Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis

The Arabidopsis genome contains approximately 200 genes that encode proteins with similarity to the nucleotide binding site and other domains characteristic of plant resistance proteins. Through a reiterative process of sequence analysis and reannotation, we identified 149 NBS-LRR-encoding genes in the Arabidopsis (ecotype Columbia) genomic sequence. Fifty-six of these genes were corrected from earlier annotations. At least 12 are predicted to be pseudogenes. As described previously, two distinct groups of sequences were identified: those that encoded an N-terminal domain with Toll/Interleukin-1 Receptor homology (TIR-NBS-LRR, or TNL), and those that encoded an N-terminal coiled-coil motif (CC-NBS-LRR, or CNL). The encoded proteins are distinct from the 58 predicted adapter proteins in the previously described TIR-X, TIR-NBS, and CC-NBS groups. Classification based on protein domains, intron positions, sequence conservation, and genome distribution defined four subgroups of CNL proteins, eight subgroups of TNL proteins, and a pair of divergent NL proteins that lack a defined N-terminal motif. CNL proteins generally were encoded in single exons, although two subclasses were identified that contained introns in unique positions. TNL proteins were encoded in modular exons, with conserved intron positions separating distinct protein domains. Conserved motifs were identified in the LRRs of both CNL and TNL proteins. In contrast to CNL proteins, TNL proteins contained large and variable C-terminal domains. The extant distribution and diversity of the NBS-LRR sequences has been generated by extensive duplication and ectopic rearrangements that involved segmental duplications as well as microscale events. The observed diversity of these NBS-LRR proteins indicates the variety of recognition molecules available in an individual genotype to detect diverse biotic challenges.     

Plant NBS-LRR proteins: adaptable guards

The majority of disease resistance genes in plants encode nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins. This large family is encoded by hundreds of diverse genes per genome and can be subdivided into the functionally distinct TIR-domain-containing (TNL) and CC-domain-containing (CNL) subfamilies. Their precise role in recognition is unknown; however, they are thought to monitor the status of plant proteins that are targeted by pathogen effectors.     

植物TIR-NB-LRR类型抗病基因各结构域的研究进展

植物抗病反应是一个多基因调控的复杂过程,在这个过程中R基因发挥了非常重要的作用。根据其氨基酸基序组成以及跨膜结构域的不同,R基因可以分为多种类型,其中NBS-LRR类型是植物基因组中最大的基因家族之一。TIR-NB-LRR类型的抗病基因又是NB-LRR类型中的一大类,也是目前抗病基因研究的热点。该文总结了TIR-NB-LRR类型抗病基因各个结构域的功能和相关的研究进展。相关研究表明,TIR结构域主要通过自身或异源的二聚体化介导抗性信号的转导,但也有部分研究表明,该结构域可能参与病原菌的特异性识别。NBS结构域常被认为具有"分子开关"的功能,它可以通过结合ADP或ATP来调节植物抗病蛋白的构象变化,从而调节下游抗病信号的传导。LRR结构域在植物与病原菌互作的过程中可以通过与病原菌的无毒蛋白直接或间接互作来特异识别病原菌。也有研究发现,LRR结构域具有调节信号传导的功能。这些信息将为研究植物抗病机理提供理论依据,也为将来通过基因编辑技术对作物进行定向抗病育种提供思路。     

A resistance gene product of the nucleotide binding site -- leucine rich repeats class can form a complex with bacterial avirulence proteins in vivo

Resistance (R) genes in plants mediate gene-for-gene disease resistance. The ligand-receptor model, which explains the gene-for-gene specificity, predicts a physical interaction between an elicitor, which is directly or indirectly encoded by an avirulence (avr) gene in the pathogen, and the corresponding R gene product. The nucleotide binding site (NBS) - leucine rich repeats (LRR) class of R genes is the largest known class of R genes. Here we report that an NBS-LRR R protein and its cognate Avr protein form a complex together in the plant cell. The Arabidopsis thaliana R genes RPS2 and RPM1 confer gene-for-gene disease resistance to strains of the phytopathogenic bacterium Pseudomonas syringae carrying the avr genes avrRpt2 and avrB, respectively. Using transient expression of these genes in Arabidopsis leaf mesophyll protoplasts, we first demonstrated that the protoplast system is appropriate for the investigation of the gene-for-gene recognition mechanism. Formation of an in vivo complex containing the RPS2 and AvrRpt2 proteins was demonstrated by co-immunoprecipitation of the proteins following expression of the genes in protoplasts. This complex contained at least one additional plant protein of approximately 75 kDa. Unexpectedly, RPS2 also formed a complex with AvrB. We speculate that complex formation between AvrRpt2 and RPS2 is productive and leads to the elicitation of the resistance response, whilst complex formation between AvrB and RPS2 is unproductive and possibly competes with complex formation between AvrRpt2 and RPS2.     

