KISS ME DEADLY F-box proteins modulate cytokinin responses by targeting the transcription factor TCP14 for degradation

Abstract

The F-box proteins KISS ME DEADLY interact with the transcription factor TCP14 and target it for degradation to fine-tune cytokinin responses in leaves and flowers.

Figure 1.

TCP14 interacts with KMDs. A, Y2H assays showing interaction of TCP14 with KMD proteins. Transformed yeast cells were grown in synthetic drop-out medium lacking tryptophan and leucine (−) WL as a transformation control and in (−) WL media lacking histidine (–) WLH, or lacking histidine and adenine (–) WLHA for interaction assays. Cotransformation with empty plasmids was used as negative control. 3-amino-1,2,4-triazole (3AT) was added at indicated concentration (mM) to better visualize positive interactions. B, Pull-down assay using E. coli–expressed GST or GST-KMD1 and BLRP-TCP14 transiently expressed in N. benthamiana. BLRP-TCP14 was used as bait and incubated with equal amount of GST or GST-KMD1. Streptavidin agarose beads were used to pull down the BLRP-TCP14 and its interacting proteins. Left panels (pull-down): equal amount of eluted proteins were loaded on two different membranes; one membrane was probed with anti-GST antibody (α-GST) to detect GST or GST-KMD1, the other membrane was probed with horseradish peroxidase-conjugated streptavidin to detect the BLRP-TCP14. C, Immunoblot analysis of protein extracts from N. benthamiana leaves agro-infiltrated with the indicated constructs (TCP14-GFP, KMD2-Myc, or GFP, numbers indicate agro-infiltration doses). TCP14-GFP and GFP (upper and bottom panel, respectively) and KMD2-Myc (middle panel) were detected using anti-GFP (α-GFP) and anti-Myc (α-Myc) antibodies, respectively. BR-biological replicate. D, Inflorescences of Col, kmd1/2/4, pAS1:FLAG-TCP14 (FLAG-TCP14), and pAS1:FLAG-TCP14 in kmd1/2/4. Bar = 1 mm. E, Immunoblot analysis of proteins extracted from young flowers. FLAG-TCP14 was detected using anti-FLAG antibody (α-FLAG). Ponceau S staining was used to evaluate protein loading.

Figure 2

The loss of KMD1/2/4 partially restored TCP14 accumulation in spy mutant. A, Rosette leaves (leaf no. 5) of WT Col, spy-3, kmd1/2/4, transgenic pAS1:FLAG-TCP14 (FLAG-TCP14), FLAG-TCP14 in spy-3, and FLAG-TCP14 in spy-3 kmd1/2/4. B, Flowers (flower no. 7) of the same lines. C, Number of trichomes on sepals of the same lines. Values are means of 20 flowers taken from five plants ± SE. Small letters above the columns represent significant differences between respective lines by Student’s t test (P < 0.05). D, Immunoblot analysis of proteins extracted from transgenic young flowers of pAS1:FLAG-TCP14, pAS1:FLAG-TCP14 spy-3, and pAS1:FLAG-TCP14 spy-3 kmd1/2/4. FLAG-TCP14 was detected using anti-FLAG antibody. Coomassie staining was used to evaluate protein loading.