Extended Data Fig. 7: Transcriptional and protein stability assays of NSP1a.
a, qRT–PCR analysis of NSP1a in uninoculated (-R) and inoculated (+R) roots of WT Wm82 and btsa btsb-1 grown under NFe and LFe conditions sampled at 1 DAI. Expression was normalized to ELF1b and values are means of three technical repetitions and SEM. The experiments were repeated for three times with similar results. Significant differences were determined by two-way ANOVA (P < 0.05). NFe: 10 mM Fe-citrite, LFe: 200 mM AA-DP. b, Cell-free degradation assay with recombinant GST–NSP1a in protein extracts from N. bethamiana leaves transiently transformed with UBIpro:BTSa–3×HA or an empty-vector control (HA). The samples were incubated for the indicated times and protein abundance was determined by western blotting. The numbers at the bottom denote the relative intensities of GST–NSP1a normalized to 0 point. The experiments were repeated for three times with similar results. c, Quantification of GST–NSP1a band intensities in (b). d, In vivo analysis of NSP1a–MYC stability in the presence or absence of BTSa–HA. UBIpro: NSP1a–4×MYC was transiently expressed in N. bethamiana leaves with UBIpro:BTSa–3×HA or vector control (HA-EV). The samples were collected 48 h after infiltration and prepared for western blotting. The numbers at the bottom denote the relative intensities of NSP1a–MYC normalized to respective HA-EV control. e, Quantification of NSP1a–MYC band intensities in (d). Data are the means ± SD of n = 4. Significant difference was determined by two-tailed Student’ t-test (**P < 0.01).
