Mla Locus Research Articles

Powdery Mildew Resistance of Czech and Slovak Spring Barley Breeding Lines in Variety Trials

In 2001–2005, resistance to powdery mildew was studied in 227 Czech and Slovak breeding lines of spring barley included in the breeding station trials or official trials. Seventeen known resistances were identified (Al, Ar, At, HH, Kr, La, Ly, Mlo, N81, Ri, Ru, Sp, St, Tu, We, Mla21, and Mlp1). Unknown resistances were found in 11 breeding lines, in five of which resistance was effective against all used pathotypes of the pathogen. Besides the identified resistances, unknown resistances were detected in another three breeding lines. Sixty-five breeding lines (= 29%) exhibited heterogeneity in the examined trait, i.e. they are composed of components with different resistances to powdery mildew. Comparison of current results with the previous ones shows a considerable increase in the proportion of breeding lines carrying the resistance Mlo (72%), on the account of the resistances located at the Mla locus, particularly Ru. The examined set is characterised by a high proportion of breeding lines resistant to all used Czech pathotypes of the given pathogen (78%), however of low diversity in the resistance.  

RAR1 Positively Controls Steady State Levels of Barley MLA Resistance Proteins and Enables Sufficient MLA6 Accumulation for Effective Resistance

The polymorphic barley (Hordeum vulgare) Mla locus harbors allelic race-specific resistance (R) genes to the powdery mildew fungus Blumeria graminis f sp hordei. The highly sequence-related MLA proteins contain an N-terminal coiled-coil structure, a central nucleotide binding (NB) site, a Leu-rich repeat (LRR) region, and a C-terminal non-LRR region. Using transgenic barley lines expressing epitope-tagged MLA1 and MLA6 derivatives driven by native regulatory sequences, we show a reversible and salt concentration-dependent distribution of the intracellular MLA proteins in soluble and membrane-associated pools. A posttranscriptional process directs fourfold greater accumulation of MLA1 over MLA6. Unexpectedly, in rar1 mutant plants that are compromised for MLA6 but not MLA1 resistance, the steady state level of both MLA isoforms is reduced. Furthermore, differential steady state levels of MLA1/MLA6 hybrid proteins correlate with their requirement for RAR1; the RAR1-independent hybrid protein accumulates to higher levels and the RAR1-dependent one to lower levels. Interestingly, yeast two-hybrid studies reveal that the LRR domains of RAR1-independent but not RAR1-dependent MLA isoforms interact with SGT1, a RAR1 interacting protein required for the function of many NB-LRR type R proteins. Our findings implicate the existence of a conserved mechanism to reach minimal NB-LRR R protein thresholds that are needed to trigger effective resistance responses.

Powdery mildew resistance genes in Latvian barley varieties

Thirty-three Latvian spring barley varieties and breeding lines and 90individual progenies from these were tested at the seedling stage for their reaction to a maximum 37 isolates of Blumeria graminis f. sp. hordei. Eighteen varieties were composed of two or three lines with different resistance. TwelveMl powdery mildew resistance genes(a1, a6, a7, a8, a9, a12, a13, at, g, k1, (Kr) and La) and four Ml genes closely linked to the Mla locus [a(Al2), a(No3), a(Em2) and a(Ru3)] were found. Mla8, Mlg and Mlk1 were the most frequent genes that occurred among the tested genotypes. The results confirm the pedigrees of all genotypes apart from cv. Agra and the breeding line L-215.

Mildew-resistant mutants induced in North American two- and six-rowed malting barley cultivars.

