Published online 22 February 2008
Published in Crop Sci 48:S-3-S-11 (2008)
© 2008 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
Defensin-like Genes: Genomic Perspectives on a Diverse Superfamily in Plants
Michelle A. Grahama,*,
Kevin A. T. Silversteinb and
Kathryn A. VandenBoschb
a USDA-ARS, Corn Insects and Crop Genetics Research Unit and Dep. of Agronomy, Iowa State Univ., Ames, IA 50010
b Dep. of Plant Biology, Univ. of Minnesota, St. Paul, MN 55108
* Corresponding author (magraham{at}iastate.edu).
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ABSTRACT
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Defensins represent a diverse set of antimicrobial peptides found in almost all multicellular organisms. These small proteins can be characterized by an N-terminal signal sequence, a highly divergent mature protein with the exception of conserved cysteine residues, presence of defensin motifs, and a tissue-specific expression pattern. Defensin expression can be induced by pathogen inoculation and environmental stress. Until recently, defensins in plants were thought to be members of small gene families. However, the advent of expressed sequence tag (EST) and genome sequencing coupled with novel bioinformatic techniques has allowed researchers to recognize the size and diversity of the family. Recent research has identified over 300 defensin-like (DEFL) genes in each of the genomes of Arabidopsis thaliana (L.) Heynh. and Medicago truncatula Gaertner. In addition, over 1000 DEFL genes have been identified from the plant EST projects. The identification of such a broad family involved in defense against pathogens and environmental stress provides new opportunities for crop improvement. This review focuses on genome level analyses of DEFL genes in plants.
Abbreviations: AFP, antifungal protein • CCPs, cysteine cluster proteins • CS
/β, cysteine-stabilized /β • DEFL, defensin-like • EST, expressed sequence tag • LRR, leucine-rich repeat • NBS, nucleotide binding site • PAMPs, pathogen-associated molecular patterns • PPRRs, pathogen pattern recognition receptors • R-genes, resistance genes • SCR, sterility-locus cysteine-rich • SRK, stigma receptor kinase • TIR, Toll/Interleukin-1 receptor-like domain
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ACKNOWLEDGMENTS
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This work was supported by the USDA-ARS, the National Science Foundation (award IOB-0516811), and the University of Minnesota College of Biological Sciences.
Received for publication April 25, 2007.
Defensin-like Genes: Genomic Perspectives on a Diverse Superfamily in Plants
Michelle A. Grahama,*,
Kevin A. T. Silversteinb and
Kathryn A. VandenBoschb
a USDA-ARS, Corn Insects and Crop Genetics Research Unit and Dep. of Agronomy, Iowa State Univ., Ames, IA 50010
b Dep. of Plant Biology, Univ. of Minnesota, St. Paul, MN 55108
* Corresponding author (magraham{at}iastate.edu).
Defensins represent a diverse set of antimicrobial peptides found in almost all multicellular organisms. These small proteins can be characterized by an N-terminal signal sequence, a highly divergent mature protein with the exception of conserved cysteine residues, presence of defensin motifs, and a tissue-specific expression pattern. Defensin expression can be induced by pathogen inoculation and environmental stress. Until recently, defensins in plants were thought to be members of small gene families. However, the advent of expressed sequence tag (EST) and genome sequencing coupled with novel bioinformatic techniques has allowed researchers to recognize the size and diversity of the family. Recent research has identified over 300 defensin-like (DEFL) genes in each of the genomes of Arabidopsis thaliana (L.) Heynh. and Medicago truncatula Gaertner. In addition, over 1000 DEFL genes have been identified from the plant EST projects. The identification of such a broad family involved in defense against pathogens and environmental stress provides new opportunities for crop improvement. This review focuses on genome level analyses of DEFL genes in plants.
