Genome sequence of the biocontrol strain Pseudomonas fluorescens F113.

Pseudomonas fluorescens F113 is a plant growth-promoting rhizobacterium (PGPR) that has biocontrol activity against fungal plant pathogens and is a model for rhizosphere colonization. Here, we present its complete genome sequence, which shows that besides a core genome very similar to those of other strains sequenced within this species, F113 possesses a wide array of genes encoding specialized functions for thriving in the rhizosphere and interacting with eukaryotic organisms.

Solanum lycopersicum cv. Heinz 1706 chromosome 6: distribution and abundance of genes and retrotransposable elements.

We studied the physical and genetic organization of chromosome 6 of tomato (Solanum lycopersicum) cv. Heinz 1706 by combining bacterial artificial chromosome (BAC) sequence analysis, high-information-content fingerprinting, genetic analysis, and BAC-fluorescent in situ hybridization (FISH) mapping data. The chromosome positions of 81 anchored seed and extension BACs corresponded in most cases with the linear marker order on the high-density EXPEN 2000 linkage map. We assembled 25 BAC contigs and eight singleton BACs spanning 2.0 Mb of the short-arm euchromatin, 1.8 Mb of the pericentromeric heterochromatin and 6.9 Mb of the long-arm euchromatin. Sequence data were combined with their corresponding genetic and pachytene chromosome positions into an integrated map that covers approximately a third of the chromosome 6 euchromatin and a small part of the pericentromeric heterochromatin. We then compared physical length (Mb), genetic (cM) and chromosome distances (microm) for determining gap sizes between contigs, revealing relative hot and cold spots of recombination. Through sequence annotation we identified several clusters of functionally related genes and an uneven distribution of both gene and repeat sequences between heterochromatin and euchromatin domains. Although a greater number of the non-transposon genes were located in the euchromatin, the highly repetitive (22.4%) pericentromeric heterochromatin displayed an unexpectedly high gene content of one gene per 36.7 kb. Surprisingly, the short-arm euchromatin was relatively rich in repeats as well, with a repeat content of 13.4%, yet the ratio of Ty3/Gypsy and Ty1/Copia retrotransposable elements across the chromosome clearly distinguished euchromatin (2:3) from heterochromatin (3:2).

High-resolution chromosome mapping of BACs using multi-colour FISH and pooled-BAC FISH as a backbone for sequencing tomato chromosome 6.

Within the framework of the International Solanaceae Genome Project, the genome of tomato (Solanum lycopersicum) is currently being sequenced. We follow a 'BAC-by-BAC' approach that aims to deliver high-quality sequences of the euchromatin part of the tomato genome. BACs are selected from various libraries of the tomato genome on the basis of markers from the F2.2000 linkage map. Prior to sequencing, we validated the precise physical location of the selected BACs on the chromosomes by five-colour high-resolution fluorescent in situ hybridization (FISH) mapping. This paper describes the strategies and results of cytogenetic mapping for chromosome 6 using 75 seed BACs for FISH on pachytene complements. The cytogenetic map obtained showed discrepancies between the actual chromosomal positions of these BACs and their markers on the linkage group. These discrepancies were most notable in the pericentromere heterochromatin, thus confirming previously described suppression of cross-over recombination in that region. In a so called pooled-BAC FISH, we hybridized all seed BACs simultaneously and found a few large gaps in the euchromatin parts of the long arm that are still devoid of seed BACs and are too large for coverage by expanding BAC contigs. Combining FISH with pooled BACs and newly recruited seed BACs will thus aid in efficient targeting of novel seed BACs into these areas. Finally, we established the occurrence of repetitive DNA in heterochromatin/euchromatin borders by combining BAC FISH with hybridization of a labelled repetitive DNA fraction (Cot-100). This strategy provides an excellent means to establish the borders between euchromatin and heterochromatin in this chromosome.

Comparative analysis indicates that alternative splicing in plants has a limited role in functional expansion of the proteome.

BACKGROUND: Alternative splicing (AS) is a widespread phenomenon in higher eukaryotes but the extent to which it leads to functional protein isoforms and to proteome expansion at large is still a matter of debate. In contrast to animal species, for which AS has been studied extensively at the protein and functional level, protein-centered studies of AS in plant species are scarce. Here we investigate the functional impact of AS in dicot and monocot plant species using a comparative approach. RESULTS: Detailed comparison of AS events in alternative spliced orthologs from the dicot Arabidopsis thaliana and the monocot Oryza sativa (rice) revealed that the vast majority of AS events in both species do not result from functional conservation. Transcript isoforms that are putative targets for the nonsense-mediated decay (NMD) pathway are as likely to contain conserved AS events as isoforms that are translated into proteins. Similar results were obtained when the same comparison was performed between the two more closely related monocot species rice and Zea mays (maize).Genome-wide computational analysis of functional protein domains encoded in alternatively and constitutively spliced genes revealed that only the RNA recognition motif (RRM) is overrepresented in alternatively spliced genes in all species analyzed. In contrast, three domain types were overrepresented in constitutively spliced genes. AS events were found to be less frequent within than outside predicted protein domains and no domain type was found to be enriched with AS introns. Analysis of AS events that result in the removal of complete protein domains revealed that only a small number of domain types is spliced-out in all species analyzed. Finally, in a substantial fraction of cases where a domain is completely removed, this domain appeared to be a unit of a tandem repeat. CONCLUSION: The results from the ortholog comparisons suggest that the ability of a gene to produce more than one functional protein through AS does not persist during evolution. Cross-species comparison of the results of the protein-domain oriented analyses indicates little correspondence between the analyzed species. Based on the premise that functional genetic features are most likely to be conserved during evolution, we conclude that AS has only a limited role in functional expansion of the proteome in plants.

