Pierre R. Fobert

The Arabidopsis NPR1/NIM1 Protein Enhances the DNA Binding Activity of a Subgroup of the TGA Family of bZIP Transcription Factors

The Arabidopsis NPR1 gene is essential in activating systemic, inducible plant defense responses. To gain a better understanding of NPR1 function, we conducted a yeast two-hybrid screening procedure and identified a differential interaction between NPR1 and all known members of the Arabidopsis TGA family of basic leucine zipper transcription factors. In the electrophoretic mobility shift assay, NPR1 substantially increased the binding of TGA2 to its cognate promoter element (as-1) as well as to a positive salicylic acid–inducible element (LS7) and a negative element (LS5) in the promoter of the pathogenesis-related PR-1 gene. Proteins encoded by npr1 mutants interacted poorly with TGA2 and did not substantially increase TGA2 binding to the as-1, LS5, or LS7 elements, thus establishing a link between the loss of disease resistance and the loss of TGA2 interaction and NPR1-enhanced DNA binding. Coupled with observations that the DNA binding activity of TGA factors is deregulated in npr1 plants, the results suggest that NPR1-mediated DNA binding of TGA2 is critical for activation of defense genes.

The Arabidopsis NPR1 Disease Resistance Protein Is a Novel Cofactor That Confers Redox Regulation of DNA Binding Activity to the Basic Domain/Leucine Zipper Transcription Factor TGA1

The Arabidopsis NPR1 protein is essential for regulating salicylic acid–dependent gene expression during systemic acquired resistance. NPR1 interacts differentially with members of the TGA class of basic domain/Leu zipper transcription factors and regulates their DNA binding activity. Here, we report that although TGA1 does not interact with NPR1 in yeast two-hybrid assays, treatment with salicylic acid induces the interaction between these proteins in Arabidopsis leaves. This phenomenon is correlated with a reduction of TGA1 Cys residues. Furthermore, site-directed mutagenesis of TGA1 Cys-260 and Cys-266 enables the interaction with NPR1 in yeast and Arabidopsis. Together, these results indicate that TGA1 relies on the oxidation state of Cys residues to mediate the interaction with NPR1. An intramolecular disulfide bridge in TGA1 precludes interaction with NPR1, and NPR1 can only stimulate the DNA binding activity of the reduced form of TGA1. Unlike its animal and yeast counterparts, the DNA binding activity of TGA1 is not redox regulated; however, this property is conferred by interaction with the NPR1 cofactor.

The Tomato Transcription Factor Pti4 Regulates Defense-Related Gene Expression via GCC Box and Non-GCC Box cis Elements

The tomato transcription factor Pti4, an ethylene-responsive factor (ERF), interacts physically with the disease resistance protein Pto and binds the GCC box cis element that is present in the promoters of many pathogenesis-related (PR) genes. We reported previously that Arabidopsis plants expressing Pti4 constitutively express several GCC box–containing PR genes and show reduced disease symptoms compared with wild-type plants after inoculation with Pseudomonas syringae pv tomato or Erysiphe orontii. To gain insight into how genome-wide gene expression is affected by Pti4, we used serial analysis of gene expression (SAGE) to compare transcripts in wild-type and Pti4-expressing Arabidopsis plants. SAGE provided quantitative measurements of >20,000 transcripts and identified the 50 most highly expressed genes in Arabidopsis vegetative tissues. Comparison of the profiles from wild-type and Pti4-expressing Arabidopsis plants revealed 78 differentially abundant transcripts encoding defense-related proteins, protein kinases, ribosomal proteins, transporters, and two transcription factors (TFs). Many of the genes identified were expressed differentially in wild-type Arabidopsis during infection by Pseudomonas syringae pv tomato, supporting a role for them in defense-related processes. Unexpectedly, the promoters of most Pti4-regulated genes did not have a GCC box. Chromatin immunoprecipitation experiments confirmed that Pti4 binds in vivo to promoters lacking this cis element. Potential binding sites for ERF, MYB, and GBF TFs were present in statistically significantly increased numbers in promoters regulated by Pti4. Thus, Pti4 appears to regulate gene expression directly by binding the GCC box and possibly a non-GCC box element and indirectly by either activating the expression of TF genes or interacting physically with other TFs.

