Although isolating microorganisms from food microbiota may appear less challenging than from the gut or environmental sources, recovering all representative species from food remains a difficult task. Here, we showed by metagenomic analysis that several abundant species had escaped isolation in a previous study of ten cheeses, including several previously uncharacterized species. This highlights the ongoing challenge of achieving a comprehensive recovery of microbes from food. To address this gap, we designed a novel strategy integrating metagenomics-based probes targeting the species of interest, coupled with an incremental culturing approach using pooled samples. As proof of concept, we applied this strategy to two cheeses containing species that were not isolated in our previous study, with the objective of isolating all species present at levels above 2% and, in particular, potential novel food species. Through this approach, we successfully performed the targeted isolation of two Psychrobacter and two Vibrio species from the first cheese, and four Halomonas and two Pseudoalteromonas species from the second one. Notably, P. undina and V. litoralis represented, as far as we know, the first cheese isolates characterized for these species. However, we were unable to isolate a novel species of Pseudoalteromonas, with no characterized representative to date, and Marinomonas foliarum, previously isolated from marine environment. Using metagenome-assembled genomes (MAGs) and metagenomic analysis, we discussed the possible reasons for their non-recovery. Finally, this strategy offers a promising approach for isolating a set of strains representative of the microbial diversity present in food ecosystems. These isolates can serve as a basis for investigating their roles in the communities, their impact on product development, safety implications and their potential in the development of starter cultures.

The white button mushroom (Agaricus bisporus), widely cultivated worldwide as an edible mushroom, is susceptible to browning, which significantly impacts its nutritional and commercial value. Extensive research has enhanced our understanding of the mechanisms underlying this browning process. Although the role of DNA methylation in regulating gene expression has been studied in many fungi, information specifically concerning DNA methylation during the browning in A. bisporus is still limited. In this study, we initially evaluated the impact of temperatures (4 ℃ and room temperature) on discoloration in A. bisporus, and samples with similar discoloration under different temperatures were collected for transcriptome and DNA methylation sequencing. The results revealed that DNA methylation was positively correlated with browning, suggesting its involvement during the browning in A. bisporus. Further analysis showed the heightened methylation levels were primarily attributed to increased methylation at CHG and CHH sites. By joint analysis of transcriptome and DNA methylome, 342 genes with significant expression changes were identified to be affected by DNA methylation, and finally 13 genes were considered as important browning genes under different signaling pathways, such as ABA/ET pathway. Notably, four DNA methyltransferases were identified and validated to play important role during browning in A. bisporus. Altogether, this study provides theoretical insights into the functions of DNMTs in A. bisporus, and offers new perspectives on the role of DNA methylation in edible mushrooms.

Calyptosporidae are heteroxenous coccidia with fish as their definitive hosts. Currently, most of this diversity is found in freshwater fish from the Brazilian Amazon region. The objective of this study was to perform an integrative taxonomy of a new species of Calyptospora in Cynoscion virescens (Cuvier, 1830), a marine and estuarine fish. Thirty specimens of C. virescens from the Eastern Amazon were collected and necropsied, of which 12 (40 %) were infected after microscopic analysis, which has revealed spherical oocysts measuring 17.8 μm in diameter and four elliptical sporocysts with 7.1 μm in length and 6.1 μm in width. The oocysts were located in the hepatopancreas, and melanomacrophagic bodies were observed in nearby regions. In phylogenetic analysis, coccidia were found in the basal clade of the Calyptosporidae family. Morphological and molecular divergence characterized a new species of Calyptospora in C. virescens, the first occurrence of coccidia in a fish of the family Sciaenidae.

As a member of the NLRs family, NLRC3 has been determined to function in the NF-κB, MAPK, and type I IFN signaling, which are crucial for the host innate immunity and inflammatory response. In this study, an NLRC3 ortholog, named as Lc-NLRC3, was cloned and identified in large yellow croaker (Larimichthys crocea). The gene characteristics analysis revealed that Lc-NLRC3 consists of 18 exons and 17 introns, with a full-length open reading frame (ORF) of 3405 bp, encoding a protein of 1134 amino acids (aa), that containing a N-terminal CARD domain, a central NACHT domain, and a C-terminal LRRs domain. It was shown that Lc-NLRC3 is predominantly found in the cytosol, and was widely distributed across various tissues/organs, with the highest expression detected in the intestine, and could be induced by poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation. Importantly, Lc-NLRC3 overexpression significantly activate NF-κB, TNFα, IL-1β, IRF3, IRF7, and IFN1 promoters, whereas when co-expressed with RIP2, STING, or TBK1, it down-regulated those promoter activation compared to their individual overexpression alone, thereby suppressing downstream antiviral and inflammatory gene expression. Interestingly, Lc-NLRC3 associated with TRAF6 in IRF3/IRF7 promoter activation, and enhanced the expression of IRF7, Mx, ISG15, and TNF-α. Co-immunoprecipitation assays also confirmed interactions of Lc-NLRC3 with RIP2, STING, TBK1, and TRAF6. The above results imply that Lc-NLRC3 is an important regulator in RIP2, STING, TBK1, and TRAF6 mediated type I IFN signaling and inflammatory response.

Hookworms infect 450 million people globally and account for the loss of 5 million disability-adjusted life years annually. Over the last decade, the Human Hookworm Vaccine (HHV) candidate N. americanus Glutathione-S-Transferase-1 (Na-GST-1) has advanced to efficacy testing. This manuscript describes the manufacture of third-stage N. americanus larvae (NaL3) as an infectious challenge agent to provide "proof-of-concept" for the efficacy of Na-GST-1 prior to more extensive and more resource-intensive vaccine field trials in hookworm endemic areas. NaL3 were produced from fecal samples of three hookworm-infected human donors by a modified Harada and Mori method that complied with current Good Manufacturing Practices (cGMP). A series of lot release tests assessed the purity (bioburden), viability (potency), and identity (speciation) of NaL3 before administration to participants in a hookworm vaccine challenge model (HVCM) in Washington, DC. Twenty-four production runs yielded an average of 947 NaL3 per lot, which were approved for clinical to inoculate of 39 participants in a Hookworm Vaccine Challenge Model. This manuscript describes the unique manufacture and testing for NaL3 in compliance with cGMP.

The white-rot fungus Phanerochaete sordida YK-624 is capable of degrading various emerging contaminants (ECs), with cytochrome P450 (CYP) enzymes playing crucial catalytic roles in the degradation process. In this study, we first identified 214 putative CYPs in P. sordida YK-624 (PsCYPs), of which 208 PsCYPs were classified into 31 CYP families, and 6 PsCYPs remained unclassified. To construct a comprehensive functional screening system for identifying PsCYPs involved in EC degradation, a heterologous co-expression system was established using Saccharomyces cerevisiae. The heterologous expressing yeasts were then used to evaluate the degradation of 7-ethoxycoumarin, a natural compound commonly used to assess CYP activity. Several expressing yeasts catalyzed the hydroxylation and O-deethylation of 7-ethoxycoumarin, confirming that these yeasts expressed active forms of PsCYPs. Subsequent degradation experiments were conducted on ECs such as carbazole, acetamiprid (ACE), bisphenol A (BPA), and loxoprofen (LOX). Metabolite analyses using LC/MS, GC/MS and NMR revealed that the PsCYPs catalyzed the hydroxylation of carbazole, BPA, and LOX, as well as the N-dealkylation of ACE. These findings not only provide strong evidence supporting our previous finding regarding degradation research mediated by P. sordida YK-624 mycelia, but also offer valuable insights for future bioprospecting of fungal CYPs in the bioremediation of EC.

