Anna Gogleva

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.

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)).

Comparative analysis of CRISPR cassettes from the human gut metagenomic contigs

BackgroundCRISPR (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.ResultsWe 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.ConclusionsThe 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.

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.

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

Significance Despite the importance of liverworts as the earliest diverging land plant lineage to support fungal symbiosis, it is unknown whether filamentous pathogens can establish intracellular interactions within living cells of these nonvascular plants. Here, we demonstrate that an oomycete pathogen invades Marchantia polymorpha and related liverworts to form intracellular infection structures inside cells of the photosynthetic layer. Plants lacking this tissue layer display enhanced resistance to infection, revealing an architectural susceptibility factor in complex thalloid liverworts. Moreover, we show that dedicated host cellular trafficking proteins are recruited to pathogen interfaces within liverwort cells, supporting the idea that intracellular responses to microbial invasion originated in nonvascular plants. 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.

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

BackgroundPlant-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.ResultsWe 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.ConclusionsThese 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.

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

Background Plant-pathogenic oomycetes are responsible for economically important losses on crops worldwide. Phytophthora palmivora, a broad-host-range tropical relative of the potato late blight pathogen, causes rotting diseases in many important 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 understanding of 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 N. benthamiana to study the P. palmivora root infections at the cellular and molecular level. 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.

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

The leaf epidermal wall is covered by a cuticle, composed of cutin and waxes, which protects against dehydration and constitutes a barrier against pathogen attack. Cutin monomers are formed by the transfer of 16- or 18-carbon fatty acids to glycerol by glycerol-3-phosphate acyltransferase (GPAT) enzymes, which facilitates their transport to the plant surface. Here we address the dual functionality of pathogen-inducible Glycerol phosphate acyltransferase 6 (GPAT6) in controlling pathogen entry and dehydration in leaves. Silencing of Nicotiana benthamiana NbGPAT6a increased leaf susceptibility to the oomycetes Phytophthora infestans and P. palmivora, whereas stable overexpression of NbGPAT6a-GFP rendered leaves more resistant to infection. A loss-of-function mutation of the orthologous gene in tomato (Solanum lycopersicum), SlGPAT6, similarly resulted in increased susceptibility of leaves to Phytophthora infection concomitant with altered intracellular infection structure morphology. Conversely, Botrytis cinerea disease symptoms were reduced. Modulation of GPAT6 expression predominantly altered the outer cell wall of leaf epidermal cells. The impaired cell wall-cuticle continuum of tomato gpat6-a mutants resulted in increased water loss and these plants had fewer stomata. Our work highlights a hitherto unknown role for GPAT6-generated cutin monomers in controlling epidermal cell properties that are integral to leaf-microbe interactions and limit dehydration.