Activation of a Bean Chitinase Promoter in Transgenic Tobacco Plants by Phytopathogenic Fungi

The temporal and spatial expression of a bean chitinase promoter has been investigated in response to fungal attack. Analysis of transgenic tobacco plants containing a chimeric gene composed of a 1.7-kilobase fragment carrying the chitinase 5B gene promoter fused to the coding region of the gus A gene indicated that the chitinase promoter is activated during attack by the fungal pathogens Botrytis cinerea, Rhizoctonia solani, and Sclerotium rolfsii. Although induction of [beta]-glucuronidase activity was observed in tissues that had not been exposed to these phytopathogens, the greatest induction occurred in and around the site of fungal infection. The increase in [beta]-glucuronidase activity closely paralleled the increase in endogenous tobacco chitinase activity produced in response to fungal infection. Thus, the chitinase 5B-gus A fusion gene may be used to analyze the cellular and molecular details of the activation of the host defense system during pathogen attack.     

Isolation and Characterization of the Genes Encoding Basic and Acidic Chitinase in Arabidopsis thaliana

Plants synthesize a number of antimicrobial proteins in response to pathogen invasion and environmental stresses. These proteins include two classes of chitinases that have either basic or acidic isoelectric points and that are capable of degrading fungal cell wall chitin. We have cloned and determined the nucleotide sequence of the genes encoding the acidic and basic chitinases from Arabidopsis thaliana (L.) Heynh. Columbia wild type. Both chitinases are encoded by single copy genes that contain introns, a novel feature in chitinase genes. The basic chitinase has 73% amino acid sequence similarity to the basic chitinase from tobacco, and the acidic chitinase has 60% amino acid sequence similarity to the acidic chitinase from cucumber. Expression of the basic chitinase is organ-specific and age-dependent in Arabidopsis. A high constitutive level of expression was observed in roots with lower levels in leaves and flowering shoots. Exposure of plants to ethylene induced high levels of systemic expression of basic chitinase with expression increasing with plant age. Constitutive expression of basic chitinase was observed in roots of the ethylene insensitive mutant (etr) of Arabidopsis, demonstrating that root-specific expression is ethylene independent. Expression of the acidic chitinase gene was not observed in normal, untreated Arabidopsis plants or in plants treated with ethylene or salicylate. However, a transient expression assay indicated that the acidic chitinase promoter is active in Arabidopsis leaf tissue.     

Cotyledon nuclear proteins bind to DNA fragments harboring regulatory elements of phytohemagglutinin genes.

The effects of deleting DNA sequences upstream from the phytohemagglutinin-L gene of Phaseolus vulgaris have been examined with respect to the level of gene product produced in the seeds of transgenic tobacco. Our studies indicate that several upstream regions quantitatively modulate expression. Between -1000 and -675, a negative regulatory element reduces expression approximately threefold relative to shorter deletion mutants that do not contain this region. Positive regulatory elements lie between -550 and -125 and, compared with constructs containing only 125 base pairs of upstream sequences (-125), the presence of these two regions can be correlated with a 25-fold and a 200-fold enhancement of phytohemagglutinin-L levels. These experiments were complemented by gel retardation assays, which demonstrated that two of the three regions bind cotyledon nuclear proteins from mid-mature seeds. One of the binding sites maps near a DNA sequence that is highly homologous to protein binding domains located upstream from the soybean seed lectin and Kunitz trypsin inhibitor genes. Competition experiments demonstrated that the upstream regions of a bean beta-phaseolin gene, the soybean seed lectin gene, and an oligonucleotide from the upstream region of the trypsin inhibitor gene can compete differentially for factor binding. We suggest that these legume genes may be regulated in part by evolutionarily conserved protein/DNA interactions.     

