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Keywords:

  • RAR1 signaling;
  • resistance specificity;
  • transposable elements;
  • resistance gene evolution;
  • Blumeria graminis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary Material
  9. References
  10. Supporting Information

Interactions between barley and the powdery mildew pathogen, Blumeria graminis f. sp. hordei, (Bgh) are determined by unique combinations of host resistance genes, designated Mildew-resistance locus (Ml), and cognate pathogen avirulence genes. These interactions occur both dependent and independent of Rar1 (required for Mla12 resistance) and Sgt1 (Suppressor of G-two allele of skp1), which are differentially required for diverse plant disease-resistance pathways. We have isolated two new functional Mla alleles, Rar1-independent Mla7 and Rar1-dependent Mla10, as well as the Mla paralogs, Mla6-2 and Mla13-2. Utilizing the inherent diversity amongst Mla-encoded proteins, we identified the only two amino acids exclusively conserved in RAR1-dependent MLA6, MLA10, MLA12, and MLA13 that differ at the corresponding position in RAR1-independent MLA1 and MLA7. Two- and three-dimensional modeling places these residues on a predicted surface of the sixth leucine-rich repeat (LRR) domain at positions distinct from those within the β-sheets hypothesized to determine resistance specificity. Site-directed mutagenesis of these residues indicates that RAR1 independence requires the presence of an aspartate at position 721, as mutation of this residue to a structurally similar, but uncharged, asparagine did not alter RAR1 dependence. These results demonstrate that a single-amino acid substitution in the sixth MLA LRR can alter host signaling but not resistance specificity to B. graminis.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary Material
  9. References
  10. Supporting Information

Plants are protected from disease by specific recognition of diverse effectors presented by invading pathogens. This recognition capacity is encoded by plant resistance (R) genes that, in turn, activate a multitude of innate defense responses. The most prevalent class of plant R genes encode putative intracellular receptors containing highly conserved motifs including an N-terminal coiled coil (CC) or Toll/Interleukin-1 receptor-like (TIR) domain, a nucleotide binding site (NBS), and C-terminal, leucine-rich repeats (LRR; Dangl and Jones, 2001). Specific residues within the LRR domains are hypervariable and targets for diversifying selection, which is a major factor in the determination of disease resistance specificity (Ellis et al., 2000; Meyers et al., 1998; Michelmore and Meyers, 1998; Mondragon-Palomino et al., 2002; Noel et al., 1999; Parniske et al., 1997; Wang et al., 1998). Typically, one member of a multigene family specifies resistance through direct or indirect recognition of a cognate pathogen avirulence (Avr) gene product. However, it has been shown that two functional members of a gene family can confer the same specificity (Dixon et al., 1996), or conversely, that one gene encodes recognition of multiple distinct pathogen signals (Axtell and Staskawicz, 2003; Grant et al., 1995; Kim et al., 2002; Mackey et al., 2003). In addition, genetic diversity within host–pathogen systems may also exist in the requirements for downstream components of disease response pathways (Shirasu and Schulze-Lefert, 2003).

In barley, specific resistance to the biotrophic pathogen, Blumeria graminis f. sp. hordei (Bgh), commonly referred to as powdery mildew, is conferred by genes designated Ml (Jørgensen, 1994; Schulze-Lefert and Vogel, 2000; Wise, 2000). Approximately 30 distinct resistance specificities have been identified at the Mla locus on chromosome 5(1H) (Jørgensen, 1994). In the susceptible cultivar Morex, the 261-kbp Mla reference sequence comprises a cluster of three families of NBS-LRR resistance gene homologs (RGH), designated RGH1, RGH2, and RGH3, containing four, two, and two members, respectively (Wei et al., 2002). Interspersed within this cluster are two nested complexes of transposable elements, plus additional non-NBS-LRR genes that are defense related. To date, the deduced amino acid sequence similarity among functional Mla alleles suggests that all variants are descendants of one ancestral RGH1 family member, which is represented in Morex by RGH1bcd (Shen et al., 2003; Wei et al., 2002). Depending on the Bgh isolate used, Mla1, Mla6, and Mla13 normally confer rapid and absolute resistance, while others, such as Mla7, Mla10, Mla12, and Mla14 confer an intermediate response (Jørgensen, 1994; Wei et al., 1999; Wise and Ellingboe, 1983). Alleles of Mla all encode R proteins of the CC-NBS-LRR class, yet may differ in their requirements for the zinc-binding protein RAR1 (Rar1 protein) (Shirasu et al., 1999a), and a subunit of the SCF ubiquitin ligase complex (a RING-type F3 ubiquitin ligase complex that contains cul1, RBX1, SKP1 and F-box protein), SGT1 (sgt1 protein) (Azevedo et al., 2002). Recently, domain swaps among segments of MLA1 and MLA6 were used to delimit the regions required for recognition specificity and RAR1 signaling independence to the LRR/C-terminus and LRRs 2–8, respectively (Shen et al., 2003).

In this study, we report the isolation and functional characterization of two new Mla alleles, Rar1-independent Mla7 and Rar1-dependent Mla10. Subsequently, we utilized the inherent diversity amongst six Mla-encoded proteins to identify amino acid residues that establish R-protein-mediated, RAR1-dependent signaling. Guided by this comparative approach, site-directed mutagenesis was used to demonstrate that a single glycine to aspartate substitution at position 721 in the sixth leucine-rich repeat (LRR) of MLA6 and MLA13 is necessary to alleviate Rar1-dependent signaling, while retaining resistance specificity to B. graminis.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary Material
  9. References
  10. Supporting Information

Cloned Mla6 confers both AvrMla6- and AvrMla14-dependent resistance to Bgh

We undertook a series of investigations, using the Mla resistance gene family, to identify molecular determinants underlying R-protein-mediated, RAR1 signaling requirements in plants. Although three Rar1-dependent alleles have been cloned (Halterman et al., 2001, 2003; Shen et al., 2003), up to now, only one Rar1-independent allele, Mla1, has been available for analysis (Zhou et al., 2001). Therefore, in order to conduct a more thorough evaluation, another Rar1-independent allele, Mla7, as well as Rar1-dependent Mla10, were targeted for isolation.