Lignified stone cell content is one of the critical factors affecting 'Dangshansuli' fruit quality. The function of MADS-box transcription factors in regulating lignin biosynthesis in pear fruit is still less. In this study, PbMADS49 gene silencing inhibited the lignin biosynthesis and stone cell secondary wall development of pear fruit mainly through reducing the expression levels of lignin monomer polymerisation key enzymes (PbPRX33 and PbPRX45). PbMADS49 was a transcriptional repressor inhibiting its transcription by binding to the CArG element in the target gene promoter. Combined with the co-expression network and promoter cis-acting element analysis, we hypothesised that PbMADS49 positively regulates the transcription of PbPRX33 through PbWRKY63. The gene silencing effect of homologous genes PbPRX33-1 and PbPRX33-2 was consistent with PbMADS49, and PbPRX33-2 was more significant than PbPRX33-1. This study shows that PbMADS49 is a positive regulator of stone cell lignification, providing new insights into the development mechanism of pear stone cells.

Ferroptosis is a regulated form of cell death driven by lipid peroxidation, with 15-lipoxygenase (15LOX) enzyme playing a critical role in catalyzing the oxygenation of polyunsaturated fatty acid-containing phospholipids, such as 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (SAPE), to initiate this process. The molecular oxygen required for this catalytic reaction is subject to continuous competition among various oxygen-consuming enzymes, which influences the efficiency of lipid peroxidation. In this study, we utilized structure-based modeling and all-atom molecular dynamics simulations to explore the oxygen diffusion pathways in 15LOX-1 under varying oxygen concentrations and in the presence of key components, including a substrate, binding partner PE-binding protein 1 (PEBP1), and the membrane environment. Extensive computational experiments were performed on various system configurations, examining the role of substrate binding, membrane presence, and PEBP1 association in oxygen acquisition. Our computational results indicate that the substrate binding induces a conformational change in 15LOX-1, facilitating the simultaneous recruitment of one or two O2 molecules, which drive peroxidation, leading predominantly to monohydroperoxide products and, less frequently, to dihydroperoxide products. A similar trend was observed in our redox lipidomics analysis. Moreover, we noticed that the presence of the membrane significantly reduces irrelevant oxygen binding spots, directing oxygen molecules toward a primary tunnel essential for the catalytic activity. We identified two primary oxygen tunnels with sequentially and structurally conserved regions across the lipoxygenase family. These findings provide novel insights into the regulation of oxygen acquisition mechanism for LOX members, shedding light on the molecular basis of ferroptosis signaling.

The development of ideal plant architecture is crucial for optimizing grain yield in crop breeding. The transition from prostrate growth habit in wild rice to erect growth habit in cultivated rice is one of the important events during rice domestication. Here, we identified a yield-related quantitative trait locus (QTL) cluster on the short arm of chromosome 7 using Teqing/W2014 (Oryza nivara) derived BC3F6 population. The introgression line TIL81 containing this QTL cluster exhibited significantly larger tiller angle, increased tiller numbers, and prostrate growth habit compared to the recipient parent Teqing. Using a segregating F2 population derived from a cross between TIL81 and Teqing, this yield-related QTL cluster was mapped to a similar position as the known rice plant architecture domestication (RPAD) locus controlling rice plant architecture domestication. CRISPR/Cas9-mediated genome (where CRISPR is clustered regularly interspaced short palindromic repeats) editing of four zinc finger transcription factors (OnZnF1, OnZnF6, OnZnF8, and OnZnF9) within the RPAD locus demonstrated their collective involvement in regulating plant architecture and yield-related traits. Notably, the knockout lines harboring all four zinc finger gene mutations exhibited plant architecture traits and grain yield per plant comparable to the control Teqing. These findings demonstrated that RPAD locus in O. nivara functions in prostrate growth habit and provided new insights into the molecular mechanism of plant architecture during rice domestication.

Aedes albopictus (Diptera: Culicidae), commonly known as the Asian tiger mosquito, is an important vector transmitting dangerous arboviruses to humans. This study investigated the phenotypic and genetic variation of this species in Thailand through wing geometric morphometric (GM) and mitochondrial cytochrome c oxidase subunit I (COI) gene sequence analyses. A total of 236 Ae. albopictus specimens from 12 populations in Thailand and 89 specimens from invasive populations in Florida, Hawaii and Brazil underwent wing GM analysis. The centroid size (CS) of Ae. albopictus populations in Thailand ranged from 2.00 mm in Bangkok to 2.36 mm in Chanthaburi, while in invasive populations, CS varied from 2.25 mm in Brazil to 2.47 mm in Florida. Pairwise comparisons of wing shape revealed significant differences for most population pairs, with distances ranging from 1.63 to 10.02. The clustering tree indicated distant relationships in wing shape between native and invasive populations. Additionally, partial COI gene sequences were amplified from 108 specimens, revealing a mean haplotype diversity of 0.842 ± 0.025 and a mean nucleotide diversity of 0.002 ± 0.001. The results from neutral Tajima's D and Fu's Fs tests indicated negative and statistically significant values (-2.159 and -33.846, respectively), suggesting population expansion. Further examination of haplotype relationships between Thailand and other countries identified two distinct groups: a Southeast Asia group, with Thai haplotypes clustered exclusively within it, and a non-Southeast Asia group. These findings highlight the phenotypic and genetic variation of Ae. albopictus in Thailand, providing essential insights for disease control strategies and tracing the mosquito's origins across regions.

The presence of leptospires in the follicular fluid has only been confirmed through molecular techniques, as culturing leptospires is extremely challenging. The lack of studies demonstrating the viability of leptospires in this site limits a deeper understanding of pathogenesis. Therefore, this study aimed to cultivate and molecularly characterize Leptospira spp. from follicular fluid and uterine tissue samples collected from naturally infected cows. A total of 85 cows from herds with leptospirosis were selected and 53 follicular fluids (FF) and 85 uterine fragments (UF) were collected after slaughter. The samples were seeded into T80/40LH, and evaluated by Darkfield Microscopy (DFM). Positive cultures were tested by lipL32-PCR to confirm the presence of pathogenic Leptospira spp. Positive tubes were submitted to serogrouping and genotyping by sequencing of the secY gene. A maximum likelihood (ML) tree was constructed. A total of 33/85 (39 %) cows were positive, 16/53 (30.2%) only in FF, 14/85 (16.4 %) only in UF, and three in both samples. It was possible to obtain one isolate from FF, serogrouped as Icterohaemorrhagiae. Six samples were sequenced by secY. All of them were identified as L. interrogans, with > 99 % identity. The ML tree revealed that all sequences belong to a group with strains close to serovar Hardjo. Herein, we highlight the presence of live L. interrogans in follicular fluid, emphasizing it as an important site of infection, as leptospires could impair embryo production. The similarity of the strains involved to highly virulent strains in humans raises concerns, posing a potential zoonotic risk.