Abbreviations: AFP, antifungal protein • CCPs, cysteine cluster proteins • CS/β, cysteine-stabilized /β • DEFL, defensin-like • EST, expressed sequence tag • LRR, leucine-rich repeat • NBS, nucleotide binding site • PAMPs, pathogen-associated molecular patterns • PPRRs, pathogen pattern recognition receptors • R-genes, resistance genes • SCR, sterility-locus cysteine-rich • SRK, stigma receptor kinase • TIR, Toll/Interleukin-1 receptor-like domain
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INTRODUCTION
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ALL SPECIES ARE CONFRONTED with a barrage of potentially harmful pathogens. Only rarely, however, does this lead to disease. In plants, defense against pathogens occurs at two main levels. Nonhost resistance, or species resistance, occurs when a plant species does not support the growth and colonization of an entire species of pathogen (Mysore and Ryu, 2004). Cultivar-specific resistance occurs when plant resistance genes (R-genes) recognize pathogen attack and mount defense responses. For excellent reviews of nonhost and cultivar-specific resistance see Jones and Dangl (2006) and Nürnberger et al. (2004).
In nonhost resistance, preformed compounds such as wax layers, secondary metabolites, and antimicrobial compounds deter potential invaders. Should penetration of the plant cell wall occur, pathogen pattern recognition receptors (PPRRs) recognize conserved pathogen-associated molecular patterns (PAMPs) such as oligosaccharides, bacterial flagellins, and fungal glucans and mount a defense response (Jones and Dangl, 2006; Nürnberger et al., 2004). Since PAMPs are essential to microbial fitness, it is unlikely that they can evolve to evade plant detection. However, microbes can use other mechanisms, such as type III effector proteins, to suppress or inhibit plant defense responses. Thus far, only two PAMP receptors have been cloned, both from Arabidopsis thaliana (L.) Heynh. FLS2 encodes a receptor-like protein kinase and binds the bacterial elicitor flagellin (Gomez-Gomez and Boller, 2000; Zipfel et al., 2006). EFR encodes a receptor-like protein kinase that recognizes the bacterial elongation factor TU (Zipfel et al., 2006). Analysis of the Arabidopsis genome suggests that many other receptor-like kinases exist, some of which could have roles in PAMP recognition (Shiu and Bleecker, 2003).
If the pathogen manages to avoid detection by PPRRs or inhibit the defense response, cultivar-specific resistance can be initiated. This response is governed by classical R-genes. The R-genes monitor pathogen targets in the plant cell or interact directly or indirectly with pathogen effector molecules (Dodds et al., 2006; Jones and Dangl, 2006). An "arms race" occurs between plant and pathogen as the pathogen evolves to escape detection and the plant evolves to recognize the pathogen (Hulbert et al., 2001).
Defense responses induced by nonhost and cultivar-specific mechanisms are surprisingly similar. These include cell wall cross-linking (Bradley et al., 1992; Brisson et al., 1994), production of reactive oxygen intermediates (Alvarez et al., 1998; Jabs et al., 1997; Kawano, 2003), and induction of signaling cascades (Klessig et al., 2000; Scheel, 1998). These signaling cascades can lead to localized cell death, also known as the hypersensitive response (Beers and McDowell, 2001; Greenberg and Yao, 2004), and the production of antimicrobial compounds including phytoalexins, pathogenesis-related proteins, and defensins. In cultivar-specific resistance, the plant may also become immune to other pathogens due to systemic acquired resistance (Dong, 2001; Durrant and Dong, 2004; Gozzo, 2003).
Not surprisingly, plants have dedicated relatively large portions of their genomes to defense against pathogens. The A. thaliana genome contains approximately 150 members of the nucleotide binding site (NBS), leucine-rich repeat (LRR) family of R-genes (Baumgarten et al., 2003; Meyers et al., 2003). Similarly, while only two PPRRs have been identified to date, over 200 LRR-kinases exist in the A. thaliana genome (Shiu and Bleecker, 2003). In contrast, antimicrobial peptides, which have direct antimicrobial effects, were encoded by small gene families and were not considered major players in resistance and defense. However, the advent of expressed sequence tag (EST) and genome sequencing paired with novel bioinformatic approaches has revealed that the number and diversity of antimicrobial peptides is much greater than originally expected (Graham et al., 2004; Schutte et al., 2002, 2005; Silverstein et al., 2007). These peptides may provide novel sources of resistance to a broad spectrum of plant pathogens. While new advances have been made in the study of all antimicrobial peptides, this review will focus specifically on plant defensins.
