In vitro tolerance to Botrytis cinerea of grapevine 41B rootstock in transgenic plants expressing the stilbene synthase Vst1 gene under the control of a pathogen-inducible PR 10 promoter.

Resveratrol is a major phytoalexin in grapevine but its synthesis in response to phytopathogen attack decreases with grape berry ripening. A chimeric gene combining an alfalfa PR 10 promoter and Vst1 (Vitis stilbene synthase 1) gene was introduced into the genome of 41B rootstock. Transgenic plants were analysed for resveratrol production in leaves infected with Botrytis using an in vitro test. Among the 50 transgenic lines analysed, some exhibited a production lower than the non-transgenic control, but others accumulated resveratrol from 5-100-fold. Moreover, in the latter clones, symptoms were highly reduced in response to infection. These results were a good indication that the combination of a pathogen-inducible promoter and a defence gene may increase tolerance against fungi in grapevine. The efficacy of this approach should be further tested by experiments conducted in the vineyard.

Defense reaction in Medicago sativa: a gene encoding a class 10 PR protein is expressed in vascular bundles.

Infiltration of Medicago sativa leaves with a suspension of Pseudomonas syringae pv. pisi elicits the accumulation of several mRNA classes. A clone, designated as MsPR10-1, encoding a polypeptide exhibiting strong similarity to the class 10 PR protein was isolated and characterized from a cDNA library prepared from leaf mRNA. The corresponding gene was shown to be developmentally regulated: Except in roots, its expression was not detectable in other analyzed organs of healthy plants (hypocotyls, cotyledons, stems, leaves, and flower buds). MsPR10-1 transcript accumulation was especially high in leaf blades during an incompatible interaction: It was already detectable 3 h after infection, reached its maximum level 24 h postinfection, and remained at a high level over a period of at least 72 h. In addition, the expression of this gene was induced by salicylic acid treatment of the leaves. Southern hybridizations showed that this gene belongs to a multigene family. Using a 5' extension technique for cDNA, we demonstrated that during the incompatible interaction with P. syringae pv. pisi several genes or allelic variants of this class were expressed. Measurements of transcript accumulation in both the infiltrated and noninfiltrated zones by Northern and in situ hybridization allowed to demonstrate the "systemic" expression pattern of the MsPR10-1. In situ hybridizations indicated that MsPR10-1 was expressed in the vascular bundles adjacent to and distant from the infection site.

Nucleotide sequences of four pathogen-induced alfalfa peroxidase-encoding cDNAs.

We constructed an alfalfa cDNA library from mRNA extracted from leaves after infection with Pseudomonas syringae (incompatible interaction). Screening with oligodeoxyribonucleotides designed from regions conserved in all known peroxidases allowed the isolation of four cDNAs (Msprx1A, 1B, 1C and 2). Sequence analysis revealed the presence of open reading frames of 351, 355, 358 and 323 amino acids, respectively, with the characteristic consensus sequences of plant peroxidases. Sequence comparison showed that the Msprx2 product is significantly different from the others and, particularly, lacks a C-terminal propeptide which might be required for vacuolar targeting.

Role of plant defence in alfalfa during symbiosis.

During effective symbiosis, rhizobia colonize their hosts, and avoid plant defence mechanisms. To determine whether the host defence responses can be elicited by the symbiotic bacteria, specific markers involved in incompatible pathogenic interactions are required. The available markers of alfalfa defence mechanisms are described and their use in the study of the symbiotic interaction discussed. As defence-related gene expression in roots is not always related to defence mechanisms, other model systems have been established allowing confirmation of an important role of bacterial surface components in alfalfa-Rhizobium meliloti interactions. Nod factors at high concentrations have been shown to elicit defence-like responses in Medicago cell suspensions and roots. Elicitation of defence mechanisms by high levels of Nod factors in Rhizobium-infected roots may be a part of the mechanism by which nodulation is feed-back regulated.

Cloning and expression of a cDNA encoding a cytoplasmic L5 ribosomal protein from alfalfa (Medicago sativa L.).

A cDNA encoding a putative cytoplasmic ribosomal protein L5 from alfalfa (MsRL5), the first sequence from higher plants, has been characterized. The derived amino acid sequence of 181 residues contains the L5 signature, is 72.2% identical to yeast ribosomal L5 and shares high identity with other RL5 peptides from eukaryotic origin. The sequence does not contain any signal or transit peptide and therefore might be cytoplasmic. In all alfalfa organs examined MsRL5 transcripts were detected at approximately equal levels.

Pathological and molecular characterizations of alfalfa interactions with compatible and incompatible bacteria, Xanthomonas campestris pv. alfalfae and Pseudomonas syringae pv. pisi.

