BdHD1, a histone deacetylase of Brachypodium distachyon, interacts with two drought-responsive transcription factors, BdWRKY24 and BdMYB22

ABSTRACT

Histone deacetylases (HDACs) play an important role in plant stress response. In Brachypodium distachyon, which is model species for molecular biology research on monocot plants, the histone deacetylase BdHD1, homologous to AtHDAC1 of the RPD3/HDA1 class, functions as a positive regulator in the plant drought stress response. AtHDAC1 has been found to interact with transcription factors to regulate gene expression. However, the drought-responsive transcription factors that interact with BdHD1 have not been identified yet. Previously, we identified BdWRKY24 and BdMYB22 as drought responsive transcription factors in Brachypodium. In this study, we used yeast two-hybrid (Y2 H) and bimolecular fluorescence complementation (BiFC) assays to show that BdHD1 interacts with BdWRKY24 and BdMYB22. Our findings provides a base to investigate BdHD1-transcription factor complexes in the context of drought stress response in Brachypodium.

Plants are frequently subjected to abiotic and biotic stressors in their environment. At the molecular level, changes in gene transcriptional regulatory networks occur in response to stress. Gene expression driven by stress cues often depends on epigenetic regulatory mechanisms, including histone acetylation.^1,2 Histone acetylation is mediated by two sets of enzymes, histone acetyltransferase (HAT) and histone deacetylase (HDAC). In Arabidopsis thaliana, a number of HDACs, including HDA6,^3,4 HDA9,⁵ and HDA19,^3,6,7 have been found to participate in regulating stress-related genes. We previously identified HDACs based on sequence similarity in the model monocot plant species, Brachypodium distachyon. BdHD1 is the closest homologous gene to AtHDAC1 in Arabidopsis and it acts as a positive regulator in abscisic acid (ABA) and drought stress responses.⁸ Understanding the mechanisms for how BdHD1 regulates drought stress response in Brachypodium requires further study. Current evidence shows that certain HDACs interact with specific transcription factors to regulate distinct sets of biological processes.^9,10 However, transcription factors that participate in BdHD1-complexes have not yet been reported. Identifying candidate proteins that interact with BdHD1 is a first step to reveal the role of the BdHD1 complex in regulating gene expression for stress response.

Some members of the WRKY and MYB transcription factor families have been found to interact with HDACs in Arabidopsis. BdWRKY24 and BdMYB22 have been identified as drought responsive transcription factors based on our previous findings^8,11 and are therefore interesting candidates for this study. BdHOS15 is a WD-40 repeat family protein and it shares 59% similarity with AtHOS15, which interacts with HDA9 and HDA19 to regulate gene expresion.^12,13 These proteins were selected as potential interacting partners in the BdHD1-complex.

To investigate interactions with BdHD1, the transcription factors BdWRKY24, BdMYB22 and BdHOS15 were subjected to a yeast two-hybrid (Y2 H) assay. The coding sequence of BdHD1 was fused to the GAL4 activation domain (AD), whereas the coding sequence of each gene, BdWRKY24, BdMYB22 and BdHOS15, was fused to the GAL4 DNA-binding (BK) domain. Yeast cells were co-transformed with BdHD1-AD and each of BdWRKY24-BK, BdMYB22-BK, BdHOS15-BK and -BK as a negative control. Yeast colonies formed on the Double Dropout (DDO) (-Trp/-Leu) medium for each co-transformation, indicating the success of co-transformation (Figure 1a,). Yeast colonies for each co-transformation were picked from the DDO medium and plated on the Quadruple Dropout (QDO) (-Trp/-Leu/-His/-Ade) medium. Yeast cells co-transformed with BdHD1-AD+BdWRKY24-BK or BdHD1-AD+BdMYB22-BK grew successfully on the QDO medium, indicating that BdHD1 interacts with both BdWRKY24 and BdMYB22, respectively (Figure 1a). However, no interaction between BdHD1 and BdHOS15 was observed. The control did not show an interaction, as expected.

Figure 1.

Interaction of BdHD1 with BdWRKY24 and BdWRKY22. a. Yeast two-hybrid (Y2 H) assays. BdHD1 was fused to the GFL4 activation domain (AD), whereas each of BdWRKY24, BdMYB22 and BdHOS15 was fused to the GAL4 DNA-binding (BK) domain. DDO indicates Double Dropout and QDO means Quadruple Dropout. b. Bimolecular fluorescence complementation (BiFC) assays. Partial yellow fluorescent protein fusion constructs containing either BdHD1 or each of BdWRKY24, BdMYB22 and BdHOS15 were transiently co-expressed in Nicotiana benthamiana leaves. YFP signals indicate protein interaction.

Bimolecular fluorescence complementation (BiFC) was conducted to confirm the results from Y2 H. The coding sequence of BdHD1 was inserted into pEarlygate201-YN and the coding sequence of each gene, BdWRKY24, BdMYB22 and BdHOS15, was inserted into pEarlygate202-YC. The vectors encode either the N- or the C-terminal fragment of yellow fluorescent protein (YFP). Leaves of Nicotiana benthamiana plants were infiltrated with BdHD1-YN and each of BdWRKY24-YC, BdMYB22-YC, BdHOS15-YC and -YC. Leaves co-infiltrated with BdHD1-YN and BdWRKY24-YC or BdHD1-YN and BdMYB22-YC expressed YFP signals in the nucleus, indicating protein-protein interactions between BdHD1 and each of BdWRKY24 and BdMYB22, respectively (Figure 1b). No YFP signals were observed from the leaves co-infiltrated with BdHD1-YN+BdHOS15-YC, which confirmed the result of Y2 H that BdHD1 does not interact with BdHOS15 (Figure 1b). The control did not show a protein interaction signal.

Together, we identified protein-protein interactions between BdHD1 and BdWRKY24, and between BdHD1 and BdMYB22. It has been reported that HDA19 interacts with two WRKY proteins, WRKY38 and WRKY62, in basal defense¹⁴ in Arabidopsis. BdMYB22 possesses two SANT domains which are often found in the chromatin modifying complex.¹⁵ A recent study has indicated that the MYB transcription factor MYB95 interacts with HDA15 to suppress negative regulators of ABA signaling in Arabidopsis.¹⁶ Moreover, BdHD1 as well as BdMYB22 and BdWRKY24 are all drought-responsive regulators in Brachypodium.^8,11 Interestingly, no interactions between BdHD1 and BdHOS15 were observed in our experiments, which is in contrast to the interaction exhibited between HDAC1 and HOS15 in Arabidopsis. This result may be due to the different gene regulatory networks between monocots and dicots.¹⁷ Our study provides a first step to explore the drought-responsive target genes of BdHD1-complexes. Future work is needed to study the biological relevance of these interactions in terms of regulating downstream genes.

Funding Statement

This work was supported by the Natural Sciences and Engineering Research Council of Canada [Discovery Grant].