Pantoea ananatis is a member of the Enterobacteriaceae family known for its broad host adaptability. This study isolated 10 P. ananatis strains from white spot (MWS)-diseased leaves of maize (Zea mays) grown in Yunnan Province, China, and analyzed their putative functions, genomic diversity, and variation. The inoculation tests revealed that none of the 10 isolates caused MWS symptoms in maize. Nine maize isolates, except for S47, induced a hypersensitive response (HR) in tobacco and caused rot symptoms in onion. Most isolates exhibited plant growth-promoting characteristics, with strains JCC14, JCY1, and S47 significantly enhancing maize seedling growth parameters. Genomic sequencing of 10 maize isolates and two rice isolates revealed that 12 isolates clustered into three groups, with an open pan-genome identified. Ancestral reconstruction indicated that the genome size increased in Group A and then decreased in Group B, with significant gains in orthologous groups at Node 14, the most recent common ancestor (MRCA) of Group A and Group B, and at Node 19, the MRCA of seven maize-isolated strains and other Group B strains. Additionally, 11 single-copy orthologous groups were under positive selection. Furthermore, the HIVir (high virulence, also known as PASVIL, P. ananatis-specific virulence locus) cluster and type VI secretion system-related genes were conserved in certain P. ananatis strains but were not related to their group divergences. This study not only reveals the diverse functions of MWS-diseased maize P. ananatis isolates, but also enhances our understanding of divergent genome evolution and environmental adaptation across P. ananatis species.IMPORTANCEPantoea ananatis is a bacterium commonly found in various agronomic crops. Maize white spot (MWS) has been one of the most destructive diseases affecting maize, leading to significant economic losses. This study clarified that P. ananatis strains colonized maize leaves but were not the causal agents of MWS in Yunnan Province, China. Moreover, most of these P. ananatis strains exhibited plant growth-promoting (PGP) activities, induced hypersensitive response (HR) activity on tobacco, and caused rot symptoms in onion. Notably, the analysis of divergence throughout the evolutionary process revealed significant genomic evolution and environmental adaptation in these P. ananatis strains. This highlights the genetic exchange that has shaped the genome of P. ananatis. These findings improve our understanding of the functional diversity of P. ananatis strains across different hosts and their positions within the evolutionary lineages of P. ananatis species.

In Thailand, the median-striped bullfrog, Kaloula mediolineata is a commercially important amphibian because it is a natural food source that is easily accessible in rural areas. However, there is currently a dearth of knowledge, especially on the population diversity of this species. This study, therefore, aims to evaluate the genetic variation of K. mediolineata in Thailand using the partial cytochrome b (Cyt-b) and 16S ribosomal RNA (16S rRNA) sequences. A total of 118 samples from 20 localities (Provinces) were collected. K. mediolineata has been classified into 52 and 31 haplotypes of Cyt-b haplotypes (Km1-Km52) and 16S rRNA haplotypes (Kr1-Kr31), respectively, which reflect high levels of genetic variation in this species. The 16S rRNA tree demonstrated a monophyletic group in our examined samples. Based on the Cyt-b sequence, the haplotype network and phylogenetic tree analyses showed that there are two separate genetic groups, which are named G1 and G2. These two divergent groups showed genetic differences with p-distance and FST values of 0.028 and 0.608 (p-value < 0.001), respectively. Genetic group G1 is strictly found in certain localities in the north, central, and east regions, whereas G2 is most widespread in this study. Two specimens showed high genetic differences from the others, which indicated that other cryptic genetic groups may exist. Thus, a comprehensive investigation of the biology, ecology, as well as genetic variation of K. mediolineata could be further conducted.

Larval settlement is of interest both for ecologists and for evolutionary biologists, who have proposed that anterior sensory systems for substrate selection provided the basis for animal brains. Nevertheless, the cellular and molecular regulation of settlement, including in Cnidaria (corals, jellyfish, sea anemones, and hydroids), is not well understood. We generated and compared anterior (aboral) transcriptomes and single-cell RNA sequencing datasets from the planula larvae of three cnidarian species: the jellyfish Clytia hemisphaerica and the corals Astroides calycularis and Pocillopora acuta. Integrating these datasets and characterizing aboral cell types, we defined common cellular features of the planula aboral end and identified clade-specific specializations in cell types. Among shared features were genes implicated in taurine uptake and catabolism expressed in distinct specialized aboral cell types. In functional assays using both Clytia and Astroides planulae, exogenous taurine inhibited settlement. These findings define the molecular and cellular architecture of the planula aboral pole and implicate localized taurine destruction in regulating settlement.

The mitochondrial translation system contains two ribosome rescue factors, ICT1 and MTRFR (C12orf65), which hydrolyze peptidyl-tRNA in stalled ribosomes. ICT1 also functions as a ribosomal protein of the mitochondrial large ribosomal subunit (mtLSU) in mice and humans, and its deletion is lethal. In contrast, MTRFR does not share this role. Although loss-of-function mutations in MTRFR have been linked to human mitochondrial diseases, data on this association in other vertebrates are lacking. Here, attempts to generate Mtrfr knockout mice were unsuccessful. However, knockout zebrafish lines were successfully generated for both ict1 and mtrfr (ict1-/- and mtrfr-/-). Both knockout lines appeared healthy and fertile. ict1-/-, mtrfr-/-, and wild-type adult caudal fin cells showed significant differences in mitochondrial morphology. The ict1 deletion affected the network properties more than the number of individuals and networks, whereas the mtrfr deletion exhibited the opposite effect. Additionally, the survival rates of the knockout line larvae were significantly lower than those of the wild-type larvae under starvation conditions. These results suggest that ict1 and mtrfr are required for survival under specific stress conditions, whereas ict1-/- and mtrfr-/- involve different compensatory mechanisms in response to loss of either factor under nonstress conditions. Ict1 proteins from all teleosts, including zebrafish, lack the N-terminal mtLSU-binding motif found in most metazoans, suggesting that Ict1 does not function as a ribosomal protein in teleosts. Thus, Mtrfr may partially compensate for the loss of Ict1. In conclusion, zebrafish appear to exemplify a limited category of vertebrates capable of enduring genetic abnormalities in ict1 or mtrfr.

In this study, we identified a novel mitovirus, designated as "Pleurotus pulmonarius duamitovirus 1" (PpDMV1), from the edible fungus Pleurotus pulmonarius. Genome sequencing revealed a 2,622-nucleotide (nt) genome containing a single 2,001-nt open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp). Full-length genome sequence comparisons using BLASTx demonstrated the closest sequence similarity (56.68% identity) to Erysiphe necator associated mitovirus 15 (EnMV15). Phylogenetic analysis positioned PpDMV1 within the genus Duamitovirus (family Mitoviridae). To our knowledge, this is the first report of a mitovirus infection in P. pulmonarius.