The genotype of Bgh isolate A27 (Giese et al., 1981; AvrMla1, AvrMla7, AvrMla10, AvrMla13, AvrMla14) was ideal for testing the functionality of candidate Mla7 and Mla10 clones, as well as single-amino acid substitution mutations among Rar1-dependent and Rar1-independent Mla alleles (see below). In Cereal Introduction (C.I.) 16151, AvrMla14-dependent resistance specificity, as evidenced by an infection type (IT) of 2-3n, co-segregates in coupling with Mla6, which typically produces an IT of 0 (Giese, 1981; Jørgensen, 1994; Wei et al., 1999). These observations led to the hypothesis that either Mla14 is a gene separate but tightly linked to Mla6, or Mla6 is able to mediate partial recognition of AvrMla14 leading to a weakened resistance phenotype. Thus, in order to utilize isolate A27 we first needed to establish if Mla6, used as a control in subsequent experiments, could impart AvrMla14-dependent recognition specificity. To test this hypothesis, we used a three-component transient assay to determine whether constructs containing Mla6, or the actively transcribed paralog, Mla6-2 (Halterman et al., 2001; Figure S1), can confer resistance to Bgh isolate A27, which contains AvrMla14, but not AvrMla6 (Giese et al., 1981; Wei et al., 1999). In this assay, green fluorescent protein (GFP)-fluorescing, leaf epidermal cells are rendered susceptible to Bgh, because of the presence of wild-type Mlo contained within the GFP-Mlo (pUGLUM) reporter plasmid, whereas neighboring non-transformed cells retain broad-spectrum mlo resistance (Panstruga, 2004; Shirasu et al., 1999b; Zhou et al., 2001). The mlo-mediated resistance of non-transformed cells makes it possible to score the infection phenotypes of the GFP-marked, single-cell transformation events, which otherwise would become masked by spreading fungal hyphae originating from neighboring susceptible cells.

Powdery mildew resistant leaves containing the mlo-5 mutation were co-bombarded with pUGLUM in combination with the 27-kbp cosmid 9589-5a (Mla6), a 15-kbp subclone containing Mla6 (9589-5a-15), cosmid 10276-15, which contains Mla6-2, or cosmids 10052-1-10 and 10907-10c containing Mla13 and Mla13-2, respectively. To compare the effects of a strong versus native promoter on resistance function, the Mla6 cDNA under the control of the maize ubiquitin promoter and the nopaline synthase (nos) terminator (Ubi:Mla6) was also tested. As illustrated in Figure 1, Bgh isolates 5874 (AvrMla6), K1 (AvrMla13), or A27 (AvrMla13, AvrMla14) were used to inoculate bombarded leaves. The 15-kbp cosmid subclone, 9589-5a-15 (Mla6), but not cosmid 10276-15 (Mla6-2), significantly reduced the number of GFP-marked, mlo-5 cells that supported Bgh hyphal growth after inoculation with both isolates 5874 and A27. Additionally, the Mla6 cDNA under the control of the maize ubiquitin promoter was capable of significantly reducing the percentage of Bgh hyphal colonies on A27- as compared to K1-challenged cells. Taken together, these results support the conclusion that Mla6 is able to successfully mediate an incompatible interaction with AvrMla14 present in isolate A27, and that conversely, the Mla14 specificity is not conferred by the Mla6-2 paralog.

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Figure 1. Recognition of AvrMla14 is conferred by Mla6 and not Mla6-2.

The percentage of challenged (GFP-positive with attached spore) mlo-5 cells supporting growth of Bgh A27 (AvrMla13, AvrMla14; closed bars), K1 (AvrMla13; open bars), and 5874 (AvrMla6; striped bars) were scored 5 days after inoculation. The plasmid or cosmid constructs bombarded in combination with pUGLUM (GFP/Mlo) are indicated below each set of bars. The data shown are means of at least two independent experiments. Error bars indicate SD between experiments.

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Cosmid 10907-10c (Mla13-2), did not significantly reduce the percentage of Bgh hyphal growth after inoculation with isolates A27 (73.2%) or K1 (57.4%), whereas GFP-marked cells bombarded with an Mla13-containing construct only supported Bgh growth 1.5 and 2.4% of the time, respectively.

Amplification and functional analysis of Mla7 and Mla10

Early attempts to PCR-amplify full-length Mla alleles from genomic DNA was difficult – likely because of the divergence of Mla-flanking sequences (Figure S1). Therefore, we developed a two-step RT-PCR method to circumvent these dissimilarities and amplify additional members of the Mla family (see Experimental procedures). Briefly, conserved primers within the previously isolated Mla1, Mla6, and Mla13 open-reading frames (ORFs) were used for reverse transcription of mRNAs from accessions containing Mla1, Mla6, and Mla13 (as controls), as well as the Mla7 and Mla10 resistance specificities. Eight RT-PCR clones from each amplification experiment were sequenced to identify uninterrupted ORFs. Gene-specific primers were then designed from the 3′ end of each transcript and used in combination with a conserved 5′ end primer to amplify full-length Mla transcripts from each barley accession by RT-PCR. Amplified products containing a complete transcript leader region (TLR) and ORF of Mla candidates were placed into a plasmid vector under the control of the maize ubiquitin promoter and the nos terminator. Mla6, Mla7, and Mla10 constructs were co-bombarded with the pUGLUM reporter plasmid into either mlo-5 or mlo-31/rar1-2 barley leaves. The demonstrated AvrMla14-dependent specificity of Mla6 made Bgh isolate A27, which contains AvrMla7, AvrMla10, and AvrMla14, ideal for testing the functionality of these candidate clones. As shown in Figure 2, RT-PCR-amplified Mla6 and Mla10 ORFs under the control of the maize ubiquitin promoter (Ubi:Mla6; Ubi:Mla10) significantly reduced the percentage of GFP-marked, mlo-5 cells that supported growth of A27 hyphal colonies, as compared to cells bombarded with pUGLUM alone. However, neither Ubi:Mla6 nor Ubi:Mla10 significantly reduced the number of hyphal colonies on mlo-31/rar1-2 cells, demonstrating the dependence of Mla6 and Mla10 on Rar1. In contrast, Ubi:Mla7 significantly reduced the number of cells that supported Bgh hyphal growth in both mlo-5 and mlo-31/rar1-2 leaves after inoculation with isolate A27. Consistent with previous genetic data (Jørgensen, 1996), this indicates that the Ubi:Mla7 construct is able to confer AvrMla7-dependent resistance specificity in a Rar1-independent manner.

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Figure 2. Functional analysis of Mla7 and Mla10.

