Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting that most effectors represent species-specific adaptations.

BackgroundProtein effectors of pathogenicity are instrumental in modulating host immunity and disease resistance. The powdery mildew pathogen of grasses Blumeria graminis causes one of the most important diseases of cereal crops. B. graminis is an obligate biotrophic pathogen and as such has an absolute requirement to suppress or avoid host immunity if it is to survive and cause disease.ResultsHere we characterise a superfamily predicted to be the full complement of Candidates for Secreted Effector Proteins (CSEPs) in the fungal barley powdery mildew parasite B. graminis f.sp. hordei. The 491 genes encoding these proteins constitute over 7% of this pathogen’s annotated genes and most were grouped into 72 families of up to 59 members. They were predominantly expressed in the intracellular feeding structures called haustoria, and proteins specifically associated with the haustoria were identified by large-scale mass spectrometry-based proteomics. There are two major types of effector families: one comprises shorter proteins (100–150 amino acids), with a high relative expression level in the haustoria and evidence of extensive diversifying selection between paralogs; the second type consists of longer proteins (300–400 amino acids), with lower levels of differential expression and evidence of purifying selection between paralogs. An analysis of the predicted protein structures underscores their overall similarity to known fungal effectors, but also highlights unexpected structural affinities to ribonucleases throughout the entire effector super-family. Candidate effector genes belonging to the same family are loosely clustered in the genome and are associated with repetitive DNA derived from retro-transposons.ConclusionsWe employed the full complement of genomic, transcriptomic and proteomic analyses as well as structural prediction methods to identify and characterize the members of the CSEPs superfamily in B. graminis f.sp. hordei. Based on relative intron position and the distribution of CSEPs with a ribonuclease-like domain in the phylogenetic tree we hypothesize that the associated genes originated from an ancestral gene, encoding a secreted ribonuclease, duplicated successively by repetitive DNA-driven processes and diversified during the evolution of the grass and cereal powdery mildew lineage.

An experiment was conducted to measure DE and ME and the apparent total tract digestibility (ATTD) of energy, N, and P in distillers dried grains with solubles (DDGS) fed to growing pigs. Ten sources of DDGS were obtained from ethanol plants in South Dakota and Minnesota, and 11 diets were formulated. One diet was based on corn (96.8%), limestone, salt, vitamins, and microminerals. Ten additional diets were formulated by mixing the corn diet and each of the 10 sources of DDGS in a 1:1 ratio. Eleven growing pigs (initial BW of 29.3 +/- 0.42 kg) were allotted to an 11 x 11 Latin square design, with 11 periods and 11 pigs. Each of the 11 diets was fed to each pig during 1 period. Pigs were placed in metabolism cages that allowed for the total, but separate, collection of feces and urine. Samples were analyzed for GE, N, and P and energy and N balances, and the ATTD of GE, N, and P were calculated for each diet. By subtracting the contribution from the corn diet to the DDGS-containing diets, the energy and N balances and the ATTD for GE, N, and P for each source of DDGS were calculated. Results of the experiment showed that the DE and ME differed (P < 0.001) among the 10 sources of DDGS (3,947 to 4,593 kcal of DE/kg of DM and 3,674 to 4,336 kcal of ME/kg of DM). The average DE and ME in DDGS were 4,140 and 3,897 kcal/kg of DM, respectively. These values were not different from the DE and ME in corn (4,088 and 3,989 kcal/kg of DM, respectively). Based on the analyzed GE and nutrient composition of DDGS and the calculated values for DE and ME, prediction equations for DE and ME were developed. These equations showed that DE and ME in DDGS may be predicted from the concentration of ash, ether extract, ADF, and GE. The retention of N from DDGS was greater (P < 0.001) than from corn, but when calculated on a percentage basis, the N retention did not differ between DDGS and corn. The ATTD of P in DDGS was 59.1% on average for the 10 samples. This value was greater (P < 0.001) than the ATTD of P in corn (19.3%). It is concluded that the DE and ME in DDGS is not different from the DE and ME in corn. However, if DDGS is included in diets fed to growing swine, a greater portion of the organic P will be digested and absorbed, thus reducing the need for adding inorganic P to the diets.

The objective of this experiment was to measure the digestibilities of energy, CP, and AA in 10 samples of corn distillers dried grain with solubles (DDGS) and in corn fed to growing pigs. Twelve growing barrows (initial BW: 34.0 +/- 1.41 kg) were allotted to an 8 x 12 Youden square design with 8 periods and 12 animals. Ten of 12 diets were based on the 10 DDGS samples (66.7%), 1 diet was based on corn (97%), and the last diet was a N-free diet based on cornstarch and sucrose. Chromic oxide (0.3%) was included in all diets as an inert marker. Pigs were provided their respective diets at a level of 3 times their estimated energy requirement for maintenance. The apparent (AID) and standardized (SID) ileal digestibilities for CP and AA were measured in the 10 samples of DDGS and in corn using the direct procedure, but the apparent total tract digestibilities for DM and GE were estimated using the difference procedure. The concentration of DE in each sample of DDGS and in corn was also calculated. The results of the experiment indicated variation among the different sources of DDGS in AID and SID for Lys, which ranged from 35.0 to 55.9% and 43.9 to 63.0%, respectively. For Met, the SID varied between 73.9 and 84.7%. However, the variability among samples in the SID for CP, and for the indispensable AA other than Lys and Met, was relatively low and ranged between 6 and 8 percentage units (i.e., from 64.0 to 70.6%, 74.1 to 80.1%, and 67.4 to 75.3% for Thr, Trp, and Ile, respectively). The SID for Trp in corn (72.8%) was lower (P < 0.05) than in DDGS, but for the remaining indispensable AA, except Arg, the SID for corn were greater (P < 0.01) than for DDGS. The DE concentration in the 10 samples of DDGS varied (P < 0.001) from 3,382 to 3,811 kcal of DE per kg of DM. For corn, the DE was 3,845 kcal per kg of DM. It is concluded that the AID and SID for Lys vary among samples of DDGS, but for most other AA the AID and SID are relatively similar and vary only 6 to 8 percentage units among different samples. Future work should focus on identifying the reasons for the variation in the digestibility of Lys to avoid processing procedures that are detrimental to Lys digestibility.

