Restrictive glycosylphosphatidylinositol anchor synthesis in cwh6/gpi3 yeast cells causes aberrant biogenesis of cell wall proteins.

We previously reported that the defects in the Saccharomyces cerevisiae cwh6 Calcofluor white-hypersensitive cell wall mutant are caused by a mutation in SPT14/GPI3, a gene involved in glycosylphosphatidylinositol (GPI) anchor biosynthesis. Here we describe the effect of cwh6/spt14/gpi3 on the biogenesis of cell wall proteins. It was found that the release of precursors of cell wall proteins from the endoplasmic reticulum (ER) was retarded. This was accompanied by proliferation of ER structures. The majority of the cell wall protein precursors that eventually left the ER were not covalently incorporated into the cell wall but were secreted into the growth medium. Despite the inefficient incorporation of cell wall proteins, there was no net effect on the protein level in the cell wall. It is postulated that the availability of GPI-dependent cell wall proteins determines the rate of cell wall construction and limits growth rate.     

Molecular and biochemical basis of the interaction between tomato and its fungal pathogen Cladosporium fulvum

The interaction between the biotrophic fungal pathogen Cladosporium fulvum and tomato complies with the genefor-gene model. Resistance, expressed as a hypersensitive response (HR) followed by other defence responses, is based on recognition of products of avirulence genes from C. fulvum (race-specific elicitors) by receptors (putative products of resistance genes) in the host plant tomato. The AVR9 elicitor is a 28 amino acid (aa) peptide and the AVR4 elicitor a 106 aa peptide which both induce HR in tomato plants carrying the complementary resistance genes Cf9 and Cf4, respectively. The 3-D structure of the AVR9 peptide, as determined by 1H NMR, revealed that AVR9 belongs to a family of peptides with a cystine knot motif. This motif occurs in channel blockers, peptidase inhibitors and growth factors. The Cf9 resistance gene encodes a membrane-anchored extracellular glycoprotein which contains leucine-rich repeats (LRRs). 125I labeled AVR9 peptide shows the same affinity for plasma membranes of Cf9+ and Cf9- tomato leaves. Membranes of solanaceous plants tested so far all contain homologs of the Cf9 gene and show similar affinities for AVR9. It is assumed that for induction of HR, at least two plant proteins (presumably CF9 and one of his homologs) interact directly or indirectly with the AVR9 peptide which possibly initiates modulation and dimerisation of the receptor, and activation of various other proteins involved in downstream events eventually leading to HR. We have created several mutants of the Avr9 gene, expressed them in the potato virus X (PVX) expression system and tested their biological activity on Cf9 genotypes of tomato. A positive correlation was observed between the biological activity of the mutant AVR9 peptides and their affinity for tomato plasma membranes. Recent results on structure and biological activity of AVR4 peptides encoded by avirulent and virulent alleles of the Avr4 gene (based on expression studies in PVX) are also discussed as well as early defence responses induced by elicitors in tomato leaves and tomato cell suspensions.     

Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis

A pair of vectors for expression of heterologous genes in Lactococcus lactis was constructed. In addition to an origin of replication that has a broad host range, these vectors contain a multiple cloning site flanked by gene expression signals originating from L. lactis subsp. cremoris Wg2. The two vectors, about 3.7 kilobase pairs in size, differ only in the type of antibiotic resistance they confer to their hosts. pMG36 carries a kanamycin resistance marker, which was replaced by an erythromycin resistance marker in pMG36e. As an example of the use of these vectors, the hen egg white lysozyme-coding sequence was inserted. A fusion protein of the expected size was detected in a transformed L. lactis subsp. lactis strain by using Western blotting (immunoblotting).     

A QTL for broad-spectrum resistance to cyst nematode species (Globodera spp.) maps to a resistance gene cluster in potato

 Broad-spectrum resistance in potato to the potato cyst nematode (PCN) species Globodera rostochiensis and G. pallida is commonly regarded as a polygenically inherited trait. Yet, by use of QTL analysis and a selected set of PCN populations, resistance to both PCN species could be ascribed to the action of locus Grp1. Grp1 confers major resistance to G. rostochiensis line Ro₅-22 and G. pallida population Pa₂-D383 and partial resistance to G. pallida population Pa₃-Rookmaker. Grp1 was mapped on chromosome 5 using previously characterized AFLP markers. Cleaved amplified polymorphic sequence (CAPS) markers available for RFLP loci GP21 and GP179 revealed that Grp1 maps on a genomic region harboring other resistance factors to viral, fungal and nematodal pathogens. The present data indicate that Grp1 is a compound locus which contains multiple genes involved in PCN resistance. Received: 10 September 1997 / Accepted: 6 October 1997     

Antimicrobial activity of lactobacilli: preliminary characterization and optimization of production of acidocin B, a novel bacteriocin produced by Lactobacillus acidophilus M46

Approximately 1000 lactobacillus strains were isolated and screened for the production of antimicrobial activity, using a target panel of spoilage organisms and pathogens. Only eight positive strains were found; two of these were studied in more detail. Lactobacillus salivarius M7 produces the new broad spectrum bacteriocin salivaricin B which inhibits the growth of Listeria monocytogenes, Bacillus cereus, Brochothrix thermosphacta, Enterococcus faecalis and many lactobacilli. A new atypical bacteriocin produced by Lact. acidophilus M46, acidocin B, combines the inhibition of Clostridium sporogenes with a very narrow activity spectrum within the genus Lactobacillus and was selected for further characterization. Acidocin B is sensitive to trypsin, heat-stable (80°C for 20 min) and can be extracted from the culture supernatant fluid with butanol. Native acidocin B occurs as a large molecular weight complex (100 kDa), while with SDS-PAGE the partly purified activity migrates as a peptide of 2·4 kDa. Optimization of the cultivation conditions resulted in an eightfold increase of the amount of acidocin B produced during growth. Growth is not necessary for acidocin B production; washed producer cells can synthesize the bacteriocin in a chemically defined production medium. The application potential of acidocin B is discussed.     