Mildew-resistant mutants were induced with sodium azide in three North American malting barley cultivars, two in the six-rowed Ursula (URS1 and URS2), one in the six-rowed Gertrud (GER1), and one in the two-rowed Prudentia (PRU1). Two of the mutants, URS1 and PRU1, showed complete resistance and were shown to have two new alleles at the mlo locus; these were designated, respectively, mlo31 and mlo32. Mutant URS2, showing partial resistance, was inherited as a dominant gene, but was not an allele at the Mla locus. The mean yield of each mutant was higher than that of its parental line, but yield levels varied across environments, although this was independent of the severity of the mildew attack. Other reasons, for example, the severity of the necrotic lesions in the mutants, may account for yield variations. The malting quality of the GER1 mutant proved similar to that of Gertrud, but both URS1 and URS2 showed lower malt extract than Ursula. This lower extract might be due to the smaller grain size of the mutants that could, in turn, result from necrotic lesions in the leaves, as implied by the effects on grain yield.

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

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.

Inheritance of Resistance to Powdery Mildew (Blumeria graminis f.sp. hordei) in Selections from Moroccan Landraces of Barley

Powdery mildew caused by the pathogen Blumeria graminis (DC.) Golovin ex Speer f. sp. hordei Em. Marchai is one of the most serious foliar diseases of barley (Hordeum vulgare L.) in Europe. This study was conducted to determine the number of genes, the types of genes action and the gene loci conferring resistance to powdery mildew in two selections E 1059-1-1, E 1067-1-2. These lines were selected from barley landraces collected were collected during expedition organized by Warsaw Agricultural University SGGW-AR in Southern Morocco (Agadir Province). Two types of crosses were made: (1) the lines were crossed with the susceptible cultivar Pallas, (2) the lines were crossed with six Pallas isolines with Mla3 (isoline P2), Mla12 (isoline P10), Mlp (isoline P19), Mlat (isoline P20), Mlg (isoline P21) and mlo5 (isoline P22) genes for resistance. The parents, F1, F2, F3 were evaluated for the powdery mildew resistance with isolate R303.1. On the basis of segregation ratios it was found that resistance in tested lines was determine by a single dominant gene at the locus Mla. Results are compared with previous study in which the presence of genes in Mla locus was postulated based on testing of these lines with 30 differential isolates of powdery mildew. The possible use of new identified sources of resistance to powdery mildew was discussed.

Cell-autonomous expression of barley Mla1 confers race-specific resistance to the powdery mildew fungus via a Rar1-independent signaling pathway.

The barley Mla locus encodes 28 characterized resistance specificities to the biotrophic fungal pathogen barley powdery mildew. We describe a single-cell transient expression assay using entire cosmid DNAs to pinpoint Mla1 within the complex 240-kb Mla locus. The MLA1 cDNA encodes a 108-kD protein containing an N-terminal coiled-coil structure, a central nucleotide binding domain, and a C-terminal leucine-rich repeat region; it also contains a second short open reading frame at the 5' end that has a possible regulatory function. Although most Mla-encoded resistance specificities require Rar1 for their function, we used the single-cell expression system to demonstrate that Mla1 triggers full resistance in the presence of the severely defective rar1-2 mutant allele. Wheat contains an ortholog of barley Mla, designated TaMla, that is tightly linked to (0.7 centimorgan) but distinct from a tested resistance specificity at the complex Pm3 locus to wheat powdery mildew. Thus, the most polymorphic powdery mildew resistance loci in barley and wheat may have evolved in parallel at two closely linked homeoloci. Barley Mla1 expressed in wheat using the single-cell transformation system failed to trigger a response to any of the wheat powdery mildew Avr genes tested, indicating that AvrMla1 is not genetically fixed in wheat mildew strains.

The MLA6 coiled-coil, NBS-LRR protein confers AvrMla6-dependent resistance specificity to Blumeria graminis f. sp. hordei in barley and wheat.

