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. 2024 Apr 1;5(7):100890. doi: 10.1016/j.xplc.2024.100890

Figure 3.

Figure 3

TRB proteins from the two clades physically and genetically interact with each other.

(A) Interactions among the five Arabidopsis TRB proteins probed in the Y2H system. Growth on selective medium lacking histidine and adenine reveals interaction between the two proteins tested. Horizontal, translational fusions with the Gal4-Activation domain (AD); vertical, translational fusion with the Gal4-DNA-binding domain (BD). BD and AD indicate the respective empty vectors.

(B) BiFC reveals protein–protein interactions (PPIs) between TRB proteins within each clade and between proteins from the TRB_I and TRB_II clades in N. benthamiana leaf cells. Maximum intensity projections of z stacks acquired with a confocal microscope are shown. PPI takes place within distinct nuclear speckles, which likely correspond to telomeres. No signal was observed when TRB4-nYFP was co-expressed with the C-terminal part of YFP alone.

(C) Representative WT, trb4-1 trb5-1 double-mutant, trb2-1 trb4-1 trb5-1 triple-mutant, and trb2-1 trb3-1 trb4-1 trb5-1 quadruple-mutant plants at 3 weeks of age. Scale bar, 1 cm.

(D) Quantification of the root length of in vitro-grown WT, trb4-1 trb5-1 double mutants, trb2-1 trb4-1 trb5-1 triple mutants, and trb2-1 trb3-1 trb4-1 trb5-1 quadruple mutants at days 3 (D3), 5 (D5), and 7 (D7) after germination. For each time point, values from two independent replicates are shown. Different letters indicate significant differences determined by Mann–Whitney test (p < 0.01) among samples from the same time point.

(E) Representative trb4-1 trb5-1 trb1-1/TRB1 mutant plant. Scale bar, 1 cm.

(F) Mean number of seeds per silique from WT, trb4-1 trb5-1, and trb4-1 trb5-1 trb1-1/TRB1 plants. Seeds from at least 27 siliques from five plants were counted. About 12% fewer seeds were present in the trb4-1 trb5-1 mother plant heterozygous for the trb1 mutation compared with the trb4-1 trb5-1 double mutants (∗∗∗p < 0.0001, t-test).

(G) Seeds from trb4-1 trb5-1 trb1-1/TRB1 plants, revealing the presence of shriveled, non-germinating seeds marked with an asterisk.