A cDNA clone for a pathogenesis-related protein 1 from barley

A barley cDNA clone (PRb-1) corresponding to an mRNA differentially induced in resistant compared to susceptible barley cultivars by powdery mildew infection was isolated and characterised. The deduced amino acid sequence revealed 24 amino acids comprising the signal peptide and 140 amino acids of the mature peptide (15 kDa). This showed close homology to PR-1-like proteins, which have been isolated from maize, tobacco, tomato and Arabidopsis thaliana. Northern blot analysis showed accumulation of the corresponding mRNA 12 h after inoculation of resistant barley cultivars with Erysiphe graminis. Increased expression of the PRb-1 gene was also observed in resistant compared with near-isogenic susceptible barley plants following treatment with ethylene, salicylic acid, methyl jasmonate and 2,6-dichloro-isonicotinic acid.     

Pathogenesis-related acidic beta-1,3-glucanase genes of tobacco are regulated by both stress and developmental signals

Three pathogenesis-related (PR) proteins of tobacco are acidic isoforms of beta-1,3-glucanase (PR-2a, -2b, -2c). We have cloned and sequenced a partial cDNA clone (lambda FJ1) corresponding to one of the PR-2 beta-1,3-glucanases. A small gene family encodes the PR-2 proteins in tobacco, and similar genes are present in a number of plant species. We analyzed the stress and developmental regulation of the tobacco PR-2 beta-1,3-glucanases by using northern and western analyses and a new technique to assay enzymatic activity. Stress caused by both thiamine and tobacco mosaic virus (TMV) infection resulted in a dramatic increase in the levels of PR-2 mRNA, protein, and enzyme activities. The increased PR-2 gene expression in upper uninoculated leaves of plants infected with TMV also suggests a role in systemic acquired resistance. During floral development, a number of beta-1,3-glucanase activities were observed in all flower tissues. However, PR-2 polypeptides were observed only in sepal tissue. In contrast, an mRNA that hybridized to the PR-2 cDNA was present in stigma/style tissue and the sepals. Primer extension analysis confirmed the identity of the PR-2 mRNA in sepals, but indicated that the beta-1,3-glucanase gene expressed in the stigma/style of flowers was distinct from the PR-2 genes. The induction of PR-2 protein synthesis by both stress and developmental signals was accompanied by a corresponding increase in the steady-state levels of PR-2 mRNA, suggesting that PR-2 gene expression is regulated, in part, at the level of mRNA accumulation.     

A gene encoding a novel isoform of the PR-1 protein family from tomato is induced upon viroid infection

A Lycopersicon esculentum cDNA clone encoding an acidic-type pathogenesis-related protein (PR-lal) was isolated, sequenced and characterized. It contains an open reading frame of 175 amino acids and the mature protein, after cleavage of the 21 amino acid signals peptide, has a pl of 5.24. The protein shows highest homology (75% identity) with the basic pathogenesis-related prb-lb protein from tobacco. The PR-lal gene shows constitutive expression in roots from tomato plants. It is expressed in leaves and stems upon viroid infection, and appears to be induced by ethylene. Comparative studies of this gene and a related basic isoform of PR-1 indicate that the expression of these two members of the PR-1 gene family in tomato may be differentially regulated upon viroid infection.The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X71592.     

Colonization of Transgenic Tobacco Constitutively Expressing Pathogenesis-Related Proteins by the Vesicular-Arbuscular Mycorrhizal Fungus Glomus mosseae

We studied the effect of constitutive expression of pathogenesis-related proteins (PRs) in tobacco plants on vesicular-arbuscular mycorrhiza. Tobacco lines genetically transformed to express various PRs constitutively under the control of the cauliflower mosaic virus 35S promoter of tobacco were examined. Immunoblot analysis and activity measurements demonstrated high levels of expression of the PRs in the root systems of the plants. Constitutive expression of the following acidic isoforms of tobacco PRs did not affect the time course or the final level of colonization by the vesicular-arbuscular mycorrhizal fungus Glomus mosseae: PR-1a, PR-3 (=PR-Q), PR-Q(prm1), PR-4, and PR-5. Similarly, constitutive expression of an acidic cucumber chitinase, of a basic tobacco chitinase with and without its vacuolar targeting peptide, of a basic (beta)-1,3-glucanase, and of combinations of PR-Q and PR-Q(prm1) or basic chitinase and basic (beta)-1,3-glucanase did not affect colonization by the mycorrhizal fungus. A delay of colonization by G. mosseae was observed in tobacco plants constitutively expressing the acidic isoform of tobacco PR-2, a protein with (beta)-1,3-glucanase activity.     

