The role of protein-protein interactions mediated by the PB1 domain of NLP transcription factors in nitrate-inducible gene expression

Background: NIN-LIKE PROTEIN (NLP) transcription factors are master regulators of nitrate-inducible gene expression in higher plants. NLP transcription factors contain a nitrate signal-responsive domain in the amino-terminal region, an RWP-RK-type DNA-binding domain in the middle, and a Phox and Bem1 (PB1) domain at the carboxy terminus. Although the PB1 domain of NLP transcription factors appears to mediate protein-protein interactions associated with nitrate-inducible gene expression in higher plants, its precise role in nitrate-inducible gene expression has not previously been characterized. Results: Yeast two-hybrid assays with the PB1 domain of the Arabidopsis transcription factor NLP7 revealed NLP-NLP interactions that required the core amino acid residues (K867, D909, D911, and E913) within the PB1 domain. Consistent with previous speculation on redundant and overlapping functions between different Arabidopsis NLP transcription factors, NLP-NLP interactions were observed between a variety of combinations of different NLP transcription factors. Furthermore, a mutated form of NLP7 that harbored amino acid substitutions at K867, D909, D911, and E913 required a far higher level of expression than wild-type NLP7 to restore nitrate-responsive gene expression and growth of nlp6 nlp7-1 double mutants. Surprisingly, however, the ability to transactivate nitrate-responsive promoters in protoplast transient expression assays was similar between wild-type and mutant forms of NLP7, suggesting that the PB1 domain was not required for transcription from naked DNA. Conclusions: Protein-protein interactions mediated by the PB1 domain of NLP transcription factors are necessary for full induction of nitrate-dependent expression of target genes in planta. The PB1 domains of NLP transcription factors may act on gene expression from chromosomal DNA via homo- and hetero-oligomerization in the presence of nitrate.