Characterization of Wound-Inducible Genes Encoding Enzymes for Terpenoid Biosynthesis in Medicago Truncatula

In addition to having numerous applications as food flavorings and pharmaceuticals, terpenoids are an important class of defensive compounds that can accumulate in plants after pathogen infection or injury by insects. Sequences of DNA encoding putative terpene synthases and an oxidosqualene synthase, isolated from insect-damaged Medicago truncatula leaves, were selected from an expressed sequence tag (EST) database. The eDNA clones were used as radiolabeled probes to analyze gene expression in leaves treated with factors known to trigger a defense response in plants. Transcript levels for all of the genes examined increased in response to artificial wounding, insect herbivory, and methyl jasmonate (meJA) treatments, whereas salicylic acid (SA) and glucose oxidase (GOX) had no measurable effects on transcript levels. Furthermore, the genome of M. truncatula was analyzed via DNA blots for an estimation of the number of copies of enzyme isoforms and indicate that each of the enzymes examined is encoded by a single-copy gene or a small gene family. The results show that M. truncatula can serve as a valuable source for novel terpene synthase clones and potentially for strong wound-inducible regulatory elements

Characterization of Wound-Inducible Genes Encoding Enzymes for Terpenoid Biosynthesis in Medicago Truncatula

In addition to having numerous applications and pharmaceuticals, terpenoids are an important class of defensive compounds that can accumulate in plants after pathogen infection or injury by insects. Sequences of DNA encoding putative terpene synthases and an oxidosqualene synthase, isolated from insect-damaged Medicago truncatula leaves, were selected from an expressed sequence tag (EST) data base. The cDNA clones were used as radio-labeled probes to analyze gene expression in leaves treated with factors known to trigger a defense response in plants. Transcript levels for all of the genes examined increased in response to artificial wounding, insect herbivory, and methyl jasmonate (meJA) treatments, whereas salicylic acid (SA) and glucose oxidase (GOA) had no measurable effects on transcript levels. Furthermore, the genome of M. truncatula was analyzed via DNA blots for an estimation of the -number of copies of enzyme isoforms and indicate that each of the enzymes examined is encoded by a single-copy gene or a small gene family. The results show that M. truncatula can serve as a valuable source for novel terpene synthase clones and potentially for strong wound-inducible regulatory elements

Expression patterns of novel wound-inducible plant genes in Medicago truncatula

Terpenoids are an important class of defensive compounds that can accumulate in plants after pathogen infection or injury by chewing insects. Clones encoding putative terpene synthases and an oxidosqualene synthase, isolated from insect-damaged Medicagotruncatula leaves, were selected from an expressed sequence tag (EST) database. The cDNA clones were used as radiolabeled probes to analyze gene expression in leaves treated by known factors that can trigger a defense response in plants. Transcript levels for all of the genes examined increased in response to artificial wounding, insect herbivory, and methyl jasmonate (meJA) treatments, whereas salicylic acid (SA) and glucose oxidase (GOX) had no measurable effects on transcript levels. Furthermore, the genome of M. truncatula was analyzed via DNA blots for an estimation of the number of copies of enzyme isoforms; these data indicate that each of the enzymes examined is encoded by a single-copy gene or a small gene family

Compositions and methods of enhancing immune responses to flagellated bacterium

Vaccines comprising fliC and CD 154 polypeptides and Salmonella enteritidis vaccine vectors comprising fliC polypeptides are provided. Also provided arc methods of enhancing an immune response against flagellated bacteria and methods of reducing morbidity associated with infection with flagellated bacteria

Compositions and methods of enhancing immune responses

Provided herein are Salmonella enteritidis 13A strains and compositions comprising these strains. Also provided are methods of enhancing an immune response against Influenza A and methods of reducing morbidity associated with an Influenza A infection. Methods of enhancing an immune response to a vaccine vector by expressing a polypeptide of CD154 capable of binding CD40 are also disclosed. Methods of developing a bacterial vaccine vector are disclosed. Methods of generating scarless site-specific mutations in a bacterium are also disclosed

Compositions and methods of enhancing immune responses

Provided herein are Salmonella enteritidis 13A strains and compositions comprising these strains. Also provided are methods of enhancing an immune response against Influenza A and methods of reducing morbidity associated with an Influenza A infection. Methods of enhancing an immune response to a vaccine vector by expressing a polypeptide of CD154 capable of binding CD40 are also disclosed. Methods of developing a bacterial vaccine vector are disclosed. Methods of generating scarless site-specific mutations in a bacterium are also disclosed

Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome

<p>Abstract</p> <p>Background</p> <p>A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into <it>Salmonella enteritidis </it>chromosome.</p> <p>Results</p> <p>The final product of this mutation strategy is the insertion of DNA encoding a foreign epitope into the <it>S. enteritidis </it>genome without the addition of any unwanted sequence. This experiment was performed by a two-step mutation process via PCR fragments, Red recombinase and counter-selection with the I-SceI enzyme site. First, the I-SceI site and kanamycin resistance gene were introduced into the genome of cells expressing Red recombinase enzymes. Next, this sequence was replaced by a chosen insertion sequence. DNA fragments used for recombination were linear PCR products which consisted of the foreign insertion sequence flanked by homologous sequences of the target gene. Described herein is the insertion of a section of the M2e epitope (LM2) of Influenza A virus, a domain of CD154 (CD154s) or a combination of both into the outer membrane protein LamB of <it>S. enteritidis</it>.</p> <p>Conclusion</p> <p>We have successfully used this method to produce multiple mutants with no antibiotic gene on the genome or extra sequence except those nucleotides required for expression of epitope regions. This method is advantageous over other protocols in that it does not require cloning or creating extra duplicate regions to facilitate homologous recombination, contains a universal construct in which an epitope of choice can be placed to check for cell surface expression, and shows high efficiency when screening for positive mutants. Other opportunities of this mutational strategy include creating attenuated mutants and site-specific, chromosomal deletion mutations. Furthermore, this method should be applicable in other gram-negative bacterial species where Red recombinase enzymes can be functionally expressed.</p

Compositions and methods of enhancing immune responses to Eimeria

Vaccines comprising TRAP polypeptides and Salmonella enteritidis vectors comprising TRAP polypeptides are provided. The vaccines may also include a CD154 polypeptide capable of binding to CD40. Also provided are methods of enhancing an immune response against Apicomplexan parasites and methods of reducing morbidity associated with infection with Apicomplexan parasites

Compositions and methods of enhancing immune responses to Eimeria

Vaccines comprising TRAP polypeptides and Salmonella enteritidis vectors comprising TRAP polypeptides are provided. The vaccines may also include a CD154 polypeptide capable of binding to CD40. Also provided are methods of enhancing an immune response against Apicomplexan parasites and methods of reducing morbidity associated with infection with Apicomplexan parasites

Compositions and methods of enhancing immune responses

Salmonella enteritidis 13A strains and compositions comprising these strains are provided. Methods of enhancing an immune response against Influenza A; of reducing morbidity associated with an Influenza A infection; of enhancing an immune response to a vaccine vector by expressing a polypeptide of CD 154 capable of binding CD40; of developing a bacterial vaccine vector are disclosed, and of generating scarless site-specific mutations in a bacterium are disclosed