Microbiotas from Humans with Inflammatory Bowel Disease Alter the Balance of Gut Th17 and RORγt+ Regulatory T Cells and Exacerbate Colitis in Mice.

Microbiota are thought to influence the development and progression of inflammatory bowel disease (IBD), but determining generalizable effects of microbiota on IBD etiology requires larger-scale functional analyses. We colonized germ-free mice with intestinal microbiotas from 30 healthy and IBD donors and determined the homeostatic intestinal T cell response to each microbiota. Compared to microbiotas from healthy donors, transfer of IBD microbiotas into germ-free mice increased numbers of intestinal Th17 cells and Th2 cells and decreased numbers of RORγt+ Treg cells. Colonization with IBD microbiotas exacerbated disease in a model where colitis is induced upon transfer of naive T cells into Rag1-/- mice. The proportions of Th17 and RORγt+ Treg cells induced by each microbiota were predictive of human disease status and accounted for disease severity in the Rag1-/- colitis model. Thus, an impact on intestinal Th17 and RORγt+ Treg cell compartments emerges as a unifying feature of IBD microbiotas, suggesting a general mechanism for microbial contribution to IBD pathogenesis.

FAIR Header Reference genome: a TRUSTworthy standard.

The lack of interoperable data standards among reference genome data-sharing platforms inhibits cross-platform analysis while increasing the risk of data provenance loss. Here, we describe the FAIR bioHeaders Reference genome (FHR), a metadata standard guided by the principles of Findability, Accessibility, Interoperability and Reuse (FAIR) in addition to the principles of Transparency, Responsibility, User focus, Sustainability and Technology. The objective of FHR is to provide an extensive set of data serialisation methods and minimum data field requirements while still maintaining extensibility, flexibility and expressivity in an increasingly decentralised genomic data ecosystem. The effort needed to implement FHR is low; FHR's design philosophy ensures easy implementation while retaining the benefits gained from recording both machine and human-readable provenance.

Allelic variations in the chpG effector gene within Clavibacter michiganensis populations determine pathogen host range.

Plant pathogenic bacteria often have a narrow host range, which can vary among different isolates within a population. Here, we investigated the host range of the tomato pathogen Clavibacter michiganensis (Cm). We determined the genome sequences of 40 tomato Cm isolates and screened them for pathogenicity on tomato and eggplant. Our screen revealed that out of the tested isolates, five were unable to cause disease on any of the hosts, 33 were exclusively pathogenic on tomato, and two were capable of infecting both tomato and eggplant. Through comparative genomic analyses, we identified that the five non-pathogenic isolates lacked the chp/tomA pathogenicity island, which has previously been associated with virulence in tomato. In addition, we found that the two eggplant-pathogenic isolates encode a unique allelic variant of the putative serine hydrolase chpG (chpGC), an effector that is recognized in eggplant. Introduction of chpGC into a chpG inactivation mutant in the eggplant-non-pathogenic strain Cm101, failed to complement the mutant, which retained its ability to cause disease in eggplant and failed to elicit hypersensitive response (HR). Conversely, introduction of the chpG variant from Cm101 into an eggplant pathogenic Cm isolate (C48), eliminated its pathogenicity on eggplant, and enabled C48 to elicit HR. Our study demonstrates that allelic variation in the chpG effector gene is a key determinant of host range plasticity within Cm populations.

Subchronic inhalation exposure to ultrafine particulate matter alters the intestinal microbiome in various mouse models.