Iron deficiency is the prevalent and most widespread nutritional shortfall for humans, affecting over 30% of the global population and leading to anemia, particularly among preschool-aged children and pregnant women in developing countries. Simultaneously, while half of the world's population depends on rice (Oryza sativa L.) as a staple food, this cereal does not provide a sufficient amount of that micronutrient to meet these people's nutritional needs: even when iron is readily available in the soil, it does not accumulate in the consumed portion of the grain, namely, the starchy endosperm, being instead retained in the aleurone layer, in the pericarp and in the embryo. In this context, the present work applies computational biology tools-such as normal mode analysis and molecular dynamics simulations-to elucidate the behavior and transport mechanism of the Vacuolar Iron Transporter 2 (OsVIT2), a central protein for iron homeostasis in rice, with the objective of laying the foundations for future OsVIT2 engineering projects that could be articulated with ongoing efforts to promote iron biofortification in rice. We shed light on the interplay between protonation state, configuration and hydration of OsVIT2's pore; on the mechanics of its opening and on the ever-shifting hydrogen bond network contained within it. We also explore the potential contribution of the "flexible arms" to the iron-capturing function performed by the cytoplasmic domain.

Treatment of chronic or non-healing wounds has faced a considerable clinical challenge and impose several detrimental effects on individuals, society, the healthcare system, and the economy. Bioactive peptides have been employed to accelerate wound healing in active wound treatment efficiently and effectively. In the current study, a novel wound-healing peptide, WHP1, was designed from 23 existing wound-healing peptides by a rational template-assisted approach. It demonstrated the ability to enhance migration and proliferation of human keratinocyte cell lines (HaCaT) without exhibiting cytotoxic effects on human red blood cells and HaCaT cells. By quantitative proteomic analysis, WHP1 exerted a multifaceted role on diverse cellular processes in human keratinocyte. Notably, it increased the expression of intracellular proteins of HaCaT cells involved in cell cycle regulation and focal adhesion, including centromeric histone H3 variant CENPA, ubiquitin-conjugating enzyme E2 C, thyroid receptor-interacting protein 6, and ribosomal components essential for cell adhesion and migration. WHP1 upregulated the key enzyme glyceraldehyde-3-phosphate dehydrogenase, orchestrating metabolic biosynthesis particularly glycolysis, cell cycle regulation, and cytoskeletal processes. An intriguing observation was the antioxidant activity of WHP1, protecting cells from reactive oxygen species-induced senescence. This is consistent with the upregulation of GAPDH expression and reduction of histone H2A.J levels. WHP1 also stimulated macrophages to secrete transforming growth factor-β (TGF-β), a crucial growth factor necessary for the remodeling phase of wound healing. This investigation highlighted the feasibility of rational design to create novel wound-healing peptides. Such advancements hold promise for improving patients' quality of life and elevating the standard of care in contemporary healthcare.

Bacillus cereus sensu lato is frequently involved in foodborne toxico-infections and is found in various foodstuff. It is unclear whether certain strains have a higher affinity for specific food matrices, which can be of interest for risk assessment. This study reports the characterization by whole-genome sequencing of 169 B. cereus isolates, isolated from 12 food types and soil over two decades. Any potential links between the food matrix of isolation, the isolate's genetic lineage and/or their (putative) virulence gene reservoir were investigated. More than 20 % of the strains contained the genes for the main potential enterotoxins (nheABC, hblCDA and cytK_2). Cereulide biosynthesis genes and genes encoding hemolysins and phospholipases, were detected in multiple isolates. Strain typing revealed a high diversity, as illustrated by 84 distinct sequence types, including 26 not previously described. This diversity was also reflected in the detection of all seven panC types and 71 unique virulence gene profiles. Core-genome MLST was used for phylogenomic investigation of the entire collection and SNP-based clustering was performed on the four most abundant sequence types, which did not reveal a clear affinity for specific B. cereus lineages or (putative) virulence genes for certain food matrices. Additionally, minimal genetic overlap was observed between soil and foodborne isolates. Clusters of closely-related isolates with common epidemiological metadata were detected. However, some isolates from different food matrices or collected several years apart were found to be genetically identical. This study provides elements that can be used for risk assessment of B. cereus in food.

Plant cell walls contain a meshwork of cellulose fibers embedded into a matrix of other carbohydrate and non-carbohydrate-based biopolymers. This composite material exhibits extraordinary properties, from stretchable and pliable cell boundaries to solid protective shells. Cellulose, a linear glucose polymer, is synthesized and secreted across the plasma membrane by cellulose synthase (CesA), of which plants express multiple isoforms. Different subsets of CesA isoforms are necessary for primary and secondary cell wall biogenesis. Here, we structurally and functionally characterize the Glycine max (soybean) primary cell wall CesAs CesA1, CesA3, and CesA6. The CesA isoforms exhibit robust in vitro catalytic activity. Cryo-electron microscopy analyses reveal their assembly into homotrimeric complexes in vitro in which each CesA protomer forms a cellulose-conducting transmembrane channel with a large lateral opening. Biochemical and co-purification analyses demonstrate that different CesA isoforms interact in vitro, leading to synergistic cellulose biosynthesis. Interactions between CesA trimers are only observed between different CesA isoforms and require the class-specific region (CSR). The CSR forms a hook-shaped extension of CesA's catalytic domain at the cytosolic water-lipid interface. Negative stain and cryo-electron microscopy analyses of mixtures of different CesA isoform trimers reveal their side-by-side arrangement into loose clusters. Our data suggest a model by which CesA homotrimers of different isoforms assemble into cellulose synthase complexes to synthesize and secrete multiple cellulose chains for microfibril formation. Inter-trimer interactions are mediated by fuzzy interactions between their CSR extensions.

Synaptic vesicle glycoprotein 2B (SV2B) gene plays a crucial role in neuromodulation and neurotransmission and is a key regulator of synaptotagmin trafficking. However, physiological functions of this gene in insects remain poorly understood. In this study, we investigated the function of the BmSV2B gene in growth and development of silkworms. Tissue expression profiling revealed that BmSV2B is highly expressed in head and midgut. A phylogenetic tree and sequence alignment demonstrated that this gene is highly conserved among lepidopteran insects. Knockout of BmSV2B using the clustered regularly interspaced small palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas9) system resulted in smaller body size compared to the wild type (WT) strain. In the BmSV2B mutants, the levels of triacylglycerol were dramatically lower than that in WT. Furthermore, we found that deletion of BmSV2B extended the developmental time of larvae and led to early larval death. High-throughput RNA sequencing and quantitative real-time polymerase chain reaction analysis showed that the expression levels of juvenile hormone-degrading genes, digestive genes, 20-hydroxyecdysone -response genes and forkhead box O (FOXO) were significantly affected by the absence of BmSV2B. Taken together, BmSV2B is essential for early larval development in silkworms and could serve as a potential target for insecticides, offering a more effective approach to pest control management.

Tick-borne pathogen infections are an increasing occurrence globally, yet many aspects of pathogen maintenance and host-tick interactions remain poorly understood. Here we consider the potential role of eastern cottontails (Sylvilagus floridanus) in the enzootic cycles of tick-borne pathogens of medical importance in Virginia. Over a 3-year period, ticks and blood were collected from rabbits acquired through passive surveillance in 21 counties in Virginia. Seven hundred seventy ticks were collected from 90 of the 121 rabbits examined in this study. Tick species collected from the rabbits included Haemaphysalis leporispalustris, Haemaphysalis longicornis, Amblyomma americanum, Dermacentor variabilis, and Ixodes spp. Ticks identified as Ixodes spp. and H. leporispalustris were tested in pools for Borrelia burgdorferi, Borrelia miyamotoi, Anaplasma phagocytophilum, and Powassan virus (POWV). Borrelia burgdorferi and A. phagocytophilum were detected in several Ixodes spp. pools yielding a pooled infection rate of 4.6% and 3.7%, respectively. These bacterial pathogens along with POWV were detected in pools of H. leporispalustris yielding pooled infection rates of 0.2%, 0.2%, and 0.5%, respectively. In addition, 3 rabbits were found to have neutralizing antibodies against POWV indicating exposure to this tick-borne flavivirus. We describe the presence of infected ticks (including juvenile ticks that could bite humans as adults) utilizing rabbits as hosts, as well as evidence of POWV infection (1.75% seroprevalence) in rabbit sera. These results provide useful information about the role of rabbits as hosts to infected ticks, though cannot ascertain their role in the maintenance or the transfer of pathogens from the rabbits to naïve ticks. Future studies are warranted to explore any additional roles these and other lagomorphs may be playing in the enzootic cycle of tick-borne pathogens.

