Luteibacter mycovicinus sp. nov., a yellow-pigmented gammaproteobacterium found as an endohyphal symbiont of endophytic Ascomycota.

We isolated and described a yellow-pigmented strain of bacteria (strain 9143T), originally characterized as an endohyphal inhabitant of an endophytic fungus in the Ascomycota. Although the full-length sequence of its 16S rRNA gene displays 99 % similarity to Luteibacter pinisoli, genomic hybridization demonstrated <30 % genomic similarity between 9143T and its closest named relatives, further supported by average nucleotide identity results. This and related endohyphal strains form a well-supported clade separate from L. pinisoli and other validly named species including the most closely related Luteibacter rhizovicinus. The name Luteibacter mycovicinus sp. nov. is proposed, with type strain 9143T (isolate DBL433), for which a genome has been sequenced and is publicly available from the American Type Culture Collection (ATCC TSD-257T) and from the Leibniz Institute DSMZ (DSM 112764T). The type strain reliably forms yellow colonies across diverse media and growth conditions (lysogeny broth agar, King's Medium B, potato dextrose agar, trypticase soy agar and Reasoner's 2A (R2A) agar). It forms colonies readily at 27 °C on agar with a pH of 6-8, and on salt (NaCl) concentrations up to 2 %. It lacks the ability to utilize sulphate as a sulphur source and thus only forms colonies on minimal media if supplemented with alternative sulphur sources. It is catalase-positive and oxidase-negative. Although it exhibits a single polar flagellum, motility was only clearly visible on R2A agar. Its host range and close relatives, which share the endohyphal lifestyle, are discussed.

A TAL effector-like protein of an endofungal bacterium increases the stress tolerance and alters the transcriptome of the host.

Symbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis of Mycetohabitans (formerly Burkholderia) rhizoxinica with the fungus Rhizopus microsporus, bacterial type III (T3) secretion is known to be essential. Proteins resembling T3-secreted transcription activator-like (TAL) effectors of plant pathogenic bacteria are encoded in the three sequenced Mycetohabitans spp. genomes. TAL effectors nuclear-localize in plants, where they bind and activate genes important in disease. The Burkholderia TAL-like (Btl) proteins bind DNA but lack the N- and C-terminal regions, in which TAL effectors harbor their T3 and nuclear localization signals, and activation domain. We characterized a Btl protein, Btl19-13, and found that, despite the structural differences, it can be T3-secreted and can nuclear-localize. A btl19-13 gene knockout did not prevent the bacterium from infecting the fungus, but the fungus became less tolerant to cell membrane stress. Btl19-13 did not alter transcription in a plant-based reporter assay, but 15 R. microsporus genes were differentially expressed in comparisons both of the fungus infected with the wild-type bacterium vs. the mutant and with the mutant vs. a complemented strain. Southern blotting revealed btl genes in 14 diverse Mycetohabitans isolates. However, banding patterns and available sequences suggest variation, and the btl19-13 phenotype could not be rescued by a btl gene from a different strain. Our findings support the conclusion that Btl proteins are effectors that act on host DNA and play important but varied or possibly host genotype-specific roles in the M. rhizoxinica-R. microsporus symbiosis.

Cruciferous Weed Isolates of Xanthomonas campestris Yield Insight into Pathovar Genomic Relationships and Genetic Determinants of Host and Tissue Specificity.

Pathovars of Xanthomonas campestris cause distinct diseases on different brassicaceous hosts. The genomic relationships among pathovars as well as the genetic determinants of host range and tissue specificity remain poorly understood despite decades of research. Here, leveraging advances in multiplexed long-read technology, we fully sequenced the genomes of a collection of X. campestris strains isolated from cruciferous crops and weeds in New York and California as well as strains from global collections, to investigate pathovar relationships and candidate genes for host- and tissue-specificity. Pathogenicity assays and genomic comparisons across this collection and publicly available X. campestris genomes revealed a correlation between pathovar and genomic relatedness and provide support for X. campestris pv. barbareae, the validity of which had been questioned. Linking strain host range with type III effector repertoires identified AvrAC (also 'XopAC') as a candidate host-range determinant, preventing infection of Matthiola incana, and this was confirmed experimentally. Furthermore, the presence of a copy of the cellobiosidase gene cbsA with coding sequence for a signal peptide was found to correlate with the ability to infect vascular tissues, in agreement with a previous study of diverse Xanthomonas species; however, heterologous expression in strains lacking the gene gave mixed results, indicating that factors in addition to cbsA influence tissue specificity of X. campestris pathovars. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Molecular Mechanism & Structure-Zooming in on Plant Immunity.