Exposure to ultrafine particles (UFPs) has been associated with multiple adverse health effects. Inhaled UFPs could reach the gastrointestinal tract and influence the composition of the gut microbiome. We have previously shown that oral ingestion of UFPs alters the gut microbiome and promotes intestinal inflammation in hyperlipidemic Ldlr-/- mice. Particulate matter (PM)2.5 inhalation studies have also demonstrated microbiome shifts in normolipidemic C57BL/6 mice. However, it is not known whether changes in microbiome precede or follow inflammatory effects in the intestinal mucosa. We hypothesized that inhaled UFPs modulate the gut microbiome prior to the development of intestinal inflammation. We studied the effects of UFP inhalation on the gut microbiome and intestinal mucosa in two hyperlipidemic mouse models (ApoE-/- mice and Ldlr-/- mice) and normolipidemic C57BL/6 mice. Mice were exposed to PM in the ultrafine-size range by inhalation for 6 h a day, 3 times a week for 10 weeks at a concentration of 300-350 µg/m3.16S rRNA gene sequencing was performed to characterize sequential changes in the fecal microbiome during exposures, and changes in the intestinal microbiome at the end. PM exposure led to progressive differentiation of the microbiota over time, associated with increased fecal microbial richness and evenness, altered microbial composition, and differentially abundant microbes by week 10 depending on the mouse model. Cross-sectional analysis of the small intestinal microbiome at week 10 showed significant changes in α-diversity, ß-diversity, and abundances of individual microbial taxa in the two hyperlipidemic models. These alterations of the intestinal microbiome were not accompanied, and therefore could not be caused, by increased intestinal inflammation as determined by histological analysis of small and large intestine, cytokine gene expression, and levels of fecal lipocalin. In conclusion, 10-week inhalation exposures to UFPs induced taxonomic changes in the microbiome of various animal models in the absence of intestinal inflammation.

Ambient long-term exposure to organophosphorus pesticides and the human gut microbiome: an observational study.

BACKGROUND: Organophosphorus pesticides (OP) have been associated with various human health conditions. Animal experiments and in-vitro models suggested that OP may also affect the gut microbiota. We examined associations between ambient chronic exposure to OP and gut microbial changes in humans. METHODS: We recruited 190 participants from a community-based epidemiologic study of Parkinson's disease living in a region known for heavy agricultural pesticide use in California. Of these, 61% of participants had Parkinson's disease and their mean age was 72 years. Microbiome and predicted metagenome data were generated by 16S rRNA gene sequencing of fecal samples. Ambient long-term OP exposures were assessed using pesticide application records combined with residential addresses in a geographic information system. We examined gut microbiome differences due to OP exposures, specifically differences in microbial diversity based on the Shannon index and Bray-Curtis dissimilarities, and differential taxa abundance and predicted Metacyc pathway expression relying on regression models and adjusting for potential confounders. RESULTS: OP exposure was not associated with alpha or beta diversity of the gut microbiome. However, the predicted metagenome was sparser and less evenly expressed among those highly exposed to OP (p = 0.04). Additionally, we found that the abundance of two bacterial families, 22 genera, and the predicted expression of 34 Metacyc pathways were associated with long-term OP exposure. These pathways included perturbed processes related to cellular respiration, increased biosynthesis and degradation of compounds related to bacterial wall structure, increased biosynthesis of RNA/DNA precursors, and decreased synthesis of Vitamin B1 and B6. CONCLUSION: In support of previous animal studies and in-vitro findings, our results suggest that ambient chronic OP pesticide exposure alters gut microbiome composition and its predicted metabolism in humans.

Mitigating Emerging and Reemerging Diseases of Fruit and Vegetable Crops in a Changing Climate.

Fruit and vegetable crops are important sources of nutrition and income globally. Producing these high-value crops requires significant investment of often scarce resources, and, therefore, the risks associated with climate change and accompanying disease pressures are especially important. Climate change influences the occurrence and pressure of plant diseases, enabling new pathogens to emerge and old enemies to reemerge. Specific environmental changes attributed to climate change, particularly temperature fluctuations and intense rainfall events, greatly alter fruit and vegetable disease incidence and severity. In turn, fruit and vegetable microbiomes, and subsequently overall plant health, are also affected by climate change. Changing disease pressures cause growers and researchers to reassess disease management and climate change adaptation strategies. Approaches such as climate smart integrated pest management, smart sprayer technology, protected culture cultivation, advanced diagnostics, and new soilborne disease management strategies are providing new tools for specialty crops growers. Researchers and educators need to work closely with growers to establish fruit and vegetable production systems that are resilient and responsive to changing climates. This review explores the effects of climate change on specialty food crops, pathogens, insect vectors, and pathosystems, as well as adaptations needed to ensure optimal plant health and environmental and economic sustainability.