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Identification of Plant Defensins
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The term plant defensin was originally used by Terras et al. (1995) to describe a novel family of small, cysteine-rich antifungal proteins (AFPs) isolated from the seeds of radish (Raphanus sativus L.) and four other crucifers including A. thaliana (Terras et al., 1993). Sequence analyses of the AFPs identified a cysteine-stabilized /β (CS/β) motif also found in insect defensins. Structural homology to pathogen-inducible insect defensins coupled with their antifungal activity, suggested the AFPs were members of the defensin superfamily.
Defensin sequences have now been found in a variety of plant species allowing researchers to identify defensin signatures (reviewed by (Thomma et al., 2002). These include (i) a similar gene structure, usually a two exon gene with an intron positioned between the signal peptide and mature protein; (ii) a small (<5 kDa), highly charged or polar mature protein; (iii) a highly divergent mature protein with the exception of the conserved cysteines; (iv) the presence of the CS/β motif common to plant, insect, arachnid, nematode, and mollusk defensins (Lay et al., 2003b; Zhu et al., 2005) and the 
-core motif common to all cysteine-containing antimicrobial proteins (Yount and Yeaman, 2004); and (v) a tissue-specific expression pattern (Thomma et al., 2002).
Constitutive expression of plant defensins is typically found in the aboveground parts of plants such as leaves (Terras et al., 1995), seeds (Chen et al., 2002; Janssen et al., 2003), flowers (Lay et al., 2003a; Park et al., 2002), or fruit (Meyer et al., 1996). Others are induced by cold stress (Carvalho et al., 2006; Koike et al., 2002), Zn stress (Mirouze et al., 2006), pathogen inoculation (Epple et al., 1997; Manners et al., 1998; Terras et al., 1998), and wounding (Manners et al., 1998; Meyer et al., 1996). Interestingly, Hanks et al. (2005) identified homologs of the Medicago sativa L. defensins MsDef1 and MsDef2 from M. truncatula that were induced by mycorrhizal or rhizobial symbiosis.
While the majority of plant defensins tested to date show broad antifungal activity (Cabral et al., 2003; Gao et al., 2000; Lay et al., 2003a; Park et al., 2002; Sudar and Kirti, 2006) defensins with activity against bacteria have also been identified (Koike et al., 2002; Osborn et al., 1995; Segura et al., 1998). Other defensins lacking antifungal or antibacterial activity have been found that inhibit -amylase (Liu et al., 2006; Osborn et al., 1995), which may confer insect resistance by inhibiting -amylase in the insect gut. Similarly, trypsin inhibitor activity, characteristic of a seed defensin from the legume Cassia fistula L. (Wijaya et al., 2000), may inhibit protease activity during insect predation. Expression of the novel sunflower (Helianthus annuus L.) defensin Ha-DEF1 is induced in roots by infection with the parasitic plant Orobanche cumana Wallr. (De Zélicourt et al., 2007). Bioassays of Ha-DEF1 revealed that it could induce browning and cell death in O. cumana seedlings. Growing evidence also suggests that defensins may be also important for general stress responses. Expression of the antifungal defensins VUDEF (Carvalho et al., 2006) and Tad1 (Koike et al., 2002) from cowpea [Vigna unguiculata (L.) Walp.] and winter wheat (Triticum aestivum L.), respectively, are induced by cold. In addition, a defensin from A. halleri (L.) O'Kane and Al-Shehbaz has been shown to confer Zn tolerance in transgenic A. thaliana plants (Mirouze et al., 2006).
