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In the human gastrointestinal tract, bacterial beta-D-glucuronidases (BG; E. C. 3.2.1.31) are involved both in xenobiotic metabolism and in some of the beneficial effects of dietary compounds. Despite their biological significance, investigations are hampered by the fact that only a few BGs have so far been studied. A functional metagenomic approach was therefore performed on intestinal metagenomic libraries using chromogenic glucuronides as probes. Using this strategy, 19 positive metagenomic clones were identified but only one exhibited strong beta-D-glucuronidase activity when subcloned into an expression vector. The cloned gene encoded a beta-D-glucuronidase (called H11G11-BG) that had distant amino acid sequence homologies and an additional C terminus domain compared with known beta-D-glucuronidases. Fifteen homologs were identified in public bacterial genome databases (38-57% identity with H11G11-BG) in the Firmicutes phylum. The genomes identified derived from strains from Ruminococcaceae, Lachnospiraceae, and Clostridiaceae. The genetic context diversity, with closely related symporters and gene duplication, argued for functional diversity and contribution to adaptive mechanisms. In contrast to the previously known beta-D-glucuronidases, this previously undescribed type was present in the published microbiome of each healthy adult/child investigated (n = 11) and was specific to the human gut ecosystem. In conclusion, our functional metagenomic approach revealed a class of BGs that may be part of a functional core specifically evolved to adapt to the human gut environment with major health implications. We propose consensus motifs for this unique Firmicutes beta-D-glucuronidase subfamily and for the glycosyl hydrolase family 2.

Artemisinin is a type of sesquiterpene lactone well known as an antimalarial drug, and is specifically produced in glandular trichomes of Artemisia annua. However, the regulatory network for the artemisinin biosynthetic pathway remains poorly understood. Exploration of trichome-specific transcription factors would facilitate the elucidation of regulatory mechanism of artemisinin biosynthesis. The WRKY transcription factor GLANDULAR TRICHOME-SPECIFIC WRKY 1 (AaGSW1) was cloned and analysed in A. annua. AaGSW1 exhibited similar expression patterns to the trichome-specific genes of the artemisinin biosynthetic pathway and AP2/ERF transcription factor AaORA. A β-glucuronidase (GUS) staining assay further demonstrated that AaGSW1 is a glandular trichome-specific transcription factor. AaGSW1 positively regulates CYP71AV1 and AaORA expression by directly binding to the W-box motifs in their promoters. Overexpression of AaGSW1 in A. annua significantly improves artemisinin and dihydroartemisinic acid contents; moreover, AaGSW1 can be directly regulated by AaMYC2 and AabZIP1, which are positive regulators of jasmonate (JA)-and abscisic acid (ABA)-mediated artemisinin biosynthetic pathways, respectively. These results demonstrate that AaGSW1 is a glandular trichome-specific WRKY transcription factor and a positive regulator in the artemisinin biosynthetic pathway. Moreover, we propose that two trifurcate feed-forward pathways involving AaGSW1, CYP71AV1 and AaMYC2/AabZIP1 function in the JA/ABA response in A. annua.

