Knowledge graph-based recommendation framework identifies drivers of resistance in EGFR mutant non-small cell lung cancer.

Resistance to EGFR inhibitors (EGFRi) presents a major obstacle in treating non-small cell lung cancer (NSCLC). One of the most exciting new ways to find potential resistance markers involves running functional genetic screens, such as CRISPR, followed by manual triage of significantly enriched genes. This triage process to identify 'high value' hits resulting from the CRISPR screen involves manual curation that requires specialized knowledge and can take even experts several months to comprehensively complete. To find key drivers of resistance faster we build a recommendation system on top of a heterogeneous biomedical knowledge graph integrating pre-clinical, clinical, and literature evidence. The recommender system ranks genes based on trade-offs between diverse types of evidence linking them to potential mechanisms of EGFRi resistance. This unbiased approach identifies 57 resistance markers from >3,000 genes, reducing hit identification time from months to minutes. In addition to reproducing known resistance markers, our method identifies previously unexplored resistance mechanisms that we prospectively validate.

The Genome of Peronospora belbahrii Reveals High Heterozygosity, a Low Number of Canonical Effectors, and TC-Rich Promoters.

Along with Plasmopara destructor, Peronosopora belbahrii has arguably been the economically most important newly emerging downy mildew pathogen of the past two decades. Originating from Africa, it has started devastating basil production throughout the world, most likely due to the distribution of infested seed material. Here, we present the genome of this pathogen and results from comparisons of its genomic features to other oomycetes. The assembly of the nuclear genome was around 35.4 Mbp in length, with an N50 scaffold length of around 248 kbp and an L50 scaffold count of 46. The circular mitochondrial genome consisted of around 40.1 kbp. From the repeat-masked genome, 9,049 protein-coding genes were predicted, out of which 335 were predicted to have extracellular functions, representing the smallest secretome so far found in peronosporalean oomycetes. About 16% of the genome consists of repetitive sequences, and, based on simple sequence repeat regions, we provide a set of microsatellites that could be used for population genetic studies of P. belbahrii. P. belbahrii has undergone a high degree of convergent evolution with other obligate parasitic pathogen groups, reflecting its obligate biotrophic lifestyle. Features of its secretome, signaling networks, and promoters are presented, and some patterns are hypothesized to reflect the high degree of host specificity in Peronospora species. In addition, we suggest the presence of additional virulence factors apart from classical effector classes that are promising candidates for future functional studies.

Conserved Biochemical Defenses Underpin Host Responses to Oomycete Infection in an Early-Divergent Land Plant Lineage.

The expansion of plants onto land necessitated the evolution of robust defense strategies to protect against a wide array of microbial invaders. Whereas host responses to microbial colonization are extensively explored in evolutionarily young land plant lineages such as angiosperms, we know relatively little about plant-pathogen interactions in early-diverging land plants thought to better represent the ancestral state. Here, we define the transcriptional and proteomic response of the early-divergent liverwort Marchantia polymorpha to infection with the oomycete pathogen Phytophthora palmivora. We uncover a robust molecular response to oomycete colonization in Marchantia that consists of conserved land plant gene families. Direct macroevolutionary comparisons of host infection responses in Marchantia and the model angiosperm Nicotiana benthamiana further reveal a shared set of orthologous microbe-responsive genes that include members of the phenylpropanoid metabolic pathway. In addition, we identify a role for the Marchantia R2R3-MYB transcription factor MpMyb14 in activating phenylpropanoid (flavonoid) biosynthesis during oomycete infection. Mpmyb14 mutants infected with P. palmivora fail to activate phenylpropanoid biosynthesis gene expression and display enhanced disease susceptibility compared to wild-type plants. Conversely, the ectopic induction of MpMyb14 led to the accumulation of anthocyanin-like pigments and dramatically enhanced liverwort resistance to P. palmivora infection. Collectively, our results demonstrate that the Marchantia response to oomycete infection displays evolutionarily conserved features indicative of an ancestral pathogen deterrence strategy centered on phenylpropanoid-mediated biochemical defenses.

Glycerol-3-phosphate acyltransferase 6 controls filamentous pathogen interactions and cell wall properties of the tomato and Nicotiana benthamiana leaf epidermis.

