Table 1.
Summary of mitochondrial processing efficiency and solubility of the processed form of pFAγ51::Nif proteins expressed in Nicotiana benthamiana, and effect of pFAγ51 nine amino acid “scar” (scar9) peptide translationally fused to individual Nif proteins on nitrogenase function tested in Escherichia coli.
| N. benthamiana construct | Mitochondrial processing | Solubility of processed form | E. coli construct | E. coli function testing | ||
|---|---|---|---|---|---|---|
| % Activity of MIT v2.1 | S.D. | n | ||||
| n/a | n/a | n/a | MIT v2.1 | 100 | 33 | 8 |
| pFAγ51::J | ++ | – | scar9::J | 257 | 72 | 4 |
| pFAγ51::H | ++ | – | scar9::H | 124 | 12 | 4 |
| pFAγ51::B | ++ | – | scar9::B | 120 | 28 | 4 |
| pFAγ51::D | n/a# | n/a# | scar9::D | 110 | 9 | 2 |
| pFAγ51::F | + | ++ | scar9::F | 109 | 6 | 4 |
| pFAγ51::Q | – | ?* | scar9::Q | 102 | 49 | 4 |
| pFAγ51::W | ++ | + | scar9::W | 57 | 26 | 4 |
| pFAγ51::U | ++ | + | scar9::U | 55 | 14 | 4 |
| pFAγ51::V | ++ | – | scar9::V | 52 | 18 | 4 |
| pFAγ51::K | ++ | – | scar9::K | 50 | 5 | 4 |
| pFAγ51::Y | + | + | scar9::Y | 44 | 12 | 4 |
| pFAγ51::Z | + | + | scar9::Z | 43 | 23 | 4 |
| pFAγ51::S | + | + | scar9::S | 39 | 5 | 4 |
| pFAγ51::N | ++ | + | scar9::N | 38 | 7 | 3 |
| pFAγ51::E | ++ | – | scar9::E | 33 | 2 | 4 |
| pFAγ51::M | + | ++ | scar9::M | 8 | 2 | 4 |
| pFAγ51::X | + | + | scar9::X | n/a | n/a | n/a |
| n/a | n/a | n/a | ΔnifM | 5 | 3 | 3 |
| n/a | n/a | n/a | NifK::HA | 0 | 1 | 3 |
| n/a | n/a | n/a | p-ori (ΔnifJDKYENJ) | 0 | 0 | 8 |
Values are presented as % acetylene reduction activity compared to MIT v2.1. pB-ori, negative control containing nifBQFUSVWZM; ▵nifM, NifM coding sequence removed from MIT v2.1; NifK::HA, HA epitope tag fused to the C-terminus of NifK; S.D., standard deviation (n=2-8). ++, efficiently processed or mostly soluble; + partially processed or partially soluble; - not processed or insoluble. #Reported in Allen et al., 2020. *Solubility of pFAγ51::NifQ in N. benthamiana may be an artifact during soluble protein extraction.