Cyclic lipopeptides (CLPs) are versatile molecules produced by a variety of bacterial genera, including plant-associated Pseudomonas spp. CLPs are composed of a fatty acid tail linked to a short oligopeptide, which is cyclized to form a lactone ring between two amino acids in the peptide chain. CLPs are very diverse both structurally and in terms of their biological activity. The structural diversity is due to differences in the length and composition of the fatty acid tail and to variations in the number, type, and configuration of the amino acids in the peptide moiety. CLPs have received considerable attention for their antimicrobial, cytotoxic, and surfactant properties. For plant-pathogenic Pseudomonas spp., CLPs constitute important virulence factors, and pore formation, followed by cell lysis, is their main mode of action. For the antagonistic Pseudomonas sp., CLPs play a key role in antimicrobial activity, motility, and biofilm formation. CLPs are produced via nonribosomal synthesis on large, multifunctional peptide synthetases. Both the structural organization of the CLP synthetic templates and the presence of specific domains and signature sequences within peptide synthetase genes will be described for both pathogenic and antagonistic Pseudomonas spp. Finally, the role of various genes and regulatory mechanisms in CLP production by Pseudomonas spp., including two-component regulation and quorum sensing, will be discussed in detail.

SummaryAnalysis of microbial genome sequences have revealed numerous genes involved in antibiotic biosynthesis. In Pseudomonads, several gene clusters encoding non-ribosomal peptide synthetases (NRPSs) were predicted to be involved in the synthesis of cyclic lipopeptide (CLP) antibiotics. Most of these predictions, however, are untested and the association between genome sequence and biological function of the predicted metabolite is lacking. Here we report the genome-based identification of previously unknown CLP gene clusters in plant pathogenic Pseudomonas syringae strains B728a and DC3000 and in plant beneficial Pseudomonas fluorescens Pf0-1 and SBW25. For P. fluorescens SBW25, a model strain in studying bacterial evolution and adaptation, the structure of the CLP with a predicted 9-amino acid peptide moiety was confirmed by chemical analyses. Mutagenesis confirmed that the three identified NRPS genes are essential for CLP synthesis in strain SBW25. CLP production was shown to play a key role in motility, biofilm formation and in activity of SBW25 against zoospores of Phytophthora infestans. This is the first time that an antimicrobial metabolite is identified from strain SBW25. The results indicate that genome mining may enable the discovery of unknown gene clusters and traits that are highly relevant in the lifestyle of plant beneficial and plant pathogenic bacteria.

Massetolide A is a cyclic lipopeptide (CLP) antibiotic produced by various Pseudomonas strains from diverse environments. Cloning, sequencing, site-directed mutagenesis, and complementation showed that massetolide A biosynthesis in P. fluorescens SS101 is governed by three nonribosomal peptide synthetase (NRPS) genes, designated massA, massB, and massC, spanning approximately 30 kb. Prediction of the nature and configuration of the amino acids by in silico analysis of adenylation and condensation domains of the NRPSs was consistent with the chemically determined structure of the peptide moiety of massetolide A. Structural analysis of massetolide A derivatives produced by SS101 indicated that most of the variations in the peptide moiety occur at amino acid positions 4 and 9. Regions flanking the mass genes contained several genes found in other Pseudomonas CLP biosynthesis clusters, which encode LuxR-type transcriptional regulators, ABC transporters, and an RND-like outer membrane protein. In contrast to most Pseudomonas CLP gene clusters known to date, the mass genes are not physically linked but are organized in two separate clusters, with massA disconnected from massB and massC. Quantitative real-time PCR analysis indicated that transcription of massC is strongly reduced when massB is mutated, suggesting that these two genes function in an operon, whereas transcription of massA is independent of massBC and vice versa. Massetolide A is produced in the early exponential growth phase, and biosynthesis appears not to be regulated by N-acylhomoserine lactone-based quorum sensing. Massetolide A production is essential in swarming motility of P. fluorescens SS101 and plays an important role in biofilm formation.

A consensus map of barley was constructed based on three reference doubled haploid (DH) populations and three recombinant inbred line (RIL) populations. Several sets of microsatellites were used as bridge markers in the integration of those populations previously genotyped with RFLP or with AFLP markers. Another set of 61 genic microsatellites was mapped for the Wrst time using a newly developed Xuorescent labelling strategy, referred to as A/T labelling. The Wnal map contains 3,258 markers spanning 1,081 centiMorgans (cM) with an average distance between two adjacent loci of 0.33 cM. This is the highest density of markers reported for a barley genetic map to date. The consensus map was divided into 210 BINs of about 5 cM each in which were placed 19 quantitative trait loci (QTL) contributing to the partial resistance to barley leaf rust (Puccinia hordei Otth) in Wve of the integrated populations. Each parental barley combination segregated for diVerent sets of QTLs, with only few QTLs shared by any pair of cultivars. Defence gene homologues (DGH) were identiWed by tBlastx homology to known genes involved in the defence of plants against microbial pathogens. Sixty-three DGHs were located into the 210 BINs in order to identify candidate genes responsible for the QTL eVects. Eight BINs were cooccupied by a QTL and DGH(s). The positional candidates identiWed are receptor-like kinase, WIR1 homologues and several defence response genes like peroxidases, superoxide dismutase and thaumatin.