The X-linked orange (O) locus in domestic cats controls an unknown molecular mechanism that causes the suppression of black-brownish pigmentation in favor of orange coloration. The alternating black-brownish and orange patches seen in tortoiseshell and calico cats are considered classic examples of the phenotypic expression of random X chromosome inactivation (XCI) occurring in female mammals. However, the O gene in the cat genome has not been identified, and the genetic variation responsible for the orange coloration remains unknown. We report here that a 5.1-kilobase (kb) deletion within an intron of the X-linked ARHGAP36 gene, encoding a Rho GTPase-activating protein, is closely and exclusively associated with orange coloration. The deleted region contains a highly conserved putative regulatory element, whose removal is presumed to alter ARHGAP36 expression. Notably, ARHGAP36 expression in cat skin tissues is linked to the suppression of many melanogenesis genes, potentially shifting pigment synthesis from eumelanin to pheomelanin. Furthermore, we find evidence that the gene undergoes XCI in female human and mouse cells and XCI-dependent CpG island methylation consistent with random XCI in female domestic cats. The 5.1-kb deletion seems widespread in domestic cats with orange coat coloration, suggesting a single origin of this coat color phenotype.

Triculamin is a ribosomally synthesized and post-translationally modified peptide (RiPP) lasso peptide with potent antimycobacterial activity, produced by an unusual, non-canonical biosynthetic gene cluster (BGC). In this study, we elucidate the biosynthetic pathway of triculamin through heterologous expression and show that the biosynthesis proceeds in the presence of a precursor (triA), macrocyclase (triC), and N-acetyltransferase (triT). Through in vitro triT acetylation and bioactivity assays, we show that acetylation functions as a resistance mechanism. Genomic searches of triculamin BGC genes across bacteria show that triculamin is more widely distributed than previously anticipated, as triculamin-like core peptides are found in at least three phyla in contrast to previously described lasso peptides that are typically restricted to one phylum. Triculamin BGCs with both canonical and non-canonical RiPP biosynthetic genes were identified. Two strains containing canonical triculamin-like BGCs were chemically characterized and shown to produce the novel triculamin-like lasso peptides palmamin and gelatinamin, the latter of which appears to have an unprecedented additional ring formation. Detailed phylogenetic investigation of the macrocyclases from triculamin-like BGCs suggests that these molecules are products of convergent evolution. These findings broaden the evolutionary and functional landscape of lasso peptides, revealing their unexpected diversification and cross-phylum distribution.

Tricholoma kakishimeji, a poisonous fungus containing the toxic compound ustalic acid, has sometimes been misidentified as closely related species (T. stans, T. matsushimeji, T. kakishimejioides) under the name T. ustale in Japan until recently. Tricholoma ustale s. str. was not found in Japan according to a recent study, and it has been only recorded in Europe. Here, we report the first comprehensive morphological comparison of ectomycorrhizae among these four Tricholoma species. Several cultured strains of these species were inoculated onto Pinus densiflora in vitro. The resulting ectomycorrhizal pine seedlings were subsequently used as mother plants to establish an ectomycorrhizal system on Fagaceae plants. Although all tested fungal strains formed ectomycorrhizae on pine, mycorrhizal colonization by T. kakishimejioides was limited. On Quercus hosts, T. matsushimeji exhibited discontinuous Hartig net development, whereas T. kakishimeji and T. stans produced distinct Hartig nets. Additionally, ectomycorrhizal biomass development on oak hosts was limited in T. stans and T. matsushimeji. These findings correspond to the habitat characteristics of these fungal species. Ectomycorrhizae of these Tricholoma species sampled from natural forests showed morphological and anatomical characteristics similar to their in vitro ectomycorrhizae, including species-specific hyphal arrangements of the mantle and rhizomorphs. We propose that the ectomycorrhizal specificity of Tricholoma can be experimentally assessed in relation to their genetic background on pine and oak hosts, as well as the phyloecological characteristics of these fungal species.

Currently, limited information is available on the molecular basis of the biosynthesis of essential oil in the Monarda citriodora plant. Given the pivotal role of the MEP pathway in the biosynthesis of monoterpenes, in the present study, DXS genes have been functionally characterized from M. citriodora, for the first time. The CDS corresponding to four McDXS genes (1-4) were cloned, and their deduced proteins displayed distinct phylogenetic positioning. Using a bacterial complementation test, we demonstrated that all four McDXS genes encode functional DXS proteins. Based on the results obtained from phylogenetic analysis, tissue-specific expression analysis, and accumulation of monoterpenes, McDXS2 was identified as the candidate gene involved in the biosynthesis of monoterpenes of essential oil in M. citriodora. Transient overexpression and silencing of McDXS2 significantly modified the content of volatile monoterpenes in M. citriodora. Constitutive expression of McDXS2 in Nicotiana tabacum resulted in increased biosynthesis of specialized diterpenoids. Further, the exogenous treatment of MeJA, ABA, and GA3 modulated the expression of McDXS2, and the content of the components of essential oil in M. citriodora. McDXS2 promoter activity was primarily restricted to the glandular trichomes of M. citriodora. The present work demonstrates that McDXS2 is primarily involved in the specialized terpenoid biosynthesis in M. citriodora.

Pulmonary lophomoniasis is an emerging disease caused by the protozoan pathogen Lophomonas spp. Recently, a conventional polymerase chain reaction (PCR) method has been developed. However, its sensitivity and specificity remain to be fully established. Therefore, this study aimed to develop in-house conventional and multiplex PCR for the detection and identification of Lophomonas infections. Additionally, we attempted to compare the diagnostic performance of these novel PCR tests with the current microscopic examination method using BAL samples.

We studied 120 bronchoalveolar lavage (BAL) specimens of the patients clinically suspected of having lophomoniasis. The specimens were examined using three methods: microscopic examination (Giemsa staining), in-house conventional PCR, and multiplex-PCR. Moreover, multiplex-PCR was used for the simultaneous identification of two species of Lophomonas.

Out of the 120 BAL specimens tested, 30 (25%) tested positive through microscopic wet mount examination. Among the three techniques, multiplex-PCR was the most sensitive (100%, 95% CI, 88.3-100), while Giemsa staining had the lowest sensitivity (86.2%, 95% CI, 69.4-94.5). The data reveal a strong agreement between multiplex-PCR and conventional PCR (κ = 0.96), while the lowest agreement was found between multiplex-PCR and microscopy methods (κ = 0.16). The study also confirmed the presence of L. blattarum species in all samples using multiplex-PCR.