Background Irinotecan is a chemotherapeutic agent used to treat a variety of tumors, including colorectal cancer (CRC). In the intestine, it is transformed into SN-38 by gut microbial enzymes, which is responsible for its toxicity during excretion. Objective Our study highlights the impact of Irinotecan on gut microbiota composition and the role of probiotics in limiting Irinotecan-associated diarrhea and suppressing gut bacterial β-glucuronidase enzymes. Material and methods To investigate the effect of Irinotecan on the gut microbiota composition, we applied 16S rRNA gene sequencing in three groups of stool samples from healthy individuals, colon cancer, and Irinotecan treated patients ( n  = 5/group). Furthermore, three Lactobacillus spp.; Lactiplantibacillus plantarum (L. plantarum) , Lactobacillus acidophilus (L. acidophilus) , Lacticaseibacillus rhamnosus ( L. rhamnosus) were used in a single and mixed form to in-vitro explore the effect of probiotics on the expression of β-glucuronidase gene from E. coli . Also, probiotics were introduced in single and mixed forms in groups of mice before the administration of Irinotecan, and their protective effects were explored by assessing the level of reactive oxidative species (ROS) as well as studying the concomitant intestinal inflammation and apoptosis. Results The gut microbiota was disturbed in individuals with colon cancer and after Irinotecan treatment. In the healthy group, Firmicutes were more abundant than Bacteriodetes, which was the opposite in the case of colon-cancer or Irinotecan treated groups. Actinobacteria and Verrucomicrobia were markedly present within the healthy group, while Cyanobacteria were noted in colon-cancer and the Irinotecan-treated groups. Enterobacteriaceae and genus Dialister were more abundant in the colon-cancer group than in other groups. The abundance of Veillonella , Clostridium , Butryicicoccus, and Prevotella were increased in Irinotecan-treated groups compared to other groups. Using Lactobacillus spp. mixture in mice models significantly relieved Irinotecan-induced diarrhea through the reduction of both β-glucuronidase expression and ROS, in addition to guarding gut epithelium against microbial dysbiosis and proliferative crypt injury. Conclusions Irinotecan-based chemotherapy altered intestinal microbiota. The gut microbiota participates greatly in determining both the efficacy and toxicity of chemotherapies, of which the toxicity of Irinotecan is caused by the bacterial ß-glucuronidase enzymes. The gut microbiota can now be aimed and modulated to promote efficacy and decrease the toxicity of chemotherapeutics. The used probiotic regimen in this study lowered mucositis, oxidative stress, cellular inflammation, and apoptotic cascade induction of Irinotecan.

BRITTLE1 (BT1), responsible for unidirectional transmembrane transport of ADP-glucose, plays a pivotal role in starch synthesis of cereal grain. In this study, we isolated three TaBT1 homoeologous genes located on chromosomes 6A, 6B, and 6D in common wheat. TaBT1 was mainly expressed in developing grains, and knockdown of TaBT1 in common wheat produced a decrease in grain size, thousand kernel weight (TKW), and grain total starch content. High diversity was detected at the TaBT1-6B locus, with 24 polymorphic sites forming three haplotypes (Hap1, Hap2, and Hap3). Association analysis revealed that Hap1 and Hap2 were preferred haplotypes in modern breeding, for their significant correlations with higher TKW. Furthermore, β-glucuronidase (GUS) staining and enzyme activity assays in developing grains of transgenic rice with exogenous promoters indicated that the promoters of Hap1 and Hap2 showed stronger driving activity than that of Hap3. Evolutionary analysis revealed that BT1 underwent strong selection during wheat polyploidization. In addition, the frequency distribution of TaBT1-6B haplotypes revealed that Hap1 and Hap2 were preferred in global modern wheat cultivars. Our findings suggest that TaBT1 has an important effect on starch synthesis and TKW, and provide two valuable molecular markers for marker assisted selection (MAS) in wheat high-yield breeding.

Plants have evolved a variety of mechanisms to adapt to N starvation. NITRATE TRANSPORTER2.4 [NRT2.4) is one of seven NRT2 family genes in Arabidopsis thaliana, and NRT2.4 expression is induced under N starvation. Green fluorescent protein and ß-glucuronidase reporter analyses revealed that NRT2.4 is a plasma membrane transporter expressed in the epidermis of lateral roots and in or close to the shoot phloem. The spatiotemporal expression pattern of NRT2A in roots is complementary with that of the major high-affinity nitrate transporter NTR2.1. Functional analysis in Xenopus laevis oocytes and in planta showed that NRT2.4 is a nitrate transporter functioning in the high-affinity range. In N-starved nrX2A mutants, nitrate uptake under low external supply and nitrate content in shoot phloem exudates was decreased. In the absence of NRT2.1 and NRT2.2, loss of function of NRT2.4 (triple mutants) has an impact on biomass production under low nitrate supply. Together, our results demonstrate that NRT2.4 is a nitrate transporter that has a role in both roots and shoots under N starvation.