A novel gene family in moss (Physcomitrella patens) shows sequence homology and a phylogenetic relationship with the TIR-NBS class of plant disease resistance genes

Plant disease resistance (R) genes encode proteins in which several motifs of the nucleotide-binding region (NBS) are highly conserved. Using degenerate primers designed according to the kinase 1 (P-loop) and hydrophobic (HD) motifs of the R gene NBS domains, homologous sequences were cloned from moss (Physcomitrella patens; phylum Bryophyta) representing an ancient nonvascular plant. A novel gene family (PpC) with at least eight homologous members was found. Expression of five members was detected. The level of expression was dependent on the developmental stage of moss, being higher in the gametophyte tissue than in the protonema tissue. The PpCs contained the conserved motifs characteristic of the NBS regions of R genes, and a kinase domain was found upstream from the NBS region. Phylogenetic analysis using the deduced NBS amino acid sequences of the PpCs and the plant genes available in databanks indicated that the PpCs show the closest relationship with the TIR-NBS class of R genes. No significant similarity to plant genes other than R genes was observed. These findings shed novel light on the evolutionary history of the R gene families, suggesting that the NBS region characteristic of the TIR-NBS class of R-like genes evolved prior to the evolutionary differentiation of vascular and nonvascular plants.     

Relative evolutionary rates of NBS-encoding genes revealed by soybean segmental duplication

It is well known that nucleotide binding site (NBS)-encoding genes are duplicate-rich and fast-evolving genes. However, there is little information on the relative importance of tandem and segmental NBS duplicates and their exact evolutionary rates. The two rounds of large-scale duplication that have occurred in soybean provide a unique opportunity to investigate these issues. Comparison of NBS and non-NBS genes on segments of syntenic homoeologs shows that NBS-encoding genes evolve at least 1.5-fold faster (~1.5-fold higher synonymous and approximately 2.3-fold higher nonsynonymous substitution rates) and lose their genes approximately twofold faster than the flanking non-NBS genes. Compared with segmental duplicates, tandem NBS duplicates are more abundant in soybean, suggesting that tandem duplication is the major driving force in the expansion of NBS genes. Notably, significant sequence exchanges along with significantly positive selection were detected in most tandem-duplicated NBS gene families. The results suggest that the rapid evolution of NBS genes may be due to the combined effects of diversifying selection and frequent sequence exchanges. Interestingly, TIR-NBS-LRR genes (TNLs) have a higher nucleotide substitution rate than non-TNLs, indicating that these types of NBS genes may have a rather different evolutionary pattern. It is important to determine the exact relative evolutionary rates of TNL, non-TNL, and non-NBS genes in order to understand how fast the host plant can adjust its response to rapidly evolving pathogens in a coevolutionary context.     