The first of three International Society for Molecular Plant-Microbe Interactions (IS-MPMI) eSymposia was convened on 12 and 13 July 2021, with the theme "Molecular Mechanism & Structure-Zooming in on Plant Immunity". Hosted by Jian-Min Zhou (Beijing, China) and Jane Parker (Cologne, Germany), the eSymposium centered on "Top 10 Unanswered Questions in MPMI" number five: Does effector-triggered immunity (ETI) potentiate and restore pattern-triggered immunity (PTI)-or is there really a binary distinction between ETI and PTI? Since the previous International Congress of IS-MPMI in 2019, substantial progress has been made in untangling the complex signaling underlying plant immunity, including a greater understanding of the structure and function of key proteins. A clear need emerged for the MPMI community to come together virtually to share new knowledge around plant immunity. Over the course of two synchronous, half days of programming, participants from 32 countries attended two plenary sessions with engaging panel discussions and networked through interactive hours and poster breakout rooms. In this report, we summarize the concerted effort by multiple laboratories to study the molecular mechanisms underlying ETI and PTI, highlighting the essential role of plant resistosomes in the formation of calcium channels during an immune response. We conclude our report by forming new questions about how overlapping signaling mechanisms are controlled.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Insular Cortex Projections to Nucleus Accumbens Core Mediate Social Approach to Stressed Juvenile Rats.

Social interactions are shaped by features of the interactants, including age, emotion, sex, and familiarity. Age-specific responses to social affect are evident when an adult male rat is presented with a pair of unfamiliar male conspecifics, one of which is stressed via two foot shocks and the other naive to treatment. Adult test rats prefer to interact with stressed juvenile (postnatal day 30, PN30) conspecifics but avoid stressed adult (PN50) conspecifics. This pattern depends upon the insular cortex (IC), which is anatomically connected to the nucleus accumbens core (NAc). The goal of this work was to test the necessity of IC projections to NAc during social affective behavior. Here, bilateral pharmacological inhibition of the NAc with tetrodotoxin (1 µm; 0.5 µl/side) abolished the preference for stressed PN30, but did not alter interactions with PN50 conspecifics. Using a combination of retrograding tracing and c-Fos immunohistochemistry, we report that social interactions with stressed PN30 conspecifics elicit greater Fos immunoreactivity in IC → NAc neurons than interactions with naive PN30 conspecifics. Chemogenetic stimulation of IC terminals in the NAc increased social exploration with juvenile, but not adult, conspecifics, whereas chemogenetic inhibition of this tract blocked the preference to investigate stressed PN30 conspecifics, which expands upon our previous finding that optogenetic inhibition of IC projection neurons mediated approach and avoidance. These new findings suggest that outputs of IC to the NAc modulate social approach, which provides new insight to the neural circuitry underlying social decision-making.SIGNIFICANCE STATEMENT Social decision-making underlies an animal's behavioral response to others in a range of social contexts. Previous findings indicate the insular cortex (IC) and the nucleus accumbens (NAc) play important roles in social behaviors, and human neuroimaging implicates both IC and NAc in autism and other psychiatric disorders characterized by aberrant social cognition. To test whether IC projections to the NAc are involved in social decision-making, circuit-specific chemogenetic manipulations demonstrated that the IC → NAc pathway mediates social approach toward distressed juvenile, but not adult, conspecifics. This finding is the first to implicate this circuit in rodent socioemotional behaviors and may be a neuroanatomical substrate for integration of emotion with social reward.

Convergent Evolution of Effector Protease Recognition by Arabidopsis and Barley.

The Pseudomonas syringae cysteine protease AvrPphB activates the Arabidopsis resistance protein RPS5 by cleaving a second host protein, PBS1. AvrPphB induces defense responses in other plant species, but the genes and mechanisms mediating AvrPphB recognition in those species have not been defined. Here, we show that AvrPphB induces defense responses in diverse barley cultivars. We also show that barley contains two PBS1 orthologs, that their products are cleaved by AvrPphB, and that the barley AvrPphB response maps to a single locus containing a nucleotide-binding leucine-rich repeat (NLR) gene, which we termed AvrPphB Response 1 (Pbr1). Transient coexpression of PBR1 with wild-type AvrPphB but not with a protease inactive mutant triggered defense responses, indicating that PBR1 detects AvrPphB protease activity. Additionally, PBR1 coimmunoprecipitated with barley and Nicotiana benthamiana PBS1 proteins, suggesting mechanistic similarity to detection by RPS5. Lastly, we determined that wheat cultivars also recognize AvrPphB protease activity and contain two putative Pbr1 orthologs. Phylogenetic analyses showed, however, that Pbr1 is not orthologous to RPS5. Our results indicate that the ability to recognize AvrPphB evolved convergently and imply that selection to guard PBS1-like proteins occurs across species. Also, these results suggest that PBS1-based decoys may be used to engineer protease effector recognition-based resistance in barley and wheat.