The barley Mla locus confers multiple resistance specificities to the obligate fungal biotroph, Blumeria (= Erysiphe) graminis f. sp. hordei. Interspersed within the 240 kb Mla complex are three families of resistance gene homologs (RGHs). Probes from the Mla-RGH1 family were used to identify three classes of cDNAs. The first class is predicted to encode a full-length CC-NBS-LRR protein and the other two classes contain alternatively spliced, truncated variants. Utilizing a cosmid that contains a gene corresponding to the full-length candidate cDNA, two single-cell expression assays were used to demonstrate complementation of AvrMla6-dependent, resistance specificity to B. graminis in barley and wheat. The first of these assays was also used to substantiate previous genetic data that the Mla6 allele requires the signaling pathway component, Rar1, for function. Computational analysis of MLA6 and the Rar1-independent, MLA1 protein reveals 91.2% identity and shows that the LRR domain is subject to diversifying selection. Our findings demonstrate that highly related CC-NBS-LRR proteins encoded by alleles of the Mla locus can dictate similar powdery mildew resistance phenotypes yet still require distinct downstream signaling components.

Chromosome landing at the Mla locus in barley (Hordeum vulgare L.) by means of high-resolution mapping with AFLP markers

The complex Mla locus of barley determines resistance to the powdery mildew pathogen Erysiphe graminis f. sp. hordei. With a view towards gene isolation, a population consisting of 950 F2 individuals derived from a cross between the near-isogenic lines ‘P01’ (Mla1) and ‘P10’ (Mla12) was used to construct a high-resolution map of the Mla region. A fluorescence-based AFLP technique and bulked segregant analysis were applied to screen for polymorphic, tightly linked AFLP markers. Three AFLP markers were selected as suitable for a chromosome-landing strategy. One of these AFLP markers and a closely linked RFLP marker were converted into sequence-specific PCR markers. PCR-based screening of approximately 70 000 yeast artificial chromosome (YAC) clones revealed three identical YACs harbouring the Mla locus. Terminal insert sequences were obtained using inverse PCR. The derived STS marker from the right YAC end-clone was mapped distal to the Mla locus.

Construction of a MluI-YAC library from barley (Hordeum vulgare L.) and analysis of YAC insert terminal regions

We describe the construction of a specific yeast artificial chromosome (YAC) library from barley (Hordeum vulgare L.) using the vector pYAC-RC. The library was generated by total digestion of high molecular weight DNA with the infrequently cutting restriction enzyme MluI. Only 10-30% of the colonies were recombinant, as visualized by red-white selection and subsequent pulsed-field gel electrophoresis analysis. About 17 000 individual recombinant YAC clones with insert sizes ranging from 50 to 700 kb, with a mean of 170 kb, were selected. No chloroplast sequences were detected and the proportion of YAC clones containing BARE-1 copia-like retroelements is about 5%. Screening of the library with a single-copy RFLP marker closely linked to the Mla locus yielded three identical clones of the same size. Insert termini of randomly chosen YAC clones were investigated with respect to their redundancy in the barley genome and compared with termini of YAC clones from an EcoRI-based YAC library, resulting in a fourfold enrichment of single-copy sequences at the MluI vector-insert junctions.

RFLP mapping of three new loci for resistance genes to powdery mildew (Erysiphe graminis f. sp. hordei) in barley

Three new major, race-specific, resistance genes to powdery mildew (Erysiphe graminis f. sp. hordei) were identified in three barley lines, 'RS42-6*O', 'RS137-28*E', and 'HSY-78*A', derived from crosses with wild barley (Hordeum vulgare ssp. spontaneum). The resistance gene origining from wild barley in line 'RS42-6*O', showed a recessive mode of inheritance, whereas the other wild barley genes were (semi)-dominant. RFLP mapping of these three genes was performed in segregating F2 populations. The recessive gene in line 'RS42-6*O', was localized on barley chromosome 1S (7HS), while the (semi)-dominant genes in lines 'RS137-28*E', and 'HSY-78*A', were localized on chromosomes 1L (7HL) and 7L (5HL), respectively. Closely linked RFLP clones mapped at distances between 2.6cM and 5.3 cM. Hitherto, specific loci for powdery mildew resistance in barley had not been located on these chromosomes. Furthermore, tests for linkage to the unlocalized resistance gene Mlp revealed free segregation. Therefore, these genes represent new loci and new designations are suggested: mlt ('RS42-6*O'), Mlf ('RS137-28*E'), and Mlj ('HSY-78*A'). Comparisons with mapped QTLs for mildew resistance were made and are discussed in the context of homoeology among the genomes of barley (H-vulgare), wheat (Triticum aestivum), and rye (Secale cereale). Duplications of RFLP bands detected in the neighbourhood of Mlf and mlt might indicate an evolutionary interrelationship to the Mla locus for mildew resistance.

