The phytopathogenic fungus Alternaria alternata f. sp. lycopersici (AAL) produces toxins that are essential for pathogenicity of the fungus on tomato (Lycopersicon esculentum). AAL toxins and fumonisins of the unrelated fungus Fusarium moniliforme are sphinganine-analog mycotoxins (SAMs), which cause inhibition of sphingolipid biosynthesis in vitro and are toxic for some plant species and mammalian cell lines. Sphingolipids can be determinants in the proliferation or death of cells. We investigated the tomato Alternaria stem canker (Asc) locus, which mediates resistance to SAM-induced apoptosis. Until now, mycotoxin resistance of plants has been associated with detoxification and altered affinity or absence of the toxin targets. Here we show that SAM resistance of tomato is determined by Asc-1, a gene homologous to the yeast longevity assurance gene LAG1 and that susceptibility is associated with a mutant Asc-1. Because both sphingolipid synthesis and LAG1 facilitate endocytosis of glycosylphosphatidylinositol-anchored proteins in yeast, we propose a role for Asc-1 in a salvage mechanism of sphingolipid-depleted plant cells. Mycotoxins are a continuous risk in the production and postharvest storage of crops, threatening animal and human health. They can be divided into the nonselective mycotoxins, phytotoxic to a broader range of organisms than the producing fungus infests and in the host-selective mycotoxins, primary determinants of phytopathogenicity (1). One class of host-selective mycotoxins is structurally related to sphinganine (2), a precursor in plant sphingolipid biosynthesis ( Fig. 1) (3). Two nonrelated fungi are well known for the production of sphinganine-analog mycotoxins (SAMs): Fusarium moniliforme, a fungal pathogen of maize that produces fumonisin B 1 (FB 1 ) (4) and Alternaria alternata f. sp. lycopersici (AAL), a tomato pathogen that produces AAL toxins (5). SAMs competitively inhibit de novo sphingolipid (ceramide) biosynthesis in vitro (see Fig. 1), which leads to a variety of cellular responses, including the accumulation of sphingoid bases in animal cells (6) and plants (7). Besides their role as structural components of biomembranes, sphingolipids can be determinants in the proliferation or death of cells, depending on the type of cells studied (6). SAMs are toxic to all tissues of sensitive tomato cultivars at low concentrations (5, 8) and induce apoptosis in sensitive tomato lines (9). SAMs also induce apoptosis in some mammalian cell lines (2) and are toxic to animals and suspected to be conditionally carcinogenic in humans (4). These data indicate the necessity to understand the mechanism of action of SAMs, which has been performed by studying SAM resistance of tomato.Until now, mycotoxin resistance of plants has been associated with detoxification and altered affinity or the absence of the toxin targets (1). Resistance or susceptibility to both AAL and SAMs in tomato is determined by the Alternaria stem canker (Asc) locus (5). It inherits in a single codominant fashion. Plants heter...
We report the cloning and nucleotide sequence of the gene encoding malonyl cocnzymc A-acyl carrier protein transacylasc of Esciwrichiu co/i. Malonyl transacylasc has been overexpressed 1.55.fold compared to a wild-type strain, Overexpression of this enzyme alters the fatty acid composition of a wild-type E. coli strain; increased amounts of ris-vacccnate arc incorporated into the mcmbranc phospholipids. Mulonyl transacylase; Malonyl-CoA: Acyl transferase; Fatty acid biosynthesis 1. 1NTRODUCTION Malonyl coenzyme A-acyl carrier protein trans-acylase (malonyl transacylase) catalyzes a key reaction of fatty acid synthesis in bacteria and plants, the conversion of malonyl-CoA to malonyl-acyl carrier protein (ACP). Malonyl-ACP then acts as the two carbon donor in the elongation steps of fatty acid synthesis. A similar reaction occurs in fatty acid synthesis in fungi and mammals except that both the malonyl transacylase and ACP moieties are domains of the polyfunctional fatty acid synthases of these organisms. E. co/i malonyl transacylase has been purified [1,2], and aspects of the enzymatic mechanism studied [3]. Mutants defective in the enzyme have also been isolated [4], In this paper, we report the cloning, DNA sequence, and overexpression of malonyl transacylase. We also report the effects of overexpression on the fatty acid composition of the membrane phospholipids of E. co/i, 2. MATERIALS AND METHODS The bacterial strains used were all derivatives of E. ru/i K-l?. Strain L48 (also called LA2-89) [4], carries a temperature-sensitive lesion in the&&I gene resulting in a malonyl transacylaseol'abnormal thermo-lability. recA derivatives of this strain wrrc made tither by PI transduction or bacterial conjugation using a T~rrfO element closely linked to rccAf. Standard recombinant DNA methods were used as were standard plasmids and host strains. DNA sequencing was done using the Sequenasc kits from United States Bibchemicals with both single-stranded and double-stranded templates. The sequence reported was obtained by complete sequencing of both DNA strands and was independently obtained in both laboratories. Protein cxpres-C~r-esfi~rrlen~ &ifzrz. sion studies were done using either the T7 polymerase system (for the truncated protein) [S], or the f~ promoter of plasmid pCKRlOl [6]. 3. RESULTS 3.1. Sequejrce of fabD The fobD gene was isolated from two different sources; lambda miniset phase 235 of the Kohara library [7] and from a minibank of E. co/i DNA fragments in plasmid pACYCl84 [El. In both cases, the gene was isolated by complementation (or recombinational repair) of theJrbl> strain LA2-89 which encodes a temperature sensitive enzyme. The gene carried by a235 was further localized by subcloning into high copy number plasmids. The minimal clone conferring growth at 42°C was sequenced and found to encode a truncated version of FabD lacking the last 21 amino acids. N-terminal sequencing of the truncated protein gave the sequence TQFAFVFPGQ, and thus the initiation me-thionine is the most upstream of the three in-fr...
