Research article
Heterologous expression of wheat WRKY transcription factor genes transcriptionally activated in hybrid necrosis strains alters abiotic and biotic stress tolerance in transgenic Arabidopsis

https://doi.org/10.1016/j.plaphy.2020.02.029Get rights and content

Highlights

  • Four wheat WRKY transcription factor genes commonly upregulated in hybrid necrosis were identified.

  • Transgenic Arabidopsis expressing the wheat WRKY gene increased biotic and abiotic stress tolerance.

  • The four wheat WRKY genes act as integrated hubs of multiple stress signaling pathways.

Abstract

Hybrid necrosis and hybrid chlorosis are sometimes observed in interspecific hybrids between the tetraploid wheat cultivar Langdon and diploid wild wheat Aegilops tauschii. Many WRKY transcription factor genes are dramatically upregulated in necrosis and chlorosis wheat hybrids. Here, we isolated cDNA clones for four wheat WRKY transcription factor genes, TaWRKY49, TaWRKY92, TaWRKY112, and TaWRKY142, that were commonly upregulated in the hybrid necrosis and hybrid chlorosis and belonged to the same clade of the WRKY gene family. Expression patterns of the four TaWRKY genes in response to several stress conditions were similar in wheat seeding leaves. The four TaWRKY-GFP fusion proteins were targeted to the nucleus in onion epidermal cells. The TaWRKY gene expression levels were increased by high salt, dehydration, darkness, and blast fungus treatment in common wheat. Expression of either of the TaWRKY genes increased salinity and osmotic stress tolerance accompanied with overexpression of STZ/Zat10, and induced overexpression of the salicylic acid-signal pathway marker gene AtPR1 in transgenic Arabidopsis. TaWRKY142 expression also induced the jasmonic acid-pathway marker gene AtPDF1.2 and enhanced resistance against the fungal pathogen Colletotrichum higginsianum in transgenic Arabidopsis. These results suggest that the four TaWRKY genes act as integrated hubs of multiple stress signaling pathways in wheat and play important roles in autoimmune response-inducing hybrid necrosis and hybrid chlorosis.

Introduction

Hybrid incompatibilities observed in interspecific crosses can act as postzygotic reproductive barriers to prevent allopolyploid speciation (Rieseberg and Willis, 2007). Allopolyploid speciation is a major evolutionary process in higher plants, including common wheat. As a typical hybrid incompatibly phenotype, hybrid necrosis is triggered by autoimmune responses due to epistatic interactions of at least two loci or alleles (Bomblies and Weigel, 2007). The causative genes of hybrid necrosis have been elucidated to encode disease resistance-related proteins in various plant species such as Arabidopsis, lettuce, and rice (Bomblies et al., 2007; Jeuken et al., 2009; Alcázar et al., 2009, 2010; Yamamoto et al., 2010; Chae et al., 2014). The hypersensitive response (HR) occurs with great frequency, and a number of defense-related genes are upregulated in hybrid necrosis.

Common wheat is an allohexaploid species with an AABBDD genome derived through spontaneous crossings of cultivated tetraploid wheat with pollen of the D-genome donor Aegilops tauschii Coss. This evolutionary process can be reproduced through artificial crosses between tetraploid wheat and Ae. tauschii (McFadden and Sears, 1946; Matsuoka, 2011). However, hybrid growth abnormalities such as hybrid necrosis are frequently observed in the interspecific crosses, and four types, including types II and III hybrid necrosis, are among the abnormal growth phenotypes found in ABD hybrids between the tetraploid wheat cultivar Langdon (Ldn) and diverse Ae. tauschii accessions (Matsuoka et al., 2007; Mizuno et al., 2010). Necrotic lesions or sectors appear on the leaf surfaces gradually starting with older tissues in type III necrosis-exhibiting ABD hybrids, whereas necrotic cell death in type II necrosis occurs under low temperature conditions (Mizuno et al., 2010, 2011). Hybrid necrosis is also observed in intraspecific crosses of common wheat and is controlled by the Ne1 and Ne2 complementary loci; this type is called type I necrosis (Tsunewaki, 1960; Chu et al., 2006). Type IV necrosis, which has a phenotype visibly similar to the symptoms in types I and III necrosis, appears in interspecific crosses of two wild diploid Triticum species, Triticum monococcum subspecies aegilopoides and Triticum urartu (Yamagishi, 1987; Takamatsu et al., 2015). Wheat hybrid necrosis is accompanied by upregulation of defense-related genes, the generation of reactive oxygen species, and a reduction of photosynthesis activity before symptoms appear in leaves (Mizuno et al., 2010, 2011; Takamatsu et al., 2015). Therefore, similar to hybrid necrosis in other plant species, the programmed cell death in type I to IV wheat necrosis could be regarded as HR-like cell death. Enhanced resistance to wheat blast fungus has previously been observed in hybrid chlorosis synthetic wheat hexaploids (Nakano et al., 2015). In addition, gene expression profiles in which a large number of defense-related genes are upregulated strongly resemble those of hybrid chlorosis and hybrid necrosis in wheat (Mizuno et al., 2011; Nakano et al., 2015; Takamatsu et al., 2015).