Variation in the ratio of physical to genetic distance in intervals adjacent to the Mla locus on barley chromosome 1H.

Variants of the pulsed-field gel electrophoresis technique were used in conjunction with two-dimensional DNA gel electrophoresis (2-DDGE) to determine the ratio of physical to genetic distance in two genetically defined intervals on barley chromosome 1H.2-DDGE analysis demonstrated that two loci that define a 0.3 cM interval, as determined by hybridization with BCD249, reside on a single 450-kb MluI fragment. This result indicates a maximum ratio of physical to genetic distance in this interval of 1500 kb/cM as compared to 3.7-4.2 Mb/cM for the barley genome as a whole. High molecular weight (HMW) DNA restricted with NotI and probed sequentially with MWG068 and BCD249 yield diffuse bands at approximately 2.8 Mb and 3.0 Mb in the C.I. 16151 and C.I. 16155 parental lines, respectively. These results suggest the maximum ratio of physical to genetic distance in the interval defined by these probes is 7.8 Mb/cM. Unique HMW DNA restriction fragment length polymorphisms (RFLP) were attributed to the presence of recombination breakpoints. Data from the recombination breakpoint analysis were used to estimate a ratio of physical to genetic distance of 2.5 Mb/cM in the Xbcd249.2-Xmwg068 interval and 0.465 Mb/cM in the Xbcd249.1-Xbcd249.2 interval. Both physical linkage and recombination breakpoint analysis indicate the Xbcd249.1-Xbcd249.2 interval is approximately five-fold smaller, physically, than the Xbcd249.2-Xmwg068 interval.

Recombination of alleles conferring specific resistance to powdery mildew at the Mla locus in barley

In barley (Hordeum vulgare L.), the Mla locus conditions reaction to the powdery mildew fungus Erysiphe graminis f.sp. hordei. Enrichment for genetic recombinants in the Mla region is possible by screening for recombination events between the flanking endosperm storage proteins hordeins C and B. Reciprocal crosses were made between the Franger (C.I. 16151) and Rupee (C.I. 16155) lines carrying the (Mla6 + Mla14) and Mla13 alleles, respectively. Recombinants were identified from F2 segregants by analyzing the extracted hordein polypeptides by sodium dodecyl sulphate - polyacrylamide gel electrophoresis. Two hundred and seventy-six recombinant gametes were identified from the 1800 seeds that were screened. Recombination of Mla alleles was analyzed by inoculating F4 recombinant lines with three isolates of E. graminis (A27, 5874, and CR3), which recognize specific Mla alleles. The linkage order established is Hor1-Mla6-Mla13-Mla14-Hor2. The genetic distances between Hor1-Mla6, Mla6-Mla13, and Mla13-Hor2, obtained using Mapmaker 3.0b F3 intercross analysis, are 3.9, 0.2, and 5.2 cM, respectively.

Phenylalanine ammonia lyase mRNA accumulation, enzyme activity and cytoplasmic responses in barley isolines, differing at Ml-a and Ml-o loci, attacked by Erysiphe graminis f.sp. hordei

