Escherichia coli (E. coli) DH5α [F^- endA glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15Δ(lacZYA-argF)U169, hsdR17(r_K ^-m_K ⁺), λ-] and A. tumefaciens (GV2260) [C58 background, rifampicin resistant with the Ti plasmid (pTiB6s3)]

1. E. coli DH5α and A. tumefaciens GV2260 strains were streaked on Luria Broth (LB) agar media supplemented with appropriate antibiotics and incubated at 37°C and 28°C, respectively.

2. Single colonies of DH5α and A. tumefaciens (GV2260) were inoculated in 50 ml of LB liquid media, incubated at 37° and 28°C, respectively, and shaken at 200 rpm for 24 hours.

3. A new 500 ml LB liquid culture was re-inoculated from the initial saturated bacterial culture to produce a final concentration of OD_{600 nm} = 0.1. The liquid cultures were incubated at 18°C and 28°C for DH5α and A. tumefaciens (GV2260), respectively, until the OD at 600 nm reached 0.6.

4. The bacterial liquid culture was subsequently chilled on ice for 20 minutes.

5. Bacterial cells were harvested by centrifugation at 6000 × g for 10 minutes at 4°C.

6. The bacterial cells were washed twice with 250 ml of ice-cold water, and then finally washed with 25 ml of 10% ice-cold glycerol.

7. The bacterial cells were pelleted by centrifugation and re-suspended in 5 ml of 10% ice-cold glycerol.

8. A 100 μl aliquot of bacterial cells was transferred to 1.5 ml microtubes and immediately frozen in liquid nitrogen.

9. The cells were stored at -80°C for at least six months without loss of competency.

N. benthamiana (PI 555478) plants were propagated in a growth chamber programmed for 16 hours light (140 μmol/m²/s cool white fluorescent irradiance) at 28°C and 8 hours dark at 24°C. Agrobacterium-mediated transient assays were conducted on three to four week old plants.

The SacB-SacR genes were amplified by PCR from plasmid pMsacB 18 using primers SacB/R Forward: 5'-AATCAGGAAGGGATGGCTGAGGGATATCGGATCGATCCTTTTTAACCCATCAC-3'and SacB/R Reverse: 5'-AGACCGGCACACTGG

CCATATCGGTGG

The iProof ™ high fidelity Taq DNA polymerase (Bio-Rad, Hercules, CA) PCR program consisted of 1 cycle at 98°C (2 min), followed by 30 cycles at 98°C (30 s), 55°C (45 s), and 72°C (1 min), and finished with a 1 cycle extension at 72°C (7 min). The PCR products were digested with BbvCI and BstXI (New England BioLabs Inc, Ipswich, MA), and gel purified using an AccuPrep™ Gel Purification Kit (Bioneer, Alameda, CA).

The destination binary plasmid pEarleyGate101 9 was obtained from the Arabidopsis Biological Resource Center at Ohio State University (Columbus, OH) and digested with BbvCI and BstXI to remove the ccdB gene. The gel-purified PCR product of the SacB-SacR gene cassette was then cloned into the BbvCI and BstXI restriction sites of pEarleyGate101. The ligation products were transformed into DH5α using a gene pulse electroporation apparatus (2.5 Kv/uM/mSec) (Bio-Rad). Successfully transformed bacteria were selected for on LB medium supplemented with kanamycin (50 mg/ml) and chloramphenicol (25 mg/ml). The derived new destination vector was re-named pEG101-SacB/R.

All plasmid DNA was isolated using an AccuPrep™ Plasmid Extraction Kit (Bioneer).

Bacterial cells that were successfully transformed with the pEG101-SacB/R plasmid were negatively selected for on LB medium supplemented with 5% sucrose as described previously 13 14 .

The open reading frame (ORF) of the Luc gene was cloned from plasmid pDesA-luc 19 with primers: Sal_LucFor:5'-caccgtcgacGGCGGTGGCTCATCTGGCGGAGGT