The percentage of challenged (GFP-positive with attached spore) mlo-5 (closed bars) and mlo-31/rar1-2 (open bars) cells supporting growth of Bgh A27 (AvrMla7, AvrMla10, and AvrMla14) were scored 5 days after inoculation. The plasmid constructs bombarded in combination with pUGLUM (GFP/Mlo) are indicated below each set of bars. The data shown are means of five independent experiments. Error bars indicate SD between experiments.

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A single-amino acid substitution in the sixth LRR of MLA6 and MLA13 alleviates RAR1 dependence

The isolation of Mla7 and Mla10 allowed us to identify conserved amino acids in the RAR1-dependent MLA6, MLA10, MLA12, and MLA13 proteins that set them apart from their RAR1-independent counterparts. As highlighted in Figure 3, only two amino acids, located within the sixth LRR, are conserved exclusively among RAR1-dependent MLA proteins. The first, an asparagine at position 712, is divergent in the RAR1-independent MLA1 and MLA7. The second is a glycine at position 721 of RAR1-dependent MLAs, as opposed to an aspartate in MLA1 and MLA7. To determine where these amino acids lie within the framework of the LRR, the secondary structure of each MLA protein was predicted using psipred (McGuffin et al., 2000). A consensus of the six secondary structures placed amino acid 712 within a coil and amino acid 721 within a helical domain (Figure 3). This prediction suggested that these two residues were distinct from those within the solvent-exposed region of β-sheets shown to determine resistance specificity (Ellis et al., 2000).

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Figure 3. Amino acid alignment of MLA LRR regions.

The one-letter abbreviation of amino acids divergent from MLA7 are shown. Dots represent amino acids identical to MLA7. The location of the xxLxLxx motifs of the 11 LRR β-sheets are shown above each line of the alignment. Stars represent the outer borders of a region responsible for RAR1 independence (Shen et al., 2003). The arrows and shaded boxes indicate the only amino acids conserved exclusively in the RAR1-dependent proteins. The order of these amino acid sequences is consistent with the phylogenetic analysis shown in Figure 5(a). Below each column of amino acids is the consensus prediction of secondary structure using psipred (McGuffin et al., 2000). c, coil; e, sheet; h, helix.

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To test the role of amino acid 712 in RAR1-mediated signaling, we used site-directed mutagenesis to change the corresponding asparagine in MLA6 and MLA13 to an aspartate. We introduced reciprocal mutations to the corresponding amino acids in MLA1 and MLA7, changing them to asparagines. Constructs encoding these proteins, under control of the maize ubiquitin promoter, were co-bombarded into mlo-5 and mlo-31/rar1-2 leaves and subsequently inoculated with Bgh isolate A27 (AvrMla1, AvrMla7, AvrMla13, AvrMla14). In addition to the pUGLUM control vector used with Mla6- and Mla7-derived constructs, a second plasmid (pUMNUGN), containing Mlo and β-glucuronidase (GUS) under control of maize ubiquitin promoters (Azevedo et al., 2002), was used as a reporter of transformed cells in experiments with Mla1- and Mla13-derived constructs. Regardless of the reporter gene used, bombarded cells were assayed for the presence of Bgh hyphae growing from GFP- or GUS-positive cells. As shown in Figure 4, mutation of MLA13 at position 712 did not compromise resistance or signaling specificity, as the number of cells supporting growth of Bgh was similar between wild-type (Ubi:Mla13) and mutant (Ubi:Mla13-N712D) constructs, on either mlo-5 or mlo-31/rar1-2 leaves. Barley mlo-5 cells co-bombarded with Ubi:Mla1-R712N supported more growth of Bgh than Ubi:Mla1, but less than the reporter plasmid alone, indicating an incomplete resistance response. Even so, mlo-31/rar1-2 cells supported similar Bgh growth when co-bombarded with either Ubi:Mla1 or Ubi:Mla1-R712N constructs, indicating that MLA1-R712N retains independence from RAR1. Mutations in Mla6 (Ubi:Mla6-N712D) and Mla7 (Ubi:Mla7-D714N) abolished the ability of these genes to confer resistance specificity to A27 as the number of bombarded cells supporting Bgh growth was not significantly less than cells bombarded with the reporter plasmid alone. Taken together, these results suggest that residue 712 is not directly involved in determining Rar1-mediated signaling specificity.

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Figure 4. A glycine to aspartate substitution at position 721 in MLA6 and MLA13 confers Rar1-independent resistance to Bgh.

The percentage of challenged mlo-5 (solid bars) and mlo-31/rar1-2 (open bars) cells supporting growth of Bgh A27 (AvrMla1, AvrMla7, AvrMla10, AvrMla13, and AvrMla14) were scored 5 days after inoculation. The wild-type and mutant Mla constructs bombarded in combination with the reporter plasmid are indicated next to each set of bars. The negative control represents bombardment with the reporter plasmid alone. The pUGLUM (Mlo-GFP) reporter plasmid was used with Mla6- and Mla7-derived constructs, and pUMNUGN (Mlo-GUS) reporter was used for Mla1- and Mla13-derived constructs. The data shown are means of at least two independent experiments. Error bars indicate SD between experiments.

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The role of amino acid 721 was tested using the same strategy to change the corresponding glycine to an aspartate in MLA6 (Ubi:Mla6-G721D) and MLA13 (Ubi:Mla13-G721D). Reciprocal mutations were introduced into Mla7 (Ubi:Mla7-D723G) and Mla1 (Ubi:Mla1-D721G). As shown in Figure 4, growth of Bgh isolate A27 on mlo-5 cells co-bombarded with Ubi:Mla6-G721D or Ubi:Mla13-G721D was not significantly different from that on cells bombarded with their respective wild-type constructs, demonstrating that these mutated genes retain resistance specificity. However, the Ubi:Mla6-G721D and Ubi:Mla13-G721D constructs also significantly reduced the growth of Bgh isolate A27 on mlo-31/rar1-2 cells as compared to the pUGLUM or pUMNUGN reporter plasmids. These results indicate that the single-amino acid changes in MLA6- and MLA13-G721D proteins alleviate dependence on wild-type RAR1 to effect both AvrMla14- and AvrMla13-dependent resistances, respectively. Analogous results were obtained with the Ubi:Mla6-G721D construct on both mlo-5 and mlo-31/rar1-2 cells when Bgh isolate 5874 (AvrMla6) was used (data not shown), indicating that RAR1 independence of the Mla6-G721D mutation extends to both AvrMla14- or AvrMla6-dependent resistance specificity. To further test whether RAR1 independence is determined by the presence of an aspartate at this position, glycine 721 of MLA6 was also changed to an asparagine, which is structurally similar to aspartate, but lacks a charge on its side chain. In contrast to Mla6-G721D, the Mla6-G721N mutant retained RAR1 dependence, suggesting that the charged aspartate is required at this position to confer RAR1 independence. Co-bombardment of Ubi:Mla1-D721G and Ubi:Mla7-D723G did not significantly reduce the growth of Bgh A27 when compared to the reporter plasmid control, indicating that these single-amino acid mutations abolish the ability of MLA1 and MLA7 to specify resistance to this isolate.

