Barley Ror1 encodes a class XI myosin required for mlo-based broad-spectrum resistance to the fungal powdery mildew pathogen.

Loss-of-function alleles of plant MLO genes confer broad-spectrum resistance to powdery mildews in many eudicot and monocot species. Although barley (Hordeum vulgare) mlo mutants have been used in agriculture for more than 40 years, understanding of the molecular principles underlying this type of disease resistance remains fragmentary. Forward genetic screens in barley have revealed mutations in two Required for mlo resistance (Ror) genes that partially impair immunity conferred by mlo mutants. While Ror2 encodes a soluble N-ethylmaleimide-sensitive factor-attached protein receptor (SNARE), the identity of Ror1, located at the pericentromeric region of barley chromosome 1H, remained elusive. We report the identification of Ror1 based on combined barley genomic sequence information and transcriptomic data from ror1 mutant plants. Ror1 encodes the barley class XI myosin Myo11A (HORVU.MOREX.r3.1HG0046420). Single amino acid substitutions of this myosin, deduced from non-functional ror1 mutant alleles, map to the nucleotide-binding region and the interface between the relay-helix and the converter domain of the motor protein. Ror1 myosin accumulates transiently in the course of powdery mildew infection. Functional fluorophore-labeled Ror1 variants associate with mobile intracellular compartments that partially colocalize with peroxisomes. Single-cell expression of the Ror1 tail region causes a dominant-negative effect that phenocopies ror1 loss-of-function mutants. We define a myosin motor for the establishment of mlo-mediated resistance, suggesting that motor protein-driven intracellular transport processes are critical for extracellular immunity, possibly through the targeted transfer of antifungal and/or cell wall cargoes to pathogen contact sites.

Transcriptomic profiles of human livers undergoing rewarming machine perfusion before transplantation-first insights.

Machine perfusion by controlled oxygenated rewarming (COR) is feasible and safe in clinical application and result in a promising outcome. This study utilizes next-generation sequencing (NGS) to investigate the transcriptome of human liver tissue undergoing COR before liver transplantation. Cold-stored livers were subjected to machine-assisted slow COR for ~120 min before transplantation. Biopsies were taken before (preCOR) and after COR (postCOR) and 1 h after reperfusion (postRep). The samples were sequenced, using RNA-seq to analyze differential transcriptional changes between the different stages and treatments of the grafts. Comparison of differential gene expression preCOR and postCOR demonstrated 10 upregulated genes. postRep 97 and 178 genes were upregulated and 7 and 13 downregulated compared to preCOR and postCOR, respectively. A shift of gene expressions by machine perfusion to the TGF-beta pathway was observed. The present study demonstrates distinct transcriptome profiles associated with machine perfusion by COR and transplantation of human livers. Such data provide a deeper understanding of the molecular mechanisms of machine perfusion technology in human liver transplantation.

Lysosomal proteome analysis reveals that CLN3-defective cells have multiple enzyme deficiencies associated with changes in intracellular trafficking.

Numerous lysosomal enzymes and membrane proteins are essential for the degradation of proteins, lipids, oligosaccharides, and nucleic acids. The CLN3 gene encodes a lysosomal membrane protein of unknown function, and CLN3 mutations cause the fatal neurodegenerative lysosomal storage disorder CLN3 (Batten disease) by mechanisms that are poorly understood. To define components critical for lysosomal homeostasis that are affected by this disease, here we quantified the lysosomal proteome in cerebellar cell lines derived from a CLN3 knock-in mouse model of human Batten disease and control cells. We purified lysosomes from SILAC-labeled, and magnetite-loaded cerebellar cells by magnetic separation and analyzed them by MS. This analysis identified 70 proteins assigned to the lysosomal compartment and 3 lysosomal cargo receptors, of which most exhibited a significant differential abundance between control and CLN3-defective cells. Among these, 28 soluble lysosomal proteins catalyzing the degradation of various macromolecules had reduced levels in CLN3-defective cells. We confirmed these results by immunoblotting and selected protease and glycosidase activities. The reduction of 11 lipid-degrading lysosomal enzymes correlated with reduced capacity for lipid droplet degradation and several alterations in the distribution and composition of membrane lipids. In particular, levels of lactosylceramides and glycosphingolipids were decreased in CLN3-defective cells, which were also impaired in the recycling pathway of the exocytic transferrin receptor. Our findings suggest that CLN3 has a crucial role in regulating lysosome composition and their function, particularly in degrading of sphingolipids, and, as a consequence, in membrane transport along the recycling endosome pathway.