First Report of Bacterial Blight of Pennycress Caused by Xanthomonas campestris in Wisconsin.

In May 2023, pennycress (Thlaspi arvense, L.) lines undergoing seed production in the Walnut Street Greenhouse at the University of Wisconsin-Madison displayed symptoms of chlorosis and black necrotic leaf spots (Fig. S1-A). Lesions eventually enlarged to 1-2 cm in diameter, became necrotic, and coalesced to cover a substantial portion of leaves. Symptoms were observed in ~30% of the pennycress lines adversely affecting overall growth and reproduction. Symptomatic leaves were surface sterilized for 30 seconds in 0.75% sodium hypochlorite, rinsed in sterile deionized water, and bacteria were isolated using three-phase streaking of symptomatic tissue onto KB medium (King et al., 1954). Single colonies of three isolates (creamy white to yellow) from this initial isolation were streaked onto KB medium to obtain pure cultures. Individual colonies were transferred for growth overnight in nutrient broth (Difco) and an equal amount of the broth was added to 30% glycerol in deionized (di) water and stored at -80 °C. To validate Koch's Postulates, bacteria were grown from these stocks on Yeast Dextrose Calcium Carbonate medium (Wilson et al., 1967) and were used to inoculate 5-week-old pennycress plants in the greenhouse. The bacteria were grown for 48 hours at 26°C, suspended in 300 ml of 0.05 M PBS buffer (pH=7.2) for inoculum preparation. Plants were inoculated with three bacterial isolates (approx. 108 CFU/ml) by piercing the mid veins or hydathodes with a sterilized toothpick dipped in the suspension. Inoculated plants were then enclosed in clear plastic bags for 24-48 hours and maintained in the greenhouse at a constant temperature of 26°C with a 16-hour photoperiod. After seven days, water-soaked lesions appeared on the inoculated leaves, eventually developing into the characteristic black spots (Fig. S1-B). DNA from the original isolates was extracted, and 16S PCR and sequencing of the positive bands was done. The negative control only produced brown spots at the site of inoculation (Fig. S1-C). The primer sequences were as follows: 27F: AGAGTTTGATCMTGGCTCAG; 1492R: GGTTACCTTGTTACGACTT (Eden et al., 1991; Weisburg et al., 1991). A BLAST analysis showed that the isolates had an E value of 0.0 to the genus Xanthomonas as well as 100% identity. Amplification and sequencing of the bacterium using gyrB amplicons revealed a 99-100% pairwise match with Xc. To enhance taxonomy resolution and confirm the identity of these isolates, the complete genomes of three samples were sequenced using NextSeq2000 Illumina platform (NCBI bioproject ID PRJNA1040293). Average Nucleotide Identity (ANI) analysis was conducted with representative strains from the Xc species (Dubrow et al., 2022), using PanExplorer (Dereeper et al., 2020) featuring integrated FastANI module (Jain et al., 2018). The isolates genomes exhibited over 98% identity and clustered with that of Xc pv. incanae and Xc pv. barbarae (Fig S2). Further work will be required to identify the pathovar of Xc identified in this study through phenotypic host range assay. This marks the first documented case of Xc in pennycress in the Midwestern US. Given the potential use of pennycress as a cover crop in the region, further investigations are warranted to assess its economic impact on production and develop management strategies.

Ambient Particulate Matter Induces In Vitro Toxicity to Intestinal Epithelial Cells without Exacerbating Acute Colitis Induced by Dextran Sodium Sulfate or 2,4,6-Trinitrobenzenesulfonic Acid.

