Project description:Basil downy mildew (BDM) caused by Peronospora Belbahrii leads to losses in sweet basil cultivation across the world. Though resistant cultivars of basil exist, the formation of sterile offspring and the introduction of unwanted phenotypic and chemotypic traits slows breeding. Previous work by the Simon lab at Rutgers University identified pair of sweet basil cultivars; one resistant to BDM, MRI, and one susceptible, SB22. They predicted that three genes in MRI confer increased BDM resistance. RNA from infected MRI and SB22 plants was harvested during the first 3 days of infection at 4 timepoints in order to capture as many early phases of plant-pathogen interaction as possible. The goal is to develop resistance markers for use in breeding experiments.
Project description:The aim of this work was to contribute to the knowledge on the chemical composition and bioactive properties of two species of the Ocimum genus, namely O. basilicum cultivar 'Cinammon' and O. × citriodorum. For this purpose, samples of these plants grown in Portugal were evaluated for their composition in phenolic and volatile compounds, and the infusion and hydroethanolic extracts were assessed for their in vitro antioxidant, antimicrobial, cytotoxic, and anti-inflammatory activities. In total, the two basil samples showed the presence of seven caffeic acid and derivatives (dimers, trimers, and tetramers) and five flavonoids, mainly glycoside derivatives of quercetin. Despite some qualitative and quantitative differences, in both samples rosmarinic acid was the major phenolic compound, and linalool the predominant volatile compound. In general, the tested extracts provided relevant bioactive properties since both basil species showed higher antioxidant activity in Thiobarbituric Acid Reactive Substances (TBARs) and Oxidative Hemolysis Inhibition (OxHLIA) assays when compared with the positive control Trolox. Despite O. × citriodorum extracts showing slightly better activity against some strains, both types of extracts evidenced similar antimicrobial activity, being more active against Gram-positive bacteria. The extracts also revealed interesting cytotoxicity, particularly the O. × citriodorum hydroethanolic extract which was also the only one exhibiting anti-inflammatory activity.
Project description:Nanocellulose (NC) is a valuable material in tissue engineering, wound dressing, and drug delivery, but its lack of antimicrobial activity is a major drawback for these applications. In this work, basil ethanolic extract (BE) and basil seed mucilage (BSM) were used to endow nanocellulose with antibacterial activity. NC/BE and NC/BE/BSM sponges were obtained from nanocellulose suspensions and different amounts of BE and BSM after freeze-drying. Regardless of the BE or BSM content, the sponges started to decompose at a lower temperature due to the presence of highly volatile active compounds in BE. A SEM investigation revealed an opened-cell structure and nanofibrillar morphology for all the sponges, while highly impregnated nanofibers were observed by SEM in NC/BE sponges with higher amounts of BE. A quantitative evaluation of the porous morphology by microcomputer tomography showed that the open porosity of the sponges varied between 70% and 82%, being lower in the sponges with higher BE/BSM content due to the impregnation of cellulose nanofibers with BE/BSM, which led to smaller pores. The addition of BE increased the specific compression strength of the NC/BE sponges, with a higher amount of BE having a stronger effect. A slight inhibition of S. aureus growth was observed in the NC/BE sponges with a higher amount of BE, and no effect was observed in the unmodified NC. In addition, the NC/BE sponge with the highest amount of BE and the best antibacterial effect in the series showed no cytotoxic effect and did not interfere with the normal development of the L929 cell line, similar to the unmodified NC. This work uses a simple, straightforward method to obtain highly porous nanocellulose structures containing antibacterial basil extract for use in biomedical applications.
Project description:The present study aimed to chemically characterize 31 accessions and seven cultivars of basil. The percentage composition of the essential oils of the accessions and cultivars was based on the 14 most abundant constituents: 1,8-cineole, linalool, methyl chavicol, neral, nerol, geraniol, geranial, methyl cinnamate, β-bourbonene, methyl eugenol, α-trans-bergamotene, germacrene-D, epi-α-cadinol, and δ-cadinene. The genetic materials were classified into eight clusters according to the chemical composition of the essential oils: Cluster 1--mostly linalool and 1,8-cineole; Cluster 2--mostly linalool, geraniol, and α-trans-bergamotene; Cluster 3--mostly linalool, methyl chavicol, methyl cinnamate, and β-bourbonene; Cluster 4--mostly linalool, methyl chavicol, epi-α-cadinol, and α-trans-bergamotene; Cluster 5--mainly linalool, methyl eugenol, α-trans-bergamotene, and epi-α-cadinol; Cluster 6--mainly linalool, geraniol, and epi-α-cadinol; Cluster 7--mostly linalool and methyl chavicol; Cluster 8--mainly geranial and neral.