We report on the interactions of alfalfa with Xanthomonas campestris pv. alfalfae and Pseudomonas syringae pv. pisi. A hypersensitive response was observed when leaves were infiltrated with P. s. pv. pisi, which remained strictly limited to the injected zone. The compatible interaction with X. c. pv. alfalfae was characterized by water-soaking symptoms and the spreading of the bacterium into the leaf blade. Analyses of transcript accumulation were conducted with cDNAs encoding enzymes involved in phytoalexin synthesis: chalcone synthase (CHS), chalcone isomerase (CHI), and isoflavone reductase (IFR). In incompatible interactions the maximum accumulation of the CHS, CHI, and IFR transcripts was observed 6 hr postinfection. In the compatible interaction, the induction of these transcripts was delayed until 25-30 hr postinfection, and the level of their accumulation was considerably lower. Extending this molecular analysis to the root system showed that the reaction of roots during an incompatible interaction was quite comparable to that of leaves. To complete these analyses, expression of genes encoding pathogenesis-related (PR) proteins in leaves was also analyzed by polymerase chain reaction. High-level accumulation of a 0.8-kb transcript encoding a PR protein was observed 6 to 30 hr postinfection in the incompatible interaction.

Differential expression of two peanut peroxidase cDNA clones in peanut plants and cells in suspension culture in response to stress.

Peanut (Arachis hypogea L.) peroxidase gene expression was analyzed by measuring the accumulation of trancripts in cultured cells and various plant parts (leaf, stem, root) and upon their treatment with ethylene or wounding, respectively. Two transcripts (prxPNC1 and prxPNC2) corresponding to two peroxidase genes are expressed at higher levels in cultured cells as compared to various plant organs. Analysis of total poly(A)(+) RNA with an oligonucleotide probe corresponding to a highly conserved region of peroxidase genes showed the expression of three peroxidase related sequences (1,000, 1,400 or 2,600 bp) in stem or leaf but barely detectable in roots. The prxPNC2 transcript transiently expressed at high levels in response to ethylene treatment of cells or wounding of leaves. This suggests that the corresponding gene(s) are expressed in response to stress.

Molecular cloning of complementary DNAs encoding two cationic peroxidases from cultivated peanut cells.

We have isolated, cloned, and characterized two cDNAs corresponding to the mRNAs for cationic peroxidases synthesized by cultured peanut cells. The first clone was obtained from a phage lambda gt11 library screened with antibodies directed against the major secreted isozyme. Its predicted amino acid sequence, deduced from the 1228-base-pair (bp) cDNA, revealed a 22-amino acid signal peptide and a 294-amino acid mature protein (Mr, 31,228). The second clone was isolated from a lambda gt10 library screened with oligonucleotides corresponding to the regions for acid/base catalysis and the fifth ligand of heme. This cDNA (1344 bp) encodes a protein (330 amino acids) with a mature peptide of 307 residues (Mr, 32,954). The two peanut peroxidases are 46% homologous. The estimated gene copy numbers of these peroxidases might be close to 1 or 2 per haploid genome. A comparison of the amino acid sequence of these peanut peroxidases with other known isozymes shows two already known regions of homology (the region for acid/base catalysis and the fifth ligand of heme). Moreover, some new characteristics appeared such as a glycosylation site identical in five of the seven isozymes, a putative antigenic determinant common to all the isozymes, and a region of the highest homology. A secondary structure prediction showed that it corresponds to a 16-amino acid helix linked to the next one by a long stretch of beta strands and coils and might represent a critical structural element.

Lectin-gene expression in pea (Pisum sativum L.) roots.

The expression of a lectin gene in pea (Pisum sativum L.) roots has been investigated using the copy DNA of a pea seed lectin as a probe. An mRNA which has the same size as the seed mRNA but which is about 4000 times less abundant has been detected in 21-d-old roots. The probe detected lectin expression as early as 4 d after sowing, with the highest level being reached at 10 d, i.e. just before nodulation. In later stages (16-d- and 21-d-old roots), expression was substantially decreased. The correlation between infection by Rhizobium leguminosarum and lectin expression in pea roots has been investigated by comparing root lectin mRNA levels in inoculated plants and in plants grown under conditions preventing nodulation. Neither growth in a nitrate concentration which inhibited nodulation nor growth in the absence of Rhizobium appreciably affected lectin expression in roots.

The pea lectin gene family contains only one functional gene.