This study demonstrates that the in-house multiplex-PCR is a robust and accurate diagnostic test for the detection and identification of Lophomonas species. Therefore, our findings suggest that this method may be a powerful tool to overcome some diagnostic pitfalls for lophomoniasis.

Ixodes auritulus Neumann, 1904 (Acari: Parasitiformes: Ixodidae) represents a species complex principally associated to birds belonging to the orders Ciconiiformes, Charadriiformes, Columbiformes, Falconiformes, Galliformes, Passeriformes, Piciformes, Pelecaniformes, Procellariiformes, Strigiformes, and Tinamiformes in both immature and adult stages. This is a cosmopolitan tick species whose distribution encompass the Afrotropical, Australasian, Nearctic and Neotropical Zoogeographic Regions, and Pacific Oceans islands. Ixodes auritulus sensu stricto was described from southern Chile, and recently new species from this complex were described based only on morphological characters. In this study, specimens of ticks determined to belong to the I. auritulus complex obtained from the Southern Cone of America in different biogeographic regions of Argentina, southern Chile, Brazil and Uruguay were analyzed. Additionally, a female paratype of Ixodes rio Apanaskevich & Labruna, 2022 from the southern from Brazil was included in the study. Morphological characters were analyzed and phylogenetic analyses were performed by obtaining partial mitochondrial DNA sequences of the 16S rRNA and cox1 genes. The specimens from Punta Arenas, Magallanes Province, southern Chile (type locality) correspond morphologically to I. auritulus s.s. and those from central and northern Argentina (Pampa and Yungas Biogeographic Provinces), Uruguay and southern Brazil (Pampa Biogeographic Province) were morphologically compatible with I. rio. The phylogenetic analysis based on mitochondrial DNA sequences support the classification of I. auritulus s.s. from southern Chile and I. rio from Argentina, Brazil and Uruguay as two distinct species. Additional morphological and molecular analyses of ticks from ecological regions other than those included here are necessary to deepen the knowledge of the diversity of the I. auritulus complex in the Southern Cone of America.

Male-killing bacterial symbionts, prevalent in arthropods, skew population sex ratios by selectively killing male progeny, profoundly impacting ecology and the evolution of their hosts. Male killing is a convergently evolved trait, with microbes evolving diverse male-killing mechanisms across host species with widely divergent sex determination pathways. A common evolutionary response to male-killing presence is the spread of suppressor mutations that restore male survival. In this study, we demonstrate the evolution of a novel male-killing mechanism that is insensitive to an existing male-killing suppressor. Hypolimnas bolina butterflies from Yogyakarta, Indonesia, showed extreme female-biased population sex ratio associated with high prevalence of a male-killing Wolbachia. This strain, wBol1Y, shared a very recent common ancestor with the previously characterized "suppressed" male-killing strain in the species, wBol1, but it retained its male-killing ability in the presence of the male-killing suppressor. The genome of wBol1Y differed from the suppressed wBol1 in carrying an additional prophage that included strong candidate genes for male killing. In vitro and in vivo data demonstrated that wBol1Y feminized splicing and expression of lepidopteran sex determination pathway genes and that the gene Hb-oscar-present on wBol1Y's unique prophage insert-was sufficient to disrupt the male sex determination pathway. Our study demonstrates that the diversity of male-killing mechanisms is a product both of interaction with varying insect sex determination systems and the evolution of male killing within a host species. Our data indicate that the male killer and host may be involved in escalating arms races, where spreading male-killing suppression drives the evolution of additional systems that reestablish male killing by the symbiont.

Scale insects (Coccidae, Hemiptera) and whiteflies (Aleyrodidae, Homoptera) are diminutive, ubiquitous, sap-sucking plant parasites, many of which are serious agricultural pests. Over the course of several years, an investigation into entomopathogenic fungi affecting scale insects and whiteflies resulted in the collection of 13 novel species of Clavicipitaceae in Yunnan and Hainan Provinces, China. Based on three-loci (nrLSU, tef-1a, and rpb1) phylogenetic analysis and morphological evidence, it was determined that two new genera, Paramoelleriella and Polymicrospora, each encompassed a new species. Additionally, two new species of Hypocrella s. str. and nine new species of Moelleriella were identified. Within the Moelleriella clade, seven new species were assigned to the Effuse clade and two to the Globose clade. Hypocrella s. str. and Samuelsia were included in the Pulvinate clade, to which the new genus Paramoelleriella is closely related, although it forms a distinct branch. Paramoelleriella species exhibited characteristics similar to those of Moelleriella, including globose to subglobose, yellow to orange teleomorphic stromata, with perithecia densely arranged and fully embedded in the stromatal tissue. Its ascospores disarticulated into short-cylindrical part-spores, and the conidiomata featured large, widely open orifices bearing fusoid conidia curved to one side. Species of the new genus Polymicrospora were characterized by thin-pulvinate, snow-white to off-white teleomorphic stromata with surface smooth. These species possessed numerous obpyriform or oval, semi-embedded, and densely arranged perithecia, cylindrical asci, and ascospores that disarticulated into small, oval part-spores in large quantities. This study introduces two new genera and 13 new species, accompanied by detailed illustrations and descriptions.

Aldehydes are reactive compounds that play crucial roles in various metabolic processes within plants. However, their accumulation can lead to toxic effects, Aldehyde dehydrogenases (ALDHs) represent a diverse family of enzymes that catalyze the oxidation of aldehydes to carboxylic acids. ALDHs help mitigate the toxic effects of these compounds and maintain cellular homeostasis in plants. In this study, a bioinformatics analysis of the Medicago truncatula genome identified 27 MtALDHs, which were classified into ten distinct groups based on their phylogenetic relationships. The distribution of these families across the chromosomes of M. truncatula is uneven, with segmental duplications being the primary factor contributing to the expansion of this gene family within the species. The gene structure and motif analysis within each ALDH family in M. truncatula, along with its orthologous genes in Arabidopsis, exhibits a high degree of conservation. The promoter region analysis of these genes reveals a rich abundance of cis-regulatory elements that respond to various environmental stresses and hormones. Furthermore, examination of the expression patterns of MtALDH genes using available microarray data indicated that several of these genes exhibit high expression levels throughout all developmental stages in M. truncatula. Additionally, some genes display tissue-specific expression and are induced in response to salt stress, suggesting a significant role for these genes in growth processes and stress responses within M. truncatula. The findings from this study provide essential insights and data necessary for the functional evaluation of each MtALDH gene during developmental stages and in response to environmental stresses.

Alfalfa is widely regarded as one of the most important forage crops globally. However, its growth and development are primarily constrained by various abiotic stresses. FIMBRINs are crucial actin-binding proteins involved in regulating cellular dynamics in plants under various stress conditions and during developmental processes. The Fimbrin (FIM) gene family has been reported only in Arabidopsis, while a comprehensive identification of the FIM gene family in alfalfa and the responses of its members to abiotic stresses remain unclear.