The Coactivator Function of Arabidopsis NPR1 Requires the Core of Its BTB/POZ Domain and the Oxidation of C-Terminal Cysteines

NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) regulates systemic acquired resistance (SAR) in Arabidopsis thaliana, and current models propose that after treatment with salicylic acid (SA), Cys-82 and Cys-216 of NPR1 are reduced, leading to nuclear import. The interaction of nucleus-localized NPR1 with TGA transcription factors results in the activation of defense genes, including the SAR marker PATHOGENESIS-RELATED-1 (PR-1), and the deployment of SAR. Little is known about how TGA factors or NPR1 regulate transcription or whether a TGA-NPR1 complex forms on DNA. We show that TGA2 and NPR1 are recruited to PR-1 independently of each other and of SA treatment. Consistent with the result that a triple knockout in TGA2/5/6 derepresses PR-1, in vivo plant transcription assays revealed that TGA2 is not an autonomous transcription activator but is a transcriptional repressor in both untreated and SA-treated cells. However, after stimulation with SA, TGA2 is incorporated into a transactivating complex with NPR1, forming an enhanceosome that requires the core of the NPR1 BTB/POZ domain (residues 80 to 91) and the oxidation of NPR1 Cys-521 and Cys-529. These Cys residues are found in a new type of transactivation domain that we term Cys-oxidized. These data further our understanding of the mechanism by which TGA2 and NPR1 activate Arabidopsis PR-1.

Arabidopsis Basic Leucine-Zipper Transcription Factors TGA9 and TGA10 Interact with Floral Glutaredoxins ROXY1 and ROXY2 and Are Redundantly Required for Anther Development

ROXY1 and ROXY2 are CC-type floral glutaredoxins with redundant functions in Arabidopsis (Arabidopsis thaliana) anther development. We show here that plants lacking the basic leucine-zipper transcription factors TGA9 and TGA10 have defects in male gametogenesis that are strikingly similar to those in roxy1 roxy2 mutants. In tga9 tga10 mutants, adaxial and abaxial anther lobe development is differentially affected, with early steps in anther development blocked in adaxial lobes and later steps affected in abaxial lobes. Distinct from roxy1 roxy2, microspore development in abaxial anther lobes proceeds to a later stage with the production of inviable pollen grains contained within nondehiscent anthers. Histological analysis shows multiple defects in the anther dehiscence program, including abnormal stability and lignification of the middle layer and defects in septum and stomium function. Compatible with these defects, TGA9 and TGA10 are expressed throughout early anther primordia but resolve to the middle and tapetum layers during meiosis of pollen mother cells. Several lines of evidence suggest that ROXY promotion of anther development is mediated in part by TGA9 and TGA10. First, TGA9 and TGA10 expression overlaps with ROXY1/2 during anther development. Second, TGA9/10 and ROXY1/2 operate downstream of SPOROCYTELESS/NOZZLE, where they positively regulate a common set of genes that contribute to tapetal development. Third, TGA9 and TGA10 directly interact with ROXY proteins in yeast and in plant cell nuclei. These findings suggest that activation of TGA9/10 transcription factors by ROXY-mediated modification of cysteine residues promotes anther development, thus broadening our understanding of how redox-regulated TGA factors function in plants.

The BTB/POZ Domain of the Arabidopsis Disease Resistance Protein NPR1 Interacts with the Repression Domain of TGA2 to Negate Its Function

TGA2 and NONEXPRESSER OF PR GENES1 (NPR1) are activators of systemic acquired resistance (SAR) and of the SAR marker gene pathogenesis-related-1 (PR-1) in Arabidopsis thaliana. TGA2 is a transcriptional repressor required for basal repression of PR-1, but during SAR, TGA2 recruits NPR1 as part of an enhanceosome. Transactivation by the enhanceosome requires the NPR1 BTB/POZ domain. However, the NPR1 BTB/POZ domain does not contain an autonomous transactivation domain; thus, its molecular role within the enhanceosome remains elusive. We now show by gel filtration analyses that TGA2 binds DNA as a dimer, tetramer, or oligomer. Using in vivo plant transcription assays, we localize the repression domain of TGA2 to the N terminus and demonstrate that this domain is responsible for modulating the DNA binding activity of the oligomer both in vitro and in vivo. We confirm that the NPR1 BTB/POZ domain interacts with and negates the molecular function of the TGA2 repression domain by excluding TGA2 oligomers from cognate DNA. These data distinguish the NPR1 BTB/POZ domain from other known BTB/POZ domains and establish its molecular role in the context of the Arabidopsis PR-1 gene enhanceosome.

Conserved and novel regulators of the plant cell cycle

Cell division is highly regulated, both spatially and temporally, during plant development. Recent evidence implicates cyclin-dependent kinases (cdks) and their associated proteins as the principal temporal regulators of cell division. It is now known that plants contain an extended family of cdks, some of which appear to be unique to this group. Positive rate-limiting regulators of cell proliferation and growth include mitotic or B-type cyclins whose transcription is restricted to the G2 and M phases. Current research suggests that MYB-related transcription factors may be responsible for this restriction. Cdk-interacting proteins, such as cdk inhibitors and suc1 homologues, have been isolated using yeast two-hybrid approaches.

