DUF581-9 weakens the interaction between KIN10 and GRIK2 in vivo. A) DUF581-9 mCherry disrupts the BiFC signal generated from the association of KIN10 Venus^N173 and GRIK2-Venus^C155 in planta. Proteins were transiently expressed in leaves of N. benthamiana using Agrobacterium infiltration. Yellow fluorescent protein (YFP) and red fluorescence (mCherry) was monitored by confocal microscopy 48 h post-infiltration. Upper panel shows the reconstituted YFP fluorescence in the absence or presence of a third partner. Middle panel shows mCherry fluorescence of DUF581-9 mCherry, DUF581-9_C47S mCherry, or KINß2 mCherry. Lower panel shows the merge of YFP and mCherry fluorescence. The homomerization of KIN10-Venus^N173 and KINß2-Venus^C155 was used as control and is not negatively affected by DUF581-9 mCherry. The scale bar represents 20 µm. B) The fluorescence signal intensities of YFP and mCherry (effector) were determined along a line drawn on the confocal images using ImageJ software. Box plots represent the mean ± Sd of n = 18 to 38 individual cells for YFP fluorescence intensity and n = 17 to 28 cells for RFP fluorescence intensity (indicated as single dots). Statistically significant differences are shown as P-value between the samples against KIN10 Venus^N173 and GRIK2-Venus^C155 or against KIN10 Venus^N173 and KINß2-Venus^C155 control as determined by 1-way ANOVA followed by Dunnett’s multiple comparisons test. Regarding the RFP fluorescence signal, box plots represent the mean ± Sd, and letters above the bars represent a statistical significance determined by 1-way ANOVA (P < 0.05). The experiment was carried out twice with similar results.