The Role of TIR-NBS and TIR-X Proteins in Plant Basal Defense Responses

Toll/interleukin receptor (TIR) domain-containing proteins encoded in the Arabidopsis (Arabidopsis thaliana) genome include the TIR-nucleotide binding site (TN) and TIR-unknown site/domain (TX) families. We investigated the function of these proteins. Transient overexpression of five TX and TN genes in tobacco (Nicotiana benthamiana) induced chlorosis. This induced chlorosis was dependent on ENHANCED DISEASE RESISTANCE1, a dependency conserved in both tobacco and Arabidopsis. Stable overexpression transgenic lines of TX and TN genes in Arabidopsis produced a variety of phenotypes associated with basal innate immune responses; these were correlated with elevated levels of salicylic acid. The TN protein AtTN10 interacted with the chloroplastic protein phosphoglycerate dehydrogenase in a yeast (Saccharomyces cerevisiae) two-hybrid screen; other TX and TN proteins interacted with nucleotide binding-leucine-rich repeat proteins and effector proteins, suggesting that TN proteins might act in guard complexes monitoring pathogen effectors.Innate immunity is a primary defense mechanism in plants that functions to protect against a variety of biotic stresses (Eitas and Dangl, 2010). The plant basal immune system comprises pattern or pathogen recognition receptors that can recognize a variety of plant pathogens by identifying specific pathogen-associated molecular patterns (PAMPs; Tsuda and Katagiri, 2010). This recognition of PAMPs by plant pattern recognition receptors triggers PAMP-triggered immunity or plant basal immunity (Jones and Dangl, 2006; Zipfel, 2008). Well-known PAMPs or microbe-associated molecular patterns recognized by plants include bacterial flagellin, cold shock proteins, and elongation factor Tu. To suppress PAMP-triggered immunity, plant pathogens secrete an array of virulence factors such as type III effector proteins, while plant resistance (R) proteins function to recognize the effector molecules (Römer et al., 2009; Lewis et al., 2010; Tsuda and Katagiri, 2010; Zhang et al., 2012). Specific recognition of a pathogen effector by a plant R protein triggers a second type of immune response called effector-triggered immunity, resulting in an incompatible reaction (Qi et al., 2011; Sohn et al., 2012; Tahir et al., 2012).The most commonly known plant R proteins are the nucleotide-binding (NB) site Leucine-rich repeat (LRR) proteins that plants use to detect effector proteins. The NB is often called NB-ARC because of sequence similarities to the human apoptotic protease-activating factor APAF1 and Caenorhabditis elegans homolog CELL DEATH PROTEIN4 (Lukasik and Takken, 2009). Plant NB-LRR proteins often also have, at the N terminus, a Toll/Interleukin-1 receptor (TIR) or coiled coil (CC) domain (Meyers et al., 2003). In animal TIR proteins, this domain is more commonly located at the C terminus and is linked by a transmembrane domain to an N-terminal LRR domain (Torto et al., 2002). In Drosophila spp. and other microbes, a TIR domain has been shown to play an important role in the activation of antifungal immune responses (Jenkins and Mansell, 2010). Toll-like receptors (TLRs) perform an integral role in the activation of antimicrobial responses in many animals (Radhakrishnan and Splitter, 2010).In plants, two additional TIR-containing protein families, TIR-NB site (TN) and TIR-unknown/random (TX), were identified, which are distinct from the longer TIR-NB-LRR (TNL) R protein homologs (Meyers et al., 2002). TN proteins contain TIR and NBS domains but lack LRRs, while TX proteins lack both NBS and LRR domains, yet often have a small and variable C-terminal domain (Meyers et al., 2002). In the Arabidopsis (Arabidopsis thaliana) ecotype Columbia (Col-0) genome, there are 30 TX genes and 21 TN genes (Meyers et al., 2003). The crystal structure of a TIR domain from an Arabidopsis TN protein (At1g72930/{"type":"entrez-protein","attrs":{"text":"NP_177436","term_id":"15218629","term_text":"NP_177436"}}NP_177436) contains a compact globular fold resembling the mammalian (TLR1 and MYELOID DIFFERENTIATION PRIMARY RESPONSE GENE88 [MYD88]) and bacterial TIR domain proteins (Chan et al., 2010). Although plant TIR domains share less than 20% sequence identity with the human TLR domains, the structures of the TIR domain in plants, mammalian TLRs, and bacterial TIR domain proteins have strong similarity (Chan et al., 2010).A high proportion of TX and TN genes were previously reported to be in complex clusters with TNL genes; these clusters were found to be duplicated to multiple locations in the genome (Meyers et al., 2002). The existence of genetically linked pairs or sets of genes such as RESISTANCE TO PERONOSPORA PARASITICA2A (RPP2A)-RPP2B, RESISTANCE TO PSEUDOMONAS SYRINGAE1 (RPS1)-RPS4, LEAF RUST RESISTANCE GENE10 (LR10)-RESISTANCE GENE ANALOGUE2 (RGA2), RICE BLAST RESISTANCE GENE AT PIK LOCUS1 (PIKM-1)-TS-PIKM2-TS, and RICE BLAST RESISTANCE GENE AT PI LOCUS1 (PI5-1)-PI5-2 in the genomes and their role in disease resistance suggests that these linked genes are required to effect a defense response in plants (Eitas and Dangl, 2010). The genomic pairing of the TNL genes with TX or TN genes suggests a role of the tightly linked TN protein in the function of its cognate TNL protein or proteins (and vice versa).The specific direct or indirect interaction between an R gene and a corresponding avirulence (Avr) gene in the characterized pairs of interaction resulted in an immune response in the form of localized programmed cell death, called the hypersensitive response (HR; Burch-Smith et al., 2007; Caplan et al., 2008). The recognition of avirulence proteins from pathogens by the cognate R proteins induces a cascade of changes that increases the levels of salicylic acid (SA), jasmonic acid (JA), phenyl ammonium lyase, and systemin (Liu et al., 2010). The production of several of these biochemical signals is known to trigger multiple convergent ‘R’-gene signaling pathways, leading to programmed cell death and further changes in gene expression patterns (Vlot et al., 2008a, 2008b). Structure function analysis of Arabidopsis R proteins RPS4 (Zhang et al., 2004) and RPP1A (Michael Weaver et al., 2006) have shown that TIR and NBS domains of the proteins without the LRR domain (TNL truncations) could be sufficient to induce HR. Studies using overexpression of plant R genes (particularly the truncated TNL genes) suggest that the TIR and NBS domains by themselves might be sufficient to induce HR and to initiate plant defense responses (Michael Weaver et al., 2006; Swiderski et al., 2009).In this study, we present experimental and computational data that are collectively consistent with a role for Arabidopsis TX and TN proteins in plant defenses. For example, the ability of the TX and TN genes to induce HR responses upon transient expression is dependent on ENHANCED DISEASE RESISTANCE1 (EDS1). This EDS1 dependency in induced HR was demonstrated in both tobacco (Nicotiana benthamiana) and in Arabidopsis. Stable transgenic overexpression in Arabidopsis of TX and TN genes resulted in a variety of phenotypes involved with basal innate immune responses that are dependent on SA. We also demonstrated the interaction of TX and TN proteins with plant pathogenic elicitor proteins and other plant signal transduction proteins.     