Inflammatory bowel disease (IBD) is an immunologically complex disorder involving genetic, microbial, and environmental risk factors. Its global burden has continued to rise since industrialization, with epidemiological studies suggesting that ambient particulate matter (PM) in air pollution could be a contributing factor. Prior animal studies have shown that oral PM10 exposure promotes intestinal inflammation in a genetic IBD model and that PM2.5 inhalation exposure can increase intestinal levels of pro-inflammatory cytokines. PM10 and PM2.5 include ultrafine particles (UFP), which have an aerodynamic diameter of <0.10 µm and biophysical and biochemical properties that promote toxicity. UFP inhalation, however, has not been previously studied in the context of murine models of IBD. Here, we demonstrated that ambient PM is toxic to cultured Caco-2 intestinal epithelial cells and examined whether UFP inhalation affected acute colitis induced by dextran sodium sulfate and 2,4,6-trinitrobenzenesulfonic acid. C57BL/6J mice were exposed to filtered air (FA) or various types of ambient PM reaerosolized in the ultrafine size range at ~300 µg/m3, 6 h/day, 3-5 days/week, starting 7-10 days before disease induction. No differences in weight change, clinical disease activity, or histology were observed between the PM and FA-exposed groups. In conclusion, UFP inhalation exposure did not exacerbate intestinal inflammation in acute, chemically-induced colitis models.

Role of enterocyte Enpp2 and autotaxin in regulating lipopolysaccharide levels, systemic inflammation, and atherosclerosis.

Conversion of lysophosphatidylcholine to lysophosphatidic acid (LPA) by autotaxin, a secreted phospholipase D, is a major pathway for producing LPA. We previously reported that feeding Ldlr-/- mice standard mouse chow supplemented with unsaturated LPA or lysophosphatidylcholine qualitatively mimicked the dyslipidemia and atherosclerosis induced by feeding a Western diet (WD). Here, we report that adding unsaturated LPA to standard mouse chow also increased the content of reactive oxygen species and oxidized phospholipids (OxPLs) in jejunum mucus. To determine the role of intestinal autotaxin, enterocyte-specific Ldlr-/-/Enpp2 KO (intestinal KO) mice were generated. In control mice, the WD increased enterocyte Enpp2 expression and raised autotaxin levels. Ex vivo, addition of OxPL to jejunum from Ldlr-/- mice on a chow diet induced expression of Enpp2. In control mice, the WD raised OxPL levels in jejunum mucus and decreased gene expression in enterocytes for a number of peptides and proteins that affect antimicrobial activity. On the WD, the control mice developed elevated levels of lipopolysaccharide in jejunum mucus and plasma, with increased dyslipidemia and increased atherosclerosis. All these changes were reduced in the intestinal KO mice. We conclude that the WD increases the formation of intestinal OxPL, which i) induce enterocyte Enpp2 and autotaxin resulting in higher enterocyte LPA levels; that ii) contribute to the formation of reactive oxygen species that help to maintain the high OxPL levels; iii) decrease intestinal antimicrobial activity; and iv) raise plasma lipopolysaccharide levels that promote systemic inflammation and enhance atherosclerosis.

High Perceived Stress is Associated With Increased Risk of Ulcerative Colitis Clinical Flares.

