Addie Nina Olsen

Structure of the conserved domain of ANAC, a member of the NAC family of transcription factors.

The structure of the DNA‐binding NAC domain of Arabidopsis ANAC (abscisic‐acid‐responsive NAC) has been determined by X‐ray crystallography to 1.9 Å resolution (Protein Data Bank codes 1UT4 and 1UT7). This is the first structure determined for a member of the NAC family of plant‐specific transcriptional regulators. NAC proteins are characterized by their conserved N‐terminal NAC domains that can bind both DNA and other proteins. NAC proteins are involved in developmental processes, including formation of the shoot apical meristem, floral organs and lateral shoots, as well as in plant hormonal control and defence. The NAC domain does not possess a classical helix–turn–helix motif; instead it reveals a new transcription factor fold consisting of a twisted β‐sheet surrounded by a few helical elements. The functional dimer formed by the NAC domain was identified in the structure, which will serve as a structural template for understanding NAC protein function at the molecular level.

Ligand mimicry? Plant-parasitic nematode polypeptide with similarity to CLAVATA3

The importance of peptides in plant intercellular signaling has become apparent during the past decade. Among recently identified peptide signals is CLAVATA3 (CLV3), which is involved in cell-fate determination in the shoot apical meristem of Arabidopsis. There is evidence that CLV3 is a ligand for CLAVATA1 (CLV1), a receptor kinase with an extracellular domain containing leucine-rich repeats (LRRs) [1xSignaling through the CLAVATA1 receptor complex. DeYoung, B.J. and Clark, S.E. Plant Mol. Biol. 2001; 46: 505–513CrossRef | PubMed | Scopus (27)See all References][1]. The Arabidopsis genome contains a large gene family, called CLE for CLAVATA3/ESR-related, encoding polypeptides with similarity to CLV3. These small polypeptides are characterized by a short, C-terminal motif and an N-terminal signal peptide or signal anchor [2xA large family of genes that share homology with CLAVATA3. Cock, J.M. and McCormick, S. Plant Physiol. 2001; 126: 939–942CrossRef | PubMed | Scopus (183)See all References][2]. Similar sequences are encoded by expressed sequence tag (EST) clones from various plants.Using motif-based database search methods, we have discovered sequence similarity between the CLE polypeptides and an esophageal gland cell polypeptide from Heterodera glycines, the soybean cyst nematode (Fig. 1Fig. 1) . The sequence similarity between the plant polypeptide family and the H. glycines polypeptide, here referred to as HgCLE, was evident from a position-specific iterative BLAST (PSI-BLAST) [3xGapped BLAST and PSI-BLAST: a new generation of protein database search programs. Altschul, S.F. et al. Nucleic Acids Res. 1997; 25: 3389–3402CrossRef | PubMed | Scopus (44567)See all References][3] database search with the CLV3 sequence. Furthermore, the motif-discovery tool MEME [4xFitting a mixture model by expectation maximization to discover motifs in biopolymers. Bailey, T.L. and Elkan, C. Proc. Int. Conf. Intell. Syst. Mol. Biol. 1994; 2: 28–36PubMedSee all References][4] was applied to the CLE sequences, and the resulting file was used as input to the MAST algorithm [5xCombining evidence using p-values: application to sequence homology searches. Bailey, T.L. and Gribskov, M. Bioinformatics. 1998; 14: 48–54CrossRef | PubMedSee all References][5] in a database search. The high-scoring sequences were CLV3-like plant polypeptides, but also included HgCLE.Fig. 1Alignment of the esophageal gland cell polypeptide from Heterodera glycines (GenBank Accession number: AAG21331, here referred to as HgCLE) with CLAVATA3 and Arabidopsis CLE sequences. Also included in the alignment is the sequence encoded by wheat EST clone BE401912, exemplifying a sequence encoding multiple CLE motifs. CLE1–CLE27 were identified by J. Mark Cock and Sheila McCormick [2xA large family of genes that share homology with CLAVATA3. Cock, J.M. and McCormick, S. Plant Physiol. 2001; 126: 939–942CrossRef | PubMed | Scopus (183)See all References][2]. CLE40 and CLE41 are included in the annotation of the Arabidopsis genome performed by The Institute for Genomic Research, Rockville, MD, USA (http://www.tigr.org); the sequences are encoded by loci At5g12990 and At3g24770, respectively. Residues highlighted in red are common to at least half of the sequences, and residues highlighted in blue are chemically similar in more than half of the sequences or similar to the dominating residue. The last line shows the consensus residues. Predicted signal peptides are underlined. CLE16, CLE25, CLE26 and CLE27 are predicted to contain an N-terminal signal anchor instead of a signal peptide [2xA large family of genes that share homology with CLAVATA3. Cock, J.M. and McCormick, S. Plant Physiol. 