Seedling leaves of two pairs of near-isogenic barley lines were inoculated with conidia of the powdery mildew fungus, Erysiphe graminis D.C. f.sp. hordei Marchal, race 3. One set of isolines (RISO 5678-R and RISO 5678-S) differed at the Ml-o locus where the recessive allele ( ml-o ) confers a high degree of race non-specific, penetration-based and papilla-associated resistance to E. graminis , while the dominant allele ( Ml-o ) allows a proportion of attacking fungal germlings to succeed in infecting. The second isoline set (Algerian-R and Algerian-S) differed at the Ml-a locus where the dominant allele confers race-specific, epidermal cell death resistance visible only by light microscopy. The recessive allele ( ml-a ) allows a proportion of attacking fungal germlings to succeed in infecting. Leaf samples were taken at 0, 2, 4, 6, 8, 10, 12, 15, 18, 21 and 24 h after inoculation to examine the timing of host epidermal cell cytoplasmic aggregate responses (visible by light microscopy) relative to phenylalanine ammonia-lyase (PAL) mRNA transcript accumulation (determined by quantitative northern blots), and PAL enzyme activity (using radiolabellcd phenylalanine). Erysiphe graminis produced primary germ-tubes (PGTs) within 2 h and appressorial germ-tubes within 6–10 h of inoculation. In all isolines, host epidermal cell cytoplasmic aggregates formed and subsequently dispersed beneath PGTs, between 2 and 10 h, and beneath appressoria between 6 and 15 h. Concurrently, biphasic patterns of PAL transcript accumulation, typified by peaks at 4 and 12 h occurred in all isolines. Temporal patterns of PAL enzyme activity were roughly similar to those of PAL transcript accumulation. Fungal germ-tube contact initiated host epidermal cell cytological responses common to all isolines, induced PAL transcript accumulation, and increased PAL activity regardless of the Mendelian inheritance of “major gene resistance factors” in the barley isoline sets. Thus, there was PAL induction associated with a general defence to infection, but no unusually strong correlation between PAL induction and major gene resistance was found.

Host response gene transcript accumulation in relation to visible cytological events during Erysiphe graminis attack in isogenic barley lines differing at the Ml-a locus.

Temporal patterns of transcript accumulation of six host response genes (β-1,3 glucanase and five of unknown function), were determined for two near-isogenic barley lines (Algerian R and Algerian S), differing at the Ml-a locus for race-specific incompatibility with Erysiphe graminis f.sp. hordei. The cDNAs of these response genes were originally cloned in other laboratories. Transcript levels of the six response genes were measured using quantitative, radioanalytical analysis of Northern blots containing RNA isolated from leaves at discrete intervals from 0-48 h after inoculation. At each time interval, leaf tissue was also fixed and stained for light microscopic observations of fungal development and host epidermal cell responses to E. graminis. Response gene transcripts accumulated in biphasic patterns corresponding to periods of cytoplasmic aggregation and papilla deposition in host cells beneath primary germ tubes at 2-10 h after inoculation and again beneath appressoria at 10-21 h. This biphasic pattern was virtually the same in both isolines up to 21 h, indicating that transcript accumulation was a general, race nonspecific response to pathogen attack. From 21 to 40 h after inoculation, transcript levels tended to remain higher in the resistant than in the susceptible isoline, possibly as a consequence of hypersensitive epidermal cell death which was visible only by light microscopy and occurred at 18-30 h in the resistant isoline.

Genetical and RFLP studies at the Mla locus conferring powdery mildew resistance in barley

The complex structure of the multigene family at the Mla locus conferring powdery mildew resistance in barley was studied by making diallel crosses between several near-isogenic lines carrying different Mla alleles. The mode of inheritance of the Mla alleles investigated was determined to be dominant for Mla1, Mla6, Mla7 and Mla13 and semidominant for Mla3, Mla12 and Mla20. F1 plants were backcrossed to the susceptible recurrent parent in order to identify susceptible and double-resistant recombinants in the BC1F1 generation. Out of 17605 progenies tested in the BC1F1 generation, two susceptible recombinants, one between Mla1 and Mla12 and one between Mla13 and Mla20 were confirmed. The former was also verified by RFLP analysis.