Rar1-independent Mla1 and Mla7have divergent evolutionary histories

As aspartates 721 in MLA1 and 723 in MLA7 appeared critical in specifying Rar1 independence, we were interested in ascertaining whether these amino acids are present as a result of independent mutation or recombination among particular gene segments. To test these hypotheses, we performed phylogenetic analysis using parsimony (paup) of full-length Mla-coding regions as well as a more focused analysis of the 150 nt encompassing residues 721/723 in the sixth LRR (Figure 5). The nucleotide sequences of the Mla1, Mla6, Mla7, Mla10, Mla12, Mla13, and Mla-RGH1bcd ORFs were aligned with two Mla-like wheat expressed sequence tag (EST) sequences (GenBank Accessions AF538039 and AF538040). In both the full-length and localized paup analyses, the results shown in Figure 5 indicate that Mla1 and Mla7 belong to separate evolutionary branches. Additionally, to detect reticulate evolution of Mla alleles, tests were performed using the plato (Grassly and Holmes, 1997), reticulate (Jakobsen and Esteal, 1996), and recpars (Hein, 1993) software (reviewed by Posada, 2002). Because of strong diversifying selection within the LRR domain (Halterman et al., 2001; Shen et al., 2003), there was no consensus for the small number of recombination breakpoints revealed by these programs. Nevertheless, no recombination was observed in the 150 nt region surrounding the sixth LRR. Thus, the examination of reticulate evolution is consistent with the overall inference from the phylogenetic analysis, suggesting that the aspartates721/723 in MLA1 and MLA7 are present as a result of separate evolutionary events.

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Figure 5. Phylogenetic analysis of Mla coding regions.

(a) Phylogram of entire Mla coding sequences and (b) 150 nt surrounding the region encoding the sixth LRR, as determined by paup with a heuristic search. Branch lengths are proportional to the number of amino acid substitutions between nodes. The trees were rooted using two partial wheat (Triticum aestivum) MLA orthologs (Accession numbers shown). Bootstrap values from 1000 replications are shown.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary Material
  9. References
  10. Supporting Information

A single-amino acid substitution in the sixth MLA LRR alters Rar1 signaling specificity

Although the structures of plant R-protein LRR domains remain unknown, the crystal structures of several other LRRs have been established (Kobe and Kajava, 2001). Comparison of these structures illustrates the tendency of LRRs to form a horseshoe-shaped molecule with β-sheets on the concave side. A central xxLxLxx motif, where ‘x’ represents any amino acid, forms the β-sheet with the leucines buried in the center of the protein and the adjacent residues exposed to the solvent (Jones and Jones, 1997; Kobe and Deisenhofer, 1994). These solvent-exposed residues are hypervariable and targets for diversifying selection in plant R proteins – a major factor in the determination of disease resistance specificity (Ellis et al., 2000; Meyers et al., 1998; Michelmore and Meyers, 1998; Mondragon-Palomino et al., 2002; Noel et al., 1999; Parniske et al., 1997; Wang et al., 1998). In contrast, amino acids within the LRR that define interactions with downstream components are likely under conservative selection as many of these proteins are required by diverse R-protein pathways (Aarts et al., 1998; Austin et al., 2002; Liu et al., 2002; Muskett et al., 2002; Parker et al., 1996; Tör et al., 2002; Tornero et al., 2002). Therefore, it is probable that these residues are located outside of the xxLxLxx motif where interactions between LRR repeats determine the overall structure of the domain (Jones and Jones, 1997; Kobe and Deisenhofer, 1995).

Only two amino acids are conserved among identified RAR1-dependent MLA proteins: an asparagine at position 712 and a glycine at position 721. Constructs harboring the MLA1-R712N mutation retained moderate resistance without altering RAR1 independence. Yet, a significant reduction in the number of hyphal colonies was observed in mlo-31/rar1-2 cells bombarded with Ubi:Mla1-R712N as compared with mlo-5 cells (Figure 4). This observation suggests that either the MLA1-R712N mutation compromises resistance specificity, possibly through an alteration in tertiary structure, or the genetic background of mlo-31/rar1-2 cells confers a slight increase in resistance. Reciprocal mutation of residue 712 in MLA13 also did not alter RAR1 dependence, suggesting that this amino acid does not have a role in determining RAR1 specificity in this case. However, the G721D substitution in MLA6 and MLA13 alleviated their requirement for RAR1, while retaining resistance specificity. Moreover, these data indicate that RAR1 independence requires the presence of an aspartate at position 721, as mutation of this residue to a structurally similar, but uncharged, asparagine did not alter RAR1 dependence in MLA6. Therefore, it appears possible that the aspartate in MLA1 and MLA7 has a greater influence in defining Rar1 independence than the glycine in MLA6 and MLA13 in determining Rar1 dependence.