A Confounding Effect of Bacterial Titer in a Type III Delivery-Based Assay of Eukaryotic Effector Function.

This letter describes a newly discovered confounding effect of bacterial titer in a previously published type III delivery-based assay of the fungal effector BEC1019. The original publication (Whigham et al. 2015) has been retracted as a consequence of this discovery. Here, we tabulate the affected and unaffected figures and conclusions in the original publication and briefly reflect on potential pitfalls to bear in mind when designing experiments that use bacterial type III secretion to characterize eukaryotic effectors.

Warfarin genotyping with hybridization-induced aggregation on a poly(ethylene terephthalate) microdevice.

Warfarin, a commonly prescribed oral anticoagulant, is burdened by a narrow therapeutic index and high inter-individual variability in response, making it the second leading cause of drug-related emergency room visits. Since genetic factors contribute significantly to warfarin sensitivity, a genotype-guided dosing strategy may reduce the occurrence of adverse events. While numerous methods have been demonstrated for warfarin genotyping, the specifications of most assays with respect to turnaround time and cost are not ideal for routine testing. Here, we present a unique method for warfarin genotyping based on multiplex PCR coupled with Hybridization-induced Aggregation (HIA), a bead-based technique for sequence-specific detection. A multiplex allele-specific PCR reaction was used to generate products corresponding to 3 genetic variants associated with warfarin sensitivity [CYP2C9 *2, CYP2C9 *3, and VKORC1 (1173C>T)] and an internal control product. The products were detected simultaneously on a poly(ethylene terephthalate) (PeT) microdevice using HIA, which provided genotyping results in approximately 15 minutes following PCR. The genotyping results of 23 patient DNA samples using this approach were in 100% concordance with the results of a validated test (WARFGENO test, ARUP laboratories). Additionally, the PCR reaction was successfully transferred to a PeT chip, which provided accurate genotyping results from patient DNA samples in under an hour. This work demonstrates a simple, rapid, and affordable approach to warfarin genotyping based on multiplex allele-specific PCR coupled with HIA detection. By demonstrating both chemistries on PeT microdevices, we show the potential for integration on a single device for sample-to-answer genotyping.

Prevalence and diversity of TAL effector-like proteins in fungal endosymbiotic Mycetohabitans spp.

Endofungal Mycetohabitans (formerly Burkholderia) spp. rely on a type III secretion system to deliver mostly unidentified effector proteins when colonizing their host fungus, Rhizopus microsporus. The one known secreted effector family from Mycetohabitans consists of homologues of transcription activator-like (TAL) effectors, which are used by plant pathogenic Xanthomonas and Ralstonia spp. to activate host genes that promote disease. These 'Burkholderia TAL-like (Btl)' proteins bind corresponding specific DNA sequences in a predictable manner, but their genomic target(s) and impact on transcription in the fungus are unknown. Recent phenotyping of Btl mutants of two Mycetohabitans strains revealed that the single Btl in one Mycetohabitans endofungorum strain enhances fungal membrane stress tolerance, while others in a Mycetohabitans rhizoxinica strain promote bacterial colonization of the fungus. The phenotypic diversity underscores the need to assess the sequence diversity and, given that sequence diversity translates to DNA targeting specificity, the functional diversity of Btl proteins. Using a dual approach to maximize capture of Btl protein sequences for our analysis, we sequenced and assembled nine Mycetohabitans spp. genomes using long-read PacBio technology and also mined available short-read Illumina fungal-bacterial metagenomes. We show that btl genes are present across diverse Mycetohabitans strains from Mucoromycota fungal hosts yet vary in sequences and predicted DNA binding specificity. Phylogenetic analysis revealed distinct clades of Btl proteins and suggested that Mycetohabitans might contain more species than previously recognized. Within our data set, Btl proteins were more conserved across M. rhizoxinica strains than across M. endofungorum, but there was also evidence of greater overall strain diversity within the latter clade. Overall, the results suggest that Btl proteins contribute to bacterial-fungal symbioses in myriad ways.

After the trap snaps in the plant immune response.

In this issue of Cell Host and Microbe, Chen et al. report that global translation is increased upon plant pathogen detection by intracellular resistance proteins. To achieve this, the conserved protein CDC123 promotes translation initiation complex assembly during the early hours of a defensive programmed cell death in Arabidopsis.

