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

Department of Horticulture, Zhejiang University, Hangzhou, People's Republic of China.
Plant physiology (Impact Factor: 6.84). 04/2012; 159(2):810-25. DOI: 10.1104/pp.112.196816
Source: PubMed

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.

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    • "ut the possibility that additional VQ were missed because of gaps in the genome assembly or incomplete / wrong gene predictions . Based on Wang et al . ( 2014 ) , also the number of grapevine WRKYs is lower when compared with Arabidopsis and rice . Similar sizes of the group I and IIc WRKYs and VQ multigenic families were reported in Arabidopsis ( Cheng et al . , 2012 ) , but despite that , interaction partnership between the WRKY and VQ proteins was found not to be highly specific . In fact , yeast two - hybrid assays showed that a single VQ protein is able to interact with multiple WRKYs , and different VQ proteins have partially overlapping pools of interacting WRKY partners . Thus , there might n"
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    ABSTRACT: WRKY proteins are a class of transcription factors (TFs) involved in the regulation of various physiological processes, including the plant response to biotic and abiotic stresses. Recent studies in Arabidopsis have revealed that some WRKY TFs interact with a class of proteins designed as VQ proteins because of their typical conserved motif (FxxxVQxLTG). So far, no information is available about the genomic organization and the function of VQ motif-containing protein in grapevine (Vitis vinifera L). In the current study, we analysed the 12X V1 prediction of the nearly homozygous V. vinifera PN40024 genotype identifying up to 18 predicted VQ genes (VvVQ). VvVQs phylogenetic and bioinformatic analyses indicated that the intron-exon structures and motif distribution are highly divergent between different members of the grapevine VQ family. Moreover the analysis of the V. vinifera cv. Corvina expression atlas revealed a tissue- and stage- specific expression of several members of the family which also showed a significant correlation with WRKY TFs. Grapevine VQ genes also exhibited altered expression in response to drought, powdery mildew infection, salicylic acid (SA) and ethylene (ET) treatments. The present study represent the first characterization of VQ genes in a grapevine genotype and it is a pivotal foundation for further studies aimed at functionally characterizing this mostly unknown grapevine multigenic family.
    Frontiers in Plant Science 05/2015; 6. DOI:10.3389/fpls.2015.00417 · 3.95 Impact Factor
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    • "The regulatory mechanisms for NbWRKY8 and OsWRKY33 DNA-binding activity via phosphorylation are still unclear; however, it has been reported that 2 VQ motif-containing proteins (SIB1 and SIB2) interact with AtWRKY33, resulting in stimulation of the W-box binding activity of AtWRKY33, and overexpression of SIB1 enhanced disease resistance to the necrotrophic pathogen Botrytis cinerea in an AtWRKY33-dependent manner [48]. In Arabidopsis, there are 34 VQ motif-containing protein genes, and expression of the majority of these is responsive to pathogen infection [49]. In the rice genome, a number of genes were found to encode VQ motif-containing proteins and our previous transcriptome data showed that some of these genes were upregulated in response to rice blast fungus infection [50]. "
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    ABSTRACT: WRKY transcription factors and mitogen-activated protein kinase (MAPK) cascades have been shown to play pivotal roles in the regulation of plant defense responses. We previously reported that OsWRKY53-overexpressing rice plants showed enhanced resistance to the rice blast fungus. In this study, we identified OsWRKY53 as a substrate of OsMPK3/OsMPK6, components of a fungal PAMP-responsive MAPK cascade in rice, and analyzed the effect of OsWRKY53 phosphorylation on the regulation of basal defense responses to a virulence race of rice blast fungus Magnaporthe oryzae strain Ina86-137. An in vitro phosphorylation assay revealed that the OsMPK3/OsMPK6 activated by OsMKK4 phosphorylated OsWRKY53 recombinant protein at its multiple clustered serine-proline residues (SP cluster). When OsWRKY53 was coexpressed with a constitutively active mutant of OsMKK4 in a transient reporter gene assay, the enhanced transactivation activity of OsWRKY53 was found to be dependent on phosphorylation of the SP cluster. Transgenic rice plants overexpressing a phospho-mimic mutant of OsWRKY53 (OsWRKY53SD) showed further-enhanced disease resistance to the blast fungus compared to native OsWRKY53-overexpressing rice plants, and a substantial number of defense-related genes, including pathogenesis-related protein genes, were more upregulated in the OsWRKY53SD-overexpressing plants compared to the OsWRKY53-overexpressing plants. These results strongly suggest that the OsMKK4-OsMPK3/OsMPK6 cascade regulates transactivation activity of OsWRKY53, and overexpression of the phospho-mimic mutant of OsWRKY53 results in a major change to the rice transcriptome at steady state that leads to activation of a defense response against the blast fungus in rice plants.
    PLoS ONE 06/2014; 9(6):e98737. DOI:10.1371/journal.pone.0098737 · 3.23 Impact Factor
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    • "However, the C-terminal WRKY domain is also responsible for mediating protein–protein interactions (Lai et al., 2011), so that these two functions overlap in this domain. Besides, in a yeast two-hybrid screen with Arabidopsis VQ and WRKY proteins, the C-terminal WRKY domain of group I WRKY proteins and the sole WRKY domain of group IIc WRKY proteins seem to be important for protein–protein interactions (Cheng et al., 2012). Group IIa WRKY proteins contain canonical LZ sequences and many other group II and III WRKYs have at least multiple leucine, isoleucine or valine residues at their N-termini, forming a similar structure of a LZ for protein–protein interactions (Chi et al., 2013). "
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    ABSTRACT: bZIPs and WRKYs are two important plant transcription factor (TF) families regulating diverse developmental and stress-related processes. Since a partial overlap in these biological processes is obvious, it can be speculated that they fulfill non-redundant functions in a complex regulatory network. Here, we focus on the regulatory mechanisms that are so far described for bZIPs and WRKYs. bZIP factors need to heterodimerize for DNA-binding and regulation of transcription, and based on a bioinformatics approach, bZIPs can build up more than the double of protein interactions than WRKYs. In contrast, an enrichment of the WRKY DNA-binding motifs can be found in WRKY promoters, a phenomenon which is not observed for the bZIP family. Thus, the two TF families follow two different functional strategies in which WRKYs regulate each other's transcription in a transcriptional network whereas bZIP action relies on intensive heterodimerization.
    Frontiers in Plant Science 04/2014; 5:169. DOI:10.3389/fpls.2014.00169 · 3.95 Impact Factor
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