Amino acid 721 in MLA6 and MLA13 lies halfway between the xxLxLxx motifs of LRRs 6 and 7. The role of this residue in determining Rar1 independence is corroborated by independent tests of MLA1/MLA6 chimeras, which delimited the region required for RAR1 independence of MLA1 to between the second and eighth LRRs (Shen et al., 2003). As hydrophilic and potentially charged residues are unlikely to be buried within the hydrophobic core of the protein, we hypothesize that this aspartate residue in MLA1 and MLA7 is solvent exposed and could be involved in intra- or intermolecular charge-based interactions. A prediction of secondary structure using psipred (McGuffin et al., 2000) suggests that residue 721 lies within a helical domain (Figure 3). In order to elucidate the role this mutation might have in defining molecular interactions, a predicted tertiary structure of this LRR region was determined by alignment to the known structure of porcine ribonuclease inhibitor (PRI), an LRR-containing protein (Kobe and Deisenhofer, 1993). Although it has been suggested that plant R protein LRRs do not share the same tertiary structure as PRI (Jones and Jones, 1997), there was sufficient homology to form a three-dimensional model of the region containing the sixth LRR. As illustrated in Figure 6, amino acid 721 in MLA6 potentially lies on the convex surface of the domain where it may have a role in intermolecular interactions between repeat motifs. Modeling of this domain against another LRR protein, RNA1P (Gtpase-activating protein for Spi1, the S. pombe ortholog of Ran) (Hillig et al., 1999), also places this residue on the outer surface of the helix where it could be involved in intramolecular interactions (data not shown). While it is difficult to determine whether a glycine to aspartate change is significant enough to alter the overall structure of the LRR, the presence of a hydrophilic and potentially charged amino acid could affect intramolecular folding as well as intermolecular interactions with other members of the MLA signal transduction pathway. As residue 721 lies within the region thought to define interactions between subunits of the LRR (Jones and Jones, 1997; Kobe and Deisenhofer, 1995), this mutation could transform the overall conformation of the MLA LRR domain and, in turn, modify its dependence on RAR1. This is consistent with the observation that RAR1 specificity does not appear to be conferred by CC or TIR domains as, in Arabidopsis, RAR1-dependent and -independent R proteins were identified from classes containing either of these motifs (Muskett et al., 2002).

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Figure 6. Model of the predicted tertiary structure of the sixth MLA LRR.

This three-dimensional structure of the sixth MLA LRR was derived by sequence comparison to the determined crystal structure of porcine ribonuclease inhibitor (Kobe and Deisenhofer, 1993). Panel (a) shows an aspartic acid (circled in gold) unique to the RAR1-independent MLA1 and MLA7 proteins. Hydrogen bonds with atoms of an isoleucine in the adjacent α-helix are shown as dashed purple lines. Panel (b) illustrates a glycine at the corresponding position in the RAR1-dependent MLA6, MLA10, MLA12, and MLA13 proteins, which has only a hydrogen atom as a side chain (shown on right). The parallel β-sheets are represented by red arrows. Predicted α-helical regions are shown in green.

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Single-amino acid changes within the LRRs of resistance gene products also can result in the loss of the resistance phenotype (Bent et al., 1994; Bryan et al., 2000; Grant et al., 1995; Mindrinos et al., 1994; Shen et al., 2003; Warren et al., 1998). The majority of these mutations occur within the solvent-exposed residues of the β-sheet/β-turn region of the LRR, with a concurrent loss in resistance specificity. However, in addition to evidence presented in this paper, two other findings suggest that the LRRs of some R proteins are additionally involved in resistance gene signaling. The rps5-1 mutation in the third LRR of RPS5 compromises resistance to Pseudomonas syringae pv. tomato DC3000 expressing avrPph3, as well as resistance mediated by several other R proteins, suggesting an interaction with a signal transduction element common to several R gene pathways (Warren et al., 1998). Introduction of an Rps5 transgene did not fully restore the resistance phenotypes of all the affected genes, indicating that the rps5-1 mutant protein may titrate out a signaling component common to multiple resistance pathways. Additionally, Banerjee et al. (2001) found that an Arabidopsis Po (Poppelsdorf)-1-derived allele of Rps2 is able to confer resistance to P. syringae expressing avrRpt2 in a Columbia (Col-0) genetic background, but not in a Po-1 background. Six amino acid polymorphisms within the LRR of Po-1 RPS2 were determined to limit the ability of this protein to utilize one or more other Po-1 resistance signaling components. The majority of these differences appear to lie outside of the putative ligand-binding, solvent-exposed residues. Notably, three of the six differences lie between LRR6 and LRR7 of the RAR1-dependent RPS2 protein – the same region we have defined as important for RAR1 dependence in MLA6 and MLA13. Taken together, this suggests that, in some cases, non-xxLxLxx residues are crucial in determining signaling component interactions.

RAR1 is required for a diverse subset of R proteins (Liu et al., 2002; Muskett et al., 2002; Tornero et al., 2002). While it is not known where RAR1 fits into each of these signaling pathways, a role in the stability of pre-formed R proteins has been implicated, as RPM1 (resistance to P. syringae) fails to accumulate in an Arabidopsis rar1 mutant (Tornero et al., 2002). In this case, RAR1 either actively facilitates the stability of RPM1 or functions to block actions designed to reduce the quantity of RPM1 protein within the cell. RAR1 could affect the ubiquitination of target proteins through its interaction with both barley and Arabidopsis SGT1, as well as subunits of the COP9 signalosome (Azevedo et al., 2002). Yeast SGT1 interacts with SKP1, a subunit of the SCF-type E3 ubiquitin ligase complex (Kitagawa et al., 1999). The COP9 signalosome is a multisubunit protein complex that regulates proteosome-mediated degradation of specific proteins (Wei et al., 1998). Based on differences in susceptibility of MLA chimeras in rar1-2 mutant barley leaves, Shen et al. (2003) have suggested a role for RAR1/SGT1 in activation of MLA-containing recognition complexes. However, in our experiments we did not observe a distinct difference between susceptibility of rar1-2 cells bombarded with Mla6 when compared to other susceptible controls, indicating that MLA6 complexes are likely not more active in the absence of RAR1. Therefore, it appears that RAR1 could function as an antagonist of SGT1-mediated ubiqitination and degradation of pre-formed R proteins. Resistance mediated independently of Rar1 in plants containing Mla1 and Mla7 could be because of alternative methods of regulating the expression of these genes, or by bypassing the requirement of RAR1 through changes in the tertiary structure of the MLA1 and MLA7 LRR domains.

In summary, our results indicate that RAR1 independence is determined by an aspartate residue at position 721 within the sixth LRR of MLA. Furthermore, residue 721 appears distinct from those hypothesized to determine resistance specificity and likely plays a role in determining inter- or intramolecular interactions that regulate resistance signal transduction.