Plant disease resistance protein signaling: NBS-LRR proteins and their partners

Most plant disease resistance (R) proteins contain a series of leucine-rich repeats (LRRs), a nucleotide-binding site (NBS), and a putative amino-terminal signaling domain. They are termed NBS-LRR proteins. The LRRs of a wide variety of proteins from many organisms serve as protein interaction platforms, and as regulatory modules of protein activation. Genetically, the LRRs of plant R proteins are determinants of response specificity, and their action can lead to plant cell death in the form of the familiar hypersensitive response (HR). A total of 149 R genes are potentially expressed in the Arabidopsis genome, and plant cells must deal with the difficult task of assembling many of the proteins encoded by these genes into functional signaling complexes. Eukaryotic cells utilize several strategies to deal with this problem. First, proteins are spatially restricted to their sub-cellular site of function, thus improving the probability that they will interact with their proper partners. Second, these interactions are architecturally organized to avoid inappropriate signaling events and to maintain the fidelity and efficiency of the response when it is initiated. Recent results provide new insights into how the signaling potential of R proteins might be created, managed and held in check until specific stimulation following infection. Nevertheless, the roles of the R protein partners in these regulatory events that have been defined to date are unclear.     

Comparative analysis of NBS domain sequences of NBS-LRR disease resistance genes from sunflower, lettuce, and chicory

Plant resistance to many types of pathogens and pests can be achieved by the presence of disease resistance (R) genes. The nucleotide binding site-leucine rich repeat (NBS-LRR) class of R-genes is the most commonly isolated class of R-genes and makes up a super-family, which is often arranged in the genome as large multi-gene clusters. The NBS domain of these genes can be targeted by polymerase chain reaction (PCR) amplification using degenerate primers. Previous studies have used PCR derived NBS sequences to investigate both ancient R-gene evolution and recent evolution within specific plant families. However, comparative studies with the Asteraceae family have largely been ignored. In this study, we address recent evolution of NBS sequences within the Asteraceae and extend the comparison to the Arabidopsis thaliana genome. Using multiple sets of primers, NBS fragments were amplified from genomic DNA of three species from the family Asteraceae: Helianthus annuus (sunflower), Lactuca sativa (lettuce), and Cichorium intybus (chicory). Analysis suggests that Asteraceae species share distinct families of R-genes, composed of genes related to both coiled-coil (CC) and toll-interleukin-receptor homology (TIR) domain containing NBS-LRR R-genes. Between the most closely related species, (lettuce and chicory) a striking similarity of CC subfamily composition was identified, while sunflower showed less similarity in structure. These sequences were also compared to the A. thaliana genome. Asteraceae NBS gene subfamilies appear to be distinct from Arabidopsis gene clades. These data suggest that NBS families in the Asteraceae family are ancient, but also that gene duplication and gene loss events occur and change the composition of these gene subfamilies over time.     

Isolation of a family of resistance gene analogue sequences of the nucleotide binding site (NBS) type from Lens species.