The onset of storage lipid biosynthesis during seed development in the oilseed crop Brassica napus (rape seed) coincides with a drastic qualitative and quantitative change in fatty acid composition. During this phase of storage lipid biosynthesis, the enzyme activities of the individual components of the fatty acid synthase system increase rapidly. We describe a rapid and simple purification procedure for the plastid-localized NADH-dependent enoyl-acyl carrier protein reductase from developing B. napus seed, based on its affinity towards the acyl carrier protein (ACP). The purified protein was N-terminally sequenced and used to raise a potent antibody preparation. Immuno-screening of a seed-specific lambda gt11 cDNA expression library resulted in the isolation of enoyl-ACP reductase cDNA clones. DNA sequence analysis of an apparently full-length cDNA clone revealed that the enoyl-ACP reductase mRNA is translated into a precursor protein with a putative 73 amino acid leader sequence which is removed during the translocation of the protein through the plastid membrane. Expression studies in Escherichia coli demonstrated that the full-length cDNA clone encodes the authentic B. napus NADH-dependent enoyl-ACP reductase. Characterization of the enoyl-ACP reductase genes by Southern blotting shows that the allo-tetraploid B. napus contains two pairs of related enoyl-ACP reductase genes derived from the two distinct genes found in both its ancestors, Brassica oleracea and B. campestris. Northern blot analysis of enoyl-ACP reductase mRNA steady-state levels during seed development suggests that the increase in enzyme activity during the phase of storage lipid accumulation is regulated at the level of gene expression.
SummaryLeaf mould disease in tomato is caused by the biotrophic fungus Cladosporium fulvum. An Ac/Ds targeted transposon tagging strategy was used to isolate the gene conferring resistance to race 5 of C. fulvum, a strain expressing the avirulence gene Avr4. An infection assay of 2-weekold seedlings yielded five susceptible mutants, of which two had a Ds element integrated in the same gene at different positions. This gene, member of a gene family, showed high sequence homology to the C. fulvum resistance genes Cf-9 and Cf-2. The gene is predicted to encode an extracellular transmembrane protein containing a divided domain of 25 leucine-rich repeats. Three mutants exhibited a genomic deletion covering most of the Lycopersicon hirsutum introgressed segment, including the Cf-4 locus. Southern blot analysis revealed that this deletion includes the tagged gene and five homologous sequences. To test whether the tagged gene confers resistance to C. fulvum via Avr4 recognition, the Avr4 gene was expressed in planta. Surprisingly, expression of the Avr4 gene still triggered a specific necrotic response in the transposon-tagged plants, indicating that the tagged resistance gene is not, or is not the only gene, involved in Avr4 recognition. Mutants harbouring the genomic deletion did not show this Avr4-specific response. The deleted segment apparently contains, in addition to the tagged gene, one or more other genes, which play a role in the Avr4 responses. The tagged gene is present at the Cf-4 locus, but it does not necessarily recognize Avr4 and is therefore designated Cf-4A.
The tomato Cf-4 and Cf-9 genes confer resistance to the leaf mould pathogen Cladosporium fulvum and map at a complex locus on the short arm of chromosome 1. It was previously shown that the gene encoding Cf-4, which recognizes the Avr4 avirulence determinant, is one of five tandemly duplicated homologous genes (Hcr9-4s) at this locus. Cf-4 was identified by molecular analysis of rare Cf-4/Cf-9 disease-sensitive recombinants and by complementation analysis. The analysis did not exclude the possibility that an additional gene(s) located distal to Cf-4 may also confer resistance to C. fulvum. We demonstrate that a number of Dissociation-tagged Cf-4 mutants, identified on the basis of their insensitivity to Avr4, are still resistant to infection by C. fulvum race 5. Molecular analysis of 16 Cf-4 mutants, most of which have small chromosomal deletions in this region, suggested the additional resistance specificity is encoded by Hcr9-4E. Hcr9-4E recognizes a novel C. fulvum avirulence determinant that we have designated Avr4E.
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