2001; 126: 939–942CrossRef | PubMed | Scopus (183)See all References][2]. The three occurrences of the conserved motif in the sequence encoded by EST clone BE401912 are highlighted in gray.View Large Image | Download PowerPoint SlideH. glycines is a sedentary plant-parasitic nematode. The infective juvenile stage penetrates the root and migrates to a site near the vascular tissue to establish a permanent feeding site. The nematode induces the transformation of plant cells into metabolically active feeding cells. Secretions from the esophageal gland cells of the nematode are released through the stylet, a mouth spear that is used to pierce plant cell walls. These secretions are thought to contain the substances that cause the transformation of root cells by altering gene expression in the cells [6xNematode parasitism genes. Davis, E.L. et al. Annu. Rev. Phytopathol. 2000; 38: 365–396CrossRef | PubMed | Scopus (186)See all References][6].HgCLE is a hypothetical polypeptide predicted from cDNA sequences cloned from esophageal gland cells [7xSignal peptide-selection of cDNA cloned directly from the esophageal gland cells of the soybean cyst nematode Heterodera glycines. Wang, X. et al. Mol. Plant–Microbe Interact. 2001; 14: 536–544CrossRef | PubMedSee all References, 8xIdentification of putative parasitism genes expressed in the esophageal gland cells of the soybean cyst nematode Heterodera glycines. Gao, B. et al. Mol. Plant–Microbe Interact. 2001; 14: 1247–1254CrossRef | PubMedSee all References]. The cDNA clone hybridized to genomic DNA of H. glycines, and expression of the gene was specifically detected in the dorsal esophageal gland cell of parasitic stages of H. glycines [7xSignal peptide-selection of cDNA cloned directly from the esophageal gland cells of the soybean cyst nematode Heterodera glycines. Wang, X. et al. Mol. Plant–Microbe Interact. 2001; 14: 536–544CrossRef | PubMedSee all References][7]. The dorsal gland cell is the predominate gland of the parasitic stages of H. glycines [6xNematode parasitism genes. Davis, E.L. et al. Annu. Rev. Phytopathol. 2000; 38: 365–396CrossRef | PubMed | Scopus (186)See all References][6]. HgCLE contains the C-terminal motif and the N-terminal signal peptide that characterize the CLE sequences (Fig. 1Fig. 1). Predicted signal peptide cleavage produces a polypeptide of 117 amino acids, but further processing cannot be excluded. It has yet to be determined whether the CLE polypeptides are processed subsequent to signal peptide cleavage [2xA large family of genes that share homology with CLAVATA3. Cock, J.M. and McCormick, S. Plant Physiol. 2001; 126: 939–942CrossRef | PubMed | Scopus (183)See all References][2]. Indeed, several EST clones from wheat encode sequences containing multiple CLE motifs (Fig. 1Fig. 1). The striking resemblance to a polyprotein precursor could suggest that the conserved motif constitutes the mature CLE peptides.Based on the similarity between a bioactive plant peptide and a plant-parasite polypeptide, we hypothesize that the nematode has co-opted the plant signaling peptide for parasitic modification of host plant cells. HgCLE might thus function as a ligand for a host LRR receptor kinase, possibly imitating or inhibiting the function of an endogenous peptide ligand. This hypothesis concurs with a model proposed by David McKenzie Bird [9xManipulation of host gene expression by root-knot nematodes. Bird, D.M. J. Parasitol. 1996; 82: 881–888CrossRef | PubMed | Scopus (52)See all References][9] to explain the development of nematode-induced feeding sites. Moreover, it has been demonstrated that a low-molecular-weight peptide component of potato cyst nematode secretions can co-stimulate the proliferation of tobacco protoplasts in the presence of phytohormones [10xNaturally induced secretions of the potato cyst nematode co-stimulate the proliferation of both tobacco leaf protoplasts and human peripheral blood mononuclear cells. Goverse, A. et al. Mol. Plant–Microbe Interact. 1999; 12: 872–881CrossRef | PubMedSee all References][10]. It would be interesting to determine the effects of HgCLE in a plant protoplast proliferation assay. Furthermore, double-stranded RNA-mediated interference of HgCLE gene expression could be applied to investigate the role of HgCLE in the plant–parasite interaction [11xIngestion of double-stranded RNA by preparasitic juvenile cyst nematodes leads to RNA interference. Urwin, P.E. et al. Mol. Plant–Microbe Interact. 2002; 15: 747–752CrossRef | PubMedSee all References][11].Database searches revealed no sequences from Caenorhabditis elegans or other non-plant organisms with the characteristics of HgCLE or of the plant CLE sequences. The intriguing similarity between host and parasite sequences could be an example of adaptive molecular mimicry, defined by Roger Hall [12xMolecular mimicry. Hall, R. Adv. Parasitol. 1994; 34: 81–132CrossRef | PubMedSee all References][12] as ‘a parasite molecule mimics a host molecule for a biological reason’. Although lateral gene transfer influences the evolution of archeal and bacterial genomes, the transfer of genes between two multicellular eukaryotes is not expected to be common [13xPhylogenetic classification and the universal tree. Doolittle, W.F. Science. 1999; 284: 2124–2128CrossRef | PubMed | Scopus (998)See all References][13]. Considering the simplicity of the features shared by HgCLE and the CLE sequences, a more likely explanation of the sequence similarity is convergent evolution. If indeed this is an example of ligand mimicry, it emphasizes the importance of peptide ligands in plant biology and constitutes a convergence of research in nematode parasitism and peptide signaling.