RFLP markers to identify the alleles on the Mla locus conferring powdery mildew resistance in barley

To identify the mildew resistance locus Mla in barley with molecular markers, closely linked genomic RFLP clones were selected with the help of near-isogenic lines having the 'Pallas' and 'Siri' background. Out of 22 polymorphic clones 3 were located around the Mla locus on chromosome 5 with a distance of 5.1 + 2.9 cM (MWG 1H068), 4.2±1.7 cM (MWG 1H060) and 0.7 ± 0.7 cM (MWG 1H036), respectively. The polymorphic clone MWG 1H036 displayed the same RFLP pattern in both 'Pallas' and 'Siri' near-isogenic lines and in different varieties digested with six restriction enzymes possessing the same mildew resistance gene. The alleles of the Mla locus were grouped in 11 classes according to their specific RFLP patterns; 3 of these groups contain the majority of Mla alleles already used in barley breeding programs in Europe.

Multigene Families of Powdery Mildew Resistance Genes in Locus Mla on Barley Chromosome 5

AbstractA review of data on powdery mildew resistance genes in the Mla locus of barley reveals that there are at least 12 clusters of genes present, each comprising one Mla gene and one or more closely linked, additional resistance genes. Tentative designations are listed for 16 additional resistance genes. Many sources of powdery mildew resistance in barley appear to harbour multigene families in the Mla region, not single ‘superior’genes.

Inheritance of resistance to powdery mildew (Erysiphe graminis f. sp. hordei) in eleven primitive barley varieties

The inheritance of the powdery mildew resistance of eleven primitive barley varieties was investigated. crosses with varieties with identified resistance genes revealed that at least three different genes are present in a group of six genotypes from Greece (‘Hor736’, ‘Hor847’, ‘Hor878’, ‘Hor1159’, ‘Hor1379’ and ‘Hor1873’) and one from Turkey (‘Hor1188’). One dominant gene is common to these seve genotypes and in the Greek accessions a second gene is present. The expression of this second gene was respectively dominant in the crosses with ‘Hor736’ and ‘Hor1159’, recessive in the crosses with ‘Hor878’ and ‘Hor1873’, and depended on the employed powdery mildew isolate in the crosses with ‘Hor847’ and ‘Hor1379’. The detected genes differ from the resistance genes of the crossing partners, viz. Ml-al2, Ml-(Ab), Ml-(CP) and Ml-(1402). The accessions from China (‘Hor824’ and ‘Hor4021’) each possess two resistance genes which differ from Ml-a12, Ml-(La) and Ml-(CP). The major gene is common to both accessions and is either identical, allelic or closely linked to a gene in the variety ‘Nigrate’. The Columbian accession ‘Hor1894’ possesses one resistance gene linked to a resistance gene in ‘Nigrate’ but this gene differs from the Ml-a locus. The variety ‘Palestine’ (Hor3997) possesses two resistance genes of which one is allelic or closely linked to Ml-(at).

Specificity of cell type and timing of events associated with papilla formation and the hypersensitive reaction in leaves of Hordeum vulgare attacked by Erysiphe graminis f.sp. hordei

Rates of haustorium formation by Erysiphe graminis f.sp. hordei were determined for epidermal tissues of primary leaves of a near-isogenic pair of barley (Hordeum vulgare) lines, AlgR and AlgS, which differed at the Mla locus for compatibility with the fungus. Epidermal cells were divided into two classes, namely shorter or longer than 450 μm. Cells near stomatal files were always short, whereas cells more distant from stomata were long on the abaxial surface, long on the adaxial surface over vascular bundles, but short on the adaxial surface when not over vascular bundles. At 24 h after inoculation, haustoria were formed in 70–80% of attacked short cells but in only 15–20% of attacked long cells. When haustoria were absent, papillae were present, so papilla-associated resistance was more frequent in long than in short cells. However, the percentage of attacked sites with papillae was the same in AlgR and AlgS, indicating that papilla-associated resistance was not related to Mla incompatibility. In AlgR, patterns of development showed that haustoria were produced before Mla incompatibility was expressed by hypersensitive cell death. Key words: papilla, hypersensitive, barley, epidermis, resistance, powdery mildew.