Table IV. Various 14-3-3 isoforms from N. benthamiana interact with recombinant HopQ1-6xHis in a phosphorylation-dependent manner.
| 14-3-3 Isoform | Nonphosphorylated HopQ1-6xHisa | In Vitro Phosphorylated HopQ1-6xHisb | In Vitro Phosphorylated HopQ1-S51A-6xHisb |
|---|---|---|---|
| a | 0 | 47c | 0 |
| b | 0 | 0d | 0 |
| c | 0 | 44 | 0 |
| d | 0 | 0 | 0 |
| e | 0 | 28 | 0 |
| f | 0 | 0 | 0 |
| g | 0 | 16 | 0 |
| h | 0 | 36 | 0 |
| i | 0 | 23 | 0 |
Recombinant HopQ1-6xHis purified from E. coli was incubated with N. benthamiana crude protein extract. After consecutive purification steps, the sample was subjected to LC-MS/MS analyses. Protein coverage for HopQ1 was 87. bRecombinant HopQ1-6xHis and HopQ1-S51A-6xHis were phosphorylated in vitro prior to incubation with N. benthamiana crude protein extract. After consecutive purification steps, the samples were subjected to LC-MS/MS analyses. Protein coverage parameters for HopQ1 and HopQ1-S51A in these experiments were 83 and 86, respectively. cProtein coverage parameters of 14-3-3 isoforms that copurified with HopQ1-6xHis calculated by Mascot software following LC-MS/MS analyses. dProtein coverage 0 refers to the 14-3-3 isoform that has not been identified by LC-MS/MS analyses in this experiment. The experiment was performed twice.