Most known plant disease-resistance genes (R genes) include in their encoded products domains such as a nucleotide-binding site (NBS) or leucine-rich repeats (LRRs). Sequences with unknown function, but encoding these conserved domains, have been defined as resistance gene analogues (RGAs). The conserved motifs within plant NBS domains make it possible to use degenerate primers and PCR to isolate RGAs. We used degenerate primers deduced from conserved motifs in the NBS domain of NBS-LRR resistance proteins to amplify genomic sequences from Lens species. Fragments from approximately 500-850 bp were obtained. The nucleotide sequence analysis of these fragments revealed 32 different RGA sequences in Lens species with a high similarity (up to 91%) to RGAs from other plants. The predicted amino acid sequences showed that lentil sequences contain all the conserved motifs (P-loop, kinase-2, kinase-3a, GLPL, and MHD) present in the majority of other known plant NBS-LRR resistance genes. Phylogenetic analyses grouped the Lens NBS sequences with the Toll and interleukin-1 receptor (TIR) subclass of NBS-LRR genes, as well as with RGA sequences isolated from other legume species. Using inverse PCR on one putative RGA of lentil, we were able to amplify the flanking regions of this sequence, which contained features found in R proteins.     

Identification and characterization of nucleotide-binding site-leucine-rich repeat genes in the model plant Medicago truncatula

The nucleotide-binding site (NBS)-Leucine-rich repeat (LRR) gene family accounts for the largest number of known disease resistance genes, and is one of the largest gene families in plant genomes. We have identified 333 nonredundant NBS-LRRs in the current Medicago truncatula draft genome (Mt1.0), likely representing 400 to 500 NBS-LRRs in the full genome, or roughly 3 times the number present in Arabidopsis (Arabidopsis thaliana). Although many characteristics of the gene family are similar to those described on other plant genomes, several evolutionary features are particularly pronounced in M. truncatula, including a high degree of clustering, evidence of significant numbers of ectopic translocations from clusters to other parts of the genome, a small number of more evolutionarily stable NBS-LRRs, and numerous truncations and fusions leading to novel domain compositions. The gene family clearly has had a large impact on the structure of the genome, both through ectopic translocations (potentially, a means of seeding new NBS-LRR clusters), and through two extraordinarily large superclusters. Chromosome 6 encodes approximately 34% of all TIR-NBS-LRRs, while chromosome 3 encodes approximately 40% of all coiled-coil-NBS-LRRs. Almost all atypical domain combinations are in the TIR-NBS-LRR subfamily, with many occurring within one genomic cluster. This analysis shows the gene family not only is important functionally and agronomically, but also plays a structural role in the genome.     

Exploring the plant transcriptome through phylogenetic profiling

Publicly available protein sequences represent only a small fraction of the full catalog of genes encoded by the genomes of different plants, such as green algae, mosses, gymnosperms, and angiosperms. By contrast, an enormous amount of expressed sequence tags (ESTs) exists for a wide variety of plant species, representing a substantial part of all transcribed plant genes. Integrating protein and EST sequences in comparative and evolutionary analyses is not straightforward because of the heterogeneous nature of both types of sequence data. By combining information from publicly available EST and protein sequences for 32 different plant species, we identified more than 250,000 plant proteins organized in more than 12,000 gene families. Approximately 60% of the proteins are absent from current sequence databases but provide important new information about plant gene families. Analysis of the distribution of gene families over different plant species through phylogenetic profiling reveals interesting insights into plant gene evolution, and identifies species- and lineage-specific gene families, orphan genes, and conserved core genes across the green plant lineage. We counted a similar number of approximately 9,500 gene families in monocotyledonous and eudicotyledonous plants and found strong evidence for the existence of at least 33,700 genes in rice (Oryza sativa). Interestingly, the larger number of genes in rice compared to Arabidopsis (Arabidopsis thaliana) can partially be explained by a larger amount of species-specific single-copy genes and species-specific gene families. In addition, a majority of large gene families, typically containing more than 50 genes, are bigger in rice than Arabidopsis, whereas the opposite seems true for small gene families.     