Allele mining in <Emphasis Type="Italic">Solanum</Emphasis>: conserved homologues of <Emphasis Type="Italic">Rpi-blb1</Emphasis> are identified in <Emphasis Type="Italic">Solanum stoloniferum</Emphasis>

Allele mining facilitates the discovery of novel resistance (R) genes that can be used in breeding programs and sheds light on the evolution of R genes. Here we focus on two R genes, Rpi-blb1 and Rpi-blb2, originally derived from Solanum bulbocastanum. The Rpi-blb1 gene is part of a cluster of four paralogues and is flanked by RGA1-blb and RGA3-blb. Highly conserved RGA1-blb homologues were discovered in all the tested tuber-bearing (TB) and non-tuber-bearing (NTB) Solanum species, suggesting RGA1-blb was present before the divergence of TB and NTB Solanum species. The frequency of the RGA3-blb gene was much lower. Interestingly, highly conserved Rpi-blb1 homologues were discovered not only in S. bulbocastanum but also in Solanum stoloniferum that is part of the series Longipedicellata. Resistance assays and genetic analyses in several F1 populations derived from the relevant late blight resistant parental genotypes harbouring the conserved Rpi-blb1 homologues, indicated the presence of four dominant R genes, designated as Rpi-sto1, Rpi-plt1, Rpi-pta1 and Rpi-pta2. Furthermore, Rpi-sto1 and Rpi-plt1 resided at the same position on chromosome VIII as Rpi-blb1 in S. bulbocastanum. Segregation data also indicated that an additional unknown late blight resistance gene was present in three populations. In contrast to Rpi-blb1, no homologues of Rpi-blb2 were detected in any material examined. Hypotheses are proposed to explain the presence of conserved Rpi-blb1 homologues in S. stoloniferum. The discovery of conserved homologues of Rpi-blb1 in EBN 2 tetraploid species offers the possibility to more easily transfer the late blight resistance genes to potato varieties by classical breeding.     

Mapping of intra-locus duplications and introgressed DNA: aids to map-based cloning of genes from complex genomes illustrated by physical analysis of the Rx locus in tetraploid potato

 We describe here novel approaches to high-resolution mapping of repeated and introgressed DNA which may prove generally useful in map-based cloning from complex genomes. These approaches were developed in order to clone the Rx locus in potato. First, we prepared a BAC library from a tetraploid plant carrying Rx in the duplex condition (Rx, Rx, rx, rx). BAC clones were then isolated with close markers on either side of Rx. However these clones did not extend across Rx: in the cloned DNA the closest markers to Rx were separated by single recombination events on either side of Rx. To bridge the gap we exploited the finding that the BAC clones on the right side of Rx contained resistance-gene homologues. Anticipating that there would be duplicated copies of these resistance gene homologues in the vicinity of Rx, we used low-stringency PCR conditions to identify additional markers. One of these markers was completely linked to Rx in our mapping population and was used to isolate a BAC (BAC77) that had not been previously identified by screening with Rx-flanking markers. Based on two criteria it was concluded that BAC77 spans Rx. There was a chromosomal recombination in one plant of our mapping population that separated the BAC77 right end from Rx. On the other side of Rx it was found that the BAC77 left end was outside the region of DNA carrying Rx that had been introgressed into potato cv Amaryl from a Solanum tuberosum ssp. andigena accession CPC1673. Received: 13 July 1998 / Accepted: 17 September 1998     

High-resolution Mapping and Analysis of the Resistance Locus <Emphasis Type="Italic">Rpi-abpt</Emphasis> Against <Emphasis Type="Italic">Phytophthora infestans</Emphasis> in Potato

Introduction of more durable resistance against Phytophthora infestans causing late blight into the cultivated potato is of importance for sustainable agriculture. We identified a new monogenically inherited resistance locus that is localized on chromosome 4. The resistance is derived from an ABPT clone, which is originally a complex quadruple hybrid in which Solanum acaule, S. bulbocastanum, S. phureja and S. tuberosum were involved. Resistance data of the original resistant accessions of the wild species and analysis of mobility of AFLP markers linked to the resistance locus suggest that the resistance locus is originating from S. bulbocastanum. A population of 1383 genotypes was screened with two AFLP markers flanking the Rpi-abpt locus and 98 recombinants were identified. An accurate high-resolution map was constructed and the Rpi-abpt locus was localized in a 0.5 cM interval. One AFLP marker was found to co-segregate with the Rpi-abpt locus. Its DNA sequence was highly similar with sequences found on a tomato BAC containing several resistance gene analogues on chromosome 4 and its translated protein sequence appeared to be homologous to several disease resistance related proteins. The results indicated that the Rpi-abpt gene is a member of an R gene cluster.