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In vitro and ex vitro Agrobacterium rhizogenes -mediated hairy root transformation (HRT) assays are key components of the plant biotechnology and functional genomics toolkit. In this report, both in vitro and ex vitro HRT were optimized in soybean using the RUBY reporter. Different parameters including A. rhizogenes strain, optical density of the bacterial cell culture (OD 600 ), co-cultivation media, soybean genotype, explant age, and acetosyringone addition and concentration were evaluated. Overall, the in vitro assay was more efficient than the ex vitro assay in terms of the percentage of induction of hairy roots and transformed roots (expressing RUBY ). Nonetheless, the ex vitro technique was deemed faster and a less complicated approach. The highest transformation of RUBY was observed on 7-d-old cotyledons of cv. Bert inoculated for 30 minutes with the R1000 resuspended in ¼ B5 medium to OD 600 (0.3) and 150 µM of acetosyringone. The parameters of this assay also led to the highest percentage of RUBY through two-step ex vitro hairy root transformation. Finally, using machine learning-based modeling, optimal protocols for both assays were further defined. This study establishes efficient and reliable hairy root transformation protocols applicable for functional studies in soybean.

Gastrodia elata , also named Tianma, is a valuable traditional Chinese herbal medicine. It has numerous important pharmacological roles such as in sedation and lowering blood pressure and as anticonvulsant and anti-aging, and it also has effects on the immune and cardiovascular systems. The whole genome sequencing of G. elata has been completed in recent years, which provides a strong support for the construction of the G. elata gene functional analysis platform. Therefore, in our research, we collected and processed 39 transcriptome data of G. elata and constructed the G. elata gene co-expression networks, then we identified functional modules by the weighted correlation network analysis (WGCNA) package. Furthermore, gene families of G. elata were identified by tools including HMMER, iTAK, PfamScan, and InParanoid. Finally, we constructed a gene functional analysis platform for G. elata 1 . In our platform, we introduced functional analysis tools such as BLAST, gene set enrichment analysis (GSEA), and cis- elements (motif) enrichment analysis tool. In addition, we analyzed the co-expression relationship of genes which might participate in the biosynthesis of gastrodin and predicted 19 mannose-binding lectin antifungal proteins of G. elata . We also introduced the usage of the G. elata gene function analysis platform (GelFAP) by analyzing CYP51G1 and GFAP4 genes. Our platform GelFAP may help researchers to explore the gene function of G. elata and make novel discoveries about key genes involved in the biological processes of gastrodin.

Background The use of probiotics is increasingly popular for health applications, and Lactiplantibacillus plantarum strains are among the most widely studied for their potential in promoting gut health. In this study, we present the first genomic characterization of L. plantarum IGMA4EH, isolated from the gut of the maguey worm ( Aegiale hesperiaris ), an edible insect with traditional significance in Mexico. Methodology Whole-genome sequencing was performed on the L. plantarum IGMA4EH strain. Bioinformatics analyses were conducted to identify probiotic-related genes, antimicrobial potential, and safety features. Genes associated with resistance to environmental stressors, adhesion, and immunomodulation were screened, and safety assessment included the detection of antibiotic resistance genes, virulence factors, and plasmids. Results The genomic analysis revealed the presence of multiple probiotic-related genes, including those associated with resistance to temperature, acidity, oxidative stress, and bile. Additionally, gene clusters related to bacteriocins, and secondary metabolites were identified, suggesting strong antimicrobial potential. Safety evaluations showed no evidence of acquired antibiotic resistance genes, virulence factors, or plasmids. Furthermore, elements related to genetic stability, such as CRISPR-Cas systems and prophage regions, were also detected. Conclusions The findings highlight the potential of L. plantarum IGMA4EH as a novel probiotic strain with promising applications.