BACKGROUND & AIMS: Although perceived stress (PS) has been associated with symptomatic flares in inflammatory bowel disease, clinical and physiological measures associated with perceived stress and flare are not known. The aim of this study was to identify physiological factors associated with perceived stress in ulcerative colitis (UC) subjects, and their relationship with flare. METHODS: Patients with UC in clinical remission (Simple Colitis Clinical Activity Index [SCCAI] score <5) underwent clinical and behavioral assessments, morning salivary cortisol measurements, autonomic nervous system activity testing (heart rate variability, electrodermal activity) at baseline with patient-reported SCCAI every 2 weeks over 1 to 2 years and fecal calprotectin at time of flare. Clinical flares (SCCAI ≥5) and biochemical flares (SCCAI ≥5 with fecal calprotectin ≥250 µg/g) were evaluated. RESULTS: One hundred ten patients with UC were enrolled, with mean follow-up of 65.6 weeks. Patients with UC with higher and lower PS were determined. Although the high PS group had 3.6 times higher odds of a clinical flare than the low PS group, no significant differences in biochemical flares were observed between the low and high PS groups. The high vs low PS group differed in tonic sympathetic arousal as indexed by significantly greater baseline electrodermal activity (4.3 vs 3.4 microsiemens; P = .026) in the high PS group, but not in terms of heart rate variability and morning cortisol levels. Increased fecal calprotectin was associated with cardioautonomic measures, suggesting lower parasympathetic activity. CONCLUSIONS: Increased PS assessed at baseline is associated with tonic sympathetic arousal and greater odds of clinical flares in patients with UC.

CRISPRi in Xanthomonas demonstrates functional convergence of transcription activator-like effectors in two divergent pathogens.

Functional analysis of large gene families in plant pathogens can be cumbersome using classical insertional mutagenesis. Additionally, Cas9 toxicity has limited the application of CRISPR-Cas9 for directed mutagenesis in bacteria. Here, we successfully applied a CRISPR interference strategy to investigate the cryptic role of the transcription activator-like effector (tale) multigene family in several plant-pathogenic Xanthomonas bacterial species, owing to their contribution to pathogen virulence. Single guide RNAs (sgRNAs) designed against Xanthomonas phaseoli pv manihotis tale conserved gene sequences efficiently silenced expression of all tales, with concomitant decrease in virulence and TALE-induced host gene expression. The system is readily translatable to other Xanthomonas species infecting rice, citrus, Brassica, and cassava, silencing up to 16 tales in a given strain using a single sgRNA. Complementation with plasmid-borne designer tales lacking the sgRNA-targeted sequence restored molecular and virulence phenotypes in all pathosystems. Our results evidenced that X. campestris pv campestris CN08 tales are relevant for symptom development in cauliflower. They also show that the MeSWEET10a sugar transporter is surprisingly targeted by the nonvascular cassava pathogen X. cassavae, highlighting a new example of TALE functional convergence between phylogenetically distant Xanthomonas. Overall, this novel technology provides a platform for discovery and rapid functional understanding of highly conserved gene families.

Comparative Genomics of Barley-Infecting Xanthomonas translucens Shows Overall Genetic Similarity but Globally Distributed Virulence Factor Diversity.

Xanthomonas translucens pv. translucens (Xtt) is a global barley patho-gen and a concern for resistance breeding and regulation. Long-read whole genome sequences allow in-depth understanding of pathogen diversity. We have completed long-read PacBio sequencing of two Minnesotan Xtt strains and an in-depth analysis of available Xtt genomes. We found that average nucleotide identity (ANI)-based approaches organize Xtt strains different from the previous standard multilocus sequencing analysis approach. According to ANI, Xtt forms a separate clade from X. translucens pv. undulosa and consists of three main groups which are represented on multiple continents. Some virulence factors, such as 17 Type III-secreted effectors, are highly conserved and offer potential targets for the elicitation of broad resistance. However, there is a high degree of variation in virulence factors, meaning that germplasm should be screened for resistance with a diverse panel of Xtt.

Comparative Genomics of Xanthomonas translucens pv. undulosa Strains Isolated from Weedy Grasses and Cultivated Wild Rice.

