ABSTRACT
Single-chain Fv (scFv) antibody (Ab) fragments were transiently produced in COS-1 cells utilizing a mammalian expression vector featuring a murine immunoglobulin (Ig) light-chain leader sequence for efficient secretion and a murine Ig kappa constant domain (IgC kappa) for detection. Several hundred milliliters of supernatants from large-scale COS cell transfections were sufficient to purify the scFv::IgC kappa fusion proteins by one-step affinity chromatography utilizing an immobilized rat anti-mouse IgC kappa monoclonal Ab. Furthermore, the murine IgC kappa domain allowed for accurate quantification of the scFv::IgC kappa fusion protein secreted into the COS cell supernatant by a sandwich enzyme-linked immunosorbent assay (S-ELISA).
Subject(s)
Immunoglobulin Variable Region/genetics , Immunoglobulin kappa-Chains/genetics , Animals , Base Sequence , Cell Line , Chlorocebus aethiops , Cloning, Molecular/methods , DNA Primers/chemistry , Gene Expression , Genes, Immunoglobulin/genetics , Molecular Sequence Data , Recombinant Fusion ProteinsABSTRACT
Two recently isolated cDNAs representing genes that are transcriptionally activated in fungus-infected parsley leaves or elicitor-treated, cultured parsley cells are shown to encode a hydroxyproline-rich glycoprotein (HRGP) and an anionic peroxidase. The deduced HRGP protein is rich in tyrosine residues, a feature also found in other pathogen- and wound-induced plant HRGPs. Expression of the peroxidase gene(s) is induced rapidly upon elicitation and precedes that of the HRGP gene. In situ hybridization experiments demonstrate the presence of HRGP and peroxidase mRNAs in parsley tissue around fungal infection sites. Peroxidase mRNA accumulation is particularly sharply restricted to plant cells directly adjacent to fungal hyphae. These results provide further evidence for an important role of specific cell wall modifications in plant defense.
Subject(s)
Genes, Plant , Glycoproteins/genetics , Magnoliopsida/genetics , Peroxidases/genetics , Plant Diseases/genetics , Plant Proteins/genetics , Amino Acid Sequence , Base Sequence , DNA, Complementary/genetics , Gene Expression Regulation, Plant , Glycoproteins/chemistry , In Situ Hybridization , Molecular Sequence Data , Mycoses/genetics , Sequence Alignment , Sequence Homology, Amino Acid , TyrosineABSTRACT
Ubiquitin is an omnipresent protein found in all eukaryotes so far analysed. It is involved in several important processes, including protein turnover, chromosome structure and stress response. Parsley (Petroselinum crispum) contains at least two active polyubiquitin (ubi4) genes encoding hexameric precursor proteins. The deduced amino acid sequences of the ubiquitin monomers are identical to one another and to ubiquitin sequences from several other plant species. Analysis of the promoter region of one ubi4 gene revealed putative regulatory elements. In parsley plants, the ubi4 mRNAs were the predominant ubiquitin mRNAs and were present at comparable levels in all plant organs tested. In cultured parsley cells, high levels of ubiquitin gene expression remained unaffected by heat shock, elicitor or light treatment.
Subject(s)
Genes, Plant , Plants/genetics , Polymers , Ubiquitins/genetics , Amino Acid Sequence , Base Sequence , Cells, Cultured , Cloning, Molecular , DNA/genetics , Gene Expression , Hot Temperature , In Vitro Techniques , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , Plant Proteins/genetics , Polyubiquitin , Protein Precursors/genetics , RNA, Messenger/genetics , Transcription, GeneticABSTRACT
A group of recently isolated parsley (Petroselinum crispum) cDNAs representing genes that are transcriptionally activated upon fungal infection or elicitor treatment have been demonstrated to encode tyrosine decarboxylase (TyrDC). The deduced TyrDC protein sequence shares extensive similarity with two functionally related enzymes, tryptophan decarboxylase from periwinkle and dopa decarboxylase from Drosophila melanogaster. Expression of TyrDC cDNA in Escherichia coli yielded catalytically active protein with high substrate specificity for tyrosine. All four identified parsley TyrDC genes have been cloned and encode at least three TyrDC isozymes. Treatment of cultured parsley cells with fungal elicitor caused very rapid and transient increases in TyrDC mRNA and enzyme activity levels.
