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.     

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.     

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

Summary 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.     

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.     

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

Summary 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.     

The conserved oligomeric Golgi complex is involved in penetration resistance of barley to the barley powdery mildew fungus

Membrane trafficking is vital to plant development and adaptation to the environment. It is suggested that post‐Golgi vesicles and multivesicular bodies are essential for plant defence against directly penetrating fungal parasites at the cell wall. However, the actual plant proteins involved in membrane transport for defence are largely unidentified. We applied a candidate gene approach and single cell transient‐induced gene silencing for the identification of membrane trafficking proteins of barley involved in the response to the fungal pathogen Blumeria graminis f.sp. hordei. This revealed potential components of vesicle tethering complexes [putative exocyst subunit HvEXO70F‐like and subunits of the conserved oligomeric Golgi (COG) complex] and Golgi membrane trafficking (COPIγ coatomer and HvYPT1‐like RAB GTPase) as essential for resistance to fungal penetration into the host cell.     

The Mla (powdery mildew) resistance cluster is associated with three NBS-LRR gene families and suppressed recombination within a 240-kb DNA interval on chromosome 5S (1HS) of barley

Powdery mildew of barley, caused by Erysiphe graminis f. sp. hordei, is a model system for investigating the mechanism of gene-for-gene interaction between large-genome cereals and obligate-fungal pathogens. A large number of loci that confer resistance to this disease are located on the short arm of chromosome 5(1H). The Mla resistance-gene cluster is positioned near the telomeric end of this chromosome arm. AFLP-, RAPD-, and RFLP-derived markers were used to saturate the Mla region in a high-resolution recombinant population segregating for the (Mla6 + Mla14) and (Mla13 + Ml-Ru3) resistance specificities. These tightly linked genetic markers were used to identify and develop a physical contig of YAC and BAC clones spanning the Mla cluster. Three distinct NBS-LRR resistance-gene homologue (RGH) families were revealed via computational analysis of low-pass and BAC-end sequence data derived from Mla-spanning clones. Genetic and physical mapping delimited the Mla-associated, NBS-LRR gene families to a 240-kb interval. Recombination within the RGH families was at least 10-fold less frequent than between markers directly adjacent to the Mla cluster.     

Silencing of copine genes confers common wheat enhanced resistance to powdery mildew

Powdery mildew, caused by the biotrophic fungal pathogen Blumeria graminis f. sp. tritici (Bgt), is a major threat to the production of wheat (Triticum aestivum). It is of great importance to identify new resistance genes for the generation of Bgt‐resistant or Bgt‐tolerant wheat varieties. Here, we show that the wheat copine genes TaBON1 and TaBON3 negatively regulate wheat disease resistance to Bgt. Two copies of TaBON1 and three copies of TaBON3, located on chromosomes 6AS, 6BL, 1AL, 1BL and 1DL, respectively, were identified from the current common wheat genome sequences. The expression of TaBON1 and TaBON3 is responsive to both pathogen infection and temperature changes. Knocking down of TaBON1 or TaBON3 by virus‐induced gene silencing (VIGS) induces the up‐regulation of defence responses in wheat. These TaBON1‐ or TaBON3‐silenced plants exhibit enhanced wheat disease resistance to Bgt, accompanied by greater accumulation of hydrogen peroxide and heightened cell death. In addition, high temperature has little effect on the up‐regulation of defence response genes conferred by the silencing of TaBON1 or TaBON3. Our study shows a conserved function of plant copine genes in plant immunity and provides new genetic resources for the improvement of resistance to powdery mildew in wheat.     

Resistance genes for rye stem rust (SrR) and barley powdery mildew (Mla) are located in syntenic regions on short arm of chromosome.