The leaf outer epidermal cell wall acts as a barrier against pathogen attack and desiccation, and as such is covered by a cuticle, composed of waxes and the polymer cutin. Cutin monomers are formed by the transfer of fatty acids to glycerol by glycerol-3-phosphate acyltransferases, which facilitate their transport to the surface. The extent to which cutin monomers affect leaf cell wall architecture and barrier properties is not known. We report a dual functionality of pathogen-inducible GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE 6 (GPAT6) in controlling pathogen entry and cell wall properties affecting dehydration in leaves. Silencing of Nicotiana benthamiana NbGPAT6a increased leaf susceptibility to infection by the oomycetes Phytophthora infestans and Phytophthora palmivora, whereas overexpression of NbGPAT6a-GFP rendered leaves more resistant. A loss-of-function mutation in tomato SlGPAT6 similarly resulted in increased susceptibility of leaves to Phytophthora infection, concomitant with changes in haustoria morphology. Modulation of GPAT6 expression altered the outer wall diameter of leaf epidermal cells. Moreover, we observed that tomato gpat6-a mutants had an impaired cell wall-cuticle continuum and fewer stomata, but showed increased water loss. This study highlights a hitherto unknown role for GPAT6-generated cutin monomers in influencing epidermal cell properties that are integral to leaf-microbe interactions and in limiting dehydration.

Phytophthora palmivora establishes tissue-specific intracellular infection structures in the earliest divergent land plant lineage.

The expansion of plants onto land was a formative event that brought forth profound changes to the earth's geochemistry and biota. Filamentous eukaryotic microbes developed the ability to colonize plant tissues early during the evolution of land plants, as demonstrated by intimate, symbiosis-like associations in >400 million-year-old fossils. However, the degree to which filamentous microbes establish pathogenic interactions with early divergent land plants is unclear. Here, we demonstrate that the broad host-range oomycete pathogen Phytophthora palmivora colonizes liverworts, the earliest divergent land plant lineage. We show that P. palmivora establishes a complex tissue-specific interaction with Marchantia polymorpha, where it completes a full infection cycle within air chambers of the dorsal photosynthetic layer. Remarkably, P. palmivora invaginates M. polymorpha cells with haustoria-like structures that accumulate host cellular trafficking machinery and the membrane syntaxin MpSYP13B, but not the related MpSYP13A. Our results indicate that the intracellular accommodation of filamentous microbes is an ancient plant trait that is successfully exploited by pathogens like P. palmivora.

SecretSanta: flexible pipelines for functional secretome prediction.

Motivation: The secretome denotes the collection of secreted proteins exported outside of the cell. The functional roles of secreted proteins include the maintenance and remodelling of the extracellular matrix as well as signalling between host and non-host cells. These features make secretomes rich reservoirs of biomarkers for disease classification and host-pathogen interaction studies. Common biomarkers are extracellular proteins secreted via classical pathways that can be predicted from sequence by annotating the presence or absence of N-terminal signal peptides. Several heterogeneous command line tools and web-interfaces exist to identify individual motifs, signal sequences and domains that are either characteristic or strictly excluded from secreted proteins. However, a single flexible secretome-prediction workflow that combines all analytic steps is still missing. Results: To bridge this gap the SecretSanta package implements wrapper and parser functions around established command line tools for the integrative prediction of extracellular proteins that are secreted via classical pathways. The modularity of SecretSanta enables users to create tailored pipelines and apply them across the whole tree of life to facilitate comparison of secretomes across multiple species or under various conditions. Availability and implementation: SecretSanta is implemented in the R programming language and is released under GPL-3 license. All functions have been optimized and parallelized to allow large-scale processing of sequences. The open-source code, installation instructions and vignette with use case scenarios can be downloaded from https://github.com/gogleva/SecretSanta. Supplementary information: Supplementary data are available at Bioinformatics online.

Time-resolved dual transcriptomics reveal early induced Nicotiana benthamiana root genes and conserved infection-promoting Phytophthora palmivora effectors.

