Isolation and characterization of two wound-regulated tomato extensin genes

Extensins comprise a family of structural cell wall hydroxyproline-rich glycoproteins in plants. Two tomato genomic clones, Tom J-10 and Tom L-4, were isolated from a tomato genomic DNA library byin situ plaque hybridization with extensin DNA probes. Tom J-10 encoded an extensin with 388 amino acid residues and a predicted molecular mass of 43 kDa. The Tom J-10 encoded extensin lacked a typical signal peptide sequence, but contained two distinct protein domains consisting of 19 tandem repeats of Ser-Pro₄-Ser-Pro-Lys-Tyr-Val-Tyr-Lys at the amino terminus which were directly followed by 8 tandem repeats of the consensus sequence Ser-Pro₄-Tyr₃-Lys-Ser-Pro₄-Ser-Pro at the carboxy terminus. RNA blot hybridization analysis with the Tom J-10 extensin probe demonstrated the presence of a 4.0 kb tomato stem mRNA which accumulated markedly in response to wounding. Tom L-4 encoded an extensin with 322 amino acid residues and a predicted molecular mass of 35 kDa. The Tom L-4 encoded extensin contained a typical signal peptide sequence at the amino terminus and was followed by at least 3 distinct domains. These domains consisted of an amino terminal domain containing several Lys-Pro and Ser-Pro₄ repeat units, a central domain with repeats of the consensus sequence Ser-Pro_2–5-Thr-Pro-Ser-Tyr-Glu-His-Pro-Lys-Thr-Pro, and a carboxy terminal domain containing repeats of the consensus sequence Ser-Ser-Pro₄-Ser-Pro-Ser-Pro₄-Thr-Tyr_1–3. RNA blot hybridization analysis with the Tom L-4 extensin probe demonstrated the presence of a 2.6 kb tomato stem mRNA which accumulated in response to wounding.     

Pharbitis class-1 knotted-like homeobox gene, PKn3, shares similar characteristics to those of class-2 knotted-like genes

Three novel homeobox genes, PKn1–3 (Pharbitis knotted-like), were isolated from Japanese morning glory (Pharbitis nil Chois, strain Violet). A sequence analysis showed that these genes belong to the knotted class-1 gene family and that PKn3 has a relatively unique sequence. All PKn genes are expressed in shoot apices, stems and roots, but not in cotyledons. Transformed tobacco with PKn1 or PKn2 displayed leaf shrinkage and a dwarf phenotype, while the ectopic expression of PKn3 gave no altered phenotypes. In situ hybridization showed that PKn3 is up-regulated in developing leaf primordia and that this expression becomes restricted in the basal region of young leaf blades, which is reminiscent of the expression pattern of the class-2 knotted gene, NTH23. These data suggest that these Pharbitis homeobox genes participate in the differentiation in shoots and suggest a unique function of PKn3 in developing leaves. Received: 16 October 1999 / Revision received: 30 November 1999 / Accepted: 11 January 2000     

Isolation and characterization of fruit vacuolar invertase genes from two tomato species and temporal differences in mRNA levels during fruit ripening

To determine the relationship between invertase gene expression and glucose and fructose accumulation in ripening tomato fruit, fruit vacuolar invertase cDNA and genomic clones from the cultivated species, Lycopersicon esculentum cv. UC82B, and a wild species, Lycopersicon pimpinellifolium, were isolated and characterized. The coding sequences of all cDNA clones examined are identical. By comparison to the known amino acid sequence of mature L. esculentum fruit vacuolar invertase, a putative signal sequence and putative amino-terminal and carboxy-terminal propeptides were identified in the derived amino acid sequence. Of the residues 42% are identical with those of carrot cell wall invertase. A putative catalytic site and a five-residue motif found in carrot, yeast, and bacterial invertases are also present in the tomato sequence. Minor differences between the nucleotide sequences of the genomic clones from the two tomato species were found in one intron and in the putative regulatory region. The gene appears to be present in one copy per haploid genome. Northern analysis suggests a different temporal pattern of vacuolar invertase mRNA levels during fruit development in the two species, with the invertase mRNA appearing at an earlier stage of fruit development in the wild species. Nucleotide differences found in the putative regulatory regions may be involved in species differences in temporal regulation of this gene, which in turn may contribute to observed differences in hexose accumulation in ripening fruit.     

Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens

Analysis of clones isolated from a cosmid DNA library indicates that the Serratia marcescens chromosome contains at least two genes, chiA and chiB, which encode distinct secreted forms of the enzyme chitinase. These genes have been characterized by inspection of chitinase activity and secreted proteins in Escherichia coli strains containing subclones of these cosmids. The two chitinase genes show no detectable homology to each other. DNA sequence analysis of one of the genes predicts an amino acid sequence with an N-terminal signal peptide typical of genes encoding secreted bacterial proteins. This gene was mutagenized by cloning a neomycin phosphotransferase gene within its coding region, and the insertion mutation was recombined into the parental S. marcescens strain. The resulting chiA mutant transconjugant showed reduced chitinase production, reduced inhibition of fungal spore germination and reduced biological control of a fungal plant pathogen.     

Isolation and characterization of the Spindly homologue from tomato

The SPINDLY (SPY) gene is a crucial component of the gibberellin signal transduction pathway in Arabidopsis thaliana (L.) Heynh. In this study, the cloning of the SPINDLY-orthologous gene (LeSpy) from tomato (Lycopersicon esculentum Mill.) and its characterization are reported. SPY and LeSpy show high sequence similarity along their entire lengths, which is reflected in the conservation of exon-intron structure and of all sequence motives previously identified. To analyse the relationship between the Arabidopsis spindly and the tomato procera mutant, which show similar phenotypes, the LeSpy gene was characterized in wild-type and procera tomato plants. These analyses as well as mapping of LeSpy revealed that LeSpy and Procera are different genes.     

Identificaton and characterization of stamen- and tapetum-specific genes from tomato

Summary Differential screening of a tomato cDNA library produced from pre-anthesis stamens resulted in the isolation of 25 cDNA clones that hybridized to probes made from stamen RNA and showed no hybridization to probes made from RNA of vegetative organs. The 25 clones were found to represent 11 noncross-hybridizing classes. The majority of these clones were derived from genes that were single or low copy in the tomato genome. Northern RNA blotting experiments of vegetative and floral organs at several stages of development demonstrated that expression in all 11 classes was confined to floral organs. Of the 11 classes 9 were found to be expressed exclusively in stamens prior to anthesis. Two classes showed expression in immature stamens and in petals, with one of these two additionally being expressed in mature stamens at anthesis. Clones from three of the classes that were expressed exclusively in stamens were used as probes for in situ localization of RNA in floral organs. These experiments demonstrated that expression of the genes corresponding to these clones was confined to the tapetal cells of the anthers. Expression of one of the three genes was found to be limited to a single cell type during the 5–6 day period from late meiosis to immature pollen formation.     

Isolation and characterization of wound-inducible carboxypeptidase inhibitor from tomato leaves

In a previous report [Mol. Gen. Genet. 228 (1991) 281], carboxypeptidase inhibitor protein (CPI) mRNA was found to accumulate in leaves of wounded tomato plants, but CPI protein could not be detected. In contrast, we found that CPI protein does accumulate in tomato leaves in response to wounding, and also in response to treatment with either systemin, methyl jasmonate (MeJ), oligogalacturonic acid, or chitosan. Identification of CPI protein was confirmed by its inhibition of metallo-carboxypeptidase A (CPAase), which was used as an assay during purification of the inhibitor from leaves of MeJ-treated tomato plants. Amino acid sequence analysis and mass spectroscopic analyses of the pure protein confirmed its identity as CPI. The pure protein inhibited CPAase in a 1:1 stoichimetric interaction. Time course analyses of the induction of CPI mRNA in tomato leaves in response to wounding indicated that the gene is a member of the group of "late genes" that code for defensive proteins synthesized in leaves in response to herbivore attack.     

Purification and characterization of two ribonucleases from developing tomato fruit

Two neutral ribonucleases have been purified from developing tomato fruit. Their activity is maximal 5 days after anthesis, declines during maturation, and then increases slightly in the mature green through breaker stages. The ribonucleases Tf1 and Tf2 have molecular weights of 59 and 29 K, respectively, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and are glycoproteins. The reduced and denatured Tf1 is composed of two subunits, 30 and 29 K, of which only the 30-K subunit displays ribonuclease activity after renaturation. Reduced and denatured Tf2 is a single 29-K polypeptide that is renaturable to an active ribonuclease. Only the 30-K, active subunit of Tf1 is immunologically cross-reactive with Tf2. Both ribonucleases are cyclyzing endoribonucleases with a strong preference for cleavage at pyrimidine residues, thus generating oligonucleotide products ending with pyrimidine 2',3'-cyclic phosphate. These tomato fruit ribonucleases share a number of properties in common with the S-glycoprotein ribonucleases that are involved in self-incompatibility reactions in some solanaceous plants.     