Isolation from alfalfa of resistance gene analogues containing nucleotide binding sites

Cloned resistance (R) genes from a broad range of plant species are known to share similarities in DNA sequence and structural motifs. Degenerate oligonucleotide primers designed from conserved regions of the nucleotide binding site (NBS), common to many R genes, were used to amplify the NBS regions from genomic DNA from alfalfa (Medicago sativa L). Sequence comparisons of the amplified fragments indicated that at least 18 families of NBS-containing R genes are present in alfalfa. Comparisons to R genes from other species suggested a polyphyletic origin of these gene families. Using the same degenerate primers, PCR analysis of cDNA prepared from a plant not challenged with a pest or pathogen revealed that many of the NBS-containing gene families were transcribed actively. Amplification of NBS regions from other Medicago species showed the presence of some NBS-containing genes not present in alfalfa. These results indicate that the NBS-containing R genes comprise a large gene family in Medicago, at least some of which are transcribed in healthy plants, and that different Medicago species carry unique NBS genes.     

Genome-wide identification of NBS-encoding resistance genes in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Nucleotide-binding site (NBS)-encoding resistance genes are key plant disease-resistance genes and are abundant in plant genomes, comprising up to 2% of all genes. The availability of genome sequences from several plant models enables the identification and cloning of NBS-encoding genes from closely related species based on a comparative genomics approach. In this study, we used the genome sequence of Brassica rapa to identify NBS-encoding genes in the Brassica genome. We identified 92 non-redundant NBS-encoding genes [30 CC-NBS-LRR (CNL) and 62 TIR-NBS-LRR (TNL) genes] in approximately 100 Mbp of B. rapa euchromatic genome sequence. Despite the fact that B. rapa has a significantly larger genome than Arabidopsis thaliana due to a recent whole genome triplication event after speciation, B. rapa contains relatively small number of NBS-encoding genes compared to A. thaliana, presumably because of deletion of redundant genes related to genome diploidization. Phylogenetic and evolutionary analyses suggest that relatively higher relaxation of selective constraints on the TNL group after the old duplication event resulted in greater accumulation of TNLs than CNLs in both Arabidopsis and Brassica genomes. Recent tandem duplication and ectopic deletion are likely to have played a role in the generation of novel Brassica lineage-specific resistance genes.     

Tandemly duplicated Arabidopsis genes that encode polygalacturonase-inhibiting proteins are regulated coordinately by different signal transduction pathways in response to fungal infection

Polygalacturonase-inhibiting proteins (PGIPs) are plant proteins that counteract fungal polygalacturonases, which are important virulence factors. Like many other plant defense proteins, PGIPs are encoded by gene families, but the roles of individual genes in these families are poorly understood. Here, we show that in Arabidopsis, two tandemly duplicated PGIP genes are upregulated coordinately in response to Botrytis cinerea infection, but through separate signal transduction pathways. AtPGIP2 expression is mediated by jasmonate and requires COI1 and JAR1, whereas AtPGIP1 expression is upregulated strongly by oligogalacturonides but is unaffected by salicylic acid, jasmonate, or ethylene. Both AtPGIP1 and AtPGIP2 encode functional inhibitors of polygalacturonase from Botrytis, and their overexpression in Arabidopsis significantly reduces Botrytis disease symptoms. Therefore, gene duplication followed by the divergence of promoter regions may result in different modes of regulation of similar defensive proteins, thereby enhancing the likelihood of defense gene activation during pathogen infection.     

A C-terminal Myb extension domain defines a novel family of double-strand telomeric DNA-binding proteins in Arabidopsis

Little is known about the protein composition of plant telomeres. We queried the Arabidopsis thaliana genome data base in search of genes with similarity to the human telomere proteins hTRF1 and hTRF2. hTRF1/hTRF2 are distinguished by the presence of a single Myb-like domain in their C terminus that is required for telomeric DNA binding in vitro. Twelve Arabidopsis genes fitting this criterion, dubbed TRF-like (TRFL), fell into two distinct gene families. Notably, TRFL family 1 possessed a highly conserved region C-terminal to the Myb domain called Myb-extension (Myb-ext) that is absent in TRFL family 2 and hTRF1/hTRF2. Immunoprecipitation experiments revealed that recombinant proteins from TRFL family 1, but not those from family 2, formed homodimers and heterodimers in vitro. DNA binding studies with isolated C-terminal fragments from TRFL family 1 proteins, but not family 2, showed specific binding to double-stranded plant telomeric DNA in vitro. Removal of the Myb-ext domain from TRFL1, a family 1 member, abolished DNA binding. However, when the Myb-ext domain was introduced into the corresponding region in TRFL3, a family 2 member, telomeric DNA binding was observed. Thus, Myb-ext is required for binding plant telomeric DNA and defines a novel class of proteins in Arabidopsis.