Genetic stocks were developed for the localization and eventual cloning of the stem rust resistance gene SrR that occurs in wheat lines carrying the 1RS translocation from Secale cereale 'Imperial' rye. We have used a mutation-based approach for molecular analysis of the SrR region in rye. Forty-one independent mutants resulting in loss of SrR resistance were isolated: many of these were deletions of various sizes that were used to locate SrR with respect to chromosome group 1S markers. The analysis of the mutants showed that markers about 1 Mb apart flanking the barley Mla locus also flank SrR. Additionally, three of the approximately 20 closely related sequences of Mla in rye are deleted in each of six interstitial deletion mutants of SrR. The results indicate that the SrR region in rye is syntenic to the Mla region in barley or that SrR is possibly orthologous to the Mla locus.     

Effect of three suppressors on the expression of powdery mildew resistance genes in barley

Three recessive mutagen-induced alleles that partially suppress the phenotypic expression of the semidominant powdery mildew resistance gene Mla12 have been studied. When each suppressor is present in homozygous condition, the infection type 0, conferred by gene Mla12 when homozygous, is changed to intermediate infection types. The three suppressor lines were crossed with seven near-isogenic lines with different powdery mildew resistance genes and one, M100, was crossed with nine additional lines. Seedlings of parents and from the F1and F2 generations were tested with powdery mildew isolates that possessed the appropriate avirulence and virulence genes. The segregation of phenotypes in the F2 generation disclosed that the three suppressors affected the phenotypic expression of three resistance genes, whereas that of four resistance genes remained unaffected. The suppressor in mutant M100 affected the phenotypic expression of 9 of the 10 additional resistance genes present. It is suggested that the three suppressors are mutationally modified genes involved in host defence processes. This implies that different resistance genes employ different, but overlapping, spectra of defence processes, or signal transduction pathways. Key words : barley, Hordeum vulgare, powdery mildew, Erysiphe graminis hordei, mutation, resistance, suppressor.     

Molecular marker analyses of powdery mildew resistance in barley

Powdery mildew, caused byEryisphe graminis f. sp.hordei, is one of the most important diseases of barley (Hordeum vulgare). A number of loci conditioning resistance to this disease have been reported previously. The objective of this study was to use molecular markers to identify chromosomal regions containing genes for powdery mildew resistance and to estimate the resistance effect of each locus. A set of 28 F₁ hybrids and eight parental lines from a barley diallel study was inoculated with each of five isolates ofE. graminis. The parents were surveyed for restriction fragment length polymorphisms (RFLPs) at 84 marker loci that cover about 1100 cM of the barley genome. The RFLP genotypes of the F₁s were deduced from those of the parents. A total of 27 loci, distributed on six of the seven barley chromosomes, detected significant resistance effects to at least one of the five isolates. Almost all the chromosomal regions previously reported to carry genes for powdery mildew resistance were detected, plus the possible existence of 1 additional locus on chromosome 7. The analysis indicated that additive genetic effects are the most important component in conditioning powdery mildew resistance. However, there is also a considerable amount of dominance effects at most loci, and even overdominance is likely to be present at a number of loci. These results suggest that quantitative differences are likely to exist among alleles even at loci which are considered to carry major genes for resistance, and minor effects may be prevalent in cultivars that are not known to carry major genes for resistance.     

Antisense expression of peach mildew resistance locus O (PpMlo1) gene confers cross-species resistance to powdery mildew in Fragaria x ananassa