atg

The Gateway^® LR clonase enzyme mix kit (Invitrogen) was used for the LR recombination reaction. In brief, the LR reaction mixture contained: 1-5 μl (100 ng) of entry clone plasmid DNA, 1 μl (100 ng) of destination vector pEG101-SacB/R plasmid DNA, 2 μl of 5 × LR clonase reaction buffer, and 2 μl of LR clonase enzyme. The reaction mixture was brought to a final volume of 10 μl with TE buffer (pH 8.0) and incubated at room temperature for 1 hour. Following the incubation, a 3-5 μl aliquot was mixed with 100 μl of competent A. tumefaciens (GV2260) cells. A gene pulse apparatus (Bio-Rad), programmed for 2.5 Kv/uM/mSec, was used to transform the cells by electroporation. Following electroporation, the mixture was resuspended in 1 ml of liquid LB and incubated shaking at 28°C/200 rpm for 3 hours. Successfully transformed cells were then selected for on LB agar supplemented with kanamycin (50 mg/ml), rifampicin (100 mg/ml), and 5% sucrose. The LB plates were incubated at 28°C for 2-3 days. After incubation, ten colonies were randomly selected for further analysis. Recombinant plasmids were confirmed by PCR with primers: 35S forward 5'-AAGAAGACGTTCCAACCACGTC-3' and NLuc reverse: 5'-AACTGCAGTCATCCATCCTTGTCAATCAAG-3' to detect the Luc gene (1.4 kb) 19 . Recombinant plasmids were also confirmed by PCR using primers 35S forward plus Aae2166 reverse: 5'- GTCGACTTCGATAGCTTTTCTGATTTTTCTCA-3' to detect the Aae2166 gene (1.1 kb). The PCR fragments were separated on a 0.8% agarose gel, stained with 0.01% ethidium bromide solution, and visualized using the Gel-Document Image System™ under UV light (Bio-Rad).

Agrobacterium-mediated transient assays in N. benthamiana plants were performed as described previously 20 . In brief, the Agrobacterium strain was streaked on LB medium supplemented with appropriate antibiotics and incubated at 28°C for 2 days. Bacterial cells were harvested and re-suspended in induction buffer composed of 10 mM MgCl₂, 10 mM MES (pH 5.6), and 100 μM acetosyringone and incubated for 3 hours at room temperature. The bacterial inoculums were adjusted to OD_{600 nm} = 0.6 and infiltrated into the stomata of fully expanded N. benthamiana leaves using a 1 ml blunt-end syringe without a needle. The inoculated plants were incubated at room temperature under continuous light for 20-48 hours before the detection of expressed proteins. Transient expression of the Luc gene was detected by applying 1 μM luciferin to the inoculation site 21 . The chemical fluorescent signals were captured with a CCD camera and visualized using the Gel-Document Image System (Bio-Rad). The fluorescent signal of the Aae2166-GFP fusion protein was monitored 20 hours after inoculation by fluorescent microscopy (Zeiss Axio Observer.A1, Carl Zeiss MicroImaging, Inc., Thornwood, NY).

In order to construct a new Gateway^®-compatible destination binary vector that can be used for selection of recombinant plasmids in A. tumefaciens, the SacB-SacR gene cassette was amplified and cloned into the BbvCI and BstXI restriction sites of pEarleyGate 101 9 . The SacB-SacR genes replaced ccdB to generate a novel destination vector, pEG101-SacB/R. The key components of pEG101-SacB/R are illustrated in Figure 1A. To test the functionality of the SacB-SacR gene cassette as an effective negative selectable marker, A. tumefaciens strain GV2260, A. tumefaciens strain GV2260 carrying pEarleyGate101, and A. tumefaciens strain GV2260 carrying pEG101-SacB/R were grown on LB agar medium supplemented with or without 5% sucrose (Figure 2). As shown in Figure 2A and 2B, A. tumefaciens grew equally well on LB medium with or without 5% sucrose, which suggested that sucrose in LB agar medium has no inhibitory effect to A. tumefaciens. The A. tumefaciens strain GV2260 carrying pEarleyGate101 also grew well on LB agar medium with or without 5% sucrose (Figure 2C and 2D). This result demonstrates that the ccdb gene cassette in pEarleyGate101 cannot be used as a negative selectable marker in A. tumefaciens. To test the functionality of the SacB-SacR gene cassette as an effective negative selectable marker in A. tumefaciens, the pEG101-SacB/R plasmid DNA was transformed into A. tumefaciens strain GV2260. After transformation, ten randomly selected clones were raised on LB medium supplemented with or without 5% sucrose. A. tumefaciens carrying pEG101-SacB/R grew well on LB agar medium without sucrose (Figure 2E); however, their growth was completely inhibited when the LB agar medium was supplemented with 5% sucrose (Figure 2F). These results show that the new destination vector, pEG101-SacB/R, can efficiently select against A. tumefaciens when grown on LB agar medium supplemented with 5% sucrose.