Transcriptome characterizations of wheat hybrid necrosis based on microarray analyses have revealed upregulation of common transcription factor genes in types I to IV (Mizuno et al., 2010, 2011; Takamatsu et al., 2015). In particular, some WRKY transcription factor-encoding genes are highly upregulated in leaves of F1 plants showing hybrid necrosis. A large number of WRKY transcription factors have been identified in various plant species, and many of them play important roles in defense responses, abiotic stress responses, senescence, and various processes of plant development (Eulgem et al., 2000; Rushton et al., 2010; Ishihama and Yoshioka, 2012). The WRKY transcription factors also control secondary metabolites including bioactive compounds in medicinal plants (Singh et al., 2017; Deng et al., 2019). More than 1000 genes are activated by each WRKY transcription factor, and the downstream target genes are at least partly overlapped by multiple WRKY transcription factors (Birkenbihl et al., 2017). Overexpression of the WRKY transcription factor genes enhances disease resistance to bacterial and fungal pathogens and tolerance to salt, drought, and osmotic stress in transgenic plants. In the common wheat genome, now more than 170 WRKY transcription factors have been identified (Okay et al., 2014; Ma et al., 2017; Ning et al., 2017). Draft genome information of T. urartu and Ae. tauschii includes, respectively, 78 and 103 WRKY transcription factors (Ma et al., 2014, 2015). Recent progresses using transgenic plants partly characterized the functions of some wheat WRKY transcription factors. Overexpression of several wheat WRKY transcription factor genes enhances drought, salt, and/or freezing stress tolerance in transgenic Arabidopsis, tobacco and wheat (Niu et al., 2012; Wang et al., 2013; Ma et al., 2017; Gao, Wang, Xu,&Zhang, 2018; Zhou et al., 2019). Some wheat WRKY transcription factors also improves defense responses and disease resistance in transgenic plants (Wang et al., 2017). In addition, TaWRKY51 controls lateral root formation through regulation of ethylene biosynthesis (Hu et al., 2018). Here, we selected four wheat WRKY transcription factor genes that are commonly upregulated in types I to IV necrosis. We assumed that the selected WRKY transcription factors should be associated with the HR-like cell death in wheat hybrid necrosis and chlorosis through regulation of the biotic and abiotic stress signal pathways. The objectives of the present study were (1) to verify the transcriptional responses of the four WRKY transcription factor genes to biotic and abiotic stress, and (2) to confirm the alteration of biotic and abiotic stress tolerance in transgenic Arabidopsis plants expressing the wheat WRKY transcription factors. Based on our results, we also discuss their critical functions in the induction of necrotic symptoms in wheat hybrids.

Section snippets

Plant materials

The common wheat (Triticum aestivum L.) cultivar Chinese Spring (CS); two synthetic hexaploid wheat lines, one derived from a cross between the durum wheat (Triticum turgidum L.) cultivar Langdon (Ldn) and Aegilops tauschii Coss. KU-2828 to generate type III hybrid necrosis, and one between Ldn and KU-2059 resulting in normal growth (Mizuno et al., 2011); and two interspecific hybrids, one from a cross between Triticum monococcum subspecies aegilopoides (Link) Thell. (syn. T. boeoticum Boiss.)