Powdery mildew (PM) is one of the major plant pathogens. The conventional method of PM control includes frequent use of sulfur-based fungicides adding to production costs and potential harm to the environment. PM remains a major scourge for Rosaceae crops where breeding approaches mainly resort to gene-for-gene resistance. We have tested an alternate source of PM resistance in Rosaceae. Mildew resistance locus O (MLO) has been well studied in barley due to its role in imparting broad spectrum resistance to PM. We identified PpMlo1 (Prunus persica Mlo) in peach and characterized it further to test if a similar mechanism of resistance is conserved in Rosaceae. Due to its recalcitrance in tissue culture, reverse genetic studies involving PpMloI were not feasible in peach. Therefore, Fragaria x ananassa LF9 line, a taxonomic surrogate, was used for functional analysis of PpMlo1. Agrobacterium-mediated transformation yielded transgenic strawberry plants expressing PpMlo1 in sense and antisense orientation. Antisense expression of PpMlo1 in transgenic strawberry plants conferred resistance to Fragaria-specific powdery mildew, Podosphaera macularis. Phylogenetic analysis of 208 putative Mlo gene copies from 35 plant species suggests a large number of duplications of this gene family prior to the divergence of monocots and eudicots, early in eudicot diversification. Our results indicate that the Mlo-based resistance mechanism is functional in Rosaceae, and that Fragaria can be used as a host to test mechanistic function of genes derived from related tree species. To the best of our knowledge, this work is one of the first attempts at testing the potential of using a Mlo-based resistance strategy to combat powdery mildew in Rosaceae.     

A mutation in a coproporphyrinogen III oxidase gene confers growth inhibition, enhanced powdery mildew resistance and powdery mildew-induced cell death in Arabidopsis

Key message

A gene encoding a coproporphyrinogen III oxidase mediates disease resistance in plants by the salicylic acid pathway.

Abstract

A number of genes that regulate powdery mildew resistance have been identified in Arabidopsis, such as ENHANCED DISEASE RESISTANCE 1 to 3 (EDR1 to 3). To further study the molecular interactions between the powdery mildew pathogen and Arabidopsis, we isolated and characterized a mutant that exhibited enhanced resistance to powdery mildew. The mutant also showed dramatic powdery mildew-induced cell death as well as growth defects and early senescence in the absence of pathogens. We identified the affected gene by map-based cloning and found that the gene encodes a coproporphyrinogen III oxidase, a key enzyme in the tetrapyrrole biosynthesis pathway, previously known as LESION INITIATION 2 (LIN2). Therefore, we designated the mutant lin2-2. Further studies revealed that the lin2-2 mutant also displayed enhanced resistance to Hyaloperonospora arabidopsidis (H.a.) Noco2. Genetic analysis showed that the lin2-2-mediated disease resistance and spontaneous cell death were dependent on PHYTOALEXIN DEFICIENT 4 (PAD4), SALICYLIC ACID INDUCTION-DEFICIENT 2 (SID2), and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1), which are all involved in salicylic acid signaling. Furthermore, the relative expression levels of defense-related genes were induced after powdery mildew infection in the lin2-2 mutant. These data indicated that LIN2 plays an important role in cell death control and defense responses in plants.     

Mapping resistance to powdery mildew in barley reveals a large-effect nonhost resistance QTL

Key message

Resistance factors against non-adapted powdery mildews were mapped in barley. Some QTLs seem effective only to non-adapted mildews, while others also play a role in defense against the adapted form.The durability and effectiveness of nonhost resistance suggests promising practical applications for crop breeding, relying upon elucidation of key aspects of this type of resistance. We investigated which genetic factors determine the nonhost status of barley (Hordeum vulgare L.) to powdery mildews (Blumeria graminis). We set out to verify whether genes involved in nonhost resistance have a wide effectiveness spectrum, and whether nonhost resistance genes confer resistance to the barley adapted powdery mildew. Two barley lines, SusBgt_SC and SusBgt_DC, with some susceptibility to the wheat powdery mildew B. graminis f.sp. tritici (Bgt) were crossed with cv Vada to generate two mapping populations. Each population was assessed for level of infection against four B. graminis ff.spp, and QTL mapping analyses were performed. Our results demonstrate polygenic inheritance for nonhost resistance, with some QTLs effective only to non-adapted mildews, while others play a role against adapted and non-adapted forms. Histology analyses of nonhost interaction show that most penetration attempts are stopped in association with papillae, and also suggest independent layers of defence at haustorium establishment and conidiophore formation. Nonhost resistance of barley to powdery mildew relies mostly on non-hypersensitive mechanisms. A large-effect nonhost resistance QTL mapped to a 1.4 cM interval is suitable for map-based cloning.