Inhibition of Arabidopsis thaliana CIN‐like TCP transcription factors by Agrobacterium T‐DNA‐encoded 6B proteins

Agrobacterium T‐DNA‐encoded 6B proteins cause remarkable growth effects in plants. Nicotiana otophora carries two cellular T‐DNAs with three slightly divergent 6b genes (TE‐1‐6b‐L, TE‐1‐6b‐R and TE‐2‐6b) originating from a natural transformation event. In Arabidopsis thaliana, expression of 2×35S:TE‐2‐6b, but not 2×35S:TE‐1‐6b‐L or 2×35S:TE‐1‐6b‐R, led to plants with crinkly leaves, which strongly resembled mutants of the miR319a/TCP module. This module is composed of MIR319A and five CIN‐like TCP (TEOSINTHE BRANCHED1, CYCLOIDEA and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR) genes (TCP2, TCP3, TCP4, TCP10 and TCP24) targeted by miR319a. The CIN‐like TCP genes encode transcription factors and are required for cell division arrest at leaf margins during development. MIR319A overexpression causes excessive growth and crinkly leaves. TE‐2‐6b plants did not show increased miR319a levels, but the mRNA levels of the TCP4 target gene LOX2 were decreased, as in jaw‐D plants. Co‐expression of green fluorescent protein (GFP)‐tagged TCPs with native or red fluorescent protein (RFP)‐tagged TE‐6B proteins led to an increase in TCP protein levels and formation of numerous cytoplasmic dots containing 6B and TCP proteins. Yeast double‐hybrid experiments confirmed 6B/TCP binding and showed that TE‐1‐6B‐L and TE‐1‐6B‐R bind a smaller set of TCP proteins than TE‐2‐6B. A single nucleotide mutation in TE‐1‐6B‐R enlarged its TCP‐binding repertoire to that of TE‐2‐6B and caused a crinkly phenotype in Arabidopsis. Deletion analysis showed that TE‐2‐6B targets the TCP4 DNA‐binding domain and directly interferes with transcriptional activation. Taken together, these results provide detailed insights into the mechanism of action of the N. otophora TE‐encoded 6b genes.