombination between the attB1 and attB2 sequences that flank the TT-PCR product (see above) and the attP1 and attP2 sequences, respectively, of pDONR207. This attB x attP recombination is mediated by the BP reaction (Invitrogen) and produces the attL1 and attL2 sequences that flank the tagged gene within the pDONR vector.

Note that unrecombined pDONR vectors should be propagated in the DB3.1 strain of E. coli (Invitrogen) carrying the gyrA462 gene which confers resistance to the ccdB gene [its protein product, a natural analog of quinolone antibiotics, binds to the DNA gyrase subunit A and turns it into a poison (Bahassi et al. 1999)]. Following Gateway recombination, ccdB is replaced by the TT-PCR product, allowing selection for the recombinant clones in bacterial strains, such as DH5a or DH10B, that do not carry gyrA462 or F' episome (which also confers resistance to ccdB).

a. BP reaction and selection for recombinant clones

BP reaction mixture

BP reaction conditions

300 ng (in 1-5 µl) TT-PCR produc
150 ng (in 1 µl) pDONR207 Invitrogen)*
2 µl 5x BP Clonase reaction buffe
2 µl BP Clonase Invitrogen)
TE buffer (pH 8.0) to total volume of 10 ยต

Overnight incubation at 25°C.
*Note that unrecombined pDONR207 is toxic
to most bacterial strains and should be
propagated in the DB3.1 strain of E. coli
(Invitrogen) in the presence of chloramphenicol
and gentamycin

Add 1 µl Proteinase K (2 µg/µl) and incubate for 10 minutes at 37°C. Then, transform 2µl of the reaction mixture into 100 µl competent cells of the E. coli strain DH5a or DH10B and select for recombinants by plating on LB agar supplemented with 7 µg/ml gentamycin.

b. Identification of recombinant colonies with TT-PCR product

Pick 4-6 colonies per construct and analyze each by PCR for the presence of the TT-PCR product. Use the either of the following attL primers in combination with one gene specific primer.

forward attL1 primer: 5'-TCGCGTTAACGCTAGCATGGATCTC-'
reverse attL2 primer: 5'-gtaacatcagagattttgagacac-'

PCR reaction mixture

PCR cycles

1 bacterial colony
1 x Taq reaction buffer
0.2 mM 4 x dNTP
0.2 µM of each primer
total volume:20 uL
Incubate 10 min at °C to realease DNA
0.02 U/µl Taq (any brand)

1 cycle:
94°C for 3 min
25 cycles:
94°C for 30 sec
52°C for 30 sec
68°C for 1 min per kb
1 cycle:
72°C for 1 min per kb

Select positive clones, i.e., those that have the correct size insert, purify their plasmid DNA and sequence the tagged genes.

In our experiments, the efficiency of the recombination of the TT-PCR products into pDONR207 is 80-90%.

4. Gateway transfer of the tagged genes into binary destination vectors

a. Gateway binary destination vectors

The binary destination vector (pAM1006) was constructed by subcloning the Gateway conversion cassette into pTF101, a binary vector from the ISU transformation facility for monocot transformation. This vector has no regulatory sequences for expression of cloned genes and, thus, is useful for producing native levels and patterns of gene expression.

Also note that un-recombined destination vectors should be propagated in the ccdB-resistant DB3.1 strain of E. coli (Invitrogen) whereas, following Gateway recombination, the recombinant clones should be propagated in the ccdB-sensitive bacterial strains such as DH5a or DH10B (see description of pDONR207 above for more details).

b. LR reaction and selection and identification of recombinant clones

The tagged gene is transferred to the binary destination vector by in vitro recombination between the attL1 and attL2 sequences that flank the TT-PCR product in the pDONR vector (see above) and the attR1 and attR2 sequences, respectively, of the destination vector (Landy 1989, see also www.invitrogen.com). This attL x attR recombination is mediated by the LR reaction (Invitrogen) and produces the attB1 and attB2 sequences that flank the tagged gene within the binary vector.

LR reaction mixture

LR reaction conditions

200 ng pDONR construct
200 ng of pAM1006
0.5 µl topoisomerase I (10 U/µl)
2 µl 5x BP Clonase reaction buffer
2 µl BP Clonase (Invitrogen)
TE buffer (pH 8.0) to total volume of 10 µl

Overnight incubation at 25°C.
*Note that unrecombined destination vectors (such as pAM1006) are toxic to most bacterial strains and should be propagated in the DB3.1 strain of E. coli (Invitrogen) in the presence of chloramphenicol and spectinomycin.

Add 1 µl Proteinase K (2 µg/µl) and incubate for 10 minutes at 37°C. Then, transform 2µl of the reaction mixture into 100 µl competent cells of the E. coli strain DH5a or DH10B and plate one part of the transformation mixture on LB agar supplemented with 100 µg/ml spectinomycin to select for pAM1006 recombinants.

Pick 4-6 colonies per construct and analyze each by PCR, using P1 and P4 primers, for the presence of the TT-PCR product. In our experiments, the efficiency of the recombination of the TT-PCR products from pDONR into the binary destination vector is 90-100%.

III. Production of transgenic Maize expressing the tagged genes.

1. Introduction of binary constructs into Agrobacterium

Transform the binary plasmid into Agrobacterium tumefaciens strain EHA101 (Selection plates are LB agar with 100 mg/L spectinomycin, 50 mg/L kanamycin and 25 mg/L chloramphenicol), re-isolate plasmid and check by restriction enzyme digestion.

2. Agrobacterium-mediated transformation of Maize

You will need to get a permit from APHIS for inter-state transport of Agrobacterium as well as a notification for getting transgenic maize plants back and growing them to seed. This can take anywhere from 1-6 months, so plan early. Send the Agrobacterium strain along with a copy of the permit to Dr. Kan Wang at the Iowa State University transformation facility.

3. Introgression of transgenic HiII maize plants expressing the tagged genes to B73.

Normally it takes 3-4 months to get the transgenic seedlings from Iowa State facility. They notify you by mail about the status of your clones. Start putting B73 seeds (I realized that B73 grows a lot slower than other varieties) to be crossed to transgenic lines. It's a good idea to plant 5-10 seeds every 3-4 days at least a month before the shipment of seedlings start coming from Iowa. After seedlings arrive, check in the microscope for presence of GFP. Grow 4-5 plants from independent lines (at least 5 independent lines). Cross them to B73. I got more seeds when I crossed the transgenic pollen to B73 ear than vice versa. Follow APHIS regulations for growing transgenic plant materials.

Bibliography

This protocol is based on one designed for Arabidopsis TTPCR tagging:

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