The allelic variation in four avirulence (Avr) and four extracellular protein (Ecp)-encoding genes of the tomato pathogen Cladosporium fulvum was analyzed for a worldwide collection of strains. The majority of polymorphisms observed in the Avr genes are deletions, point mutations, or insertions of transposon-like elements that are associated with transitions from avirulence to virulence, indicating adaptive evolution of the Avr genes to the cognate C. fulvum resistance genes that are deployed in commercial tomato lines. Large differences in types of polymorphisms between the Avr genes were observed, especially between Avr2 (indels) and Avr4 (amino-acid substitutions), indicating that selection pressure favors different types of adaptation. In contrast, only a limited number of polymorphisms were observed in the Ecp genes, which mostly involved synonymous modifications. A haplotype network based on the polymorphisms observed in the effector genes revealed a complex pattern of evolution marked by reticulations that suggests the occurrence of genetic recombination in this presumed asexual fungus. This, as well as the identification of strains with identical polymorphisms in Avr and Ecp genes but with opposite mating-type genes, suggests that development of complex races can be the combined result of positive selection and genetic recombination.

SUMMARY Race-specific resistance in plants against microbial pathogens is governed by several distinct classes of resistance (R) genes. This review focuses on the class that consists of the plasma membrane-bound leucine-rich repeat proteins known as receptor-like proteins (RLPs). The first isolated resistance genes of the RLP class are the tomato Cf genes, which confer resistance to the fungal pathogen Cladosporium fulvum. To date, several other RLP genes are known to be implicated in resistance in other plant-pathogen interactions. These include HcrVf2 from apple, Ve1 and Ve2 from tomato, and RPP27 from Arabidopsis, which are involved in resistance to Venturia, Verticillium and Peronospora, respectively. Furthermore, the tomato RLP gene LeEix initiates defence responses upon elicitation with a fungal ethylene-inducing xylanase (EIX) of non-pathogenic Trichoderma. The tomato Cf genes, which are the most intensively studied RLP resistance genes, are usually found in clusters of several homologues. Whereas some of these homologues are functional Cf resistance genes, others have no known function in resistance. Different evolutionary processes contribute to variation in functional Cf genes, and functional as well as non-functional homologues may provide a source for the generation of novel Cf resistance genes. To date, little is known of the proteins that interact with Cf proteins to initiate defence responses. In contrast to the LeEix protein and the corresponding EIX elicitor, for which a direct interaction was found, no direct interaction between Cf proteins and the corresponding C. fulvum elicitors has been demonstrated. Analogous to the CLAVATA signalling complex, which comprises an RLP, a receptor-like kinase (RLK) and a small proteineous ligand, Cf proteins may form a complex with RLKs and thus initiate signalling upon recognition of the corresponding elicitors. The presence of RLP resistance genes in diverse plant species suggests that these genes play an important role in the extracellular recognition of plant pathogens.

The present study explores the potential contribution of the energy requirements associated with nocturnal carbohydrate export to (1) the fraction of dark respiration correlating with leaf nitrogen concentration and (2) the dark respiration of mature source leaves. To this end, we determined the nocturnal carbohydrate-export rates from leaves with an optimal nitrogen supply, and the correlation between the nitrogen concentration and the dark respiration of leaves. The specific energy costs of carbohydrate export from starch-storing source leaves were determined both experimentally and theoretically. The present estimate of the specific energy cost involved in carbohydrate export as obtained by linear regression (0. 70 mol C0 2 [mol sucrose] -1 ), agrees well with both literature data obtained by different methods (0.4 7 to 1.26 mol C0 2 [mol sucrose] -1 ) and the theoretically calculated range for starch-storing species (0.40 to 1.20 mol C0 2 [mol sucrose] -1 ). The conversion of starch in the chloroplast to sucrose in the cytosol is a major energy-requiring process. Maximally 42 to '107'% of the slope of the relationship between respiration rate and organic nitrogen concentration of primary bean leaves, may be ascribed to the energy costs associated with nocturnal export of carbohydrates. Total energy costs associated with export were derived from the product of the specific costs of carbohydrate export and the export rates, either measured on full-grown (primary) leaves of potato and bean or derived from the literature. These export costs account, on average, for 29% of the dark respiration rate in starch-storing species. We conclude that nocturnal carbohydrate export is a major energy-requiring process in starch-storing species.

Protein turnover is generally regarded as a major maintenance process, but experimental evidence to support this contention is scarce. Here we quantify the component of dark respiration rate associated with overall protein turnover of tissues in vivo. The effect of an inhibitor of cytosolic protein synthesis (cycloheximide, CHM) on dark respiration was tested on a cell suspension from potato (Solanum tuberosum L.) and quantified on leaf discs of expanding and full‐grown primary leaves of bean (Phaseolus vulgaris L.). The in vivo effect of CHM on protein biosynthesis was assessed by monitoring the inhibition of the induction of the ethylene‐forming enzyme (EFE) activity. The present method yields the energy costs of turnover of the total pool of proteins irrespective of their individual turnover rates. Average turnover rates were derived from the respiratory costs and the specific costs for turnover. Inhibition of respiration by CHM was readily detectable in growing‐cell suspensions and discs of expanding leaves, The derived respiratory costs of protein turnover in expanding leaves were maximally 17–37% of total respiration. Turnover costs in full‐grown primary leaves of bean amounted to 17–21% of total dark respiration. The maximum degradation constants (i.e. Kd‐values) derived for growing and full‐grown leaves were up to 2.42 × 10−6 and 1.12 × l0−6 s−1, respectively.