In plants, highly expressed genes are the least compact.

In both the monocot rice and the dicot Arabidopsis, highly expressed genes have more and longer introns and a larger primary transcript than genes expressed at a low level: higher expressed genes tend to be less compact than lower expressed genes. In animal genomes, it is the other way round. Although the length differences in plant genes are much smaller than in animals, these findings indicate that plant genes are in this respect different from animal genes. Explanations for the relationship between gene configuration and gene expression in animals might be (or might have been) less important in plants. We speculate that selection, if any, on genome configuration has taken a different turn after the divergence of plants and animals.

Evolutionary history of the phl gene cluster in the plant-associated bacterium Pseudomonas fluorescens.

Pseudomonas fluorescens is of agricultural and economic importance as a biological control agent largely because of its plant association and production of secondary metabolites, in particular 2,4-diacetylphloroglucinol (2,4-DAPG). This polyketide, which is encoded by the eight-gene phl cluster, has antimicrobial effects on phytopathogens, promotes amino acid exudation from plant roots, and induces systemic resistance in plants. Despite its importance, 2,4-DAPG production is limited to a subset of P. fluorescens strains. Determination of the evolution of the phl cluster and understanding the selective pressures promoting its retention or loss in lineages of P. fluorescens will help in the development of P. fluorescens as a viable and effective inoculant for application in agriculture. In this study, genomic and sequence-based approaches were integrated to reconstruct the phylogeny of P. fluorescens and the phl cluster. It was determined that 2,4-DAPG production is an ancestral trait in the species P. fluorescens but that most lineages have lost this capacity through evolution. Furthermore, intragenomic recombination has relocated the phl cluster within the P. fluorescens genome at least three times, but the integrity of the cluster has always been maintained. The possible evolutionary and functional implications for retention of the phl cluster and 2,4-DAPG production in some lineages of P. fluorescens are discussed.

Comparative BAC end sequence analysis of tomato and potato reveals overrepresentation of specific gene families in potato.

BACKGROUND: Tomato (Solanum lycopersicon) and potato (S. tuberosum) are two economically important crop species, the genomes of which are currently being sequenced. This study presents a first genome-wide analysis of these two species, based on two large collections of BAC end sequences representing approximately 19% of the tomato genome and 10% of the potato genome. RESULTS: The tomato genome has a higher repeat content than the potato genome, primarily due to a higher number of retrotransposon insertions in the tomato genome. On the other hand, simple sequence repeats are more abundant in potato than in tomato. The two genomes also differ in the frequency distribution of SSR motifs. Based on EST and protein alignments, potato appears to contain up to 6,400 more putative coding regions than tomato. Major gene families such as cytochrome P450 mono-oxygenases and serine-threonine protein kinases are significantly overrepresented in potato, compared to tomato. Moreover, the P450 superfamily appears to have expanded spectacularly in both species compared to Arabidopsis thaliana, suggesting an expanded network of secondary metabolic pathways in the Solanaceae. Both tomato and potato appear to have a low level of microsynteny with A. thaliana. A higher degree of synteny was observed with Populus trichocarpa, specifically in the region between 15.2 and 19.4 Mb on P. trichocarpa chromosome 10. CONCLUSION: The findings in this paper present a first glimpse into the evolution of Solanaceous genomes, both within the family and relative to other plant species. When the complete genome sequences of these species become available, whole-genome comparisons and protein- or repeat-family specific studies may shed more light on the observations made here.

Genetic and functional characterization of the gene cluster directing the biosynthesis of putisolvin I and II in Pseudomonas putida strain PCL1445.

Pseudomonas putida PCL1445 secretes two cyclic lipopeptides, putisolvin I and putisolvin II, which possess a surface-tension-reducing ability, and are able to inhibit biofilm formation and to break down biofilms of Pseudomonas species including Pseudomonas aeruginosa. The putisolvin synthetase gene cluster (pso) and its surrounding region were isolated, sequenced and characterized. Three genes, termed psoA, psoB and psoC, were identified and shown to be involved in putisolvin biosynthesis. The gene products encode the 12 modules responsible for the binding of the 12 amino acids of the putisolvin peptide moiety. Sequence data indicate that the adenylation domain of the 11th module prioritizes the recognition of Val instead of Leu or Ile and consequently favours putisolvin I production over putisolvin II. Detailed analysis of the thiolation domains suggests that the first nine modules recognize the d form of the amino acid residues while the two following modules recognize the l form and the last module the l or d form, indifferently. The psoR gene, which is located upstream of psoA, shows high similarity to luxR-type regulatory genes and is required for the expression of the pso cluster. In addition, two genes, macA and macB, located downstream of psoC were identified and shown to be involved in putisolvin production or export.