Isolation and characterization of two T-box genes from sponges,the phylogenetically oldest metazoan taxon

It is now well established that all metazoan phyla derived from one common ancestor, the hypothetical Urmetazoa. Due to the basal position of Porifera (Demospongiae) in the phylogenetic tree of Metazoa, studies on the mechanisms controlling the development of these animals can provide clues on the understanding of the origin of multicellular animals and on how the first organization of the body plan evolved. In this report we describe the isolation and genomic characterization of two T-box genes from the siliceous sponge Suberites domuncula. The phylogenetic analysis classifies one into the subfamily of Brachyury, Sd-Bra, and the second into the Tbx2 subfamily, Sd-Tbx2. Analyses of the Sd-Bra and Sd-Tbx2 sequences and their intron-exon structures demonstrate their basal position in the phylogeny of the T-box family, and allows us to hypothesize a model of the phylogenetic evolution of all T-box genes. Furthermore, we report the presence of two different products of alternative splicing of Sd-Bra, and demonstrate that they exist in different phosphorylation and glycosylation states in the sponge tissue. Sd-Bra expression in tissue and 3D-cell aggregates (primmorphs) is analyzed, suggesting that Sd-Bra might also have a role in Porifera morphogenesis.Edited by N. SatohThe sequences from Suberites domuncula reported here are deposited in the EMBL/GenBank data base: the cDNA of Brachyury (Sd-Bra; accession number AJ544242) as well as the second T-box gene Sd-Tbx2 (AJ544241).     

Isolation and characterization of hemolytic genes from Actinobacillus actinomycetemcomitans

Abstract Periodontopathic Actinobacillus actinomycetemcomitans produces hemolysin and other leukotoxins. In the present study, two distinct clones which lysed horse erythrocytes were isolated by screening genomic DNA libraries of A. actinomycetemcomitans ATCC 43718 on blood agar plates. DNA hybridization analysis indicates that there were two distinct hemolytic genes present. Sonicated extracts from both Escherichia coli clones possessed hemolytic activities on horse, sheep and human erythrocytes, but not those of rabbit. Rabbit antiserum to A. actinomycetemcomitans ATCC 43718 whole cells inhibited the hemolytic activities of these clones.     

Isolation and characterization of two pathogen- and salicylic acid-induced genes encoding WRKY DNA-binding proteins from tobacco

A pathogen- and salicylic acid (SA)-induced DNA-binding activity has been recently identified in tobacco that is related to a previously identified class of WRKY DNA-binding proteins. To identify members of the WRKY gene family associated with this DNA-binding activity, we have attempted to isolate those WRKY genes that are induced by pathogen infection. Using a domain-specific differential display procedure, we have isolated two tobacco WRKY genes, tWRKY3 and tWRKY4, that are rapidly induced in resistant tobacco plants after infection by tobacco mosaic virus (TMV). Both tWRK3 and tWRKY4 encode proteins with a single WRKY domain that contain the conserved WRKYGQK sequence. Unlike other isolated WRKY proteins that contain the Cys₂His₂ zinc motif, tWRKY3 and tWRKY4 appear to contain the Cys₂HisCys zinc motif. Nonetheless, both tWRKY3 and tWRKY4 are capable of binding DNA molecules with the W-box (TTGAC) element recognized by other WRKY proteins. Expression of the tWRKY3 and tWRKY4 genes could be rapidly induced not only by TMV infection but also by SA or its biologically active analogues that are capable of inducing pathogenesis-related genes and enhanced resistance. Interestingly, induction of both genes by TMV infection was still observed in resistant tobacco plants expressing the bacterial salicylate hydroxylase gene (nahG), although the levels of induction appeared to be reduced. Identification of pathogen- and SA-induced genes encoding WRKY DNA-binding proteins should facilitate future studies on the regulation and functions of this novel group of DNA-binding proteins.