Assessing approaches to learning with nonparametric multidimensional scaling.

This article reports on a trace-based assessment of approaches to learning used by middle school aged children who interacted with NASA Mars Mission science, technology, engineering and mathematics (STEM) games in Whyville, an online game environment with 8 million registered young learners. The learning objectives of two games included awareness and knowledge of NASA missions, developing knowledge and skills of measurement and scaling, applying measurement for planetary comparisons in the solar system. Trace data from 1361 interactions were analysed with nonparametric multidimensional scaling methods, which permitted visual examination and statistical validation, and provided an example and proof of concept for the multidimensional scaling approach to analysis of time-based behavioural data from a game or simulation. Differences in approach to learning were found illustrating the potential value of the methodology to curriculum and game-based learning designers as well as other creators of online STEM content for pre-college youth. The theoretical framework of the method and analysis makes use of the Epistemic Network Analysis toolkit as a post hoc data exploration platform, and the discussion centres on issues of semantic interpretation of interaction end-states and the application of evidence centred design in post hoc analysis.

Transcriptional Profiles of a Foliar Fungal Endophyte (Pestalotiopsis, Ascomycota) and Its Bacterial Symbiont (Luteibacter, Gammaproteobacteria) Reveal Sulfur Exchange and Growth Regulation during Early Phases of Symbiotic Interaction.

Symbiosis with bacteria is widespread among eukaryotes, including fungi. Bacteria that live within fungal mycelia (endohyphal bacteria) occur in many plant-associated fungi, including diverse Mucoromycota and Dikarya. Pestalotiopsis sp. strain 9143 is a filamentous ascomycete isolated originally as a foliar endophyte of Platycladus orientalis (Cupressaceae). It is infected naturally with the endohyphal bacterium Luteibacter sp. strain 9143, which influences auxin and enzyme production by its fungal host. Previous studies have used transcriptomics to examine similar symbioses between endohyphal bacteria and root-associated fungi such as arbuscular mycorrhizal fungi and plant pathogens. However, currently there are no gene expression studies of endohyphal bacteria of Ascomycota, the most species-rich fungal phylum. To begin to understand such symbioses, we developed methods for assessing gene expression by Pestalotiopsis sp. and Luteibacter sp. when grown in coculture and when each was grown axenically. Our assays showed that the density of Luteibacter sp. in coculture was greater than in axenic culture, but the opposite was true for Pestalotiopsis sp. Dual-transcriptome sequencing (RNA-seq) data demonstrate that growing in coculture modulates developmental and metabolic processes in both the fungus and bacterium, potentially through changes in the balance of organic sulfur via methionine acquisition. Our analyses also suggest an unexpected, potential role of the bacterial type VI secretion system in symbiosis establishment, expanding current understanding of the scope and dynamics of fungal-bacterial symbioses. IMPORTANCE Interactions between microbes and their hosts have important outcomes for host and environmental health. Foliar fungal endophytes that infect healthy plants can harbor facultative endosymbionts called endohyphal bacteria, which can influence the outcome of plant-fungus interactions. These bacterial-fungal interactions can be influential but are poorly understood, particularly from a transcriptome perspective. Here, we report on a comparative, dual-RNA-seq study examining the gene expression patterns of a foliar fungal endophyte and a facultative endohyphal bacterium when cultured together versus separately. Our findings support a role for the fungus in providing organic sulfur to the bacterium, potentially through methionine acquisition, and the potential involvement of a bacterial type VI secretion system in symbiosis establishment. This work adds to the growing body of literature characterizing endohyphal bacterial-fungal interactions, with a focus on a model facultative bacterial-fungal symbiosis in two species-rich lineages, the Ascomycota and Proteobacteria.

Rapid sperm lysis and novel screening approach for human male DNA via colorimetric loop-mediated isothermal amplification.

Correct identification of probative samples is the first crucial step in the analysis of sexual assault kits (SAKs). We report a nucleic acid-based approach, as an alternative to the widely utilized p30 assay, to screening male DNA from SAKs collected from female victims by combining a rapid lysis protocol with an isothermal amplification method. The enzymatic lysis protocol efficiently digests biological material to release nuclear DNA in 10 min in a single closed tube, including resilient cell types such as sperm cells. The amplification and detection of human male specific DNA is achieved through loop-mediated isothermal amplification (LAMP) accompanied with hydroxynaphthol blue, a colorimetric indicator, producing a visually-distinctive color change in the presence of male DNA. The Y-screen approach demonstrated high specificity to human male DNA, can reliably detect target DNA as low as 50 pg, and correctly identified all probative samples from 14 single-blind mock sexual assault samples. In contrast with the widely used p30 assay which requires at least 2 h incubation time and manual application to a lateral flow pad, this Y-screen assay can be completed in half the time, and can be performed in a 96-well format without the need for a fluorescence detector, making facile high-throughput sample screening possible.