Background Agroinfiltration is a simple and effective method of delivering transgenes into plant cells for the rapid production of recombinant proteins and has become the preferred transient expression platform to manufacture biologics in plants. Despite its popularity, few studies have sought to improve the efficiency of agroinfiltration to further increase protein yields. This study aimed to increase agroinfiltration-based transient gene expression in Nicotiana benthamiana by improving all levels of transgenesis. Results Using the benchmark pEAQ-HT deconstructed virus vector system and the GUS reporter enzyme, physical, chemical, and molecular features were independently assessed for their ability to enhance Agrobacterium -mediated transformation and improve protein production capacities. Optimal Agrobacterium strain, cell culture density and co-cultivation time for maximal transient GUS (β-glucuronidase) expression were established. The effects of chemical additives in the liquid infiltration media were investigated and acetosyringone (500 μM), the antioxidant lipoic acid (5 μM), and a surfactant Pluronic F-68 (0.002%) were all shown to significantly increase transgene expression. Gene products known to suppress post-transcriptional gene silencing, activate cell cycle progression and confer stress tolerance were also assessed by co-expression. A simple 37 °C heat shock to plants, 1–2 days post infiltration, was shown to dramatically increase GUS reporter levels. By combining the most effective features, a dual vector delivery system was developed that provided approximately 3.5-fold higher levels of absolute GUS protein compared to the pEAQ-HT platform. Conclusions In this paper, different strategies were assessed and optimised with the aim of increasing plant-made protein capacities in Nicotiana benthamiana using agroinfiltration. Chemical additives, heat shock and the co-expression of genes known to suppress stress and gene silencing or stimulate cell cycle progression were all proven to increase agroinfiltration-based transient gene expression. By combining the most effective of these elements a novel expression platform was developed capable of producing plant-made protein at a significantly higher level than a benchmark hyper-expression system.

Plant viruses have been used as rapid and cost-effective expression vectors for heterologous protein expression in genomic studies. However, delivering large or multiple foreign proteins in monocots and insect pests is challenging. Here, we recovered a recombinant plant cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), for use as a versatile expression platform in cereals and the small brown plan-thopper (SBPH, Laodelphax striatellus) insect vector. We engineered BYSMV vectors to provide versatile expression platforms for simultaneous expression of three foreign proteins in barley plants and SBPHs. Moreover, BYSMV vectors could express the c. 600-amino-acid β-glucuronidase (GUS) protein and a red fluorescent protein stably in systemically infected leaves and roots of cereals, including wheat, barley, foxtail millet, and maize plants. Moreover, we have demonstrated that BYSMV vectors can be used in barley to analyze biological functions of gibberellic acid (GA) biosynthesis genes. In a major technical advance, BYSMV vectors were developed for simultaneous delivery of CRISPR/Cas9 nuclease and single guide RNAs for genomic editing in Nicotiana benthamiana leaves. Taken together, our results provide considerable potential for rapid screening of functional proteins in cereals and planthoppers, and an efficient approach for developing other insect-transmitted negative-strand RNA viruses.