BACKGROUND: Plant-pathogenic oomycetes are responsible for economically important losses in crops worldwide. Phytophthora palmivora, a tropical relative of the potato late blight pathogen, causes rotting diseases in many tropical crops including papaya, cocoa, oil palm, black pepper, rubber, coconut, durian, mango, cassava and citrus. Transcriptomics have helped to identify repertoires of host-translocated microbial effector proteins which counteract defenses and reprogram the host in support of infection. As such, these studies have helped in understanding how pathogens cause diseases. Despite the importance of P. palmivora diseases, genetic resources to allow for disease resistance breeding and identification of microbial effectors are scarce. RESULTS: We employed the model plant Nicotiana benthamiana to study the P. palmivora root infections at the cellular and molecular levels. Time-resolved dual transcriptomics revealed different pathogen and host transcriptome dynamics. De novo assembly of P. palmivora transcriptome and semi-automated prediction and annotation of the secretome enabled robust identification of conserved infection-promoting effectors. We show that one of them, REX3, suppresses plant secretion processes. In a survey for early transcriptionally activated plant genes we identified a N. benthamiana gene specifically induced at infected root tips that encodes a peptide with danger-associated molecular features. CONCLUSIONS: These results constitute a major advance in our understanding of P. palmivora diseases and establish extensive resources for P. palmivora pathogenomics, effector-aided resistance breeding and the generation of induced resistance to Phytophthora root infections. Furthermore, our approach to find infection-relevant secreted genes is transferable to other pathogen-host interactions and not restricted to plants.

Comparative analysis of CRISPR cassettes from the human gut metagenomic contigs.

BACKGROUND: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a prokaryotic adaptive defence system that provides resistance against alien replicons such as viruses and plasmids. Spacers in a CRISPR cassette confer immunity against viruses and plasmids containing regions complementary to the spacers and hence they retain a footprint of interactions between prokaryotes and their viruses in individual strains and ecosystems. The human gut is a rich habitat populated by numerous microorganisms, but a large fraction of these are unculturable and little is known about them in general and their CRISPR systems in particular. RESULTS: We used human gut metagenomic data from three open projects in order to characterize the composition and dynamics of CRISPR cassettes in the human-associated microbiota. Applying available CRISPR-identification algorithms and a previously designed filtering procedure to the assembled human gut metagenomic contigs, we found 388 CRISPR cassettes, 373 of which had repeats not observed previously in complete genomes or other datasets. Only 171 of 3,545 identified spacers were coupled with protospacers from the human gut metagenomic contigs. The number of matches to GenBank sequences was negligible, providing protospacers for 26 spacers.Reconstruction of CRISPR cassettes allowed us to track the dynamics of spacer content. In agreement with other published observations we show that spacers shared by different cassettes (and hence likely older ones) tend to the trailer ends, whereas spacers with matches in the metagenomes are distributed unevenly across cassettes, demonstrating a preference to form clusters closer to the active end of a CRISPR cassette, adjacent to the leader, and hence suggesting dynamical interactions between prokaryotes and viruses in the human gut. Remarkably, spacers match protospacers in the metagenome of the same individual with frequency comparable to a random control, but may match protospacers from metagenomes of other individuals. CONCLUSIONS: The analysis of assembled contigs is complementary to the approach based on the analysis of original reads and hence provides additional data about composition and evolution of CRISPR cassettes, revealing the dynamics of CRISPR-phage interactions in metagenomes.

Methylophilus glucosoxydans sp. nov., a restricted facultative methylotroph from rice rhizosphere.

