Evidence for an Important Role of WRKY DNA Binding Proteins in the Regulation of NPR1 Gene Expression

The Arabidopsis NPR1 gene is a positive regulator of inducible plant disease resistance. Expression of NPR1 is induced by pathogen infection or treatment with defense-inducing compounds such as salicylic acid (SA). Transgenic plants overexpressing NPR1 exhibit enhanced resistance to a broad spectrum of microbial pathogens, whereas plants underexpressing the gene are more susceptible to pathogen infection. These results suggest that regulation of NPR1 gene expression is important for the activation of plant defense responses. In the present study, we report the identification of W-box sequences in the promoter region of the NPR1 gene that are recognized specifically by SA-induced WRKY DNA binding proteins from Arabidopsis. Mutations in these W-box sequences abolished their recognition by WRKY DNA binding proteins, rendered the promoter unable to activate a downstream reporter gene, and compromised the ability of NPR1 to complement npr1 mutants for SA-induced defense gene expression and disease resistance. These results provide strong evidence that certain WRKY genes act upstream of NPR1 and positively regulate its expression during the activation of plant defense responses. Consistent with this model, we found that SA-induced expression of a number of WRKY genes was independent of NPR1.

[1]  A. Cashmore,et al.  TGA1 and G-box binding factors: two distinct classes of Arabidopsis leucine zipper proteins compete for the G-box-like element TGACGTGG. , 1992, The Plant cell.

[2]  Enwu Liu,et al.  The Arabidopsis NPR1/NIM1 Protein Enhances the DNA Binding Activity of a Subgroup of the TGA Family of bZIP Transcription Factors , 2000, Plant Cell.

[3]  Xin Li,et al.  Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Jane Glazebrook,et al.  The Arabidopsis NPR1 Gene That Controls Systemic Acquired Resistance Encodes a Novel Protein Containing Ankyrin Repeats , 1997, Cell.

[5]  B. Fan,et al.  An oligo selection procedure for identification of sequence-specific DNA-binding activities associated with the plant defence response. , 1998, The Plant journal : for cell and molecular biology.

[6]  H. Sano,et al.  Rapid systemic accumulation of transcripts encoding a tobacco WRKY transcription factor upon wounding , 2000, Molecular and General Genetics MGG.

[7]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[8]  J. Ryals,et al.  Recent advances in systemic acquired resistance research--a review. , 1996, Gene.

[9]  J. Ryals,et al.  Arabidopsis signal transduction mutant defective in chemically and biologically induced disease resistance. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[10]  M. Bevan,et al.  GUS fusions: beta‐glucuronidase as a sensitive and versatile gene fusion marker in higher plants. , 1987, The EMBO journal.

[11]  I. Somssich,et al.  Interaction of elicitor‐induced DNA‐binding proteins with elicitor response elements in the promoters of parsley PR1 genes. , 1996, The EMBO journal.

[12]  E. Lam,et al.  NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acid. , 2000, Molecular plant-microbe interactions : MPMI.

[13]  G. Pelletier,et al.  In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. , 1998, Methods in molecular biology.

[14]  C. Pieterse,et al.  Enhancement of induced disease resistance by simultaneous activation of salicylate- and jasmonate-dependent defense pathways in Arabidopsis thaliana. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[15]  F. Ausubel,et al.  Isolation of Arabidopsis mutants with enhanced disease susceptibility by direct screening. , 1996, Genetics.

[16]  Xin Li,et al.  Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[17]  K. Kasschau,et al.  A Counterdefensive Strategy of Plant Viruses Suppression of Posttranscriptional Gene Silencing , 1998, Cell.

[18]  Xin Li,et al.  Identification and Cloning of a Negative Regulator of Systemic Acquired Resistance, SNI1, through a Screen for Suppressors of npr1-1 , 1999, Cell.

[19]  B. Fan,et al.  A pathogen- and salicylic acid-induced WRKY DNA-binding activity recognizes the elicitor response element of the tobacco class I chitinase gene promoter , 1999 .

[20]  K. Cox.  Analysis of plant gene expression , 1988 .

[21]  A. Avrova,et al.  A potato gene encoding a WRKY-like transcription factor is induced in interactions with Erwinia carotovora subsp. atroseptica and Phytophthora infestans and is coregulated with class I endochitinase expression. , 2000, Molecular plant-microbe interactions : MPMI.

[22]  T. Eulgem,et al.  The transcriptome of Arabidopsis thaliana during systemic acquired resistance , 2000, Nature Genetics.

[23]  Xinnian Dong,et al.  Characterization of an Arabidopsis Mutant That Is Nonresponsive to Inducers of Systemic Acquired Resistance. , 1994, The Plant cell.

[24]  F. Skoog,et al.  A revised medium for the growth and bioassay with tobacco tissue culture , 1962 .

[25]  I. Somssich,et al.  Transcriptional control of plant genes responsive to pathogens. , 1998, Current opinion in plant biology.

[26]  E. A. van der Biezen,et al.  Plant disease-resistance proteins and the gene-for-gene concept. , 1998, Trends in biochemical sciences.

[27]  G. L. Le Breton,et al.  Anti-peptide antibodies against the human blood platelet thromboxane A2/prostaglandin H2 receptor. Production, purification and characterization. , 1993 .

[28]  D. Klessig,et al.  Signal perception and transduction in plant defense responses. , 1997, Genes & development.

[29]  T. Eulgem,et al.  Early nuclear events in plant defence signalling: rapid gene activation by WRKY transcription factors , 1999, The EMBO journal.

[30]  K. Singh,et al.  Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP), an ethylene-inducible, GCC box DNA-binding protein interacts with an ocs element binding protein. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[31]  D. Klessig,et al.  Characterization of a salicylic acid-insensitive mutant (sai1) of Arabidopsis thaliana, identified in a selective screen utilizing the SA-inducible expression of the tms2 gene. , 1997, Molecular plant-microbe interactions : MPMI.

[32]  M. Cho,et al.  Identification of rice blast fungal elicitor-responsive genes by differential display analysis. , 2000, Molecular plant-microbe interactions : MPMI.

[33]  G. Pelletier,et al.  In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. , 1998, Methods in molecular biology.

[34]  J. Ryals,et al.  The Arabidopsis NIM1 protein shows homology to the mammalian transcription factor inhibitor I kappa B. , 1997, The Plant cell.

[35]  C. Pieterse,et al.  Salicylic acid-independent plant defence pathways. , 1999, Trends in plant science.

[36]  Xinnian Dong,et al.  Nuclear Localization of NPR1 Is Required for Activation of PR Gene Expression , 2000, Plant Cell.

[37]  T. Eulgem,et al.  The WRKY superfamily of plant transcription factors. , 2000, Trends in plant science.

[38]  S. Kay,et al.  Sequence‐specific interactions of a pea nuclear factor with light‐responsive elements upstream of the rbcS‐3A gene. , 1987, The EMBO journal.