The mode of action of many plant defensins remains unknown. Perhaps the best examined are the antifungal and anti-insect defensins. DmAMP1 (Dahlia merckii Lehm.) and RsAFP2 (radish) interact with fungal membrane sphingolipids and glucosylceramides, respectively, resulting in membrane permeation (Thevissen et al., 2000, 2005). The M. sativa defensin MsDef1 is thought to bind a Ca2+ channel and block its activity (Spelbrink et al., 2004). Docking experiments of the anti-insect defensin VrD1 [Vigna radiata (L.) R. Wilczek] suggest the binding of VrD1 to the Tenebrio molitor L. -amylase blocks its active site preventing digestion of starch (Liu et al., 2006). Interestingly, VrD1 can also inhibit protein translation in bacteria and fungi (Chen et al., 2002), presumably by different mechanisms.
Unlike typical R-genes, a single defensin may provide resistance to multiple pathogens or races of pathogens. For example, the floral defensin NaD1 inhibits the growth of fungal pathogens Botrytis cinerea Pers.:Fr. and Fusarium oxysporum Schltdl.:Fr. and insect pathogens Helicoverpa armigera Häbner and H. punctigera Wallengren (Lay et al., 2003a, 2003b). Defensins are an attractive alternative to R-genes for providing resistance to crop pathogens and pests because they provide resistance to multiple pathogens and can work synergistically with other antimicrobial compounds. Several transgenic approaches have been used to test the utility of defensins in crop protection. In rice (Oryza sativa L.), the wasabi defensin gene from horseradish [Wasabia japonica (Miq.) Matsum.] was shown to confer durable resistance to rice blast [caused by (Magnaporthe grisea (T.T. Hebert) Yaegashi and Udagawa] over multiple generations (Kanzaki et al., 2002). A defensin from M. sativa was used to confer resistance to Verticillium dahliae Kleb. in field grown potatoes (Solanum tuberosum L.) (Gao et al., 2000). A defensin from Mirabilis jalapa L. (Mj-Amp1) was used to enhance resistance to early blight caused by Alternaria solani Sorauer in tomato (Solanum lycopersicum L.) (Schaefer et al., 2005). Identification of a defensin involved in Zn tolerance (Mirouze et al., 2006) suggests defensins may also have a role in stress adaptation.
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Identification of Defensin-like Genes
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Three different research groups independently identified a large family of cysteine cluster proteins (CCPs) from nodule ESTs of the model legume M. truncatula (Fedorova et al., 2002; Graham et al., 2004; Mergaert et al., 2003). In addition, Graham et al. (2004) identified a small family of related seed-specific CCPs from ESTs of M. truncatula and soybean. In total, 369 expressed CCPs were identified, largely from nodules of M. truncatula. When BLAST homology searches (Altschul et al., 1997) failed to identify sequences homologus to CCPs with known function, Graham et al. (2004) divided the CCPs into sequence-based subgroups that could be used to create motif models (Fig. 1
). These sequence models were used to iteratively search databases of known proteins. While motifs from some groups failed to find informative hits, others had hits to insect and plant defensins, Na channel–blocking scorpion toxins, gamma thionins, and protease inhibitors, all of which have the knottin fold, a structural motif common to defensins.
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Figure 1. Sequence alignment of selected defensin-like (DEFL) proteins with four core conserved cysteines: (A) classical defensins, (B) seed specific DEFL proteins, (C) nodule specific cysteine cluster proteins (CCPs) with six cysteines, (D) nodule specific CCPs with four cysteines. Adjacent labeled groups differ primarily in a single cysteine pair. Identical residues are shaded black while conserved residues are shaded gray. A box labeled "SP" designates the size and position of the signal peptide. Boxes labeled "C" designate conserved cysteine residues within each group. The four core cysteines common to all groups are shaded black. The positions of cysteines in the cysteine stabilized /β motif are shown below the sequences. Sequences from Medicago truncatula are prefixed Mt while sequences from Arabidopsis thaliana are prefixed At. Medicago truncatula sequences are available through the Dana-Farber Cancer Institute plant gene indices (http://compbio.dfci.harvard.edu/tgi/plant.html; verified 23 Jan. 2008) and A. thaliana sequences are available through the Arabidopsis Information Resource (http://www.arabidopsis.org; verified 23 Jan. 2008).