Plant viruses encode RNA silencing suppressor (RSS) proteins to counter the induced antiviral defense, an RNAi silencing mechanism of the host. Indian citrus ringspot virus (ICRSV) causes the ringspot disease, which leads to significant yield loss of kinnow orange. The ICRSV genome contains six open reading frames (ORFs), however, the ORF encoding the potential RSS is not yet known. In this study, we have attempted to identify the RSS protein of ICRSV. To this end, ORF 2,3,4,5 and 6 were cloned into pCAMBIA1302 (35s-GFP) vector, followed by transformation of Agrobacterium tumefaciens and agro-infiltration into leaves of  Nicotiana benthamiana 16c line. Only the leaves infiltrated with 35s-GFP/ORF5 showed a GFP fluorescence signal similar to 35s-GFP/P19, a well-studied positive RSS. Usually, the induced host RNAi silencing is supposed to cleave the expressed GFP-RNA. However, it is suspected that ORF5-encoded protein was able to suppress the host silencing mechanism, leading to the retention of the GFP fluorescence signal. This finding was further supported by beta-glucuronidase (GUS) histochemical assays by infiltrating the construct expressing ORF5-GUS under 35s promoter in the leaves of N. benthamiana . Leaves infiltrated with 35s-GUS/ORF5 formed diX-indigo precipitate similar to leaves infiltrated with, indicating the RSS activity of ICRSV. Later, semi-quantitative PCR and quantitative reverse transcription PCR  ( qRT-PCR) assays showed a higher expression of GFP and GUS in ORF5 agro-infiltrated leaves. Together, these results suggest that ORF5 encoded protein has the potential RSS function of ICRSV which successfully suppresses host RNAi silencing mechanism.

Key messageMusaSNAC1 function in H2O2 mediated stomatal closure and promote drought tolerance by directly binding to CGT[A/G] motif in regulatory region of multiple stress-related genes.Drought is a abiotic stress-condition, causing reduced plant growth and diminished crop yield. Guard cells of the stomata control photosynthesis and transpiration by regulating CO2 exchange and water loss, thus affecting growth and crop yield. Roles of NAC (NAM, ATAF1/2 and CUC2) protein in regulation of stress-conditions has been well documented however, their control over stomatal aperture is largely unknown. In this study we report a banana NAC protein, MusaSNAC1 which induced stomatal closure by elevating H2O2 content in guard cells during drought stress. Overexpression of MusaSNAC1 in banana resulted in higher number of stomata closure causing reduced water loss and thus elevated drought-tolerance. During drought, expression of GUS (β-glucuronidase) under PMusaSNAC1 was remarkably elevated in guard cells of stomata which correlated with its function as a transcription factor regulating stomatal aperture closing. MusaSNAC1 is a transcriptional activator belonging to SNAC subgroup and its 5′-upstream region contain multiple Dof1 elements as well as stress-associated cis-elements. Moreover, MusaSNAC1 also regulate multiple stress-related genes by binding to core site of NAC-proteins CGT[A/G] in their 5′-upstream region. Results indicated an interesting mechanism of drought tolerance through stomatal closure by H2O2 generation in guard cells, regulated by a NAC-protein in banana.

Summary In Arabidopsis, the miR171‐GRAS module has been clarified as key player in meristem maintenance. However, the knowledge about its role in fruit crops like tomato (Solanum lycopersicum) remains scarce. We previously identified tomato SlGRAS24 as a target gene of Sly‐miR171. To study the role of this probable transcription factor, we generated transgenic tomato plants underexpressing SlGRAS24, overexpressing SlGRAS24, overexpressing Sly‐miR171 and expressing β‐glucuronidase (GUS) under the SlGRAS24 promoter (proSlGRAS24‐GUS). Plants overexpressing SlGRAS24 (SlGRAS24‐OE) had pleiotropic phenotypes associated with multiple agronomical traits including plant height, flowering time, leaf architecture, lateral branch number, root length, fruit set and development. Many GA/auxin‐related genes were down‐regulated and altered responsiveness to exogenous IAA/NAA or GA3 application was observed in SlGRAS24‐OE seedlings. Moreover, compromised fruit set and development in SlGRAS24‐OE was also observed. These newly identified phenotypes for SlGRAS24 homologs in tomato were later proved to be caused by impaired pollen sacs and fewer viable pollen grains. At anthesis, the comparative transcriptome results showed altered expression of genes involved in pollen development and hormone signalling. Taken together, our data demonstrate that SlGRAS24 participates in a series of developmental processes through modulating gibberellin and auxin signalling, which sheds new light on the involvement of hormone crosstalk in tomato development.