Fungus- and wound-induced accumulation of mRNA containing a class II chitinase of the pathogenesis-related protein 4 (PR-4) family of maize

Pathogenesis-related (PR) proteins are plant proteins that are induced in response to pathogen attack. PR proteins are grouped into independent families based on their sequences and properties. The PR-4 family comprises class I and class II chitinases. We have isolated a full-length cDNA encoding a chitinase from maize which shares a high degree of nucleotide and amino acid sequence homology with the class II chitinases of the PR-4 family of PR proteins. Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by the fungus Fusarium moniliforme, increase the level of ZmPR4 mRNA. In situ mRNA hybridization analysis in sections obtained from fungus-infected germinating embryos revealed that ZmPR4 mRNA accumulation occurs in those cell types that first establish contact with the pathogen. ZmPR4 mRNA accumulation is also stimulated by treatment with silver nitrate whereas the application of the hormones gibberellic acid or acetylsalicylic acid has no effect. Wounding, or treatment with abscisic acid or methyl jasmonate, results in accumulation of ZmPR4 mRNA in maize leaves. Furthermore, the ZmPR4 protein was expressed in Escherichia coli, purified and used to obtain polyclonal antibodies that specifically recognized ZmPR4 in protein extracts from fungus-infected embryos. Accumulation of ZmPR4 mRNA in fungus-infected maize tissues was accompanied by a significant accumulation of the corresponding protein. The possible implications of these findings as part of the general defence response of maize plants against pathogens are discussed.     

Biochemical and molecular biological studies on infection (Ascochyta rabiei)-induced thaumatin-like proteins from chickpea plants (Cicer arietinum L.)

A pathogenesis-related protein induced by infection with Ascochyta rabiei was purified from intercellular washing fluid of chickpea (Cicer arietinum L.) leaves. Amino-terminal sequencing identified the protein, named PR-5a, as a thaumatin-like protein. The isoelectric point was determined with 6.5 and the molecular mass is 16 kDa. Therefore, chickpea PR-5a is the first dicot member of a TLP subgroup containing small TLPs with a molecular weight between 15 and 18 kDa. PR-5a shows no antifungal activity towards A. rabiei. Screening of a chickpea cDNA library led to the isolation of a cDNA clone (p5a-241) for this protein. A second cDNA clone (ELR112) encoding a TLP was isolated using differential hybridisation of cDNA libraries obtained from elicited and water treated cell suspension cultures of chickpea. The deduced protein (PR-5b) has a molecular mass of 22 kDa. PR-5b is postulated to be located in the vacuole due to the presence of a respective N-terminal signal peptide and a carboxy-terminal extension. Southern blot analyses showed that ELR112 and p5a-241 represent single copy genes. During fungal infection of chickpea plants expression of both genes proceeds much faster in an A. rabiei resistant cultivar than in a susceptible one.     

Isolation and characterization of cDNA clones encoding pathogenesis-related proteins from tobacco mosaic virus infected tobacco plants.

Infection of the tobacco cultivar Samsun NN by tobacco mosaic virus (TMV) results in a hypersensitive response. During this defense reaction several host encoded proteins, known as pathogenesis-related proteins (PR-proteins), are induced. Poly(A)+ RNA from TMV infected tobacco plants was used to construct a cDNA library. Thirty two cDNA clones were isolated and after digestion with different restriction endonucleases, twenty clones were found to code for PR-1a, six clones for PR-1b, and four clones for PR-1c. Two independent cDNA clones of each class were further characterized by DNA sequence analysis. All clones analyzed contained the 138 amino acid coding regions of their respective mature proteins, but only partial sequences of the signal peptides. Minor differences between the nucleotide sequences for clones belonging to the same class were detected. Comparison of the amino acid sequence for PR-1a deduced from its nucleotide sequence with published data obtained by Edman degradation of the protein showed four differences. Analysis of the 3' ends of the cDNA clones indicates that various alternate poly(dA) addition sites are used. Southern blot analysis using these cDNAs as probes suggests the presence of multiple PR-protein genes in the genomes of tobacco and tomato plants.     

Characterization of a cDNA encoding a proline-rich 14 kDa protein in developing cortical cells of the roots of bean (Phaseolus vulgaris) seedlings

A cDNA clone, corresponding to mRNAs preferentially expressed in the roots of bean (Phaseolus vulgaris L.) seedlings, was isolated. This clone contains a 381 bp open reading frame encoding a polypeptide of 13.5 kDa, designated PVR5 (Phaseolus vulgaris root 5). The amino acid sequence of this clone is rich in proline (13.5%) and leucine (12.7%) and shares significant amino acid sequence homology with root-specific and proline-rich proteins from monocots (maize and rice), and proline-rich proteins from dicots (carrot, oilseed rape, and Madagascar periwinkle). The precise biological roles of these polypeptides are unknown. PVR5 mRNA accumulation is developmentally regulated within the root, with high levels at the root apex and declining levels at distances further from the root tip. In situ hybridization shows that PVR5 mRNA specifically accumulates in the cortical ground meristem in which maximal cell division occurs. Southern blot analysis suggests that genomic DNA corresponding to PVR5 cDNA is encoded by a single gene or a small gene family.     