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Other features also suggested the CCPs were similar to defensins (Graham et al., 2004). First, they encoded proteins having a N-terminal signal sequence and a small, highly charged or polar mature protein sequence. Second, they had a characteristic arrangement of 4, 6, 8, or 10 cysteine residues. Third, they were expressed in a tissue-specific manner. Fourth, all the sequences were extremely divergent with the exception of the signal peptide and the conserved cysteine residues. Fifth, the gene structures and the intron positions of the CCP genes were similar to those of known defensins in A. thaliana. Sixth, like many defensins in human and mouse (Maxwell et al., 2003; Semple et al., 2003), the CCP genes appeared to be clustered in the genome and evolving by duplication and positive selection (Graham et al., 2004). Given these similarities, the CCP genes were renamed defensin-like (DEFL) genes by Silverstein et al. (2005).
These findings were especially surprising as defensins were thought to belong to small gene families (Thomma et al., 2002). The Arabidopsis genome, which was virtually complete, was known to have 15 defensins (Thomma et al., 2002). The mouse and human genomes each had less than 50 known defensins (Schutte et al., 2002). However, it seemed unlikely that M. truncatula would be the only plant species to have so many defensins. Therefore, Silverstein et al. (2005) used the DEFL motif models developed by Graham et al. (2004) to iteratively screen the nearly complete A. thaliana genome. In addition to developing new motifs for DEFL genes, the search yielded 317 DEFL genes including the previously identified A. thaliana defensins (Silverstein et al., 2005). The A. thaliana DEFL genes had all the characteristics of defensins including defensin motifs, tissue-specific expression, gene structure, and genome organization.
Defensin-like motif searches of the Institute for Genomic Research plant gene indices identified approximately 1100 expressed DEFL genes from 26 different plant species. While some DEFL subgroups were conserved across different taxonomic clades, over 60% of DEFL subgroups were restricted to a single taxonomic clade. In subsequent work, a particularly large difference (greater than threefold) in DEFL gene numbers was observed when comparing the near-complete genomes of the dicot A. thaliana and the monocot rice (Silverstein et al., 2007). While A. thaliana had 317 DEFL genes, rice had only 92 (Silverstein et al., 2007). Similarly, analyses of human, mouse, rat, and dog genomic sequences identified rodent-specific, dog- and rodent-specific, and primate- and dog-specific β-defensins missing or unrecognizable in the other genomes (Patil et al., 2005). Analyses of insect defensins also found examples of mosquito- or fly-specific defensins (Dassanayake et al., 2007).
Taxonomic expansion of DEFL genes is most clear in the case of nodule-specific DEFL genes from legumes. Unlike most plants, legumes are virtually unique in their ability to form N-fixing nodules with symbiotic rhizobium bacteria (Choi et al., 2004; Doyle and Luckow, 2003). In their searches of the available M. truncatula EST data, (Fedorova et al., 2002; Graham et al., 2004; Mergaert et al., 2003) collectively identified over 300 nodule-specific DEFL genes. Similar nodule-specific sequences had been previously identified from Galega orientalis Lam. (Kaijalainen et al., 2002), Vicia faba L. (Frühling et al., 2000), and M. sativa (Scheres et al., 1990). Lack of homology to sequences outside of these species led Mergaert et al. (2003) to hypothesize the nodule-specific DEFL genes were taxonomically restricted to galegoid legumes. We recently used the DEFL hidden Markov models (Durbin et al., 1998) developed by Graham et al. (2004) and Silverstein et al. (2005) to search the available shotgun sequence of the soybean genome (Jackson et al., 2006). Based on the size of the soybean genome, the number of available sequence reads, and an average sequence length, the sequences available represented a 2.95- to 4.15-fold genome coverage (R. Nelson, USDA-ARS, Ames, IA, personal communication, April 2007). Using the DEFL motifs, no nodule-specific DEFL genes could be identified with an E-value more significant than 10–4, further supporting a dramatic expansion of nodule-specific DEFL genes in galegoid legumes (Graham et al., 2004; Graham et al., unpublished data, April 2007).