Fungi produce diverse secondary metabolites with agricultural and medical potential. Xylaria grammica EL000614 synthesizes grammicin, an isomer of patulin, which shows nematicidal activity against root-knot nematodes with low cytotoxicity. However, the genetic basis of grammicin biosynthesis remains unclear. Here, we characterized two genes, xgpatK and xgpatL , encoding a 6-methylsalicylic acid synthase XgPatK and a Zn(II)2Cys6 transcription factor XgPatL, respectively, within a putative grammicin biosynthetic cluster. Comparative genomic analysis identified 11 cluster genes in X. grammica that are also conserved in patulin-producing fungi, including Aspergillus clavatus and Penicillium expansum . Deletion of xgpatK or xgpatL abolished grammicin production, while complementation restored it, confirming their essential roles. Furthermore, constitutive overexpression of xgpatL increased grammicin production three-fold compared with the wild-type. These findings provide the first genetic and functional evidence of grammicin biosynthesis in X. grammica and demonstrate the regulatory role of XgPatL. Our study advances the understanding of fungal secondary metabolism and highlights grammicin as a potential agent for sustainable biological control of plant-parasitic nematodes.

E3 ubiquitin ligases are vital components of the ubiquitin–proteasome system (UPS), responsible for maintaining protein balance and controlling cellular functions. E3 ligases target specific proteins for degradation or modify their activities through ubiquitin attachment. One prominent E3 ligase family is the ATL family, which comprises 100 members in Arabidopsis thaliana and has significantly expanded in plant genomes. All ATLs share a common domain architecture, featuring a transmembrane domain at the amino-terminal region, a distinct RING-H2 finger domain, and the GLD motif. The RING domain facilitates interactions between E3 ligases, E2-conjugating enzymes, and target proteins, enabling the transfer of ubiquitin molecules. The amino-terminal and carboxy-terminal regions introduce sequence diversity and potentially mediate interactions with other components that assist in UPS function or target recognition. ATLs had been classified within groups, each group encompasses specific ATLs with defined roles in various biological processes. For example, group C-ATLs are implicated in drought tolerance, flower development, phosphate homeostasis, and immune signaling. G-ATLs are associated with carbon/nitrogen stress, immune signaling, salt stress, ABA responses, cadmium tolerance, and sugar-mediated plant growth. A-ATLs participate in early elicitor-response, salt and drought responses, and flowering time regulation. Lastly, D-ATLs are involved in the regulation of programmed cell death. This review let perceive ATLs as a cohesive group of E3 ligases, shedding light on their functional diversifity and redundancy, specifically examining their participation in diverse biological processes, explore their evolutionary history shaped by gene duplication events, and appraise their interactions with key proteins and targets of ubiquitination. This comprehensive overview aims to offer insights into the role of ATLs in plant adaptation, defense mechanisms, and stress tolerance, while also underlying molecular and evolutionary mechanisms and regulatory networks that govern these processes.

Lodging resistance of rice (Oryza sativa L.) has always been a hot issue in agricultural production. A brittle stem mutant, osbc17, was identified by screening an EMS (Ethylmethane sulfonate) mutant library established in our laboratory. The stem segments and leaves of the mutant were obviously brittle and fragile, with low mechanical strength. Examination of paraffin sections of flag leaf and internode samples indicated that the number of cell layers in mechanical tissue of the mutant was decreased compared with the wild type, Pingtangheinuo, and scanning electron microscopy revealed that the mechanical tissue cell walls of the mutant were thinner. Lignin contents of the internodes of mature-stage rice were significantly lower in the mutant than in the wild type. By the MutMap method, we found candidate gene OsBC17, which was located on rice chromosome 2 and had a 2433 bp long coding sequence encoding a protein sequence of 810 amino acid residues with unknown function. According to LC-MS/MS analysis of intermediate products of the lignin synthesis pathway, the accumulation of caffeyl alcohol in the osbc17 mutant was significantly higher than in Pingtangheinuo. Caffeyl alcohol can be polymerized to the catechyl lignin monomer by laccase ChLAC8; however, ChLAC8 and OsBC17 are not homologous proteins, which suggests that the osbc17 gene is involved in this process by regulating laccase expression.

