The Maize TFome--development of a transcription factor open reading frame collection for functional genomics

Abstract

Establishing the architecture of the gene regulatory networks (GRNs) responsible for controlling the transcription of all genes in an organism is a natural development that follows elucidation of the genome sequence. Reconstruction of the GRN requires the availability of a series of molecular tools and resources that so far have been limited to a few model organisms. One such resource consists of collections of transcription factor (TF) open reading frames (ORFs) cloned into vectors that facilitate easy expression in plants or microorganisms. In this study, we describe the development of a publicly available maize TF ORF collection (TFome) of 2034 clones corresponding to 2017 unique gene models in recombination-ready vectors that make possible the facile mobilization of the TF sequences into a number of different expression vectors. The collection also includes several hundred co-regulators (CoREGs), which we classified into well-defined families, and for which we propose here a standard nomenclature, as we have previously done for TFs. We describe the strategies employed to overcome the limitations associated with cloning ORFs from a genome that remains incompletely annotated, with a partial full-length cDNA set available, and with many TF/CoREG genes lacking experimental support. In many instances this required the combination of genome-wide expression data with gene synthesis approaches. The strategies developed will be valuable for developing similar resources for other agriculturally important plants. Information on all the clones generated is available through the GRASSIUS knowledgebase (http://grassius.org/).

Keywords: GRASSIUS; Zea mays; gene regulatory network; grasses; maize; recombination-ready; transcription factor; yeast one-hybrid; yeast two-hybrid.

Figure 1

Flowchart used for the generation of the maize TFome and CoREGome. (a) Flowchart describing the strategy for template identification for PCR amplification of transcription factor (TF) open reading frames (ORFs). FL, full length; AGI, Arizona Genomics Institute.(b) Distribution of template sources for the ORFs in the maize TF ORF collection (TFome).

Figure 2

Summary of the gene content of the maize transcription factor (TF) open reading frame (ORF) collection (TFome).(a) Maize TFome families.(b) Maize co-regulator open reading frame collection (CoREGome) families. The black bars indicate the number of gene models currently assigned to each protein family and the gray bars indicate the current state of progress towards cloning all of the these genes into Gateway® entry vectors. Further descriptions of each TF family may be found at the GRASSIUS website (http://www.grassius.org).

Figure 3

Schematic diagram of the strategy and constructs utilized for protein fusion expression experiments in pJ69.4a yeast cells or maize protoplasts.pENTR®SD/D-TOPO® vectors harboring synthesized open reading frames with stop codons were used as entry constructs for recombination into the p1511 and pBD-GAL4 vectors using LR Clonase, to generate GFP and GAL4-BD protein fusions, respectively.

Figure 4

Impact of codon usage on transcription factor (TF) expression in yeast and maize. (a) Yeast cells were transformed with different constructs on SD-Trp to evaluate growth depending on the different codon usage. Western blot of yeast protein extracts was done from the strains expressing GAL4-BD fused TFs. The blot was probed with the GAL4-BD monoclonal antibody (SC510). Ponceau Red staining was used as a loading control. #34, GRMZM2G162434; #75, GRMZM2G001875; #93, GRMZM2G051793; M, maize optimized open reading frame (ORF); Y, yeast optimized ORF; YM, a hybrid optimized construct for both maize and yeast; R^1–252, the N-terminal region of maize R.(b) Fluorescent micrographs showing the expression and localization of N-terminal GFP fusions of different constructs optimized for expression in yeast (Y), maize (M) and hybrid (YM) for each of the following transcription factors: GRMZM2G162434, GRMZM2G001875, GRMZM2G051793.(c) Graph bar showing the relative GFP intensity level for each construct tested.

Figure 5

Example of an alternative splice variant of the GRMZM2G475303 transcript that encodes ZmNLP6. In the gene diagram the white bars indicate exons and the lines represent introns. The gray and black shaded regions represent the RWP-RK DNA-binding motif (pfam02042) and a PB1 protein–protein interaction motif (pfam00564), respectively. The removal of a 85-bp intron in the alternative splice variant is predicted to result in an earlier stop codon and a deletion of the PB1 domain in the translated protein. aa, amino acids.