In this study, six MsFIM genes were identified in the alfalfa genome, distributed across three chromosomes. Phylogenetic analysis grouped these genes into four clades, all containing the conserved CH domain. Gene duplication events suggested that large fragment duplications contribute to gene amplification. Furthermore, cis-regulatory element analysis highlighted their pivotal roles in plant development and response to external abiotic stresses. RT-qPCR analyses revealed that the MsFIM genes exhibited differential expression across various tissues, with predominant expression in flowers, stems, and leaves. The MsFIM genes showed elevated expression under abiotic stresses (drought, cold, and salt) as well as hormone treatment (abscisic acid, ABA), suggesting that they served as positive regulators in alfalfa's resistance to abiotic stresses and its growth and development.

This study investigates the MsFIM genes in alfalfa, further analyzing their potential roles in plant development and response to abiotic stresses. These findings will provide novel insights into the molecular mechanisms of alfalfa's stress response.

Many challenges arise when monitoring organisms with cryptic life-histories. For example, some cryptic life-stages are hard to identify or sample due to their microscopic nature, which creates unknowns surrounding an organism's population dynamics. Environmental DNA (eDNA) is a non-invasive sampling technique used to monitor cryptic species when traditional survey methods are challenging. Generally, eDNA has been used to quantify the presence/absence of species in various habitats. However, recent advances in high-throughput amplicon sequencing techniques have enabled researchers to detect intraspecific genetic diversity with eDNA. In this study, we present two complementary R packages that can be used to estimate the number of individuals in an eDNA sample. The first package (Amplicomsat) cleans high-throughput amplicon microsatellite sequences and counts the observed alleles identified in eDNA. Our second package (GenotypeQuant) then uses a numerical maximum likelihood estimator (NMLE) to estimate the number of contributors most likely to have produced the sequenced panel of microsatellite alleles amplified from eDNA. We first present simulations to characterise the accuracy and precision of the method. We then estimated densities of Nereocystis luetkeana (bull kelp) microscopic gametophytes from eDNA collected from an experiment with a manipulated number of gametophytes. Finally, we analysed benthic eDNA from kelp forest habitats. We found that gametophyte estimates produced by the NMLE varied within +3/-2 individuals when processing eDNA from rocks with 8 seeded gametophytes. We estimated 500 to 800 gametophytes·m-2 densities in July, five or more months since spore germination and before the current year's spore release. Gametophyte abundance scaled with the sampling area and numbers were higher than total sporophyte densities.

Development of an effective vaccine against Lumpy Skin Disease Virus (LSDV) is crucial for protecting livestock. The current study outlines a web-based platform developed to aid the scientific community in designing effective peptide-based vaccines against LSDV. First, we generated all possible overlapping (K-mer value 9 and 15) peptides from the proteins of 73 LSDV strains. Second, after removing redundancy, the obtained peptides were utilized for predicting B-cell and T-cell epitopes. Third, the predicted B-cell and T-cell epitopes were screened for immunogenicity, allergenicity, and toxicity. Finally, the LSDV candidate vaccine database was developed utilizing 3913 unique B-cell (Linear 3344 and conformational 569) and 6473 unique T-cell (MHC-I 3200 and MHC-II 3273) epitopes. The three-dimensional structure of 156 LSDV proteins from reference (AF325528.1) LSDV genome was predicted using I-TASSER software and implemented in the database. Additionally, tools for genome analysis like DotPlot, Gblocks, BLAST, and gRNA designing were incorporated into the database. In summary, LSDVvac has been developed, which integrates information about predicted potential vaccine candidates along with useful computational tools. LSDVvac database is available at http://45.248.163.59/bic/lsdb/.

Fatty acid esters are widely used in fragrance compounds, solvents, lubricants, and biofuels. Enzymatic synthesis of these esters in aqueous phase is an environmentally friendly approach. In this study, an esterase RasEst3 from Rasamsonia emersonii was identified for fatty acid ester synthesis through sequence alignment. The gene encoding RasEst3 was heterologously expressed in Escherichia coli BL21(DE3), and its enzymatic properties were analyzed. The enzyme exhibited optimal activity at pH 3.5 and 30 °C, with a preference for medium-chain substrates. Structurally, RasEst3 contains a lid domain and a catalytic domain, with a catalytic triad composed of Ser146-His227-Asp214. The smaller pocket spatial site resistance and the hydrophobicity of the substrate channel facilitate effective substrate binding to the active center. Site-directed mutagenesis and molecular dynamics simulations revealed that the oxygen anion holes formed by Gly69 and Thr70, along with the π-bond stacking formed by Tyr112 and Tyr145, play crucial roles in catalysis. After removing a loop region from RasEst3, its ethyl octanoate synthesis activity increased by 253.22 % compared to the wild-type enzyme. This study not only clarifies the structure-function relationship of RasEst3 but also provides valuable insights for developing novel biocatalysts in green chemistry.

Actin is an intrinsically dynamic protein, the function and state of which are modulated by actin-binding proteins. Actin-depolymerizing factors (ADF)/cofilins are ubiquitous actin-binding proteins that accelerate actin turnover. Malaria is an infectious disease caused by parasites of the genus Plasmodium, which belong to the phylum Apicomplexa. The parasites require two hosts to complete their life cycle: the definitive host, or the vector, an Anopheles spp. mosquito, and a vertebrate intermediate host, such as humans. Here, the malaria vector Anopheles gambiae ADF (AgADF) crystal structure is reported. AgADF has a conserved ADF/cofilin fold with six central β-strands surrounded by five α-helices with a long β-hairpin loop protruding out of the structure. The G- and F-actin-binding sites of AgADF are conserved, and the structure shows features of potential importance for regulation by membrane binding and redox state. AgADF binds monomeric ATP- and ADP-actin with a high affinity, having a nanomolar Kd, and binds effectively also to actin filaments.

The RNA N6-adenosine methylation, resulting in N6-methyl adenosine (m6A), is one of the most important post-transcriptional modification events in the eukaryotic transcriptome, which is dynamically regulated by methyltransferases (writers), recognition proteins (readers) and demethylases (erasers). Human has five m6A readers namely YTHDC1, YTHDC2, YTHDF1, YTHDF2 and YTHDF3 that specifically recognize and bind to the methylated m6A residue of RNA through their YT521-B homology (YTH) domains, which have been involved in the pathogenesis of diabetes mellitus and its diverse complications such as diabetic nephropathy. Instead of the native N6-methylation, we herein attempted to explore the molecular effect of various non-native N6-substitutions on adenosine (A) binding behavior to YTH domains. A systematic interaction profile of 40 reported N6-substituted adenosine (x6A) mononucleotides with 5 human reader YTH domains was created computationally. Heuristic clustering of the profile divided these YTH domains and these x6A mononucleotides into two subfamilies and three classes, respectively; they represent distinct intrinsic interaction modes between the domains and mononucleotides. Statistical survey unraveled that the volume (Vg) and hydrophobicity (Hg) of N6-substituted chemical groups exhibit linear and nonlinear correlations with the binding energy (ΔGttl) of x6A mononucleotides to YTH domains, respectively; N6-substitutions with moderate size and weak polarity are favorable for the x6A binding. From the profile the N6-bromomethyl adenosine (brm6A) was identified as a potent binder of YTHDF2 YTH domain; its affinity was improved significantly by 77.2-fold from A and considerably by 19.5-fold from m6A. Structural modeling observed that the N6-bromomethyl group of brm6A is tightly packed against an aromatic cage defined by the Trp432-Trp486-Trp491 triad of YTHDF2 YTH domain. Electron-correlation analysis revealed that the bromine atom can form geometrically and energetically satisfactory halogen-π interactions with the aromatic cage, thus conferring considerable affinity and specificity to the domain-brm6A interaction.