Allene oxide synthase (AOS) is a key enzyme of the jasmonic acid (JA) signaling pathway. The AOS gene was previously found to be upregulated in an Asian chestnut species resistant to infection by the oomycete Phytophthora cinnamomi ( Castanea crenata ), while lower expression values were detected in the susceptible European chestnut ( Castanea sativa ). Here, we report a genetic and functional characterization of the C. crenata AOS (CcAOS) upon its heterologous gene expression in a susceptible ecotype of Arabidopsis thaliana , which contains a single AOS gene. It was found that Arabidopsis plants expressing CcAOS delay pathogen progression and exhibit more vigorous growth in its presence. They also show upregulation of jasmonic acid and salicylic acid-related genes. As in its native species, heterologous CcAOS localized to plastids, as revealed by confocal imaging of the CcAOS-eGFP fusion protein in transgenic Arabidopsis roots. This observation was confirmed upon transient expression in Nicotiana benthamiana leaf epidermal cells. To further confirm a specific role of CcAOS in the defense mechanism against the pathogen, we performed crosses between transgenic CcAOS plants and an infertile Arabidopsis AOS knockout mutant line. It was found that plants expressing CcAOS exhibit normal growth, remain infertile but are significantly more tolerant to the pathogen than wild type plants. Together, our results indicate that CcAOS is an important player in plant defense responses against oomycete infection and that its expression in susceptible varieties may be a valuable tool to mitigate biotic stress responses.

Kitchen waste was mainly composed of carbohydrates, lipids, and proteins. Anaerobic digestion (AD) of kitchen waste usually occurred acidification and further deteriorated. In our previous study, alkali pretreatment combined with bentonite (AP/Be) treatment was proved to enhance high solid AD of kitchen waste. However, effects of AP time on AP/Be were not yet studied. This study investigated the effects of AP time on AP/Be treatment on enhancing high solid AD. The results showed that compared with the control group, the cumulative methane production rate could be increased by 3.30 times (149.7 mL CH 4 /g VS) and the volatile solids (VS) reduction rate increased by 63.36%. Microbial community analysis showed that the relative abundance of Methanosarcina and Methanosaeta were increased from 6.49 and 7.83% to 47.14 and 16.39% respectively. Predictive functional analysis showed that AP/Be treatment increased the abundance of energy production and conversion, coenzyme transport, and metabolism. This study revealed the potential mechanism of AP/Be enhanced kitchen waste AD performance and AP/Be was a potential strategy to strengthen AD.

has garnered increasing attention for its role in improving fermentation results and promoting the biosynthesis of aromatic compounds. This study investigated the effects of exogenously introduced on the microbial community structure, functional gene expression, and volatile aroma profiles during the fermentation of cigar filler leaves. The results demonstrated that significantly enhanced the accumulation of alcohols and ketones. LEfSe analysis identified as a key differential genus in the inoculated group. Spearman correlation analysis revealed positive associations between and , as well as with key aroma compounds such as 1-methyl-4-(2-methyloxiranyl)-7-oxabicyclo [4.1.0] heptane and cis-6-nonenal. EGGNOG functional annotation indicated upregulation of carbohydrate and amino acid metabolism pathways. Additionally, CAZy analysis revealed increased abundance of glycosyltransferases and carbohydrate-binding modules, which may facilitate sugar conversion and utilization. These findings provide a theoretical basis for the application of exogenous microorganisms in cigar fermentation and offer insights into the regulation of microbial community structure for quality improvement.

Vector-encoded fluorescent proteins (FPs) facilitate unambiguous identification or sorting of gene-modified cells by fluorescence-activated cell sorting (FACS). Exploiting this feature, we have recently developed lentiviral gene ontology (LeGO) vectors ( www.LentiGO-Vectors.de ) for multi-gene analysis in different target cells. In this study, we extend the LeGO principle by introducing 10 different drug-selectable FPs created by fusing one of the five selection marker (protecting against blasticidin, hygromycin, neomycin, puromycin and zeocin) and one of the five FP genes (Cerulean, eGFP, Venus, dTomato and mCherry). All tested fusion proteins allowed both fluorescence-mediated detection and drug-mediated selection of LeGO-transduced cells. Newly generated codon-optimized hygromycin- and neomycin-resistance genes showed improved expression as compared with their ancestors. New LeGO constructs were produced at titers >10 6 per ml (for non-concentrated supernatants). We show efficient combinatorial marking and selection of various cells, including mesenchymal stem cells, simultaneously transduced with different LeGO constructs. Inclusion of the cytomegalovirus early enhancer/chicken beta-actin promoter into LeGO vectors facilitated robust transgene expression in and selection of neural stem cells and their differentiated progeny. We suppose that the new drug-selectable markers combining advantages of FACS and drug selection are well suited for numerous applications and vector systems. Their inclusion into LeGO vectors opens new possibilities for (stem) cell tracking and functional multi-gene analysis.