To test the stability of destination vector pEG101-SacB/R in E. coli, the restriction patterns of pEG101-SacB/R were compared before and after repeated sub-culturing. A single E. coli clone carrying pEG101-SacB/R was grown overnight in LB liquid medium supplemented with both kanamycin and chloramphenicol. The overnight bacterial culture was diluted (5000 ×) with LB medium and sub-cultured for an additional 24 hours. This process was repeated five times as described previously 22 . The plasmid DNA of pEG101-SacB/R was isolated after each generation and digested with BstXI and BbvCI to remove a 1.9 kb fragment corresponding to the inserted SacB-SacR gene cassette. As shown in Figure 3, the restriction digestion patterns of pEG101-SacB/R from each generation were identical. All plasmid DNAs were further transformed into both E. coli and A. tumefaciens and the derived clones were re-plated on LB agar medium supplemented with or without 5% sucrose. All transformed bacteria colonies failed to grow on LB agar medium supplemented with 5% sucrose, confirming the functionality of the SacB-SacR genes. These results demonstrate that pEG101-SacB/R can be stably propagated in E. coli grown on LB agar medium without sucrose.

The functionality of pEG101-SacB/R for Gateway^® LR cloning was tested in A. tumefaciens using two entry clones carrying the Luc and Aae2166 genes, respectively. A. tumefaciens strain GV2260 was directly transformed with the LR reaction mixture for each gene by electroporation. Putative recombinant clones were selected on LB agar medium supplemented with kanamycin (50 mg/ml) and 5% sucrose. Ten randomly selected A. tumefaciens clones were analyzed by PCR using either Luc or Aae2166 gene specific primers, or a combination of gene specific primers with a primer designed using the 35S promoter in the vector pEG101-SacB/R as a template (Figure 1A). As shown in Figure 4, all colonies tested were positive, suggesting a high cloning efficiency of targeted genes into pEG101-SacB/R in A. tumefaciens. We also used the vector to clone twenty two bacterial type III effector genes of Acidovorax avenae pv. citrulli, which gave a similar cloning efficiency (data not shown).

As shown in Figure 1A, the cloned Luc and Aae2166 genes are driven by the 35S promoter. The ORF of Aae2166 was cloned, without a stop codon, into the pEntry/D vector. After LR cloning, the lack of a stop codon allowed the Aae2166 ORF to be fused in-frame with the N-terminus of the GFP gene in the recombinant pEG101-Aae2166 plasmid. Conversely, a stop codon was added to the C-terminus of the Luc gene; therefore, GFP fusion would not have occurred.

To examine the expression of the Luc and Aae2166 genes cloned in pEG101-SacB/R, A. tumefaciens colonies carrying the recombinant plasmids pEG101-Luc or pEG101-Aae2166 were inoculated into the leaves of N. benthamiana. As shown in Figure 5B, A. tumefaciens colonies carrying pEG101-Luc (Figure 5 B-2) but not pEarleyGate 101 (Figure 5 B-1) triggered strong luminescence after treatment with luciferin, thereby confirming the expression of luciferase. As a negative control, the same A. tumefaciens strain, carrying pEG101-Luc, was spotted onto either the leaf surface of N. benthamiana or onto a LB agar plate. After treatment with luciferin, the samples were examined under a CCD camera and no luminescence signal was detected (data not shown). This result confirmed that the luminescence signal in Figure 5B-2 was from the transiently transformed N. benthamiana plant cells. When N. benthamiana plants were inoculated with A. tumefaciens colonies carrying pEG101-Aae2166, cell death was observed at 48 hours after inoculation (Figure 5C-2). As a negative control, an A. tumefaciens strain carrying an un-modified pEarleyGate101 was inoculated into the leaves of N. benthamiana, which did not trigger cell death at 48 hours after inoculation (Figure 5C-1). Since we modified Aae2166 to contain a C-terminal GFP tag (Figure 1A), pEG101-Aae2166 expressed an Aae2166-GFP fusion protein. As shown in Figure 5E, the fluorescent signal of the Aae2166-GFP fusion protein was visible by fluorescent microscopy. The picture in Figure 5E was taken at 20 hours after inoculation, at which point there was no cell death observed at this time. This eliminated the possibility of autofluorescent signals from dead cells. Aae2166-GFP proteins were predominantly localized in the cytosol of transformed cells, although this observation requires further confirmation by an independent method. As a negative control, A. tumefaciens carrying an un-modified pEarleyGate101 did not incite a fluorescent signal in N. benthamiana (Figure 5D). These results demonstrated that two different genes could be efficiently cloned into pEG101-SacB/R using the Gateway LR reaction with direct selection in A. tumefaciens. The two genes were functionally expressed when assayed by Agrobacterium-mediated transient assays.