The mlo resistance alleles to powdery mildew infection in barley trigger a developmentally controlled defence mimic phenotype

Recessive mlo resistance alleles of the Mlo locus in barley control a non race-specific resistance response to infection by the obligate biotrophic fungus Erysiphe graminis f.sp. hordei. All the mlo alleles analysed stop fungal growth at the same developmental stage within a subcellularly restricted, highly localized cell wall apposition directly beneath the site of abortive fungal penetration. We report that near-isogenic lines carrying the alleles mlo ₁, mlo ₃ or mlo ₅ undergo dramatic spontaneous formation of cell wall appositions, not only in the absence of the fungal pathogen but also in sterile grown plants. A comparative study of spontaneous and infection-triggered cell wall appositions reveals a high degree of similarity with respect to structure, chemical composition and distinct localization within plant tissue. We show that the rate of spontaneous apposition formation is dependent on the genetic background of the plant and that its onset is under developmental control. Furthermore, spontaneous formation of wall appositions is specifically triggered by mlo alleles, since it is unaffected in the presence of the race-specific resistance allele Mlg. We propose a model for the function of the Mlo locus that suggests that both Mlo and mlo alleles control qualitatively the same apposition-based resistance mechanism, which, in the presence of the wild-type Mlo allele, is merely less efficient to provide protection against the currently common races of E. graminis f.sp. hordei.     

Construction of immune lines with complex resistance to leaf rust and powdery mildew in common spring wheat cultivar Saratovskaya 29

Immune lines resistant both to leaf rust and to powdery mildew were constructed on the basis of common wheat cultivar Saratovskaya 29. Synthetic wheat Triticum timopheevii/Aegilops squarrosa (AAGGDD, 2n = 42) of Savov (Bulgaria) was used as a source of resistance genes. Using cytological analysis of BC2, we selected resistant plants (21") free from meiosis 1 (M1) defects. With these plants and continuous selection, BC8-BC9 immune lines were obtained. The lines were shown to carry new resistance genes differing from the known ones, and were proposed as donors of immunity to the diseases.     

Metabolic regulation in diseased leaves. I. The respiratory rise in barley leaves infected with powdery mildew

Photosynthetic and respiratory activities have been measured in leaves of Hordeum vulgare L. var. Manchuria (barley) after infection with Erysiphe graminis var. hordei (powdery mildew). Two isogenic lines, one resistant to infection and the other highly susceptible, were examined.

These isogenic lines showed very different physiological responses following infection. Photosynthesis and the chlorophyll content of resistant leaves was unaffected by infection. Respiration increased slightly and this was accompanied by small increases in activities of enzymes of glycolysis, the pentose-P pathway and the tricarboxylic acid cycle.

The infection of susceptible leaves resulted in a slight increase in photosynthesis 48 hours after inoculation, but subsequently there was a progressive decrease in the photosynthesis of these leaves compared with that of noninfected leaves. The capacity of infected leaves for partial reactions of photosynthesis such as the Hill reaction and the photoreduction of nicotinamide adenine dinucleotide phosphate (NADP¹) decreased during the later stages of infection. The levels of chlorophyll, NADPH-diaphorase and aldolase also declined. There was no detectable difference in the respiration of infected and noninfected leaves until 48 hours after inoculation. After this time, the infected leaves showed a higher respiration, the maximum difference occurring about 144 hours after inoculation. The respiratory increase was not accompanied by significant changes in the levels of enzymes of glycolysis and the tricarboxylic acid cycle with the exception of malate dehydrogenase which was lower in infected leaves. In contrast, the activities of glucose-6-P dehydrogenase and 6-P-gluconate dehydrogenase showed changes similar to that observed for respiration.