BackgroundPowdery mildews are biotrophic pathogenic fungi infecting a number of economically important plants. The grass powdery mildew, Blumeria graminis, has become a model organism to study host specialization of obligate biotrophic fungal pathogens. We resolved the large-scale genomic architecture of B. graminis forma specialis hordei (Bgh) to explore the potential influence of its genome organization on the co-evolutionary process with its host plant, barley (Hordeum vulgare).ResultsThe near-chromosome level assemblies of the Bgh reference isolate DH14 and one of the most diversified isolates, RACE1, enabled a comparative analysis of these haploid genomes, which are highly enriched with transposable elements (TEs). We found largely retained genome synteny and gene repertoires, yet detected copy number variation (CNV) of secretion signal peptide-containing protein-coding genes (SPs) and locally disrupted synteny blocks. Genes coding for sequence-related SPs are often locally clustered, but neither the SPs nor the TEs reside preferentially in genomic regions with unique features. Extended comparative analysis with different host-specific B. graminis formae speciales revealed the existence of a core suite of SPs, but also isolate-specific SP sets as well as congruence of SP CNV and phylogenetic relationship. We further detected evidence for a recent, lineage-specific expansion of TEs in the Bgh genome.ConclusionsThe characteristics of the Bgh genome (largely retained synteny, CNV of SP genes, recently proliferated TEs and a lack of significant compartmentalization) are consistent with a “one-speed” genome that differs in its architecture and (co-)evolutionary pattern from the “two-speed” genomes reported for several other filamentous phytopathogens.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4750-6) contains supplementary material, which is available to authorized users.

SummaryPenetration resistance is often the first line of defence against fungal pathogens. Subsequently induced defences are mediated by the programmed cell death (PCD) reaction pathway and the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signalling pathways. We previously demonstrated that full penetration resistance in Arabidopsis against the non-host barley powdery mildew fungus (Blumeria graminis f.sp. hordei) requires the syntaxin SYP121 (PEN1). Here we report that SYP121, together with SYP122, functions as a negative regulator of subsequently induced defence pathways. The SA level in the syntaxin double mutant syp121-1 syp122-1 is dramatically elevated, resulting in necrosis and dwarfism. This phenotype is partially rescued by introducing the SA-signalling mutations eds1-2, eds5-3, sid2-1 and npr1-1 as well as the NahG transgene. These partially rescued triple mutants have an unknown defence to Pseudomonas syringae pv. tomato, and have increased HR-like responses to non-host and host powdery mildew fungi. The HR-like responses cause efficient resistance to the latter. These defence pathways are SA-independent. Furthermore, the JA/ET signalling marker, PDF1.2, is highly upregulated in the triple mutants. Thus SYP121 and SYP122 are negative regulators of PCD, SA, JA and ET pathways through a molecular function distinct from that of SYP121 in penetration resistance. Our data suggest that individual cells preferentially express either penetration resistance or the subsequently induced defences.

BackgroundPowdery mildew and rust fungi are widespread, serious pathogens that depend on developing haustoria in the living plant cells. Haustoria are separated from the host cytoplasm by a plant cell-derived extrahaustorial membrane. They secrete effector proteins, some of which are subsequently transferred across this membrane to the plant cell to suppress defense.ResultsIn a cDNA library from barley epidermis containing powdery mildew haustoria, two-thirds of the sequenced ESTs were fungal and represented ~3,000 genes. Many of the most highly expressed genes encoded small proteins with N-terminal signal peptides. While these proteins are novel and poorly related, they do share a three-amino acid motif, which we named "Y/F/WxC", in the N-terminal of the mature proteins. The first amino acid of this motif is aromatic: tyrosine, phenylalanine or tryptophan, and the last is always cysteine. In total, we identified 107 such proteins, for which the ESTs represent 19% of the fungal clones in our library, suggesting fundamental roles in haustoria function. While overall sequence similarity between the powdery mildew Y/F/WxC-proteins is low, they do have a highly similar exon-intron structure, suggesting they have a common origin. Interestingly, searches of public fungal genome and EST databases revealed that haustoria-producing rust fungi also encode large numbers of novel, short proteins with signal peptides and the Y/F/WxC-motif. No significant numbers of such proteins were identified from genome and EST sequences from either fungi which do not produce haustoria or from haustoria-producing Oomycetes.ConclusionIn total, we identified 107, 178 and 57 such Y/F/WxC-proteins from the barley powdery mildew, the wheat stem rust and the wheat leaf rust fungi, respectively. All together, our findings suggest the Y/F/WxC-proteins to be a new class of effectors from haustoria-producing pathogenic fungi.

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