Quantitative proteomics of synaptosome S-nitrosylation in Alzheimer's disease.

Increasing evidence suggests that both synaptic loss and neuroinflammation constitute early pathologic hallmarks of Alzheimer's disease. A downstream event during inflammatory activation of microglia and astrocytes is the induction of nitric oxide synthase type 2, resulting in an increased release of nitric oxide and the post-translational S-nitrosylation of protein cysteine residues. Both early events, inflammation and synaptic dysfunction, could be connected if this excess nitrosylation occurs on synaptic proteins. In the long term, such changes could provide new insight into patho-mechanisms as well as biomarker candidates from the early stages of disease progression. This study investigated S-nitrosylation in synaptosomal proteins isolated from APP/PS1 model mice in comparison to wild type and NOS2-/- mice, as well as human control, mild cognitive impairment and Alzheimer's disease brain tissues. Proteomics data were obtained using an established protocol utilizing an isobaric mass tag method, followed by nanocapillary high performance liquid chromatography tandem mass spectrometry. Statistical analysis identified the S-nitrosylation sites most likely derived from an increase in nitric oxide (NO) in dependence of presence of AD pathology, age and the key enzyme NOS2. The resulting list of candidate proteins is discussed considering function, previous findings in the context of neurodegeneration, and the potential for further validation studies.

Loss of CLN7 results in depletion of soluble lysosomal proteins and impaired mTOR reactivation.

Defects in the MFSD8 gene encoding the lysosomal membrane protein CLN7 lead to CLN7 disease, a neurodegenerative lysosomal storage disorder belonging to the group of neuronal ceroid lipofuscinoses. Here, we have performed a SILAC-based quantitative analysis of the lysosomal proteome using Cln7-deficient mouse embryonic fibroblasts (MEFs) from a Cln7 knockout (ko) mouse model. From 3335 different proteins identified, we detected 56 soluble lysosomal proteins and 29 highly abundant lysosomal membrane proteins. Quantification revealed that the amounts of 12 different soluble lysosomal proteins were significantly reduced in Cln7 ko MEFs compared with wild-type controls. One of the most significantly depleted lysosomal proteins was Cln5 protein that underlies another distinct neuronal ceroid lipofuscinosis disorder. Expression analyses showed that the mRNA expression, biosynthesis, intracellular sorting and proteolytic processing of Cln5 were not affected, whereas the depletion of mature Cln5 protein was due to increased proteolytic degradation by cysteine proteases in Cln7 ko lysosomes. Considering the similar phenotypes of CLN5 and CLN7 patients, our data suggest that depletion of CLN5 may play an important part in the pathogenesis of CLN7 disease. In addition, we found a defect in the ability of Cln7 ko MEFs to adapt to starvation conditions as shown by impaired mammalian target of rapamycin complex 1 reactivation, reduced autolysosome tubulation and increased perinuclear accumulation of autolysosomes compared with controls. In summary, depletion of multiple soluble lysosomal proteins suggest a critical role of CLN7 for lysosomal function, which may contribute to the pathogenesis and progression of CLN7 disease.

Investigation of Oligodendrocyte Precursor Cell Differentiation by Quantitative Proteomics.

Oligodendrocytes, the myelinating cells of the central nervous system, are essential for correct brain function. They originate from oligodendrocyte precursor cells through a differentiation process which is only incompletely understood and impaired in a variety of demyelinating diseases. Better knowledge of this differentiation holds the promise to develop novel therapies for these disorders. The differentiation of rat oligodendrocyte precursor cells to oligodendrocytes in vitro is investigated. After confirmation of differentiation by immunohistochemical analysis using cell type-specific marker proteins, a quantitative proteomics study using tandem mass tags (TMT) is conducted. Four time points of differentiation covering early, intermediate, and late stages are investigated. Data analysis by Mascot and MaxQuant identified 5259 protein groups of which 471 are not described in the context of cells of the oligodendroglial lineage before. Quantitative analysis of the dataset revealed distinct regulation patterns for proteins of different functional categories including metabolic processes, regulation of the cell cycle, and transcriptional control of protein expression. The present data confirm a significant number of proteins known to play a role in oligodendrocytes and myelination. Furthermore, novel candidate proteins are identified which may play an important role in this differentiation process providing a valuable resource for oligodendrocyte research.

Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota.