DNA binding by the plant-specific NAC transcription factors in crystal and solution: a firm link to WRKY and GCM transcription factors.

NAC (NAM/ATAF/CUC) plant transcription factors regulate essential processes in development, stress responses and nutrient distribution in important crop and model plants (rice, Populus, Arabidopsis), which makes them highly relevant in the context of crop optimization and bioenergy production. The structure of the DNA-binding NAC domain of ANAC019 has previously been determined by X-ray crystallography, revealing a dimeric and predominantly β-fold structure, but the mode of binding to cognate DNA has remained elusive. In the present study, information from low resolution X-ray structures and small angle X-ray scattering on complexes with oligonucleotides, mutagenesis and (DNase I and uranyl photo-) footprinting, is combined to form a structural view of DNA-binding, and for the first time provide experimental evidence for the speculated relationship between plant-specific NAC proteins, WRKY transcription factors and the mammalian GCM (Glial cell missing) transcription factors, which all use a β-strand motif for DNA-binding. The structure shows that the NAC domain inserts the edge of its core β-sheet into the major groove, while leaving the DNA largely undistorted. The structure of the NAC-DNA complex and a new crystal form of the unbound NAC also indicate limited flexibility of the NAC dimer arrangement, which could be important in recognizing suboptimal binding sites.

Preliminary crystallographic analysis of the NAC domain of ANAC, a member of the plant-specific NAC transcription factor family

The NAC domain (residues 1-168) of ANAC, encoded by the abscisic acid-responsive NAC gene from Arabidopsis thaliana, was recombinantly produced in Escherichia coli and crystallized in hanging drops. Three morphologically different crystal forms were obtained within a relatively narrow range of conditions: 10-15% PEG 4000 and 0.1 M imidazole/malic acid buffer pH 7.0 in the reservoir, 3.2-7.7 mg ml(-1) protein stock and a 1:1 ratio of reservoir to protein solution in the hanging drop. One of the crystal forms, designated crystal form III, was found to be suitable for further X-ray analysis. Form III crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 62.0, b = 75.2, c = 80.8 A at 100 K. The unit-cell volume is consistent with two molecules in the asymmetric unit and a peak in the native Patterson map suggests the presence of a non-crystallographic twofold axis parallel to a crystallographic axis. Size-exclusion chromatography of the NAC domain showed that the dimeric state is also the preferred state in solution and probably represents the biologically active form. Data sets were collected from four potential heavy-atom derivatives of the form III crystals. The derivatized crystals are reasonably isomorphous with the non-derivatized crystals and the four data sets are being evaluated for use in structure determination by multiple isomorphous replacement.