Local coexpression domains in the genome of rice show no microsynteny with Arabidopsis domains.

Chromosomal coexpression domains are found in a number of different genomes under various developmental conditions. The size of these domains and the number of genes they contain vary. Here, we define local coexpression domains as adjacent genes where all possible pair-wise correlations of expression data are higher than 0.7. In rice, such local coexpression domains range from predominantly two genes, up to 4, and make up approximately 5% of the genomic neighboring genes, when examining different expression platforms from the public domain. The genes in local coexpression domains do not fall in the same ontology category significantly more than neighboring genes that are not coexpressed. Duplication, orientation or the distance between the genes does not solely explain coexpression. The regulation of coexpression is therefore thought to be regulated at the level of chromatin structure. The characteristics of the local coexpression domains in rice are strikingly similar to such domains in the Arabidopsis genome. Yet, no microsynteny between local coexpression domains in Arabidopsis and rice could be identified. Although the rice genome is not yet as extensively annotated as the Arabidopsis genome, the lack of conservation of local coexpression domains may indicate that such domains have not played a major role in the evolution of genome structure or in genome conservation.

The CLAVATA3/ESR motif of CLAVATA3 is functionally independent from the nonconserved flanking sequences.

It is believed that CLAVATA3 (CLV3) encodes a peptide ligand that interacts with the CLV1/CLV2 receptor complex to limit the number of stem cells in the shoot apical meristem of Arabidopsis thaliana; however, the exact composition of the functional CLV3 product remains a mystery. A recent study on CLV3 shows that the CLV3/ESR (CLE) motif, together with the adjacent C-terminal sequence, is sufficient to execute CLV3 function when fused behind an N-terminal sequence of ERECTA. Here we show that most of the sequences flanking the CLE motif of CLV3 can be deleted without affecting CLV3 function. Using a liquid culture assay, we demonstrate that CLV3p, a synthetic peptide corresponding to the CLE motif of CLV3, is able to restrict the size of the shoot apical meristem in clv3 seedlings but not in clv1 seedlings. In accordance with this decrease in meristem size, application of CLV3p to in vitro-grown clv3 seedlings restricts the expression of the stem cell-promoting transcription factor WUSCHEL. Thus, we propose that the CLE motif is the functional region of CLV3 and that this region acts independently of its adjacent sequences.

Local coexpression domains of two to four genes in the genome of Arabidopsis.

Expression of genes in eukaryotic genomes is known to cluster, but cluster size is generally loosely defined and highly variable. We have here taken a very strict definition of cluster as sets of physically adjacent genes that are highly coexpressed and form so-called local coexpression domains. The Arabidopsis (Arabidopsis thaliana) genome was analyzed for the presence of such local coexpression domains to elucidate its functional characteristics. We used expression data sets that cover different experimental conditions, organs, tissues, and cells from the Massively Parallel Signature Sequencing repository and microarray data (Affymetrix) from a detailed root analysis. With these expression data, we identified 689 and 1,481 local coexpression domains, respectively, consisting of two to four genes with a pairwise Pearson's correlation coefficient larger than 0.7. This number is approximately 1- to 5-fold higher than the numbers expected by chance. A small (5%-10%) yet significant fraction of genes in the Arabidopsis genome is therefore organized into local coexpression domains. These local coexpression domains were distributed over the genome. Genes in such local domains were for the major part not categorized in the same functional category (GOslim). Neither tandemly duplicated genes nor shared promoter sequence nor gene distance explained the occurrence of coexpression of genes in such chromosomal domains. This indicates that other parameters in genes or gene positions are important to establish coexpression in local domains of Arabidopsis chromosomes.

Optimization of the expression of equistatin in Pichia pastoris.

To improve the expression of equistatin, a proteinase inhibitor from the sea anemone Actinia equina, in the yeast Pichia pastoris, we prepared gene variants with yeast-preferred codon usage and lower repetitive AT and GC content. The full gene optimization approximately doubled the level of steady-state mRNA and protein accumulated in the culture medium. The removal of a short stretch of 12 additional nucleotides from the multiple cloning site (MCS) sequence in the vector pPIC9 had an enhancement effect similar to full gene optimization (factor 1.5) at the mRNA level. However, at the protein level, this increase was 4- to 10-fold. The optimized gene without the MCS sequence yielded 1.66 g/L active protein in a bioreactor and was purified by a new two-step procedure with a recovery of activity that was >95%. This production level constitutes an overall improvement of about 20-fold relative to our previously published results. The characteristics of the MCS sequence element are discussed in the light of its apparent ability to act as negative expression regulator.