The plant root defines the interface between a multicellular eukaryote and soil, one of the richest microbial ecosystems on Earth. Notably, soil bacteria are able to multiply inside roots as benign endophytes and modulate plant growth and development, with implications ranging from enhanced crop productivity to phytoremediation. Endophytic colonization represents an apparent paradox of plant innate immunity because plant cells can detect an array of microbe-associated molecular patterns (also known as MAMPs) to initiate immune responses to terminate microbial multiplication. Several studies attempted to describe the structure of bacterial root endophytes; however, different sampling protocols and low-resolution profiling methods make it difficult to infer general principles. Here we describe methodology to characterize and compare soil- and root-inhabiting bacterial communities, which reveals not only a function for metabolically active plant cells but also for inert cell-wall features in the selection of soil bacteria for host colonization. We show that the roots of Arabidopsis thaliana, grown in different natural soils under controlled environmental conditions, are preferentially colonized by Proteobacteria, Bacteroidetes and Actinobacteria, and each bacterial phylum is represented by a dominating class or family. Soil type defines the composition of root-inhabiting bacterial communities and host genotype determines their ribotype profiles to a limited extent. The identification of soil-type-specific members within the root-inhabiting assemblies supports our conclusion that these represent soil-derived root endophytes. Surprisingly, plant cell-wall features of other tested plant species seem to provide a sufficient cue for the assembly of approximately 40% of the Arabidopsis bacterial root-inhabiting microbiota, with a bias for Betaproteobacteria. Thus, this root sub-community may not be Arabidopsis-specific but saprophytic bacteria that would naturally be found on any plant root or plant debris in the tested soils. By contrast, colonization of Arabidopsis roots by members of the Actinobacteria depends on other cues from metabolically active host cells.

Lrp1/LDL Receptor Play Critical Roles in Mannose 6-Phosphate-Independent Lysosomal Enzyme Targeting.

Most lysosomal enzymes require mannose 6-phosphate (M6P) residues for efficient receptor-mediated lysosomal targeting. Although the lack of M6P residues results in missorting and hypersecretion, selected lysosomal enzymes reach normal levels in lysosomes of various cell types, suggesting the existence of M6P-independent transport routes. Here, we quantify the lysosomal proteome in M6P-deficient mouse fibroblasts (PT(ki)) using Stable Isotope Labeling by Amino acids in Cell culture (SILAC)-based comparative mass spectrometry, and find unchanged amounts of 20% of lysosomal enzymes, including cathepsins D and B (Ctsd and Ctsb). Examination of fibroblasts from a new mouse line lacking both M6P and sortilin, a candidate for M6P-independent transport of lysosomal enzymes, revealed that sortilin does not act as cargo receptor for Ctsb and Ctsd. Using fibroblast lines deficient for endocytic lipoprotein receptors, we could demonstrate that both LDL receptor and Lrp1 mediate the internalization of non-phosphorylated Ctsb and Ctsd. Furthermore, the presence of Lrp1 inhibitor increased the secretion of Ctsd from PT(ki) cells. These findings establish Lrp1 and LDL receptors in M6P-independent secretion-recapture targeting mechanism for lysosomal enzymes.

In silico analysis of the core signaling proteome from the barley powdery mildew pathogen (Blumeria graminis f.sp. hordei).

BACKGROUND: Compared to other ascomycetes, the barley powdery mildew pathogen Blumeria graminis f.sp. hordei (Bgh) has a large genome (ca. 120 Mbp) that harbors a relatively small number of protein-coding genes (ca. 6500). This genomic assemblage is thought to be the result of numerous gene losses, which likely represent an evolutionary adaptation to a parasitic lifestyle in close association with its host plant, barley (Hordeum vulgare). Approximately 8% of the Bgh genes are predicted to encode virulence effectors that are secreted into host tissue and/or cells to promote pathogenesis; the remaining proteome is largely uncharacterized at present. RESULTS: We provide a comparative analysis of the conceptual Bgh proteome, with an emphasis on proteins with known roles in fungal development and pathogenicity, for example heterotrimeric G proteins and G protein coupled receptors; components of calcium and cAMP signaling; small monomeric GTPases; mitogen-activated protein cascades and transcription factors. The predicted Bgh proteome lacks a number of proteins that are otherwise conserved in filamentous fungi, including two proteins that are required for the formation of anastomoses (somatic hyphal connections). By contrast, apart from minor modifications, all major canonical signaling pathways are retained in Bgh. A family of kinases that preferentially occur in pathogenic species of the fungal clade Leotiomyceta is unusually expanded in Bgh and its close relative, Blumeria graminis f.sp. tritici. CONCLUSIONS: Our analysis reveals characteristic features of the proteome of a fungal phytopathogen that occupies an extreme habitat: the living plant cell.