The respiration and the activities of glucose-6-P dehydrogenase and 6-P-gluconate dehydrogenase did not increase in infected leaves of etiolated plants, even when excellent growth of the fungus was established by growing the plants in White's basal medium supplemented with sucrose. The respiration of a susceptible mutant barley (the yellow-green virescent mutant of the variety Himalaya) when grown in the light at 11° was not changed by infection although the characteristic respiratory rise occurred in plants grown at 15°. At the lower temperature chloroplasts fail to develop in this mutant, although development is normal at 15°.

It is suggested that the pathogen is not directly responsible for the increase in respiration in green leaves, rather that this is a response in the host cells to a loss of photosynthetic capacity.

     

The streptococcal Blr and Slr proteins define a family of surface proteins with leucine-rich repeats: camouflaging by other surface structures

Regions with tandemly arranged leucine-rich repeats (LRRs) have been found in many prokaryotic and eukaryotic proteins, in which they provide a remarkably versatile framework for the formation of ligand-binding sites. Bacterial LRR proteins include the recently described Slr protein of Streptococcus pyogenes, which is related to internalin A of Listeria monocytogenes. Here, we show that strains of the human pathogen Streptococcus agalactiae express a protein, designated Blr, which together with Slr defines a family of internalin A-related streptococcal LRR proteins. Analysis with specific antibodies demonstrated that Blr is largely inaccessible on S. agalactiae grown in vitro, but surface exposure was increased approximately 100-fold on mutants lacking polysaccharide capsule. In S. pyogenes, surface exposure of Slr was not affected in a mutant lacking hyaluronic acid capsule but was increased >20-fold in mutants lacking M protein or protein F. Thus, both Blr and Slr are efficiently camouflaged by other surface structures on bacteria grown in vitro. When Blr and Slr exposed on the bacterial surface were compared, they exhibited only little immunological cross-reactivity, in spite of extensive residue identity, suggesting that their surface-exposed parts have been under evolutionary pressure to diverge functionally and/or antigenically. These data identify a family of immunologically diverse streptococcal LRR proteins that show unexpected complexity in their interactions with other bacterial surface components.     

The mlo resistance alleles to powdery mildew infection in barley trigger a developmentally controlled defence mimic phenotype

Recessive mlo resistance alleles of the Mlo locus in barley control a non race-specific resistance response to infection by the obligate biotrophic fungus Erysiphe graminis f.sp. hordei. All the mlo alleles analysed stop fungal growth at the same developmental stage within a subcellularly restricted, highly localized cell wall apposition directly beneath the site of abortive fungal penetration. We report that near-isogenic lines carrying the alleles mlo ₁, mlo ₃ or mlo ₅ undergo dramatic spontaneous formation of cell wall appositions, not only in the absence of the fungal pathogen but also in sterile grown plants. A comparative study of spontaneous and infection-triggered cell wall appositions reveals a high degree of similarity with respect to structure, chemical composition and distinct localization within plant tissue. We show that the rate of spontaneous apposition formation is dependent on the genetic background of the plant and that its onset is under developmental control. Furthermore, spontaneous formation of wall appositions is specifically triggered by mlo alleles, since it is unaffected in the presence of the race-specific resistance allele Mlg. We propose a model for the function of the Mlo locus that suggests that both Mlo and mlo alleles control qualitatively the same apposition-based resistance mechanism, which, in the presence of the wild-type Mlo allele, is merely less efficient to provide protection against the currently common races of E. graminis f.sp. hordei.     

The effects of three film-forming polymers, with and without a polyamine biosynthesis inhibitor, on powdery mildew infection of barley seedlings

Powdery mildew infection of barley seedlings was significantly reduced using either pre-inoculation or post-inoculation sprays of the film-forming polymers Nu-Film P, Emerald or Vapor Gard. The greatest reduction in mildew infection was obtained with a pre-inoculation spray of the polymer Vapor Gard. Although the addition of 1 mm α-difluoromethylornithine (DFMO) to a 2% solution of Emerald or Vapor Gard resulted in better control of powdery mildew infection than that obtained using the polymer or DFMO alone, the reductions in infection were not significant. Reduction in mildew infection was greatest when the Vapor Gard + DFMO mixture was applied 1 day prior to inoculation with the fungus. Although substantial reduction in mildew infection was achieved using as little as 0.1 mm DFMO with 2% Vapor Gard, or reducing the polymer concentration to 1% in combination with 1 mm DFMO, best control was achieved using 2% Vapor Gard plus 1 mm DFMO.     