Bacterial leaf streak (BLS) of wheat (Triticum aestivum), caused by Xanthomonas translucens pv. undulosa, is a disease of major concern in the Northern Great Plains. The host range for X. translucens pv. undulosa is relatively broad, including several small grains and perennial grasses. In Minnesota, X. translucens pv. undulosa was isolated from weedy grasses in and around wheat fields that exhibited BLS symptoms and from cultivated wild rice (Zizania palustris) with symptomatic leaf tissue. Currently, no genomic resources are available for X. translucens pv. undulosa strains isolated from non-wheat hosts. In this study, we sequenced and assembled the complete genomes of five strains isolated from weedy grass hosts, foxtail barley (Hordeum jubatum), green foxtail (Setaria viridis), and wild oat (Avena fatua), and from cultivated wild rice and wheat. These five genomes were compared with the publicly available genomes of seven X. translucens pv. undulosa strains originating from wheat and one genome of an X. translucens pv. secalis strain originating from rye (Secale cereale). Global alignments of the genomes revealed little variation in genomic structures. Average nucleotide identity-based phylogeny and life identification numbers revealed that the strains share ≥99.25% identity. We noted differences in the presence of Type III secreted effectors, including transcription activator-like effectors. Despite differences between strains, we did not identify unique features distinguishing strains isolated from wheat and non-wheat hosts. This study contributes to the availability of genomic data for X. translucens pv. undulosa from non-wheat hosts, thus increasing our understanding of the diversity within the pathogen population.

Bacterial Leaf Streak Diseases of Plants: Symptom Convergence in Monocot Plants by Distant Pathogenic Xanthomonas Species.

Bacterial leaf streak (BLS) is a disease of monocot plants caused by Xanthomonas translucens on small grains, X. vasicola on maize and sorghum, and X. oryzae on rice. These three pathogens cause remarkably similar symptomology in their host plants. Despite causing similar symptoms, BLS pathogens are dispersed throughout the larger Xanthomonas phylogeny. Each aforementioned species includes strain groups that do not cause BLS and instead cause vascular disease. In this commentary, we hypothesize that strains of X. translucens, X. vasicola, and X. oryzae convergently evolved to cause BLS due to shared evolutionary pressures. We examined the diversity of secreted effectors, which may be important virulence factors for BLS pathogens and their evolution. We discuss evidence that differences in gene regulation and abilities to manipulate plant hormones may also separate BLS pathogens from other Xanthomonas species or pathovars. BLS is becoming an increasing issue across the three pathosystems. Overall, we hope that a better understanding of conserved mechanisms used by BLS pathogens will enable researchers to translate findings across production systems and guide approaches to control this (re)emerging threat.

Development of Genome-Driven, Lifestyle-Informed Markers for Identification of the Cereal-Infecting Pathogens Xanthomonas translucens Pathovars undulosa and translucens.

Bacterial leaf streak, bacterial blight, and black chaff caused by Xanthomonas translucens pathovars are major diseases affecting small grains. Xanthomonas translucens pv. translucens and X. translucens pv. undulosa are seedborne pathogens that cause similar symptoms on barley, but only X. translucens pv. undulosa causes bacterial leaf streak of wheat. Recent outbreaks of X. translucens have been a concern for wheat and barley growers in the Northern Great Plains; however, there are limited diagnostic tools for pathovar differentiation. We developed a multiplex PCR based on whole-genome differences to distinguish X. translucens pv. translucens and X. translucens pv. undulosa. We validated the primers across different Xanthomonas and non-Xanthomonas strains. To our knowledge, this is the first multiplex PCR to distinguish X. translucens pv. translucens and X. translucens pv. undulosa. These molecular tools will support disease management strategies enabling detection and pathovar incidence analysis of X. translucens. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Complete Genome Sequencing of Three Clade-1 Xanthomonads Reveals Genetic Determinants for a Lateral Flagellin and the Biosynthesis of Coronatine-Like Molecules in Xanthomonas.