Identification of functional cis-regulatory elements by sequential enrichment from a randomized synthetic DNA library.

BACKGROUND: The identification of endogenous cis-regulatory DNA elements (CREs) responsive to endogenous and environmental cues is important for studying gene regulation and for biotechnological applications but is labor and time intensive. Alternatively, by taking a synthetic biology approach small specific DNA binding sites tailored to the needs of the scientist can be generated and rapidly identified. RESULTS: Here we report a novel approach to identify stimulus-responsive synthetic CREs (SynCREs) from an unbiased random synthetic element (SynE) library. Functional SynCREs were isolated by screening the SynE libray for elements mediating transcriptional activity in plant protoplasts. Responsive elements were chromatin immunoprecipitated by targeting the active Ser-5 phosphorylated RNA polymerase II CTD (Pol II ChIP). Using sequential enrichment, deep sequencing and a bioinformatics pipeline, candidate responsive SynCREs were identified within a pool of constitutively active DNA elements and further validated. These included bonafide biotic/abiotic stress-responsive motifs along with novel SynCREs. We tested several SynCREs in Arabidopsis and confirmed their response to biotic stimuli. CONCLUSIONS: Successful isolation of synthetic stress-responsive elements from our screen illustrates the power of the described methodology. This approach can be applied to any transfectable eukaryotic system since it exploits a universal feature of the eukaryotic Pol II.

Fine mapping and chromosome walking towards the Ror1 locus in barley (Hordeum vulgare L.).

KEY MESSAGE: The Ror1 gene was fine-mapped to the pericentric region of barley chromosome 1HL. Recessively inherited loss-of-function alleles of the barley (Hordeum vulgare) Mildew resistance locus o (Mlo) gene confer durable broad-spectrum disease resistance against the obligate biotrophic fungal powdery mildew pathogen Blumeria graminis f.sp. hordei. Previous genetic analyses revealed two barley genes, Ror1 and Ror2, that are Required for mlo-specified resistance and basal defence. While Ror2 was cloned and shown to encode a t-SNARE protein (syntaxin), the molecular nature or Ror1 remained elusive. Ror1 was previously mapped to the centromeric region of the long arm of barley chromosome 1H. Here, we narrowed the barley Ror1 interval to 0.18 cM and initiated a chromosome walk using barley yeast artificial chromosome (YAC) clones, next-generation DNA sequencing and fluorescence in situ hybridization. Two non-overlapping YAC contigs containing Ror1 flanking genes were identified. Despite a high degree of synteny observed between barley and the sequenced genomes of the grasses rice (Oryza sativa), Brachypodium distachyon and Sorghum bicolor across the wider chromosomal area, the genes in the YAC contigs showed extensive interspecific rearrangements in orientation and order. Consequently, the position of a Ror1 homolog in these species could not be precisely predicted, nor was a barley gene co-segregating with Ror1 identified. These factors have prevented the molecular identification of the Ror1 gene for the time being.

Mouse models for hereditary spastic paraplegia uncover a role of PI4K2A in autophagic lysosome reformation.

Hereditary spastic paraplegia (HSP) denotes genetically heterogeneous disorders characterized by leg spasticity due to degeneration of corticospinal axons. SPG11 and SPG15 have a similar clinical course and together are the most prevalent autosomal recessive HSP entity. The respective proteins play a role for macroautophagy/autophagy and autophagic lysosome reformation (ALR). Here, we report that spg11 and zfyve26 KO mice developed motor impairments within the same course of time. This correlated with enhanced accumulation of autofluorescent material in neurons and progressive neuron loss. In agreement with defective ALR, tubulation events were diminished in starved KO mouse embryonic fibroblasts (MEFs) and lysosomes decreased in neurons of KO brain sections. Confirming that both proteins act in the same molecular pathway, the pathologies were not aggravated upon simultaneous disruption of both. We further show that PI4K2A (phosphatidylinositol 4-kinase type 2 alpha), which phosphorylates phosphatidylinositol to phosphatidylinositol-4-phosphate (PtdIns4P), accumulated in autofluorescent deposits isolated from KO but not WT brains. Elevated PI4K2A abundance was already found at autolysosomes of neurons of presymptomatic KO mice. Immunolabelings further suggested higher levels of PtdIns4P at LAMP1-positive structures in starved KO MEFs. An increased association with LAMP1-positive structures was also observed for clathrin and DNM2/dynamin 2, which are important effectors of ALR recruited by phospholipids. Because PI4K2A overexpression impaired ALR, while its knockdown increased tubulation, we conclude that PI4K2A modulates phosphoinositide levels at autolysosomes and thus the recruitment of downstream effectors of ALR. Therefore, PI4K2A may play an important role in the pathogenesis of SPG11 and SPG15.Abbreviations: ALR: autophagic lysosome reformation; AP-5: adaptor protein complex 5; BFP: blue fluorescent protein; dKO: double knockout; EBSS: Earle's balanced salt solution; FBA: foot base angle; GFP: green fluorescent protein; HSP: hereditary spastic paraplegia; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; SQSTM1/p62: sequestosome 1; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RFP: red fluorescent protein; SPG: spastic paraplegia gene; TGN: trans-Golgi network; WT: wild type.