Pathogenesis-related protein 1 (PR1), a hallmark of plant disease resistance, plays pivotal roles in defense signaling. In this study, we identified 16 intronless PR1 genes in Populus deltoides, all classified within the CAP superfamily (cysteine-rich secretory protein, antigen 5, and pathogenesis-related 1) and characterized by conserved N-terminal signal peptides, caveolin-binding motifs, and CAP-derived peptides. Phylogenomic reconstruction of 231 PR1 homologs across 15 plant species traced their origin to Chara braunii, with lineage-specific expansions driven by gene duplication. Evolutionary analyses revealed strong purifying selection acting on ancestral PR1 paralogs to confer a selective advantage for disease resistance. Integrated transcriptomic profiling and quantitative RT-PCR analyses identified PdePR1_10 as a key marker gene for defense activation, exhibiting significant induction at two days post-inoculation in resistant poplars. Co-expression network analysis indicated that PdePR1_10 interacts with several defense-related genes, including NBS-LRR resistance genes, signaling kinases, and hormone biosynthesis enzymes. Specifically, the W-box cis-regulatory element in the PdePR1_10 promoter is hypothesized to interact with WRKY transcription factors, activating PdePR1_10 expression through a salicylic acid (SA)-dependent signaling pathway. Transgenic poplars overexpressing PdePR1_10 exhibited significantly enhanced rust resistance, confirming its critical in defense response. In summary, we thoroughly elucidated the biological functions and regulatory mechanisms of PR1 genes in rust resistance and provided a valuable transgenic poplar line for future studies.

Effective disease management is crucial for sustainable aquaculture, particularly for economically important species like golden pompano (Trachinotus ovatus). Streptococcus agalactiae represents a major threat to this species, leading to severe health issues and significant economic losses. Understanding the immune mechanisms involved is essential to address this challenge. The IκB and IKK genes are known to be key regulators of immune responses, playing pivotal roles in modulating inflammatory pathways during infections. However, their specific roles in golden pompano immunity are not well characterized. In this study, we used bioinformatics analysis and tissue-specific expression profiling to investigate the roles of IκB and IKK genes in golden pompano during bacterial infection. The results demonstrated that ToIKK was significantly upregulated during the early stages of infection, indicating rapid immune activation, while ToIκB showed an initial decrease followed by recovery, suggesting its involvement in inflammation modulation. These genes were found to regulate the NF-κB signaling pathway, which is crucial for coordinating the immune response to bacterial infection. This research provides valuable insights into the molecular basis of golden pompano immune response against S. agalactiae, offering a foundation for developing targeted anti-infection strategies and improving disease resistance and health management practices in aquaculture.

LysM effectors are suppressors of chitin-triggered plant immunity in biotrophic and hemibiotrophic fungi. In necrotrophic fungi, LysM effectors might induce a mechanism to suppress host immunity during the short asymptomatic phase they establish before these fungi activate plant defenses and induce host cell death leading to necrosis. Here, we characterize a secreted LysM protein from a major necrotrophic fungus, Botrytis cinerea, called BcLysM1. Transcriptional induction of BcLysM1 gene was observed in multicellular appressoria, called infection cushions, in unicellular appressoria and in the early phase of infection on bean leaves. We confirmed that BcLysM1 protein binds chitin in the fungus cell wall and protects hyphae against degradation by external chitinases. This effector is also able to suppress the chitin-induced ROS burst in Arabidopsis thaliana, suggesting sequestration of chitooligosaccharides in apoplast during infection. Moreover, contribution of BcLysM1 in infection initiation and in adhesion to bean leaf surfaces were demonstrated. Our data show for the first time that a LysM effector can play a dual role in mycelial adhesion and suppression of chitin-triggered host immunity, both of which occur during the early asymptomatic phase of infection by necrotrophic fungi.

Two new ɑ-galactosidases PpAgl27B and PpAgl27C from Penicillium parvum 4-14 were functionally investigated in this study. Based on the analysis of catalytic domain and phylogenetic tree, PpAgl27B (435 aa) and PpAgl27C (543 aa) belong to glycoside hydrolase (GH) 27 family. After expression in Pichia pastoris, the recombinant PpAgl27B and PpAgl27C showed the highest activities at pH 3.5 and 65 °C, or 4.0 and 45 °C, respectively. Using p-nitrophenyl-α-d-galactopyranoside (pNPGal) as substrate, the Michaelis constant were 0.90 mM for PpAgl27B and 2.54 mM for PpAgl27C. PpAgl27C had a low catalytic activity toward pNPGal and negligible activities on various natural substrates. Differently, PpAgl27B efficiently released galactose from the artificial substrate, raffinose family oligosaccharides, or galactomannans. Hydrolysis of corn bran arabinoxylan (CBAX) 1 or 2 were conducted by PpAgl27B alone or in combination with the enzyme blend E_CBAX1. PpAgl27B released a small amount of galactose (1.7-3.0 mg/g) from the both substrates. Compared with the individual enzymes, the liberations of galactose, xylose and arabinose from the substrates were significantly enhanced by combing PpAgl27B and E_CBAX1. The degrees of synergy of the enzyme combination for the saccharification of CBAX1 or CBAX2 were 1.20 and 1.13, respectively. PpAgl27B showed promising potential for the valorization of galactose-rich feedstocks as well as CBAX.

Two novel endornaviruses were found in Phytophthora cactorum isolated from black lesions on Boehmeria nivea var. nipononivea plants in a Japanese forest. These two endornaviruses were named Phytophthora cactorum alphaendornavirus 4 (PcAEV4) and Phytophthora cactorum alphaendornavirus 5 (PcAEV5) and have site-specific nick structures in their positive RNA strands, which are hallmarks of alphaendornaviruses. Ribavirin and cycloheximide treatment of the protoplasts effectively cured the host oomycete (strain Kara1) of the viruses. The resultant virus-free strain (Kara1-C) displayed abundant mycelial growth with less zoosporangia formation as compared to the Kara1 strain. Remarkably, the Kara1-C strain exhibited a reduced ability to form black lesions on B. nivea leaves, suggesting that the presence of PcAEV4 and PcAEV5 in the Kara1 strain led to enhanced virulence in host plants. Under osmotic pressure and cell wall synthesis inhibition, the Kara1 strain exhibited less growth inhibition compared with the Kara1-C strain. In contrast, the Kara1 strain showed more growth inhibition in the presence of membrane-permeable surfactant compared with the Kara1-C strain, indicating that the two endornaviruses can alter the susceptibility of the host oomycete to abiotic stresses. Co-localization and cell fractionation analyses showed that PcAEV4 and PcAEV5 localized to intracellular membranes, particularly the endoplasmic reticulum membrane fraction. Furthermore, infection with these two endornaviruses was found to affect the host's response to exogenous sterols, which enhanced vegetative growth and zoosporangia formation, as well as virulence of the host oomycete. These results provide insights into the effects of endornavirus infection in Phytophthora spp. and also highlight the usefulness of protoplast-based methods in advancing Phytophthora virus studies.