Mla6 confers AvrMla6- and AvrMla14-dependent resistance specificity to Bgh

In some host–pathogen interactions, a single resistance protein can confer specificity to more than one pathogen effector (Axtell and Staskawicz, 2003; Grant et al., 1995; Kim et al., 2002; Mackey et al., 2003). In this regard, genetic evidence indicates that barley lines harboring distinct Mla alleles are also able to confer alternate resistance specificities to multiple Bgh isolates, each containing unique AvrMla genes (Brown, 2002). In C.I. 16151, AvrMla14-dependent resistance is defined as an intermediate IT that co-segregates with a rapid and absolute response conferred by Mla6 (Jørgensen, 1994; Wei et al., 1999). We have shown that the cloned Mla6 ORF, expressed transiently under control of a maize ubiquitin or native promoter can specify Rar1-dependent resistance equally to Bgh isolates 5784 and A27, which contain AvrMla6 and AvrMla14, respectively (Giese et al., 1981; Torp et al., 1978). Furthermore, the single-amino acid change in the MLA6-G721D protein alleviates dependence on RAR1 to effect both AvrMla6- and AvrMla14-dependent resistance responses. This presents two possible scenarios: either the MLA6 protein is capable of monitoring the presence of two diverse avirulence gene products, or the weakened infection phenotype induced by Bgh isolates that contain AvrMla14 is caused by an alternate AvrMla6 allele whose product is only partially recognized by MLA6. To our knowledge, there are no clear data that indicate AvrMla6 and AvrMla14 segregate independently (Jensen et al., 1995). Thus, it remains possible that these historically designated Avr genes are either (i) allelic variants, (ii) separate but closely linked, or (iii) that the genetic backgrounds of Bgh 5874 and A27 influence the phenotype of AvrMla6 in interactions with barley accessions that harbor Mla6.

There are, however, several other documented instances of barley lines with putative, tightly linked Ml genes that provide alternate responses to various Bgh isolates that harbor unique Avr genes (Caffier et al., 1996; Giese, 1981; Jørgensen, 1994; Jensen et al., 1995). For example, the C.I. 16155 (Mla13) accession displays an intermediate IT in response to Bgh isolate R189, historically designated as the Ml-Ru3 specificity (Caffier et al., 1996; Giese, 1981). This second specificity co-segregates with Mla13 in a mapping population of 3600 gametes, just as Mla14 co-segregates with Mla6 (Wei et al., 1999). Previous data corroborate the existence of a second Avr gene in Bgh that elicits the Ml-Ru3 phenotype (Brown, 2002; Brown and Simpson, 1994; Caffier et al., 1996; Jensen et al., 1995). Thus, it could similarly be that Mla13, being the only documented functional Mla copy in C.I. 16155, recognizes either a second Avr gene or an AvrMla13 allele to confer Ml-Ru3-mediated resistance. It remains uncertain whether the recognition of multiple Bgh genotypes by Mla is determined on the plant or pathogen side, or some combination of the two, thus, the molecular isolation of additional Ml genes and their cognate AvrMl genes will be necessary to complete our understanding of these dynamic host–pathogen interactions.

Parallel evolution of the Mla family

Previously, we developed a working model for the evolution of the Mla complex using a 261-kbp DNA region spanning the locus in the barley cultivar Morex (Wei et al., 2002). In the process of isolating additional Mla paralogs (Mla6-2 and Mla13-2) and alleles (Mla7 and Mla10), we identified distinct Insertion/deletion (InDel), retrotransposon and long interspersed nuclear element (LINE) insertions, in addition to the (AT)n simple sequence repeat (SSR) within the third intron (Tables S1 and S2; Halterman et al., 2003; Shen et al., 2003; Zhou et al., 2001). These polymorphisms were used as markers within and flanking Mla family members to construct a model for the diversification of Mla resistance haplotypes (Figure S2). This analysis was consistent with the interpretation that the present-day distribution of the Mla gene family has been the result of at least two evolutionary pathways. One proposed branch of the pathway contains Mla1, in addition to Morex RGH1bcd, whereas the other branch contains Mla6 and Mla13.

The timing of retrotransposon insertions within and flanking Mla ORFs suggests that an ancient Mla progenitor was duplicated prior to insertion of two LINEs at the 5′ end of these two haplotypes (Figure S2). Only one of these haplotypes was host for the differential amplification of an (AT)n microsatellite within the third intron. As the (AT)n microsatellite is present in the Mla1, Mla6, and Mla13 alleles, as well as the corresponding third intron of the non-functional Mla6-2 and Mla13-2 paralogs, it was most likely propagated very early, but after duplication of the ancestral Mla gene. Subsequent to the initial duplication, the evolution of functional Mla resistance specificities appears to branch into two pathways – one leading to Rar1-independent Mla1 (branch A), and another leading to Rar1-dependent Mla6, and Mla13 (branch B). Initially, this suggested that Rar1 signaling specificity may have diverged early in the evolution of Mla. However, when integrated with the analysis of phylogeny and reticulate evolution, the data are consistent with the conclusion that Rar1 dependence, or independence, is not limited to one evolutionary course.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary Material
  9. References
  10. Supporting Information

Fungal isolates

Blumeria graminis f. sp. hordei isolates 5874 (Torp et al., 1978; Wei et al., 1999; AvrMla1, AvrMla6), K1 (Zhou et al., 2001; AvrMla1, AvrMla7, AvrMla13) and A27 (Giese et al., 1981; AvrMla1, AvrMla7, AvrMla10, AvrMla13, AvrMla14) were propagated on Hordeum vulgare cv. Manchuria (C.I. 2330) in separate growth chambers at 18°C (16 h light/8 h darkness).

Cosmid library construction, screening, and sequencing

Cosmid library construction was performed in cooperation with Cell & Molecular Technologies, Inc. (Phillipsburg, NJ, USA). High-molecular weight genomic DNA from C.I. 16151 and C.I. 16155 was partially digested with Sau3A, size selected for fragments ranging between 50 and 75 kbp, and ligated into the BamHI site of digested cosmid SuperCos-1 (Stratagene, La Jolla, CA, USA). Plasmid libraries of partially digested (Sau3A) cosmid DNA was constructed in pBluescript (Stratagene). DNA sequencing and oligonucleotide synthesis were performed by the Iowa State University DNA Sequencing and Synthesis Facility.

Eleven contiguous cosmids were identified from the C.I. 16151 (Mla6) cosmid library that did not harbor Mla6, but contained restriction fragments that hybridized to a Mla6 cDNA probe (Halterman et al., 2001). Likewise, 12 contiguous cosmids were identified from a C.I. 16155 (Mla13) library that did not include Mla13, but hybridized to a Mla13 cDNA probe (Halterman et al., 2003). Cosmid clones 10276-15 (C.I. 16151) and 10907-10c (C.I. 16155) contained complete Mla6-2 and Mla13-2 paralogs that are 94.9 and 95.1% similar to Mla6 and Mla13, respectively. In addition, both putative ORFs contain two copia-like elements, designated HORPIA (Hordeumcopia-like LTR retrotransposon)-4 and HORPIA-5 (Table S1; Figures S1 and S2). Consistent with the results of DNA gel blot hybridization (not shown), no additional Mla copies were identified from either library, indicating that there may be a maximum of two recently diverged copies of Mla6 and Mla13 in these accessions.