Evolutionarily, early-branching xanthomonads, also referred to as clade-1 xanthomonads, include major plant pathogens, most of which colonize monocotyledonous plants. Seven species have been validly described, among them the two sugarcane pathogens Xanthomonas albilineans and Xanthomonas sacchari, as well as Xanthomonas translucens, which infects small-grain cereals and diverse grasses but also asparagus and pistachio trees. Single-gene sequencing and genomic approaches have indicated that this clade likely contains more, yet-undescribed species. In this study, we sequenced representative strains of three novel species using long-read sequencing technology. Xanthomonas campestris pv. phormiicola strain CFBP 8444 causes bacterial streak on New Zealand flax, another monocotyledonous plant. Xanthomonas sp. strain CFBP 8443 has been isolated from common bean, and Xanthomonas sp. strain CFBP 8445 originated from banana. Complete assemblies of the chromosomes confirmed their unique phylogenetic position within clade 1 of Xanthomonas. Genome mining revealed novel genetic features, hitherto undescribed in other members of the Xanthomonas genus. In strain CFBP 8444, we identified genes related to the synthesis of coronatine-like compounds, a phytotoxin produced by several pseudomonads, which raises interesting questions about the evolution and pathogenicity of this pathogen. Furthermore, strain CFBP 8444 was found to contain a second, atypical flagellar gene cluster in addition to the canonical flagellar gene cluster. Overall, this research represents an important step toward better understanding the evolutionary history and biology of early-branching xanthomonads.

Comparative Genomics Identifies Conserved and Variable TAL Effectors in African Strains of the Cotton Pathogen Xanthomonas citri pv. malvacearum.

Strains of Xanthomonas citri pv. malvacearum cause bacterial blight of cotton, a potentially serious threat to cotton production worldwide, including in sub-Saharan countries. Development of disease symptoms, such as water soaking, has been linked to the activity of a class of type 3 effectors, called transcription activator-like (TAL) effectors, which induce susceptibility genes in the host's cells. To gain further insight into the global diversity of the pathogen, to elucidate their repertoires of TAL effector genes, and to better understand the evolution of these genes in the cotton-pathogenic xanthomonads, we sequenced the genomes of three African strains of X. citri pv. malvacearum using nanopore technology. We show that the cotton-pathogenic pathovar of X. citri is a monophyletic lineage containing at least three distinct genetic subclades, which appear to be mirrored by their repertoires of TAL effectors. We observed an atypical level of TAL effector gene pseudogenization, which might be related to resistance genes that are deployed to control the disease. Our work thus contributes to a better understanding of the conservation and importance of TAL effectors in the interaction with the host plant, which can inform strategies for improving resistance against bacterial blight in cotton.

First Report of Bacterial Leaf Streak of Barley (Hordeum vulgare) Caused by Xanthomonas translucens pv. translucens in British Columbia, Canada.