Subject(s)
Fungi , Genes, Plant , Isoenzymes/genetics , Plant Diseases , Plants/genetics , Tyrosine Decarboxylase/genetics , Amino Acid Sequence , Aromatic-L-Amino-Acid Decarboxylases/chemistry , Conserved Sequence , DNA/chemistry , Dopa Decarboxylase/chemistry , Escherichia coli/genetics , Gene Expression , Isoenzymes/chemistry , Kinetics , Magnoliopsida/enzymology , Magnoliopsida/genetics , Molecular Sequence Data , Plants/enzymology , Sequence Homology, Amino Acid , Tyrosine Decarboxylase/chemistryABSTRACT
We cloned and sequenced cDNAs encoded by a novel plant defense gene, ELI3, from parsley and Arabidopsis thaliana. The predicted product shares no homology to known sequences. ELI3 mRNA accumulates in A. thaliana leaves in response to challenge with phytopathogenic Pseudomonas syringae strains. The timing and magnitude of this response are dictated by the genetics of the plant-pathogen interaction being analyzed. During incompatible interactions, where resistance in the plant genotype Col-0 is dictated by the dominant RPM1 locus, ELI3 mRNA accumulates to high levels 5-10 h post-inoculation. This kinetic behavior is also generated by the presence of a cloned bacterial avirulence gene, in otherwise virulent bacteria, which triggers resistance mediated via RPM1 action. The phenotypic outcome is a hypersensitive resistance reaction visible 8-15 h post-infiltration. Thus, the induction kinetics of ELI3 mRNA accumulation are consistent with a functional role for the ELI3 gene product in establishing the resistant phenotype. In contrast, during compatible interactions with the susceptible plant genotype Nd-0, which is homozygous recessive at the rpm1 locus, ELI3 mRNA accumulates significantly only after 15 h. We show genetically that ELI3 activation is strictly dependent on the presence of dominant alleles at RPM1 using an assay generalizable to any pathogen induced plant defense phenomena.
Subject(s)
Arabidopsis/genetics , Gene Expression Regulation , Plant Proteins/genetics , Amino Acid Sequence , Arabidopsis/immunology , Base Sequence , Cloning, Molecular , DNA , Homozygote , Immunity, Innate/genetics , Kinetics , Molecular Sequence Data , Pseudomonas/pathogenicity , RNA, Messenger/geneticsABSTRACT
Treatment of cultured parsley (Petroselinum crispum) cells with fungal elicitor rapidly activates transcription of many genes encoding specific steps in pathogen defense-related pathways. We report evidence that three cDNAs corresponding to such genes represent two key enzymes of the activated methyl cycle. Two cDNAs are derived from distinct members of the S-adenosyl-L-methionine synthetase gene family, based on extensive similarity of the deduced polypeptides with authentic enzymes from Arabidopsis thaliana, rat, yeast, and Escherichia coli. The third cDNA exhibits large similarity with a functionally related gene, encoding S-adenosyl-L-homocysteine hydrolase, from rat and a slime mold. Marked differences in the mRNA levels occurred in different organs of parsley plants. Elicitor treatment strongly induced both mRNAs in cultured cells as well as intact leaves and led to marked increases in S-adenosyl-L-homocysteine hydrolase enzyme activity. These results suggest a close metabolic link between pathogen defense and an increased turnover of activated methyl groups.
Subject(s)
Hydrolases/genetics , Methionine Adenosyltransferase/genetics , Plants/genetics , RNA, Messenger/metabolism , Adenosylhomocysteinase , Amino Acid Sequence , Animals , Cells, Cultured , DNA/genetics , DNA/isolation & purification , Fungi , Gene Library , Molecular Sequence Data , Plants/enzymology , Poly A/genetics , Poly A/isolation & purification , RNA/genetics , RNA/isolation & purification , RNA, Messenger/genetics , Rats , Sequence Homology, Nucleic AcidABSTRACT
We have analysed three nearly full-length cDNAs complementary to mRNAs encoding two PR1 (pathogenesis-related, class 1) proteins in parsley (Petroselinum crispum). Furthermore, one selected genomic clone containing the PcPR1-1 gene was investigated in detail. The structural organization and possible regulatory elements in the 5' flanking region of this gene are presented. In situ RNA hybridization in fungus-infected parsley leaf tissue demonstrated rapid and massive PR1 mRNA accumulation around infection sites.