Structure and expression of a Manduca sexta larval cuticle gene homologous to Drosophila cuticle genes

A genomic clone was isolated from the tobacco hornworm, Manduca sexta, by virtue of its similarity to a Drosophila larval cuticle gene. RNA analysis shows that this clone, B311, is expressed at times appropriate for a larval cuticle gene. Hybrid-selection experiments using B311 DNA show that it encodes a 14 x 10(3) Mr protein, LCP-14, which is precipitated by an antiserum to Manduca larval cuticle. We have sequenced both genomic and cDNA clones for the LCP-14 gene. A conceptual translation of the cDNA sequence shows that the LCP-14 protein is similar not only to another Manduca cuticle protein, but also to Drosophila, Sarcophaga and Hyalophora cecropia cuticle proteins. Since these proteins are found in flexible cuticle and have similar sequences, we conclude they are encoded by homologous genes.     

Differential Regulation of beta-1,3-Glucanase Messenger RNAs in Response to Pathogen Infection

The acidic, extracellular, glucan endo-1,3-β-glucosidases (EC 3.2.1.39; β-1,3-glucanases), pathogenesis-related proteins-2, -N, and -O (i.e. PR-2, PR-N, and PR-O) were purified from Nicotiana tabacum (tobacco) and their partial amino acid sequences determined. Based on these data, complementary DNA (cDNA) clones encoding the proteins were isolated. Additional cDNAs were isolated that encoded proteins approximately 90% identical with PR-2, PR-N, and PR-O. Although the proteins encoded by these cDNAs have not been identified, their deduced amino acid sequences have slightly basic or neutral calculated isoelectric points, as well as carboxy-terminal extensions. These physical characteristics are shared by the vacuolar form of β-1,3-glucanase and other vacuolar localized analogs of PR proteins, suggesting that the unidentified proteins may be similarly localized. A preliminary evolutionary model that separates the β-1,3-glucanase gene family from tobacco into at least five distinct subfamilies is proposed. The expression of β-1,3-glucanase messenger RNAs (mRNAs) in response to infection by tobacco mosaic virus was examined. Messages for the acidic glucanases were induced similarly to the mRNAs for other PR proteins. However, the basic glucanase showed a different response, suggesting that different isoforms are differentially regulated by tobacco mosaic virus infection at the mRNA level.     

Characterization and expression of an mRNA encoding a wound-induced (Win) protein from ethylene-treated tomato leaf abscission zone tissue

A cDNA clone (TAB7) encoding a putative woundinduced (Win) proteinhas been isolated from a tomato (Lycopersicon esculentum Mill.cv. Ailsa Craig) leaf abscission zone cDNA library using a differentialscreening strategy. The clone has a high degree of homologyat the amino acid level to both the potato win1 and 2 genes,Hevea brasiliensis hevein and Nicotiana tabacum PR-4a and PR-4bproteins. The mRNA encoded by TAB7 is up-regulated within 12h of exposure to ethylene (10µl l^–1) and its expressionincreases steadily within the cells comprising the leaf abscissionzone and to a lesser extent in the adjacent non-zone tissue.This rise precedes the onset of cell separation. Southern analysisindicates that the mRNA is encoded by either a single gene ora small gene family. The role of the protein during abscissionis discussed. Key words: Lycopersicon esculentum, abscission zone, ethylene, tomato, wound-induced proteins     

WIP1, a wound-inducible gene from maize with homology to Bowman-Birk proteinase inhibitors

We have cloned and sequenced a wound-inducible cDNA clone designated WIP1 (for wound-induced protein) from maize coleoptiles. It was isolated by differential screening of a cDNA library prepared from excised maize coleoptile segments. The deduced amino acid sequence predicts a secretory, cysteine-rich protein of 102 residues with a calculated molecular mass of 11 kDa and a typical N-terminal signal sequence. The protein has about 30% identity with various Bowman-Birk type proteinase inhibitors. Most interestingly, it is novel in that it is double-headed with exclusive specificity for chymotrypsin. WIP1 is strongly wound-induced in contrast to other members of the Bowman-Birk proteinase inhibitor family, which occur in seeds and are regulated during development. The response is fast, similar to defenceinduced genes, and measurable as early as 30 min after wounding. Induction can also be evoked in the intact coleoptiles and the signal is systemically transmitted in the coleoptile to adjacent regions of the wounded area. Isolation and analysis of the corresponding genomic clone reveals that WIP1 contains an intron of 90 nucleotides.     

Structure of tobacco genes encoding pathogenesis-related proteins from the PR-1 group.

Infection of Samsun NN tobacco with tobacco mosaic virus (TMV) was found to induce the synthesis of mRNA encoding a basic protein with a 67% amino acid sequence homology to the known acidic pathogenesis-related (PR) proteins 1a, 1b and 1c. By Southern blot hybridization it was shown that the tobacco genome contains at least eight genes for acidic PR-1 proteins and a similar number of genes encoding the basic homologues. Clones corresponding to three of the genes for acidic PR-1 proteins were isolated from a genomic library of Samsun NN tobacco. The nucleotide sequence of these genes and their flanking sequences were determined. One clone was found to correspond to the PR-1a gene; the two other clones do not correspond to known TMV-induced PR-1 mRNA's and may represent silent genes. Compared to the PR-1a gene, these genes contain an insertion or deletion in the putative promoter region and mutations affecting the PR-1 reading frame.