Bacterial leaf streak (BLS) of barley is caused by the Gram-negative bacterial pathogen Xanthomonas translucens (Sapkota et al. 2020). In 2021, we observed multiple hill plots with BLS symptomatic plants in a barley stripe rust nursery in Vancouver, BC, Canada. We collected 29 leaf samples showing typical BLS symptoms (e.g. necrotic lesions; Fig. S1) and stored at 4 oC until bacterial isolation. Samples were surface-sterilized in 10% NaOCl for 20 sec and rinsed twice. About 1 cm2 of leaf tissue containing BLS characteristic lesions was macerated in 200 µL sterile H2O on a petri dish, incubated for 15 min, and 10 µl of the homogenates was streaked onto Wilbrink's - Boric Acid - Cephalexin (WBC) agar medium. Plates were incubated at 28-30 oC for 48 hrs. Four single colonies were obtained: BC10-1-2a (USask BC10-2a), BC10-1-2b (USask BC10-2b), UBC026 and UBC028. Colonies were grown in WBC broth and gDNA was extracted using E.Z.N.A. Bacterial DNA Kit (Omega Bio-Tek) or DNeasy Plant Pro Kit® (Qiagen) following manufacturer protocols. Genus-level identification was achieved using 16S rRNA sequencing with 27F/1492R primers (Lane 1991) of UBC026 (1,399 bp; NCBI # OP327375) and UBC028 (1,415 bp; NCBI #OP327376). Complete 16S rRNA sequences (1,533bp) of BC10-2a and BC10-2b (1,533 bp) were extracted from the draft whole-genome sequences (WGS) generated in this study. The 16S rRNA sequence homology values of 99.0-100% were recorded between the 4 strains. BLAST analyses of the 16S rRNA sequences to GenBank entries exhibited 99.5-100% similarity values (100% coverage) with the pathotype strains of Xtt DSM 18974T (LT604072) and X. translucens pv. undulosa (Xtu) CFBP 2055 (CP074361). Whole genomes of BC10-2a (JANUQY01) and BC10-2b (JANUQZ01) were sequenced (150-bp; reads 33.1 million; mean coverage 2125x) using NovaSeq Illumina, assembled (Unicycler v0.4.8; Wick et al. 2017) and analyzed to identify the strains to the species-level (Tambong et al. 2021). WGS of strains USask BC10-2a and USask BC10-2b exhibited genome-based DNA-DNA hybridization (dDDH; Meier-Kolthoff et al. 2013) and BLAST-based average nucleotide identity (ANIb; Richter et al. 2015) of 100%. The two strains also showed dDDH and ANIb of 90.4% (species-leel cut-off of 70%) and 98.780% and 98.80% (cut-off of 96%), respectively, with Xtt DSM 18974T (LT604072). In contrast, the WGS of BC10-2a and BC10-2b exhibited only 78.2% dDDH homology values with Xtu CFBP 2055T, suggesting that the strains are genetically more similar to Xtt. The assignment of these strains to Xtt is corroborated by phylogenomic analysis (Fig. S2; Meier-Kolthoff and Göker 2019) that showed the two strains clustering together (100% bootstrap) with the type strain DSM 18974T. These data suggest that these strains are taxonomically members of Xtt. Identification was also confirmed to the genus-level by LAMP assay using published X. translucens primers (Langlois et al. 2017). Pathovar-level identification was confirmed using a cbsA and S8.pep multiplex PCR diagnostic assay (Roman-Reyna et al. 2022). Koch's postulates were verified by greenhouse inoculation via leaf infiltration of UBC026 and UBC028 on 21-day old barley plants (line HB522) using an inoculum of 108 CFU ml-1 followed by re-isolation of the bacteria on WBC. The inoculated plants showed typical BLS symptoms similar to those observed in the field (Fig. S1). Water-inoculated plants had no symptoms. To our knowledge, this is the first published report of BLS of barley in British Columbia.

Oxidized phospholipids cause changes in jejunum mucus that induce dysbiosis and systemic inflammation.

We previously reported that adding a concentrate of transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to a Western diet (WD) ameliorated systemic inflammation. To determine the mechanism(s) responsible for these observations, Ldlr-/- mice were fed chow, a WD, or WD plus Tg6F. We found that a WD altered the taxonomic composition of bacteria in jejunum mucus. For example, Akkermansia muciniphila virtually disappeared, while overall bacteria numbers and lipopolysaccharide (LPS) levels increased. In addition, gut permeability increased, as did the content of reactive oxygen species and oxidized phospholipids in jejunum mucus in WD-fed mice. Moreover, gene expression in the jejunum decreased for multiple peptides and proteins that are secreted into the mucous layer of the jejunum that act to limit bacteria numbers and their interaction with enterocytes including regenerating islet-derived proteins, defensins, mucin 2, surfactant A, and apoA-I. Following WD, gene expression also decreased for Il36γ, Il23, and Il22, cytokines critical for antimicrobial activity. WD decreased expression of both Atoh1 and Gfi1, genes required for the formation of goblet and Paneth cells, and immunohistochemistry revealed decreased numbers of goblet and Paneth cells. Adding Tg6F ameliorated these WD-mediated changes. Adding oxidized phospholipids ex vivo to the jejunum from mice fed a chow diet reproduced the changes in gene expression in vivo that occurred when the mice were fed WD and were prevented with addition of 6F peptide. We conclude that Tg6F ameliorates the WD-mediated increase in oxidized phospholipids that cause changes in jejunum mucus, which induce dysbiosis and systemic inflammation.

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.