Evolution of spliceosomal introns following endosymbiotic gene transfer.

BACKGROUND: Spliceosomal introns are an ancient, widespread hallmark of eukaryotic genomes. Despite much research, many questions regarding the origin and evolution of spliceosomal introns remain unsolved, partly due to the difficulty of inferring ancestral gene structures. We circumvent this problem by using genes originated by endosymbiotic gene transfer, in which an intron-less structure at the time of the transfer can be assumed. RESULTS: By comparing the exon-intron structures of 64 mitochondrial-derived genes that were transferred to the nucleus at different evolutionary periods, we can trace the history of intron gains in different eukaryotic lineages. Our results show that the intron density of genes transferred relatively recently to the nuclear genome is similar to that of genes originated by more ancient transfers, indicating that gene structure can be rapidly shaped by intron gain after the integration of the gene into the genome and that this process is mainly determined by forces acting specifically on each lineage. We analyze 12 cases of mitochondrial-derived genes that have been transferred to the nucleus independently in more than one lineage. CONCLUSIONS: Remarkably, the proportion of shared intron positions that were gained independently in homologous genes is similar to that proportion observed in genes that were transferred prior to the speciation event and whose shared intron positions might be due to vertical inheritance. A particular case of parallel intron gain in the nad7 gene is discussed in more detail.

Transcriptome and Parasitome Analysis of Beet Cyst Nematode Heterodera schachtii.

Beet cyst nematodes depend on a set of secretory proteins (effectors) for the induction and maintenance of their syncytial feeding sites in plant roots. In order to understand the relationship between the beet cyst nematode H. schachtii and its host, identification of H. schachtii effectors is crucial and to this end, we sequenced a whole animal pre-infective J2-stage transcriptome in addition to pre- and post-infective J2 gland cell transcriptome using Next Generation Sequencing (NGS) and identified a subset of sequences representing putative effectors. Comparison between the transcriptome of H. schachtii and previously reported related cyst nematodes and root-knot nematodes revealed a subset of esophageal gland related sequences and putative effectors in common across the tested species. Structural and functional annotation of H. schachtii transcriptome led to the identification of nearly 200 putative effectors. Six putative effector expressions were quantified using qPCR and three of them were functionally analyzed using RNAi. Phenotyping of the RNAi nematodes indicated that all tested genes decrease the level of nematodes pathogenicity and/or the average female size, thereby regulating cyst nematode parasitism. These discoveries contribute to further understanding of the cyst nematode parasitism.

MIC26 and MIC27 cooperate to regulate cardiolipin levels and the landscape of OXPHOS complexes.

Homologous apolipoproteins of MICOS complex, MIC26 and MIC27, show an antagonistic regulation of their protein levels, making it difficult to deduce their individual functions using a single gene deletion. We obtained single and double knockout (DKO) human cells of MIC26 and MIC27 and found that DKO show more concentric onion-like cristae with loss of CJs than any single deletion indicating overlapping roles in formation of CJs. Using a combination of complexome profiling, STED nanoscopy, and blue-native gel electrophoresis, we found that MIC26 and MIC27 are dispensable for the stability and integration of the remaining MICOS subunits into the complex suggesting that they assemble late into the MICOS complex. MIC26 and MIC27 are cooperatively required for the integrity of respiratory chain (super) complexes (RCs/SC) and the F1Fo-ATP synthase complex and integration of F1 subunits into the monomeric F1Fo-ATP synthase. While cardiolipin was reduced in DKO cells, overexpression of cardiolipin synthase in DKO restores the stability of RCs/SC. Overall, we propose that MIC26 and MIC27 are cooperatively required for global integrity and stability of multimeric OXPHOS complexes by modulating cardiolipin levels.