Identification of TaWRKY genes upregulated in wheat crosses showing hybrid necrosis

Based on the data from our previous transcriptome analyses using a wheat 38K microarray, we found that four WRKY-type transcription factor-encoding genes, TaWRKY49, TaWRKY92, TaWRKY112, and TaWRKY142, were highly and commonly upregulated in the leaves of interspecific hybrids and synthetic hexaploid wheat lines showing hybrid necrosis/chlorosis phenotypes (Mizuno et al., 2010, 2011; Nakano et al., 2015; Takamatsu et al., 2015). The four TaWRKY genes belonging to the wheat 38K microarray were

Discussion

Defense-related genes, including some WRKY transcription genes, are dramatically upregulated in various wheat hybrids showing necrosis and chlorosis (Mizuno et al., 2010, 2011; Nakano et al., 2015; Takamatsu et al., 2015). Here, we characterized four wheat WRKY transcription factor-encoding genes that were commonly upregulated in necrosis and chlorosis wheat hybrids. As of now, more than 170 WRKY transcription factors have been identified in the common wheat genome (Ma et al., 2017; Ning et

CRediT authorship contribution statement

Yasunobu Kuki: Data curation, Formal analysis. Ryoko Ohno: Data curation, Formal analysis. Kentaro Yoshida: Formal analysis. Shigeo Takumi: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing - original draft.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank Drs. Yasuo Niwa of the University of Shizuoka and Yoshitaka Takano of Kyoto University for kindly providing us the 35S::GFP plasmid vector and the C. higginsianum strain, respectively. This work was supported by a Grant-in-Aid for Scientific Research (C) No. 26450480 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan to RO, a Grant-in-Aid for Scientific Research (B) No. 16H04862 from MEXT to ST, and by a Grant-in-Aid for Scientific Research on

References (56)

  • R. Alcázar et al.

    Incremental steps toward incompatibility revealed by Arabidopsis epistatic interactions modulating salicylic acid pathway activation

    Proc. Natl. Acad. Sci. U.S.A.

    (2009)
  • R. Alcázar et al.

    Natural variation at Strubbelig Receptor Kinase 3 drives immune-triggered incompatibilities between Arabidopsis thaliana accessions

    Nat. Genet.

    (2010)
  • R.P. Birkenbihl et al.

    Induced genome-wide binding of three Arabidopsis WRKY transcription factors during early MAMP-triggered immunity

    Plant Cell

    (2017)
  • K. Bomblies et al.

    Hybrid necrosis: autoimmunity as a potential gene-flow barrier in plant species

    Nat. Rev. Genet.

    (2007)
  • K. Bomblies et al.

    Autoimmune response as a mechanism for Dobzhansky-Muller-type incompatibility syndrome in plants

    PLoS Biol.

    (2007)
  • J. Castresana

    Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis

    Mol. Biol. Evol.

    (2000)
  • J. Chen et al.

    NPR1 promotes its own and target gene expression in plant defense by recruiting CDK8

    Plant Physiol

    (2019)
  • C.G. Chu et al.

    Molecular mapping of hybrid necrosis genes Ne1 and Ne2 in hexaploid wheat using microsatellite markers

    Theor. Appl. Genet.

    (2006)
  • R.C. Edgar

    MUSCLE: multiple sequence alignment with high accuracy and high throughput

    Nucleic Acids Res.

    (2004)
  • J. Gao et al.

    WRKY transcription factors associated with NPR1-mediated acquired resistance in barley are potential resources to improve wheat resistance to Puccinia triticina

    Front. Plant Sci.

    (2018)
  • S. Guindon et al.

    New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0

    Syst. Biol.

    (2010)
  • Z. Hu et al.

    TaWRKY51 promotes lateral root formation through negative regulation of ethylene biosynthesis in wheat (Triticum aestivum L.)

    Plant J.

    (2018)
  • M.J.W. Jeuken et al.

    Rin4 causes hybrid necrosis and race-specific resistance in an interspecific lettuce hybrid

    Plant Cell

    (2009)
  • K. Kawaura et al.

    Genome-wide analysis for identification of salt-responsive genes in common wheat

    Funct. Integr. Genom.

    (2008)
  • J. Ma et al.

    Genome-wide analysis of the WRKY transcription factors in Aegilops tauschii

    Cytogenet. Genome Res.

    (2014)
  • J. Ma et al.

    Identification and analysis of WRKY transcription factors in Triticum urartu

    Acta Agron. Sin.

    (2015)
  • J. Ma et al.

    Overexpression of TaWRKY146 increases drought tolerance through inducing stomatal closure in Arabidopsis thaliana

    Front. Plant Sci.

    (2017)
  • J.M. Manners et al.

    The promoter of the plant defensin gene PDF1.2 from Arabidopsis is systemically activated by fungal pathogens and responds to methyl jasmonate but not to salicylic acid

    Plant Mol. Biol.

    (1998)
  • Cited by (0)

    1

    These authors contributed equally to this work.

    View full text