Table 2. The effect of vir gene mutations.
| Agrobacterium straina | No. yeast coloniesc | Output yeastd 10–4 | Output Agroe 10–6 | Excision efficiencyf | |
|---|---|---|---|---|---|
| Not diluted | 10–2 Dilution | ||||
| A. On Cre::VirE2 protein translocation from Agrobacterium into yeastb | |||||
| 1100 (Cre) | 6 | 0 | 25 | 5 | 2 × 10–5 |
| 1100 (Cre::VirE2) | Full | 11 | 24 | 73 | 5 × 10–3 |
| virA (Cre::VirE2) | 12 | 0 | 30 | 58 | 4 × 10–5 |
| virB4 (Cre::VirE2) | 26 | 0 | 37 | 7 (10–5) | 7 × 10–5 |
| virB7 (Cre::VirE2) | 5 | 0 | 17 | 66 | 3 × 10–5 |
| virG (Cre::VirE2) | 5 | 0 | 28 | 61 | 2 × 10–5 |
| virC2 (Cre::VirE2) | Full | 12 | 15 | 54 | 8 × 10–3 |
| virD1 (Cre::VirE2) | 5 | 0 | 16 | 98 | 3 × 10–5 |
| 3′virD2 (Cre::VirE2) | 43 | 0 | 26 | 34 | 2 × 10–4 |
| virD4 (Cre::VirE2) | 5 | 0 | 18 | 16 | 3 × 10–5 |
| virE2 (Cre::VirE2) | Full | 23 | 19 | 39 | 1 × 10–2 |
| virF (Cre::VirE2) | Full | 8 | 25 | 72 | 3 × 10–3 |
| virD1+pD3, D4 (Cre::VirE2) | Full | 13 | 46 | 72 | 3 × 10–3 |
| 3′virD2+pD3, D4 (Cre::VirE2) | Full | 11 | 30 | 40 | 4 × 10–3 |
| B. On NLS::Cre::VirFΔ42N protein translocation from Agrobacterium into yeastb | |||||
| 1100 (Cre) | 9 | 0 | 241 | 5 | 4 × 10–6 |
| 1100 (NLS::Cre::VirFΔ42N) | Full | 80 | 74 | 25 | 1 × 10–2 |
| virA (NLS::Cre::VirFΔ42N) | 1 | 0 | 43 | 15 | 2 × 10–6 |
| virB4 (NLS::Cre::VirFΔ42N) | 8 | 0 | 89 | 2 (10–5) | 9 × 10–6 |
| virB7 (NLS::Cre::VirFΔ42N) | 3 | 0 | 53 | 26 | 6 × 10–6 |
| virG (NLS::Cre::VirFΔ42N) | 27 | 0 | 387 | 74 | 7 × 10–6 |
| virC2 (NLS::Cre::VirFΔ42N) | Full | 51 | 74 | 12 | 7 × 10–3 |
| virD1 (NLS::Cre::VirFΔ42N) | 12 | 0 | 68 | 18 | 2 × 10–5 |
| 3′virD2 (NLS::Cre::VirFΔ42N) | 301 | 0 | 55 | 29 | 5 × 10–4 |
| virD4 (NLS::Cre::VirFΔ42N) | 1 | 0 | 104 | 10 | 1 × 10–6 |
| virE2 (NLS::Cre::VirFΔ42N) | Full | 26 | 56 | 8 (10–5) | 5 × 10–3 |
| virF (NLS::Cre::VirFΔ42N) | Full | 102 | 76 | 18 | 1 × 10–2 |
| virE3 (NLS::Cre::VirFΔ42N) | Full | 80 | 81 | 30 | 1 × 10–2 |
| 1100+pD3, D4 (NLS::Cre::VirFΔ42N) | Full | 106 | 104 | 24 | 1 × 10–2 |
| virD1+pD3, D4 (NLS::Cre::VirFΔ42N) | Full | 131 | 115 | 19 | 1 × 10–2 |
| 3′virD2+pD3, D4 (NLS::Cre::VirFΔ42N) | Full | 152 | 107 | 12 | 1 × 10–2 |
aAgrobacterium wild type strain LBA1100 (disarmed pTi) and the vir mutants carried a non-mobilisable plasmid expressing Cre alone (negative control), Cre::VirE2 (A) or NLS::Cre::VirFΔ42N (B). All vir mutants, except for virF and virE3, which are deletion mutants, were obtained by Tn2hoho insertion. Plasmid pD3,D4 expresses the VirD3 and VirD4 proteins.
bThe recipient yeast strain contains a floxed URA3 gene at chromosome V. Agrobacterium and yeast were co-cultivated for 6 days at 22°C and plated on medium containing 0.1% FOA.
cAfter co-cultivation 100 µl of cells were plated (not diluted and 10–2) on medium containing 0.1% FOA. The number of yeast Ura– colonies was determined after 4 days at 30°C. Full, too many colonies to count.
d,eThe output number of yeast and Agrobacterium cells was determined on LC and MY medium, respectively. For three Agrobacterium strains the output number was determined for a dilution of 10–5.
fThe Cre-excision efficiency is determined by the number of yeast colonies on medium containing FOA per number of surviving yeast colonies (output yeast).