Amplification of new Mla specificities

RNA was isolated from uninoculated barley leaf tissue using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). RT-PCR was performed using the SuperScript™ One-Step RT-PCR with Platinum® Taq System (Invitrogen, Carlsbad, CA, USA) using the Mla gene-specific primer DH9-4 and a poly-T primer as shown in Table 1. RT-PCR products were ligated into the pGEM-T Easy vector (Promega, Madison, WI, USA) and sequenced. Gene-specific reverse primers for Mla1 (C.I. 16137), Mla6 (C.I. 16151), Mla7 (C.I. 16147), Mla10 (C.I. 16149), and Mla13 (C.I. 16155), were designed from the 3′ end of the transcript. These primers, in conjunction with the Mla forward primer (MlFORtpo), were used to amplify the Mla TLRs and ORFs.

Table 1.  Primers used for RT-PCR and site-directed mutagenesis
Primer namePrimer sequences (5′ [RIGHTWARDS ARROW] 3′)Function
MlFORtpoCACCGTCATTCCAGAGATATGCCAGRT-PCR
Mla1revTCAGTTCTCCTCCTCGTCCTCACACRT-PCR
Mla6revTTAGTTCTCCTCCTCGCCCTCACACRT-PCR
Mla7revTCAGAAATCAGTTCTCCTCCTCTCCTCACACRT-PCR
Mla10revTCACATTAAATCGTCATCTTGAGCRT-PCR
Mla13revTCAGAAATCAGTTCTCCTCCTCTCCRT-PCR
DH9-4CATAGAAGTGTTGAGGGGTATCRT-PCR
DH225GGATTTGTATGAAGATTTCGTGAAGTCMla6/13 G721D
DH226GGATTTGTATGAAGGTTTGCTGAATTCCCMla7 D723G
DH227GGATTTGTATAAAGGTTTCGTGAAGTCMla1 D721G
6G721NGACTTCACGAAATTTTCATACAAATCCMla6 G721N
6N712DGCTTGAGATTCGCTTCGATGATGGTAGTTTGGMla6/13 N712D
7D714NGCTTGAGATTCACTTCAATGATGGTAGTTTGGMla7 D714N
1R712NCCAAACTAGCATCATTGAAGCAAATATCAAGCMla1 R712N

Plasmid constructs and site-directed mutagenesis

The UbiNOS vector used for transient expression of Mla genes has been described previously by Shirasu et al. (1999b). This vector was converted to a Gateway™ destination vector using the Gateway™ Vector Conversion System (Invitrogen). Candidate Mla genes amplified using RT-PCR were inserted into the pENTR/D-TOPO® vector (Invitrogen) and then transferred to the modified UbiNOS Gateway™ vector. Site-directed mutagenesis was carried out using the Stratagene QuickChange™ site-directed mutagenesis kit. Nucleotide 2162 was changed from a G to an A in Mla6 and Mla13, resulting in an aspartate at amino acid residue 721. Nucleotides 2161 and 2162 we changed from GG to AA in Mla6 to change amino acid 721 from a glycine to asparagine. Conversely, nucleotides 2162 and 2168 were changed from an A to a G in Mla1 and Mla7, respectively, resulting in a glycine at the corresponding positions. To change amino acid 712 from asparagine to aspartate in Mla6 and Mla13, nucleotide 2134 was changed from an A to a G. In Mla7, nucleotide 2140 was changed from a G to an A to change amino acid 714 from aspartate to asparagine. Amino acid 712 of MLA1 was changed from arginine to asparagine by mutating nucleotides 2135 and 2136 from GG to AT. Primers used for mutagenesis are shown in Table 1. Mutagenized plasmid constructs were verified by sequencing.

Functional analysis of candidate genes via single-cell transient assay

Biolistic bombardment of leaves was carried out according to Shirasu et al. (1999b) using a biolistic PDS-1000/He system (Bio-Rad, Hercules, CA, USA). Detached leaves of 5–7-day-old barley seedlings were placed onto 1% agarose (BioWhittaker Molecular Applications, Rockland, ME, USA) plates supplemented with 10% sucrose (w/v) and allowed to recover for 1 h at room temperature. Gold particles (Bio-Rad) were coated with plasmid and/or cosmid DNA at a plasmid:cosmid molar ratio of 2 : 3, or plasmid:plasmid molar ratio of 1 : 1, and delivered to the leaves using 450 psi rupture disks. The leaves were then transferred to 1% agarose/85 µm benzimidazole prior to fungal inoculation. The inoculated leaves were incubated at 18°C (16 h light/8 h darkness) for 5 days. Barley cells expressing GFP were visualized using a microscope with an excitation filter of 450–490 nm (Chroma #41001). Visualization of cells expressing GUS and attached Bgh spores and hyphae was performed as described previously by Halterman et al. (2001). Data shown for each figure are the combined results of two to five replicated experiments. Each replicated experiment included examination of 50–200 interactions among Bgh conidia and transformed cells.

Secondary and tertiary structure modeling

The protein sequences illustrated in Figure 3 were submitted to the psipred protein structure prediction server (McGuffin et al., 2000; http://bioinf.cs.ucl.ac.uk/psipred/). The consensus (>51%) structure of the six MLA LRRs was determined using macvector (Accelrys, San Diego, CA, USA).

The amino acid sequence of the MLA6 LRR region (amino acids 579–956) was submitted to the swiss-model comparative modeling server (http://www.expasy.org/swissmod/SWISS-MODEL.html) using the First Approach mode with either porcine ribonuclease inhibitor (PDB 2BNH) or rna1p (1YRG) as a modeling template. The model output was viewed using swiss-pdbviewer (Guex and Peitsch, 1997; http:/us.expasy.org/spdbv/). The G721D mutation was introduced to this structure using swiss-pdbviewer. Figures were finalized using the persistence of vision raytracer (http://www.povray.org).