A new class of tobacco chitinases homologous to bacterial exo-chitinases displays antifungal activity

A novel chitinase gene of tobacco was isolated and characterized by DNA sequence analysis of a genomic clone and a cDNA clone. Comparative sequence analysis of both clones showed an identity of 94%. The proteins encoded by these sequences do not correspond to any of the previously characterized plant chitinases of classes I–IV and are designated as class V chitinases. Comparison of the chitinase class V peptide sequence with sequences in the Swiss Protein databank revealed significant sequence similarity with bacterial exo-chitinases from Bacillus circulans, Serratia marcescens and Streptomyces plicatus. It was demonstrated that class V chitinase gene expression is induced after treatment of tobacco with different forms of stress, like TMV-infection, ethylene treatment, wounding or ultraviolet irradiation. Two related chitinase class V proteins of 41 and 43 kDa were purified from Samsun NN tobacco leaves inoculated with tobacco mosaic virus. The proteins were purified by Chelating Superose chromatography and gel filtration. In vitro assays demonstrated that class V chitinases have endo-chitinase activity and exhibit antifungal activity toward Trichoderma viride and Alternaria radicina. In addition, it was shown that class V chitinase acts synergistically with tobacco class I β-1,3-glucanase against Fusarium solani germlings.     

A potato Sus3 sucrose synthase gene contains a context-dependent 3' element and a leader intron with both positive and negative tissue-specific effects.

To examine which sequences are involved in regulating the potato sucrose synthase gene Sus3-65, we examined a series of deletion and substitution constructs in transgenic potato and tobacco plants. In a construct containing 3.9 kb of 5' flanking region, substitution of the native 3' sequence with the nopaline synthase 3' sequence and deletion of the leader intron did not significantly affect expression in vegetative tissues. However, in a construct containing only 320 bp of 5' flanking region, these changes had marked effects. Replacing the native 3' sequences with nopaline synthase 3' sequences caused a six- to 20-fold increase in expression in vascular tissue, and removing the leader intron almost completely abolished expression in potato plants. Surprisingly, removal of the leader intron from either the full-length construct or a construct containing only 320 bp of 5' flanking sequence reduced expression in vascular tissue of tobacco anthers at later stages of development but increased expression in pollen by more than 100-fold.     

Identification of chitinase mRNA in abscission zones from bean (Phaseolus vulgaris Red Kidney) during ethylene-induced abscission

Abstract. Total RNA was extracted from bean leaf abscission zones at different times after the induction of abscission by ethylene. The RNA was translated in the wheat germ system and the products analysed by SDS-PAGE. Products of molecular weight (raw) 42, 32 and 17 kD were seen to accumulate substantially during the induction. An attempt was made to establish that the mRNA species which produced the 32 kD product, which was coded for the ethylene-regulated enzyme chitinase. Mature chitinase (30 kD) was purifed from ethylene-treated abscission zones and used to raise monospecific antibodies in rabbits. These antibodies recognized the 32 kD product and mature chitinase. The 2 kD difference in molecular weight was due to the presence of the signal sequence which could be removed by microsomal membranes. Chitinase was also detected by enzymatic assay and immunoblotting of crude homogenates from ethylene-treated abscission zones. Chitinase appears to be ubiquitous in bean plants and probably does not have a direct role in abscission.     

Ethylene-Induced Gene Expression of Osmotin-Like Protein, a Neutral Isoform of Tobacco PR-5, is Mediated by the AGCCGCC eft-Sequence

Osmotin-like protein (OLP) is a neutral isoform in the group5 pathogenesis-related (PR) tobacco proteins. The OLP gene,like the basic PR protein genes, is constitutively expressedin tobacco roots and cultured cells. OLP is not naturally presentin intact healthy leaves, but ethylene treatment induces a highaccumulation there. To study the mechanism of OLP gene expressionas induced by ethylene, we cloned the gene from Nicotiana sylvestris,an ancestor of N. tabacum. Sequence analysis showed that ithas no intron and that its promoter region contains two AGCCGCCsequences that are conserved in most basic PR-protein genes.The function of the AGCCGCC sequences in transgenic tobaccoplants that harbor the wild and mutated OLP promoter::GUS fusiongenes was analyzed. Mutation in the AGCCGCC sequences clearlyinhibited the GUS expression induced by ethylene, indicativethat the AGCCGCC sequence(s) is a DNA element(s) responsiveto ethylene. An EREBP2 protein, isolated as one of the proteinsbinding the AGCCGCC sequence of the tobacco rß-1,3-glucanasegene, also was found to bind to the AGCCGCC sequence(s) of OLPgene. These results suggest that the ethylene-induced expressionof OLP is regulated by trans-acting factor(s) common to basicPR-proteins. (Received November 13, 1995; Accepted January 17, 1996)