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Recognition specificity and RAR1/SGT1 dependence in barley Mla disease resistance genes to the powdery mildew fungus - PubMed

Recognition specificity and RAR1/SGT1 dependence in barley Mla disease resistance genes to the powdery mildew fungus

Qian-Hua Shen et al. Plant Cell. 2003 Mar.

Abstract

A large number of resistance specificities to the powdery mildew fungus Blumeria graminis f. sp. hordei map to the barley Mla locus. This complex locus harbors multiple members of three distantly related gene families that encode proteins that contain an N-terminal coiled-coil (CC) structure, a central nucleotide binding (NB) site, a Leu-rich repeat (LRR) region, and a C-terminal non-LRR (CT) region. We identified Mla12, which encodes a CC-NB-LRR-CT protein that shares 89 and 92% identical residues with the known proteins MLA1 and MLA6. Slow Mla12-triggered resistance was altered dramatically to a rapid response by overexpression of Mla12. A series of reciprocal domains swaps between MLA1 and MLA6 identified in each protein recognition domain for cognate powdery mildew fungus avirulence genes (AvrMla1 and AvrMla6). These domains were within different but overlapping LRR regions and the CT part. Unexpectedly, MLA chimeras that confer AvrMla6 recognition exhibited markedly different dependence on Rar1, a gene required for the function of some but not all Mla resistance specificities. Furthermore, uncoupling of MLA6-specific function from RAR1 also uncoupled the response from SGT1, a protein known to associate physically with RAR1. Our findings suggest that differences in the degree of RAR1 dependence of different MLA immunity responses are determined by intrinsic properties of MLA variants and place RAR1/SGT1 activity downstream of and/or coincident with the action of resistance protein-containing recognition complexes.

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Figures

Figure 6.
Figure 6.

Schemes of the Morex Mla Locus and Genomic Regions Containing Identified Mla Resistance Genes. DNA sequences encompassing the Morex Mla locus (261 kb, in reverse orientation) (Wei et al., 2002) are represented schematically and drawn to scale in the top line (relevant sequences only). Available genomic sequences of Mla1, Mla6, and Mla12 and flanking regions are shown below. Coding sequences of functional Mla R genes and RGHs are boxed and highlighted in black and blue, respectively. A conserved upstream open reading frame (uORF) and a simple [AT]n microsatellite are shared among functional Mla R genes. Green boxes denote retrotransposon sequences: a BARE1 solo LTR in intron 3 of RGH1bcd, HORPIA2 immediately 3′ of RGH1bcd, and ALEXANDRA 5′ of RGH1bcd. Dark gray areas denote sequences showing >90% identity, and light gray areas denote sequences showing >75% identity. A possible inversion event could explain the altered relative orientations of homologous genes Mla1-2 and RGH1f as indicated. Note that RGH1e/f and RGH3a/b are extremely similar and located within a 40-kb duplicated region (Wei et al., 2002). For this reason, the indicated homologies exist between RGH1e and RGH1f and between RGH3a and RGH3b. Arrows indicate the relative orientations of genes (5′ to 3′). Borders of Morex sequences are indicated in kb according to accession AF427791.

Figure 1.
Figure 1.

Amino Acid Sequence Alignment of Deduced Products of the Mla1, Mla6, and Mla12 Genes. Residues identical to those in MLA1 are shown as dots, and deletions are shown as hyphens. A predicted CC structure is underlined. The starts of the NB, LRR, and CT regions are indicated with arrows and are operational according to Zhou et al. (2001). Boldface letters in the NB domain indicate amino acid motifs conserved among known NB-LRR proteins. Boxes indicate amino acid exchanges identified in three susceptible Mla12 mutants, and affected residues are shaded in black.

Figure 2.
Figure 2.