Molecular mapping of a powdery mildew resistance gene in common wheat landrace Baihulu and its allelism with Pm24

Powdery mildew is one of the most devastating diseases of wheat in areas with cool and maritime climates. Chinese wheat landrace Baihulu confers a high level of resistance against a wide range of Blumeria graminis DC f. sp. tritici (Bgt) races, especially those currently prevailing in Shaanxi. The objectives of this study were to determine the chromosome bin location of the mlbhl gene from Baihulu and its allelism with Pm24. To investigate the inheritance of powdery mildew resistance and detect adjacent molecular markers, we constructed a segregating population of 301 F₂ plants and corresponding F_2:3 families derived from Baihulu/Shaanyou 225. Genetic analysis revealed that a single dominant gene was responsible for seedling stage powdery mildew resistance in Baihulu. A genetic map comprising Xgwm106, Xgwm337, Xgwm1675, Xgwm603, Xgwm789, Xbarc229, Xgpw4503, Xcfd72, Xcfd83, Xcfd59, Xcfd19, and mlbhl spanned 28.2?cM on chromosome 1D. Xgwm603/Xgwm789 and Xbarc229 were flanking markers tightly linked to mlbhl at genetic distances of 1.5 and 1.0?cM, respectively. The mlbhl locus was located in chromosome bin 1DS 0.59–1.00 delimited by the SSR markers Xgwm337 and Xbarc229. When tested with a differential array of 23 Bgt isolates Baihulu displayed a response pattern that was clearly distinguishable from that of Chiyacao and varieties or lines possessing documented Pm genes. Allelism analysis indicated that mlbhl is a new gene, either allelic or closely linked with Pm24. The new gene was designated Pm24b.     

The specificity of polygalacturonase-inhibiting protein (PGIP): a single amino acid substitution in the solvent-exposed beta-strand/beta-turn region of the leucine-rich repeats (LRRs) confers a new recognition capability

Two members of the pgip gene family (pgip-1 and pgip-2) of Phaseolus vulgaris L. were expressed separately in Nicotiana benthamiana and the ligand specificity of their products was analysed by surface plasmon resonance (SPR). Polygalacturonase-inhibiting protein-1 (PGIP-1) was unable to interact with PG from Fusarium moniliforme and interacted with PG from Aspergillus niger; PGIP-2 interacted with both PGs. Only eight amino acid variations distinguish the two proteins: five of them are confined within the beta-sheet/beta-turn structure and two of them are contiguous to this region. By site-directed mutagenesis, each of the variant amino acids of PGIP-2 was replaced with the corresponding amino acid of PGIP-1, in a loss-of-function approach. The mutated PGIP-2s were expressed individually in N.benthamiana, purified and subjected to SPR analysis. Each single mutation caused a decrease in affinity for PG from F.moniliforme; residue Q253 made a major contribution, and its replacement with a lysine led to a dramatic reduction in the binding energy of the complex. Conversely, in a gain-of-function approach, amino acid K253 of PGIP-1 was mutated into the corresponding amino acid of PGIP-2, a glutamine. With this single mutation, PGIP-1 acquired the ability to interact with F.moniliforme PG.     

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.     

Genetic and comparative genomics mapping reveals that a powdery mildew resistance gene Ml3D232 originating from wild emmer co-segregates with an NBS-LRR analog in common wheat (Triticum aestivum L.)