Chronology of retrotransposon insertion

To calculate the approximate chronology of insertion for new HORPIA elements (Table S1), we used the base substitution rate between the two LTRs of each element (SanMiguel et al., 1998; Wei et al., 2002) as compared to the average base substitution rate derived from the grass (Poaceae) adh1-adh2 region of 6.5 × 10−9 per site per year (Gaut et al., 1996).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary Material
  9. References
  10. Supporting Information

The authors thank Drs J. Wendel and G. Burleigh for assistance with the evolutionary analysis, K. Shirasu for the gift of the Mlo-GUS reporter plasmid, S. Whitham, A. Bogdanove, and T. Baum for critical review of the manuscript and P. Schulze-Lefert for sharing results prior to publication. This research was supported by USDA-NRI/CGP grants 98-35300-6169 and 00-35300-9213 to R.P.W. D.A.H. was supported in part by a USDA-ARS Post-doctoral Research Associateship. Joint contribution of the Corn Insects & Crop Genetics Research Unit, USDA-Agricultural Research Service and the Iowa Agriculture and Home Economics Experiment Station.

Supplementary Material

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary Material
  9. References
  10. Supporting Information

Table S1 Chronology of HORPIA element insertions

Table S2 Position of InDels in Mla ORFs

Figure S1. Comparative sequence analysis of the Mla family.

Nucleotide sequences from RGH1bcd (AF427791), Mla6 (AJ302293), Mla1 (AY009938), Mla1-2, Mla13 (AF523678) were compared to Mla6-2 (AY266442) and Mla13-2 (AY266443) cosmid sequences. Similar colors represent significant similarity between sequences. Different colors represent little similarity between sequences. Green and red arrows indicate transcription start and stop sites, respectively. ORFs are located between the dashed lines. Spaces in lines indicate gaps in the sequence. Thin lines denote the location of introns. In Morex, the putative end of the non-LTR Alexandra LINE element lies 320 bp upstream of the RGH1bcd ATG. In C.I. 16151, there appears to be a cross-over event within Alexandra, 1226 bp upstream of the Mla6 ATG, where significant similarity switches from Alexandra to corresponding regions of Mla1 (C.I. 16137) and Mla13 (C.I. 16155). Mla1-2 contains some remnants of Alexandra upstream of its ATG while the remaining upstream sequence has no similarity to the corresponding region of any other Mla or RGH gene. A newly identified non-LTR LINE element, designated Lauren, is 4959 bp in length and lies 1594 bp upstream of the Mla1 ATG. This element is also present upstream of Mla13, Mla6-2, and Mla13-2. However, in Mla6-2 and Mla13-2, a contiguous 986 bp has been deleted within the Lauren element. The remaining RGH1 paralogs, RGH1e/f and RGH1a, contain no similarity to any other Mla or RGH gene upstream of their ATGs (not shown). ORFs of each functional Mla gene as well as the Mla6-2 and Mla13-2 paralogs are highly similar, including the (AT)n SSR within the third intron. RGH1bcd as well as the Mla1-2 and RGH1e/f paralogs differ significantly from the functional Mla genes within the third intron, and as such, are lacking the (AT)n SSR. Intron 5 of Mla6 and Mla13 is located within the 3′ UTR and is 1102 bp in length. Mla1 and RGH1bcd lack this intron, and are divergent from Mla6 and Mla13 56 bp downstream of the stop codon. After this point, the sequence of Mla1 is 99.9% similar to the corresponding region downstream of RGH1bcd, which contains HORPIA-2. The last 347 bp of intron 5 and the remaining 3′ UTRs of Mla6 and Mla13 are 82.1 and 82.5% similar to a region of RGH1bcd downstream of HORPIA-2, respectively. However, this similarity ends shortly after the transcription stop sites in Mla6 and Mla13.

Figure S2. Model for the diversification of Mla haplotypes.

This evolutionary model incorporates sequence comparisons between the genomic sequences of the functional Mla1, Mla6, and Mla13 genes and the corresponding regions of Mla1-2, Mla6-2, Mla13-2, and RGH1bcd (Figure S1). The sequence comparisons include the ORFs as well and the flanking genomic sequence. Black triangles represent various insertion elements. When possible, the approximate time of insertion is shown above each element. The yellow bar within the Mla genes represents an (AT)n microsatellite. Cross-over events are represented by uppercase Xs. Subsequent to initial duplication, the evolution of the functional Mla resistance specificities appears to branch into two pathways – one leading to Rar1-independent Mla1 (branch A), and another leading to Rar1-dependent Mla6, and Mla13 (branch B). In branch A, a copia-like retroelement, HORPIA-2, inserted into the 5′ end of the (AT)n-containing Mla-type gene approximately 0.48 Ma (Table S1). This gene appears to be a direct precursor of Mla1 in C.I. 16137. An unequal cross-over between the Alexandra- and HORPIA-2-containing genes led to the formation of RGH1bcd in cultivar Morex. In branch B, the (AT)n-containing gene appears to have been duplicated once more. One of these duplicates is the putative ancestor of Mla6 and Mla13, although a recombination event has occurred upstream of the Mla6 ORF. HORPIA-4 and HORPIA-5 were inserted into the ORF of the other duplicated gene 2.35 and 1.60 million years ago, respectively, leading to the formation of Mla6-2 and Mla13-2. A 3-bp InDel (Table S2) in the region encoding the 10th LRR, is conserved in Mla1, Mla1-2, RGH1bcd of branch A, as well as Mla6-2 and Mla13-2 of branch B, suggesting that this event occurred very early in haplotype diversification, prior to the retrotransposon insertions. In contrast, the duplication event leading to the formation of the Mla6-2 and Mla13-2 paralogs is most likely of recent origin because the sequence of the HORPIA-4 and HORPIA-5 elements within the 10276-15 (Mla6-2) and 10907-10c (Mla13-2) cosmids are identical. Thus, it appears that the present-day diversification of the Mla gene family has been the result of at least two evolutionary pathways.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary Material
  9. References
  10. Supporting Information
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Sequence data reported in this paper has been deposited in GenBank and assigned Accession numbers AY266442 (Mla6-2), AY266443 (Mla13-2), AY266444 (Mla7), and AY266445 (Mla10).

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary Material
  9. References
  10. Supporting Information

Table S1.  Chronology of HORPIA element insertions.

Table S2.  Position of InDels in Mla open reading frames.

Figure S1.  Comparative sequence analysis of the Mla family.

Figure S2.  Model for the diversification of Mla halotypes.

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