An insular view of the social decision-making network.

Social animals must detect, evaluate and respond to the emotional states of other individuals in their group. A constellation of gestures, vocalizations, and chemosignals enable animals to convey affect and arousal to others in nuanced, multisensory ways. Observers integrate social information with environmental and internal factors to select behavioral responses to others via a process call social decision-making. The Social Decision Making Network (SDMN) is a system of brain structures and neurochemicals that are conserved across species (mammals, reptiles, amphibians, birds) that are the proximal mediators of most social behaviors. However, how sensory information reaches the SDMN to shape behavioral responses during a social encounter is not well known. Here we review the empirical data that demonstrate the necessity of sensory systems in detecting social stimuli, as well as the anatomical connectivity of sensory systems with each node of the SDMN. We conclude that the insular cortex is positioned to link integrated social sensory cues to this network to produce flexible and appropriate behavioral responses to socioemotional cues.

A polyketide synthase gene cluster associated with the sexual reproductive cycle of the banana pathogen, Pseudocercospora fijiensis.

Disease spread of Pseudocercospora fijiensis, causal agent of the black Sigatoka disease of banana, depends on ascospores produced through the sexual reproductive cycle. We used phylogenetic analysis to identify P. fijiensis homologs (PKS8-4 and Hybrid8-3) to the PKS4 polyketide synthases (PKS) from Neurospora crassa and Sordaria macrospora involved in sexual reproduction. These sequences also formed a clade with lovastatin, compactin, and betaenone-producing PKS sequences. Transcriptome analysis showed that both the P. fijiensis Hybrid8-3 and PKS8-4 genes have higher expression in infected leaf tissue compared to in culture. Domain analysis showed that PKS8-4 is more similar than Hybrid8-3 to PKS4. pPKS8-4:GFP transcriptional fusion transformants showed expression of GFP in flask-shaped structures in mycelial cultures as well as in crosses between compatible and incompatible mating types. Confocal microscopy confirmed expression in spermagonia in leaf substomatal cavities, consistent with a role in sexual reproduction. A disruption mutant of pks8-4 retained normal pathogenicity on banana, and no differences were observed in growth, conidial production, and spermagonia production. GC-MS profiling of the mutant and wild type did not identify differences in polyketide metabolites, but did identify changes in saturated fatty acid methyl esters and alkene and alkane derivatives. To our knowledge, this is the first report of a polyketide synthase pathway associated with spermagonia.

Familiarity modulates social approach toward stressed conspecifics in female rats.

Familiarity between conspecifics may influence how social affective cues shape social behaviors. In a social affective preference test, experimental rats, when given the choice to explore an unfamiliar stressed or a naive adult, will avoid interaction with a stressed conspecific. To determine if familiarity would influence social interactions with stressed conspecifics, male and female test rats underwent 2 social affective preference tests in isosexual triads where an experimental rat was presented with a naïve and a stressed target conspecific who were either familiar (cagemate) or unfamiliar. Male and female experimental rats avoided stressed unfamiliar conspecifics. However, experimental female rats demonstrated a preference to interact with their stressed, familiar cagemates. Male and female rats exhibited more self-grooming and immobility behavior in the presence of stressed conspecifics, which may indicate emotion contagion. These findings suggest a sex-specific role of familiarity in social approach and avoidance, and warrant further mechanistic exploration.

Insular cortex mediates approach and avoidance responses to social affective stimuli.

Social animals detect the affective states of conspecifics and utilize this information to orchestrate social interactions. In a social affective preference text in which experimental adult male rats could interact with either naive or stressed conspecifics, the experimental rats either approached or avoided the stressed conspecific, depending upon the age of the conspecific. Specifically, experimental rats approached stressed juveniles but avoided stressed adults. Inhibition of insular cortex, which is implicated in social cognition, and blockade of insular oxytocin receptors disrupted the social affective behaviors. Oxytocin application increased intrinsic excitability and synaptic efficacy in acute insular cortex slices, and insular oxytocin administration recapitulated the behaviors observed toward stressed conspecifics. Network analysis of c-Fos immunoreactivity in 29 regions identified functional connectivity between insular cortex, prefrontal cortex, amygdala and the social decision-making network. These results implicate insular cortex as a key component in the circuit underlying age-dependent social responses to stressed conspecifics.