Fig. 3: TaNHLP1-A interacts with TaRACK1A to regulate tiller number in wheat through ABA signaling.

a Interaction between TaNHLP1-A and TaRACK1A-A was demonstrated using a split-ubiquitin yeast two-hybrid assay. Yeast cells were first selected on synthetic dropout (SD) medium lacking Leu and Trp (SD-LW) and then transferred to SD-Leu/-Trp/-His/-Ade (SD-LWHA) selective medium with 25 mM 3-AT for protein interaction analysis. b Luciferase complementation imaging assays conducted in N. benthamiana leaves confirmed the interaction between TaNHLP1-A and TaRACK1A-A. c In vivo interaction between MYC-TaNHLP1-A and HA-TaRACK1A-A was revealed through co-immunoprecipitation assays. Following immunoprecipitation with anti-MYC magnetic beads, the precipitated proteins were probed using anti-HA and anti-MYC antibodies, respectively. Two independent experiments were performed with similar results. d Morphological observations of the shoot base and tiller buds (highlighted with red lines), which were utilized for RNA-seq and analysis of ABA levels. Scale bar, 10 mm. e Quantification of ABA contents in the shoot base and tiller buds of the Fielder and the Tanhlp1-1 mutant (T₃ generation, n = 4). f Heatmap depicting the expression levels of differentially expressed genes in the shoot base and tiller buds shared between the wheat variety Fielder (negative control) and the Tanhlp1-1 mutant. g Subcellular localization of TaRACK1A-A-mCherry with or without TaNHLP1-A-GFP in N. benthamiana leaves. White and blue arrows indicate nucleus and punctate dots, respectively. Three independent experiments were performed with similar results. Scale bars, 20 μm. Representative phenotypes h and tiller number i of Fielder (negative control) and CRISPR/Cas9-generated Tarack1a mutants. p = 0.8392 (Tarack1a#1 vs. Tarack1a#2), 0.9621 (Tarack1a#1 vs. Tarack1a#3), 0.9856 (Tarack1a#2 vs. Tarack1a#3), <0.0001 for other comparisons. The sample size (n) for each group is listed at the bottom of the rectangular column; scale bar in (h) is 15 cm. j Quantification of ABA contents in the shoot base and tiller buds of the Fielder and the Tarack1a#1 mutant (T₃ generation, n = 4). Data are presented as mean ± SD. The P-values in (e) and (j) were determined using a two-tailed unpaired Student’s t test, and the different letters indicate significant differences between groups in i, as determined by one way ANOVA with Tukey’s multiple comparisons range tests (p < 0.01). Source data are provided as a Source Data file.