Structural and Functional Analysis of VQ Motif-Containing Proteins in Arabidopsis as Interacting Proteins of WRKY Transcription Factors    

Abstract

WRKY transcription factors are encoded by a large gene superfamily with a broad range of roles in plants. Recently, several groups have reported that proteins containing a short VQ (FxxxVQxLTG) motif interact with WRKY proteins. We have recently discovered that two VQ proteins from Arabidopsis (Arabidopsis thaliana), SIGMA FACTOR-INTERACTING PROTEIN1 and SIGMA FACTOR-INTERACTING PROTEIN2, act as coactivators of WRKY33 in plant defense by specifically recognizing the C-terminal WRKY domain and stimulating the DNA-binding activity of WRKY33. In this study, we have analyzed the entire family of 34 structurally divergent VQ proteins from Arabidopsis. Yeast (Saccharomyces cerevisiae) two-hybrid assays showed that Arabidopsis VQ proteins interacted specifically with the C-terminal WRKY domains of group I and the sole WRKY domains of group IIc WRKY proteins. Using site-directed mutagenesis, we identified structural features of these two closely related groups of WRKY domains that are critical for interaction with VQ proteins. Quantitative reverse transcription polymerase chain reaction revealed that expression of a majority of Arabidopsis VQ genes was responsive to pathogen infection and salicylic acid treatment. Functional analysis using both knockout mutants and overexpression lines revealed strong phenotypes in growth, development, and susceptibility to pathogen infection. Altered phenotypes were substantially enhanced through cooverexpression of genes encoding interacting VQ and WRKY proteins. These findings indicate that VQ proteins play an important role in plant growth, development, and response to environmental conditions, most likely by acting as cofactors of group I and IIc WRKY transcription factors.WRKY proteins are a relatively recently identified class of sequence-specific DNA-binding transcription factors found almost exclusively in plants (Rushton et al., 2010). The characteristic structural feature of WRKY proteins is the highly conserved WRKY domain, which contains the almost invariant WRKYGQK sequence at the N terminus followed by a Cx 4-5 Cx 22-23 HxH or Cx 7 Cx 23 HxC zinc-finger motif (Rushton et al., 2010). Genes encoding WRKY proteins have been identified in low photosynthetic and nonphotosynthetic eukaryotes, but they have greatly proliferated and form large superfamilies only in higher plants with more than 70 members in Arabidopsis (Arabidopsis thaliana; Zhang and Wang, 2005). Based on the number and structures of the conserved WRKY zinc-finger motifs, WRKY proteins were initially classified into three groups (Eulgem et al., 2000). The first group contains two Cx 4 Cx 22-23 HxH zinc-finger motifs, the second group contains one Cx 4-5 Cx 23 HxH zinc-finger motif, and the third group contains one Cx 7 Cx 23 HxC zinc-finger motif. More recent analyses, however, have shown that group II WRKY proteins can be further divided into IIa, IIb, IIc, IId, and IIe subgroups (Zhang and Wang, 2005;Rushton et al., 2010). In the green alga Chlamydomonas reinhardtii as well as in the nonphotosynthetic slime mo...

The VQ Motif-Containing Protein Family of Plant-Specific Transcriptional Regulators

“…A loss-of-function mutant harboring a transposon tag in vq8 displayed a pale-green and stunted growth phenotype throughout its life cycle (Cheng et al, 2012). It is likely that VQ8 regulates chloroplast development or photosystem assembly, consistent with its predicted localization to plastids.…”

“…In addition, VQ22 may interact with WRKY28 and WRKY51 to negatively control JA-mediated defense (Hu et al, 2013a). Two systematic yeast two-hybrid assays showed that a large proportion of VQ proteins associate physically with members of group I or group IIc WRKY transcription factors (Table I; Rushton et al, 2010;Cheng et al, 2012;Pecher et al, 2014). This interaction with WRKY members was further validated for VQ4 in Arabidopsis protoplasts .…”

“…Most Arabidopsis VQ proteins are predicted to localize to the nucleus, some localize to plastids, and a few localize to mitochondria solely from bioinformatic prediction ( Fig. 2; Cheng et al, 2012). Experimental evidence is required to confirm the subcellular localization in future studies.…”

“…Transcription regulators that do not bind DNA often work in concert with transcription factors via protein-protein interactions to fine-tune the regulatory machinery in response to surrounding environments, such as biotic stress and light stimuli (Wray et al, 2003;Yamaguchi-Shinozaki and Shinozaki, 2006;Jiao et al, 2007;Buscaill and Rivas 2014). A group of proteins designated as VQ motif-containing proteins (referred to here as VQ proteins) was recently identified (Xie et al, 2010;Cheng et al, 2012). Since the discovery of the first VQ protein, SIGMA FACTOR-BINDING PROTEIN1 (SIB1, also known as VQ23; Morikawa et al, 2002), nine VQ proteins have been functionally characterized.…”

A systems biology perspective on the role of WRKY transcription factors in drought responses in plants

“…Recently, vQ proteins were reported to act as co-factors that positively or negatively regulate gene expression (Cheng et al 2012). Also, chromatin remodeling has also started to emerge as an important mode whereby the regulatory mechanism modulates expression of target gene expression during stress responses (Trivedi et al 2012;wang et al 2012a).…”