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Genome Organization of Defensins and DEFL Genes
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Silverstein et al. (2005) computationally mapped the 317 A. thaliana DEFL genes onto the five A. thaliana chromosomes. While some DEFL genes appeared as single genes, others appeared to cluster in the genome. Cluster sizes ranged from 2 to 14 genes within a 100,000 base-pair region. Introns and regions up and downstream of DEFL genes were used to track the obvious DEFL duplications within a cluster. In addition, clear evidence of segmental duplication between A. thaliana chromosomes was observed. Similar clustering and duplication of M. truncatula DEFL genes were observed when examining the available M. truncatula genome. However, the extent of clustering could not be fully explored with the existing partial genome sequence (Graham et al., 2004; Graham et al., unpublished data, April 2007).
Clustering of DEFL genes has also been observed in nonplant systems. Schutte et al. (2002) used bioinformatic analyses to identify five clusters of β-defensins from syntenic regions of human and mouse. A total of 23 and 48 new β-defensins were identified from human and mouse, respectively. In human, six of the new β-defensins came from the previously identified β-defensin cluster on chromosome 8p23-p22. The number of β-defensin genes within this cluster is highly polymorphic in the human population and has lead to assembly and annotation problems (Taudien et al., 2004). Interestingly, a low copy number of the HBD-2 β-defensin, present in 2 to 10 copies per genome, is linked to colonic Crohn's disease (Fellermann et al., 2006). Low levels of the HBD-2 β-defensin, caused by low gene copy number, are thought to allow normal intestinal bacterial flora to bind to the epithelium of the colon, leading to chronic inflammation.
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Evolution of Defensins and DEFL Genes
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As mentioned previously, defensin and DEFL genes exist as single genes or as clusters within genomes. Clearly, duplication has resulted in the expansion of defensin and DEFL gene clusters. Examination of defensins and DEFL genes within clusters and between orthologs has revealed that the first exon, encoding the signal peptide, tends to be conserved. Meanwhile the second exon, encoding the mature protein, is often extremely divergent (Graham et al., 2004; Silverstein et al., 2005). Analysis of Arabidopsis DEFL gene clusters has found that the mature proteins of some clusters are under purifying selection while in others there is evidence of diversifying selection (Silverstein et al., 2005). Similarly, examination of the major β-defensin locus in human and the orthologous loci in other primates and mouse found that diversifying selection frequently acted following duplication. Subsequently however, different lineages had evidence of both purifying and diversifying selection (Semple et al., 2005). The position of sites undergoing a later round of diversifying selection often overlapped the sites that were under diversifying selection immediately following duplication. This suggests these sites were altered to provide novel function.
The evolution of defensins and DEFL genes bears striking similarity to the evolution of the NBS and LRR families of R-genes. R-genes, defensins, and DEFL genes have each been found both as single genes and clusters throughout plant genomes (for review, see Hulbert et al., 2001). Like defensins, individual clusters of NBS and LRR R-genes have arisen through duplication, unequal recombination, and diversifying selection acting mainly on the LRR. Clusters of NBS and LRR genes have spread throughout the genome through microscale chromosomal duplications and translocations (Meyers et al., 2003) and/or through segmental duplication and rearrangement of chromosomal regions (Baumgarten et al., 2003). Furthermore, the NBS and LRR family of R-genes also shows taxon-specific expansion. The NBS/LRR family can be subdivided into two groups based on the presence or absence of a Toll/Interleukin-1 receptor–like domain (TIR). TIR, NBD, and LRR genes are completely lacking in cereal genomes suggesting diversification and expansion of the TIR, NBD, and LRR families in dicots (Pan et al., 2000). These similarities are compatible with an involvement of DEFL genes in defense against pathogens.