Complementation of Susceptible Mla12 Mutants by Overexpression of Mla12 Resistance. Relative single cell resistance/susceptibility upon delivery of various Mla transgenes at 48 h after spore inoculation is indicated by haustorium indices of attacked β-glucuronidase (GUS)–expressing cells (%). Data shown were obtained by bombardment of plasmid DNAs into epidermal cells of detached barley leaves (described by Shirasu et al., 1999b; Zhou et al., 2001). A β-glucuronidase reporter gene was used to identify transformed cells. (A) The indicated transgenes were tested in detached leaves of barley cv Ingrid harboring mlo-3 Rar1. In this line, broad-spectrum mlo-3 resistance was complemented by cobombardment with a plasmid expressing wild-type Mlo; this renders cells supersusceptible to all tested Bgh isolates (Zhou et al., 2001; Kim et al., 2002). Results obtained with the Bgh isolate K1 (AvrMla1) are shown by closed columns, and results obtained with isolate A6 (AvrMla6 and AvrMla12) are shown by open columns in downward orientation. The data shown are means of at least three independent experiments (

sd

indicated). Each experiment involved light microscopic examination of at least 100 interaction sites between a single Bgh sporeling and an attacked epidermal cell. (B) The indicated transgenes and an empty vector control were delivered into epidermal cells of Sultan 5 containing Mla12 Mlo Rar1. Experimental conditions and symbols are identical to those in (A). (C) Transgene Mla12 or an empty vector control was delivered into epidermal cells of two susceptible Mla12 mutant lines (M66 and M22). Transgene Mla6 or Mla12 or an empty vector control also was delivered into the rar1-2 mutant line M100. Experimental conditions and symbols are identical to those in (A).

Figure 3.
Figure 3.

Context-Dependent Functions of Conserved MLA Residues Leu-631 and Lys-916. Mean values of single cell resistance/susceptibility (%) are shown at left after delivery of Mla1, Mla6, or Mla12 into the genetic background of cv Ingrid (mlo-3 Rar1). Results obtained with L631R variants of Mla1, Mla6, and Mla12 are shown in the middle. Results obtained with Mla1, Mla6, and Mla12 variants each containing a K to M substitution at the indicated positions are shown at right. Experimental conditions and designations are identical to those in Figure 2. GUS, β-glucuronidase.

Figure 4.
Figure 4.

Domain Swaps between MLA1 and MLA6 Reveal Determinants for Recognition Specificity and RAR1 Dependence. (A) Schemes of MLA6 (yellow), MLA1 (green), and 10 chimeras are shown. The relative positions of the CC, NB, LRR, and CT parts are indicated at top, and acronyms for each chimera are shown at left. The stars indicate gene expression driven by native 5′ flanking sequences; the strong ubiquitin promoter drove the expression of all other genes. (B) All genes shown in (A) were expressed in the Rar1 wild-type background, and mean values of single cell resistance/susceptibility were scored microscopically upon challenge inoculation with Bgh isolates A6 or K1. Experimental conditions and designations are identical to those in Figure 2. GUS, β-glucuronidase. (C) All genes shown in (A) were expressed in the rar1-2 mutant background, and mean values of single cell resistance/susceptibility were scored microscopically upon challenge inoculation with Bgh isolates A6 or K1. Experimental conditions and designations are identical to those in Figure 2.

Figure 5.
Figure 5.

Single Cell Silencing of Sgt1 by dsRNAi. Wild-type Mla6 or chimeras retaining AvrMla6-dependent recognition specificity were coexpressed with a HvSgt1 dsRNAi-silencing plasmid (Azevedo et al., 2002) in the Rar1 wild-type background using a modified single cell transient gene expression assay (Azevedo et al., 2002). After delivery of plasmid DNAs into epidermal cells, detached barley leaves were incubated for 48 h (open bars) or 96 h (closed bars). Subsequently, leaves were inoculated with spores of Bgh isolate A6 (AvrMla6) and incubated for another 48 h. Microscopic scoring of single interaction sites was identical to that described for Figure 2. Asterisks indicate haustorium indices that are significantly different (P < 0.05) from bombardments using empty dsRNAi vector controls. GUS, β-glucuronidase; n.d., not determined.

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