Powdery mildew caused by Blumeria graminis f. sp. tritici is one of the most important wheat diseases worldwide and breeding for resistance using diversified disease resistance genes is the most promising approach to prevent outbreaks of powdery mildew. A powdery mildew resistance gene, originating from wild emmer wheat (Triticum turgidum var. dicoccoides) accessions collected from Israel, has been transferred into the hexaploid wheat line 3D232 through crossing and backcrossing. Inoculation results with 21 B. graminis f. sp. tritici races indicated that 3D232 is resistant to all of the powdery mildew isolates tested. Genetic analyses of 3D232 using an F₂ segregating population and F₃ families indicated that a single dominant gene, Ml3D232, confers resistance in the host seedling stage. By applying molecular markers and bulked segregant analysis (BSA), we have identified polymorphic simple sequence repeats (SSR), expressed sequence tags (EST) and derived sequence tagged site (STS) markers to determine that the Ml3D232 is located on chromosome 5BL bin 0.59–0.76. Comparative genetic analyses using mapped EST markers and genome sequences of rice and Brachypodium established co-linearity of the Ml3D232 genomic region with a 1.4 Mb genomic region on Brachypodium distachyon chromosome 4, and a 1.2 Mb contig located on the Oryza sativa chromosome 9. Our comparative approach enabled us to develop new EST–STS markers and to delimit the genomic region carrying Ml3D232 to a 0.8 cM segment that is collinear with a 558 kb region on B. distachyon. Eight EST markers, including an NBS-LRR analog, co-segregated with Ml3D232 to provide a target site for fine genetic mapping, chromosome landing and map-based cloning of the powdery mildew resistance gene. This newly developed common wheat germplasm provides broad-spectrum resistance to powdery mildew and a valuable resource for wheat breeding programs.     

The cell-mediated immune response to Neospora caninum during pregnancy in the mouse is associated with a bias towards production of interleukin-4

Neospora caninum is an obligate intracellular protozoan parasite which is efficiently transmitted transplacentally in cattle where it may cause abortion. A pregnant mouse model was used to characterise the immune response following N. caninum infection; the response in non-pregnant and pregnant mice was compared. Spleen cells from both infected/non-pregnant and infected/pregnant mice produced interferon-gamma, interleukin-12 and tumour necrosis factor alpha; however, the levels of these Th1 cytokines were lower in infected/pregnant mice. Infected/non-pregnant and infected/pregnant mice also produced the Th2 cytokine interleukin-10; however, there was no trend toward a decrease of this in pregnant mice. Interleukin-4 was exclusively produced at high levels by infected/pregnant mice and thus appears responsible for the observed decline in Th1 cytokine production in pregnant mice. A bias towards Th2 cytokines such as IL-4 and IL-10 is normally associated with the maintenance of a viable pregnancy, and not with the control of protozoal infections. Consequently, the importance and role of cytokines and cell-mediated immunity in the control of transplacental transmission and foetal loss due to N. caninum infection are discussed.     

The breakpoint region of the most common isochromosome, i(17q), in human neoplasia is characterized by a complex genomic architecture with large, palindromic, low-copy repeats

Although a great deal of information has accumulated regarding the mechanisms underlying constitutional DNA rearrangements associated with inherited disorders, virtually nothing is known about the molecular processes involved in acquired neoplasia-associated chromosomal rearrangements. Isochromosome 17q, or "i(17q)," is one of the most common structural abnormalities observed in human neoplasms. We previously identified a breakpoint cluster region for i(17q) formation in 17p11.2 and hypothesized that genome architectural features could be responsible for this clustering. To address this hypothesis, we precisely mapped the i(17q) breakpoints in 11 patients with different hematologic malignancies and determined the genomic structure of the involved region. Our results reveal a complex genomic architecture in the i(17q) breakpoint cluster region, characterized by large ( approximately 38-49-kb), palindromic, low-copy repeats, strongly suggesting that somatic rearrangements are not random events but rather reflect susceptibilities due to the genomic structure.