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DEFL Genes May Have Roles Independent of Their Antimicrobial Activity
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It is possible that the DEFL genes identified thus far may also have roles unrelated to antimicrobial activity. The sterility-locus cysteine-rich (SCR) gene from the Brassicaceae encodes a small, cysteine-rich protein with tissue-specific expression in pollen (Nasrallah, 2002). The SCR protein adopts the same three-dimensional fold as many defensins (Chookajorn et al., 2004; Mishima et al., 2003). Binding of the SCR ligand to the stigma-expressed receptor kinase (SRK) is the foundation of sporophytic self-incompatibility in the Brassicaceae (Nasrallah, 2002). Silverstein et al. (2005) noted that almost 40% of newly identified DEFL genes in A. thaliana were also identified by Vanoosthuyse et al. (2001) in their search for SCR and pollen coat protein homologs. Initially, these findings suggested that DEFL genes could also be involved in sporophytic self-incompatibility. However, it is important to compare the phylogenetic distribution of DEFL genes and of sporophytic self-incompatibility. Silverstein et al. identified 40 and 80 expressed DEFL sequences from tomato and potato, respectively (Silverstein et al., 2005, 2007). Tomato and potato are members of the Solanaceae and use gametophytic self-incompatibility to recognize self pollen (McClure, 2004). Gametophytic self-incompatibility occurs by distinct mechanisms not involving SCR or the SRK. Therefore, it is unlikely that DEFL genes found in these species are involved in self-incompatibility.The identification of such a broad family involved in defense against pathogens and environmental stress provides new opportunities for crop improvement.
Interestingly, the majority of DEFL genes identified from M. truncatula were specifically expressed in nodules. Since this tissue is restricted to legumes and a few closely related taxa (Doyle and Luckow, 2003), it suggests the DEFL genes in M. truncatula and other galegoid legumes have been adopted for a novel function in the nodule. Mergaert et al. (2006) hypothesized that nodule-specific cysteine rich genes (also known as DEFL genes, CCPs) are plant-derived signals that trigger bacteroid differentiation in the host cell. Graham et al. (2004) hypothesized the CCPs protect the C-rich nodule from potential pathogens while defense responses are shut down to allow the rhizobial symbiosis. Given the large number of nodule-specific DEFL genes present in M. truncatula, identifying their function through transgenic technologies will be difficult.
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CONCLUSIONS
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The findings of Graham et al. (2004) and Silverstein et al. (2005, 2007) suggest that the numbers of DEFL genes in plants have been significantly underpredicted. In the case of A. thaliana with a near complete genome, underprediction was caused by a built-in minimum size requirement of 110 amino acids for predicted proteins in the genome annotation pipeline (Silverstein et al., 2005). For other species, difficulty in identifying DEFL genes comes from their extreme sequence diversity. Traditional molecular means such as hybridization or polymerase chain reaction only identified closely related sequences. The number and diversity of DEFL genes expressed in nodules of M. truncatula provided a broad base for the identification of DEFL genes from other species.
The identification of such a broad family involved in defense against pathogens and environmental stress provides new opportunities for crop improvement. Analyses of DEFL clusters will help determine how sequence differences affect pathogen specificity. The diverse DEFL sequences described here constitute natural products that could impact not only plant health, but also biomedical research and ultimately mammalian health.
This work was supported by the USDA-ARS, the National Science Foundation (award IOB-0516811), and the University of Minnesota College of Biological Sciences.
Received for publication April 25, 2007.
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TOP
ABSTRACT
INTRODUCTION