Genome history in the symbiotic hybrid Euglena gracilis.

Euglena gracilis has a chimeric gene collection in which some genes were inherited from its heterotrophic host and others were acquired from a photoautotrophic endosymbiont during secondary endosymbiosis. The evolutionary reconstruction of such a hybrid genome poses a challenge for standard phylogenetic tools that produce bifurcating trees because genome evolution by endosymbiotic gene transfer is a non tree-like process. We sequenced 2770 ESTs from E. gracilis, of which 841 have homologues in a sample of other eukaryotes. Most of these homologues are found in all of the eukaryotes in our sample, but 117 of them are specific to photoautotrophic eukaryotes. A phylogenetic tree fails to account for this observation but the distribution of homologues and a phylogenetic network clearly show the common origin of E. gracilis from both kinetoplastid and photoautotrophic ancestors.

Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements.

The complete sequence of the 1,267,782 bp genome of Wolbachia pipientis wMel, an obligate intracellular bacteria of Drosophila melanogaster, has been determined. Wolbachia, which are found in a variety of invertebrate species, are of great interest due to their diverse interactions with different hosts, which range from many forms of reproductive parasitism to mutualistic symbioses. Analysis of the wMel genome, in particular phylogenomic comparisons with other intracellular bacteria, has revealed many insights into the biology and evolution of wMel and Wolbachia in general. For example, the wMel genome is unique among sequenced obligate intracellular species in both being highly streamlined and containing very high levels of repetitive DNA and mobile DNA elements. This observation, coupled with multiple evolutionary reconstructions, suggests that natural selection is somewhat inefficient in wMel, most likely owing to the occurrence of repeated population bottlenecks. Genome analysis predicts many metabolic differences with the closely related Rickettsia species, including the presence of intact glycolysis and purine synthesis, which may compensate for an inability to obtain ATP directly from its host, as Rickettsia can. Other discoveries include the apparent inability of wMel to synthesize lipopolysaccharide and the presence of the most genes encoding proteins with ankyrin repeat domains of any prokaryotic genome yet sequenced. Despite the ability of wMel to infect the germline of its host, we find no evidence for either recent lateral gene transfer between wMel and D. melanogaster or older transfers between Wolbachia and any host. Evolutionary analysis further supports the hypothesis that mitochondria share a common ancestor with the alpha-Proteobacteria, but shows little support for the grouping of mitochondria with species in the order Rickettsiales. With the availability of the complete genomes of both species and excellent genetic tools for the host, the wMel-D. melanogaster symbiosis is now an ideal system for studying the biology and evolution of Wolbachia infections.

Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism.

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.

Mutational decay and age of chloroplast and mitochondrial genomes transferred recently to angiosperm nuclear chromosomes.

Transfers of organelle DNA to the nucleus established several thousand functional genes in eukaryotic chromosomes over evolutionary time. Recent transfers have also contributed nonfunctional plastid (pt)- and mitochondrion (mt)-derived DNA (termed nupts and numts, respectively) to plant nuclear genomes. The two largest transferred organelle genome copies are 131-kb nuptDNA in rice (Oryza sativa) and 262-kb numtDNA in Arabidopsis (Arabidopsis thaliana). These transferred copies were compared in detail with their bona fide organelle counterparts, to which they are 99.77% and 99.91% identical, respectively. No evidence for purifying selection was found in either nuclear integrant, indicating that they are nonfunctional. Mutations attributable to 5-methylcytosine hypermutation have occurred at a 6- to 10-fold higher rate than other point mutations in Arabidopsis numtDNA and rice nuptDNA, respectively, revealing this as a major mechanism of mutational decay for these transferred organelle sequences. Short indels occurred preferentially within homopolymeric stretches but were less frequent than point mutations. The 131-kb nuptDNA is absent in the O. sativa subsp. indica or Oryza rufipogon nuclear genome, suggesting that it was transferred within the O. sativa subsp. japonica lineage and, as revealed by sequence comparisons, after its divergence from the indica chloroplast lineage. The time of the transfer for the rice nupt was estimated as 148,000 (74,000--296,000) years ago and that for the Arabidopsis numtDNA as 88,000 (44,000--176,000) years ago. The results reveal transfer and integration of entire organelle genomes into the nucleus as an ongoing evolutionary process and uncover mutational mechanisms affecting organelle genomes recently transferred into a new mutational environment.