Two restricted facultatively methylotrophic strains, designed B(T) and P, were isolated from rice roots. The isolates were strictly aerobic, Gram-negative, asporogenous, mesophilic, neutrophilic, motile rods that multiplied by binary fission and were able to synthesize indole-3-acetate. The cellular fatty acid profiles of the two strains were dominated by C(16:0), C(16:1)ω7c and C(16:0) 2-OH. The major ubiquinone was Q-8. The predominant phospholipids were phosphatidylethanolamine and phosphatidylglycerol. Cardiolipin (diphosphatidylglycerol) was absent. The two strains assimilated methanol carbon at the level of formaldehyde via the ribulose monophosphate cycle (2-keto-3-deoxy-6-phosphogluconate variant). They lacked α-ketoglutarate dehydrogenase and glutamate dehydrogenase. They assimilated ammonium via the glutamate cycle enzymes glutamine synthetase and glutamate synthase. The DNA G+C contents of strains B(T) and P were 52.5 and 51.5 mol% (T(m)), respectively. The level of DNA-DNA reassociation between these strains was 78%, indicating that they belong to one species. Phylogenetic analysis of strain B(T) based on 16S rRNA and methanol dehydrogenase (mxaF) gene sequences showed a high level of similarity to members of the genus Methylophilus. As the two isolates were clearly distinct from all recognized members of the genus Methylophilus based on phenotypic data and levels of DNA-DNA relatedness (30-46%), they are considered to represent a novel species, for which the name Methylophilus glucosoxydans sp. nov. is proposed; the type strain is B(T) (=VKM B-1607(T)=CCUG 59685(T)=DSM 5898(T)).

Methylobacillus arboreus sp. nov., and Methylobacillus gramineus sp. nov., novel non-pigmented obligately methylotrophic bacteria associated with plants.

Two newly isolated obligate methanol-utilizing bacteria (strains Iva(T) and Lap(T)) with the ribulose monophosphate pathway of C(1) assimilation are described. The isolates are strictly aerobic, Gram negative, asporogenous, motile rods multiplying by binary fission, mesophilic and neutrophilic, synthesize indole-3-acetate. The prevailing cellular fatty acids are straight-chain saturated C(16:0) and unsaturated C(16:1) acids. The major ubiquinone is Q-8. The predominant phospholipids are phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Ammonia is assimilated by glutamate dehydrogenase. The DNA G+C contents of strains Iva(T) and Lap(T) are 54.0 and 50.5mol% (T(m)), respectively. Based on 16S rRNA gene sequence analysis and DNA-DNA relatedness (38-45%) with type strains of the genus Methylobacillus, the novel isolates are classified as the new species of this genus and named Methylobacillus arboreus Iva(T) (VKM B-2590(T), CCUG 59684(T), DSM 23628(T)) and Methylobacillus gramineus Lap(T) (VKM B-2591(T), CCUG 59687(T), DSM 23629(T)). The GenBank accession numbers for the 16S rRNA gene and mxaF gene sequences of the strains Iva(T) and Lap(T) are GU937479, GU937478 and HM030736, HM030735, respectively.

Methylophilus flavus sp. nov. and Methylophilus luteus sp. nov., aerobic, methylotrophic bacteria associated with plants.

Novel yellow, obligately methylotrophic and restricted facultatively methylotrophic bacteria, respectively designated strains Ship(T) and Mim(T), with the ribulose monophosphate pathway of C(1) assimilation are described. Cells were strictly aerobic, Gram-negative, asporogenous, non-motile rods that multiply by binary fission, were mesophilic and neutrophilic and synthesized indole-3-acetic acid and exopolysaccharide. The predominant cellular fatty acids were C(16 : 0) and C(16 : 1). The major ubiquinone was Q-8. The predominant phospholipids were phosphatidylethanolamine and phosphatidylglycerol; diphosphatidylglycerol was absent. The two strains lacked α-ketoglutarate dehydrogenase and glutamate dehydrogenase. They assimilated ammonium via the glutamate cycle enzymes glutamine synthetase and glutamate synthase. The DNA G+C contents of strains Ship(T) and Mim(T) were 50.7 and 54.5 mol% (T(m)), respectively. The level of 16S rRNA gene sequence similarity between these strains was very high (99.8 %) but they shared a low level of DNA-DNA relatedness (44 %). Based on 16S rRNA gene sequence analysis and low levels of DNA-DNA relatedness with the type strains of recognized species of the genus Methylophilus (31-36 %), strains Ship(T) and Mim(T) are considered to represent novel species of the genus Methylophilus, for which the names Methylophilus flavus sp. nov. (type strain Ship(T) =DSM 23073(T) =VKM B-2547(T) =CCUG 58411(T)) and Methylophilus luteus sp. nov. (type strain Mim(T) =DSM 22949(T) =VKM B-2548(T) =CCUG 58412(T)) are proposed.