Molecular hybridization experiments have shown that the pea genome contains four regions which hybridize with pea lectin cDNA (Kaminski, Buffard, and Strosberg, 1986. Plant Science 46, 111-116). The complete organization of the pea lectin gene family was investigated. Four partial EcoRI genomic libraries were screened with a lectin cDNA (pPS 15-50) covering the entire coding region. Four positive recombinant phages, λI 101, λI 52, λIII 51 and λIV 22, were isolated and the DNA sequences of the subclones, designated respectively PSL1, PSL2, PSL3 and PSL4, were determined. PSL2, PSL3 and PSL4 are incomplete genes; the presence of several stop codons in the correct reading frames indicate that these genes cannot code for a functional lectin protein. The sequences of PSL1 and pPS 15-50 have identical coding regions. The pea lectin gene has no intervening sequences and is flanked at its 5' region by a sequence containing an exceptionally high A+T content (73%). Eucaryotic consensus sequences such as a TATA box and a polyadenylation signal are also found in the flanking regions of the PSL1 clone.

Rapid large-scale purification of plasmid DNA by medium or low pressure gel filtration. Application: construction of thermoamplifiable expression vectors.

This paper describes a new method of plasmid DNA purification which is fast and reliable enough for most purposes in recombinant DNA technology. The present method does not require the use of toxic chemicals such as phenol or ethidium bromide, costly ultracentrifugation procedures or other processes which can modify the supercoiled structure of the plasmids, such as adsorption on glass fiber. This method is based on the principle of gel filtration chromatography, at low pressure (1 bar) or medium pressure (between 5 and 10 bars), using Sephacryl S1000 or Superose 6B. It permits recovery of plasmids: in preparative quantities (from 300 micrograms to 4 mg), exempt from RNA, DNA and protein contamination, and suitable for various common genetic engineering procedures immediately after purification. To test the reliability of the technique as well as the degree of purification, the plasmids were used to construct thermoamplifiable vectors, carrying the lacUV5 promoter and the 5' end of the beta-galactosidase gene with a single EcoR1 site in each of the three possible translational phases. This set of vectors is designed for the expression of foreign genes as hybrid proteins in Escherichia coli.

Changes in the polyadenylated messenger RNA population during differentiation of Vicia faba root cells.

Using cDNA probes, the polysomal polyadenylated RNA populations of Vicia faba meristematic, elongating, and mature root cells were analysed and compared, with respect to complexity, abundance distribution, and sequence representation. These cells express 12 300, 15 800, and 15 200 sequences, respectively, of an average size of 1300 nucleotides, distributed in three frequency classes. Transition from the meristematic to the elongating stage is coordinated with the disappearance of 25-30% of the abundant RNA species (2000 copies per cell) and with the appearance of new transcripts corresponding to 3000-3500 genes expressed at an intermediate level (50-60 copies per cell) and belonging to the rare class (about 4 copies per cell). These new transcripts represent 12.5% of the mass of the mRNA during the elongating stage and are quantitatively modulated during the transition to the mature stage. Thus 80 to 85% of the polysomal polyadenylated RNA species expressed in elongating or mature cells are common to the three cell types.

Gibberellic-acid-regulated expression of α-amylase and six other genes in wheat aleurone layers.

The effect of gibberellic acid (GA3) on gene expression in wheat aleurone cells has been characterised. In-vitro translation of polyadenylated RNA indicated that α-amylase and other messenger-RNA (mRNA) species increase in relative concentration in GA3-treated tissue. At least one mRNA species declines in relative level in response to GA3. There is also a GA3-dependent, four-fold increase in the level of polyadenylated RNA. This effect is largely the result of increased levels of many mRNA species which are also present in untreated tissue. Seven GA3-induced polyadenylated RNA species including the Amyl α-amylase gene product have been cloned as complementary DNA in the plasmid pBR322. These cloned DNAs have been used as hybridisation probes to show that the GA3-induced increase in α-amylase mRNA is more prolonged than the accumulation of the other GA3-regulated mRNA species. A polyadenylated-RNA sequence showing reduced concentration in GA3-treated tissue has also been cloned.

Complexity of polysomal polyadenylated RNAs in Vicia faba meristematic root cells.

As a first step in quantifying gene expression during differentiation of the Vicia faba root cells we have analysed the mRNA population from meristematic cells. Using cDNA X mRNA hybridizations we have shown that this population can be divided into three abundance classes (abundant, intermediate and rate) representing 37%, 34% and 29% of the mRNAs and containing 26, 610 and 11700 sequences respectively. The total base-sequence complexity of the mRNA population, as determined by cDNA X mRNA hybridization, was found to be 1.6 x 10(7) nucleotides. This estimate was confirmed by the determination of the amount of single-copy genomic DNA hybridizing to the mRNA. Expressed as the number of structural gene transcripts, this complexity corresponds to about 13000 mRNA sequences.