Project description:Phenylpropenes, a large group of plant volatile compounds that serve in multiple roles in defense and pollinator attraction, contain a propenyl side chain. Eugenol synthase (EGS) catalyzes the reductive displacement of acetate from the propenyl side chain of the substrate coniferyl acetate to produce the allyl-phenylpropene eugenol. We report here the structure determination of EGS from basil (Ocimum basilicum) by protein x-ray crystallography. EGS is structurally related to the short-chain dehydrogenase/reductases (SDRs), and in particular, enzymes in the isoflavone-reductase-like subfamily. The structure of a ternary complex of EGS bound to the cofactor NADP(H) and a mixed competitive inhibitor EMDF ((7S,8S)-ethyl (7,8-methylene)-dihydroferulate) provides a detailed view of the binding interactions within the EGS active site and a starting point for mutagenic examination of the unusual reductive mechanism of EGS. The key interactions between EMDF and the EGS-holoenzyme include stacking of the phenyl ring of EMDF against the cofactor's nicotinamide ring and a water-mediated hydrogen-bonding interaction between the EMDF 4-hydroxy group and the side-chain amino moiety of a conserved lysine residue, Lys132. The C4 carbon of nicotinamide resides immediately adjacent to the site of hydride addition, the C7 carbon of cinnamyl acetate substrates. The inhibitor-bound EGS structure suggests a two-step reaction mechanism involving the formation of a quinone-methide prior to reduction. The formation of this intermediate is promoted by a hydrogen-bonding network that favors deprotonation of the substrate's 4-hydroxyl group and disfavors binding of the acetate moiety, akin to a push-pull catalytic mechanism. Notably, the catalytic involvement in EGS of the conserved Lys132 in preparing the phenolic substrate for quinone methide formation through the proton-relay network appears to be an adaptation of the analogous role in hydrogen bonding played by the equivalent lysine residue in other enzymes of the SDR family.
Project description:Applying a metatranscriptomic analysis pipeline (Guo et al. 2016 Frontiers in Plant Science), we are the first to analyze the host-pathogen metatranscriptome of the basil downy mildew system. RNA-sequencing technology was utilized to gain access to the full array of expressed transcripts from both O. basilicum and P. belbahrii. This RNA-seq workflow has allowed us to identity nearly 3,000 candidate P. belbahrii genes expressed in planta, as well as 1,267 and 2,798 candidate O. basilicum genes induced or suppressed respectively under P. belbahrii infection (five days post inoculation). Up-regulated candidate genes are highly enriched for biological processes such as biotic and abiotic stress responses whereas down-regulated genes are enriched for metabolism and photosynthesis, suggesting that basil plants actively respond to pathogen infection with transcriptome reprogramming.
Project description:Sweet basil (Ocimum basilicum) is an economically important herb and its global production is threatened by basil downy mildew caused by the obligate biotrophic oomycete Peronospora belbahrii. Effective tools are required for functional understanding of its genes involved in synthesis of valuable secondary metabolites in essential oil and disease resistance, and breeding for varieties with improved traits. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene editing technology has revolutionized crop breeding and functional genomics. The applicability and efficacy of this genomic tool in the allotetraploid sweet basil were tested by editing a potential susceptibility (S) gene ObDMR1, the basil homolog of Arabidopsis DMR1 (Downy Mildew Resistant 1) whose mutations conferred nearly complete resistance against Arabidopsis downy mildew pathogen, Hyaloperonospora arabidopsidis. Two single guide RNAs targeting two different sites of the ObDMR1 coding sequence were designed. A total of 56 transgenic lines were obtained via Agrobacterium-mediated stable transformation. Mutational analysis of 54 T0 transgenic lines identified 92.6% lines carrying mutations at target 1 site, while a very low mutation frequency was detected at target 2 site. Deep sequencing of six T0 lines revealed various mutations at target 1 site, with a complete knockout of all alleles in one line. ObDMR1 homozygous mutant plants with some being transgene free were identified from T1 segregating populations. T2 homozygous mutant plants with 1-bp frameshift mutations exhibited a dwarf phenotype at young seedling stage. In summary, this study established a highly efficient CRISPR/Cas9-mediated gene editing system for targeted mutagenesis in sweet basil. This system has the capacity to generate complete knockout mutants in this allotetraploid species at the first generation of transgenic plants and transgene-free homozygous mutants in the second generation. The establishment of this system is expected to accelerate basil functional genomics and breeding.