A novel role for protein farnesylation in plant innate immunity

Plants utilize tightly regulated mechanisms to defend themselves against pathogens. Initial recognition results in activation of specific Resistance (R) proteins that trigger downstream immune responses, in which the signaling networks remain largely unknown. A point mutation in SUPPRESSOR OF NPR1 CONSTITUTIVE1 (SNC1), a RESISTANCE TO PERONOSPORA PARASITICA4 R gene homolog, renders plants constitutively resistant to virulent pathogens. Genetic suppressors of snc1 may carry mutations in genes encoding novel signaling components downstream of activated R proteins. One such suppressor was identified as a novel loss-of-function allele of ENHANCED RESPONSE TO ABSCISIC ACID1 (ERA1), which encodes the β-subunit of protein farnesyltransferase. Protein farnesylation involves attachment of C15-prenyl residues to the carboxyl termini of specific target proteins. Mutant era1 plants display enhanced susceptibility to virulent bacterial and oomycete pathogens, implying a role for farnesylation in basal defense. In addition to its role in snc1-mediated resistance, era1 affects several other R-protein-mediated resistance responses against bacteria and oomycetes. ERA1 acts partly independent of abscisic acid and additively with the resistance regulator NON-EXPRESSOR OF PR GENES1 in the signaling network. Defects in geranylgeranyl transferase I, a protein modification similar to farnesylation, do not affect resistance responses, indicating that farnesylation is most likely specifically required in plant defense signaling. Taken together, we present a novel role for farnesyltransferase in plant-pathogen interactions, suggesting the importance of protein farnesylation, which contributes to the specificity and efficacy of signal transduction events.

Long distance movement of DIR1 and investigation of the role of DIR1-like during systemic acquired resistance in Arabidopsis

DIR1 is a lipid transfer protein (LTP) postulated to complex with and/or chaperone a signal(s) to distant leaves during Systemic Acquired Resistance (SAR) in Arabidopsis. DIR1 was detected in phloem sap-enriched petiole exudates collected from wild-type leaves induced for SAR, suggesting that DIR1 gains access to the phloem for movement from the induced leaf. Occasionally the defective in induced resistance1 (dir1-1) mutant displayed a partially SAR-competent phenotype and a DIR1-sized band in protein gel blots was detected in dir1-1 exudates suggesting that a highly similar protein, DIR1-like (At5g48490), may contribute to SAR. Recombinant protein studies demonstrated that DIR1 polyclonal antibodies recognize DIR1 and DIR1-like. Homology modeling of DIR1-like using the DIR1-phospholipid crystal structure as template, provides clues as to why the dir1-1 mutant is rarely SAR-competent. The contribution of DIR1 and DIR1-like during SAR was examined using an Agrobacterium-mediated transient expression-SAR assay and an estrogen-inducible DIR1-EGFP/dir1-1 line. We provide evidence that upon SAR induction, DIR1 moves down the leaf petiole to distant leaves. Our data also suggests that DIR1-like displays a reduced capacity to move to distant leaves during SAR and this may explain why dir1-1 is occasionally SAR-competent.

Transgenic increases in seed oil content are associated with the differential expression of novel Brassica-specific transcripts

BackgroundSeed oil accumulates primarily as triacylglycerol (TAG). While the biochemical pathway for TAG biosynthesis is known, its regulation remains unclear. Previous research identified microsomal diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) as controlling a rate-limiting step in the TAG biosynthesis pathway. Of note, overexpression of DGAT1 results in substantial increases in oil content and seed size. To further analyze the global consequences of manipulating DGAT1 levels during seed development, a concerted transcriptome and metabolome analysis of transgenic B. napus prototypes was performed.ResultsUsing a targeted Brassica cDNA microarray, about 200 genes were differentially expressed in two independent transgenic lines analyzed. Interestingly, 24–33% of the targets showing significant changes have no matching gene in Arabidopsis although these represent only 5% of the targets on the microarray. Further analysis of some of these novel transcripts indicated that several are inducible by ABA in microspore-derived embryos. Of the 200 Arabidopsis genes implicated in lipid biology present on the microarray, 36 were found to be differentially regulated in DGAT transgenic lines. Furthermore, kinetic reverse transcriptase Polymerase Chain Reaction (k-PCR) analysis revealed up-regulation of genes encoding enzymes of the Kennedy pathway involved in assembly of TAGs. Hormone profiling indicated that levels of auxins and cytokinins varied between transgenic lines and untransformed controls, while differences in the pool sizes of ABA and catabolites were only observed at later stages of development.ConclusionOur results indicate that the increased TAG accumulation observed in transgenic DGAT1 plants is associated with modest transcriptional and hormonal changes during seed development that are not limited to the TAG biosynthesis pathway. These might be associated with feedback or feed-forward effects due to altered levels of DGAT1 activity. The fact that a large fraction of significant amplicons have no matching genes in Arabidopsis compromised our ability to draw concrete inferences from the data at this stage, but has led to the identification of novel genes of potential interest.