Plant-pathogenic Streptomyces spp. cause scab disease on economically important root and tuber crops, the most important of which is potato. Key virulence determinants produced by these species include the cellulose synthesis inhibitor, thaxtomin A, and the secreted Nec1 protein that is required for colonization of the plant host. Recently, the genome sequence of Streptomyces scabies 87-22 was completed, and a biosynthetic cluster was identified that is predicted to synthesize a novel compound similar to coronafacic acid (CFA), a component of the virulence-associated coronatine phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae. Southern analysis indicated that the cfa-like cluster in S. scabies 87-22 is likely conserved in other strains of S. scabies but is absent from two other pathogenic streptomycetes, S. turgidiscabies and S. acidiscabies. Transcriptional analyses demonstrated that the cluster is expressed during plant-microbe interactions and that expression requires a transcriptional regulator embedded in the cluster as well as the bldA tRNA. A knockout strain of the biosynthetic cluster displayed a reduced virulence phenotype on tobacco seedlings compared with the wild-type strain. Thus, the cfa-like biosynthetic cluster is a newly discovered locus in S. scabies that contributes to host-pathogen interactions.

BackgroundThere is interest in improving the flavor of commercial strawberry (Fragaria × ananassa) varieties. Fruit flavor is shaped by combinations of sugars, acids and volatile compounds. Many efforts seek to use genomics-based strategies to identify genes controlling flavor, and then designing durable molecular markers to follow these genes in breeding populations. In this report, fruit from two cultivars, varying for presence-absence of volatile compounds, along with segregating progeny, were analyzed using GC/MS and RNAseq. Expression data were bulked in silico according to presence/absence of a given volatile compound, in this case γ-decalactone, a compound conferring a peach flavor note to fruits.ResultsComputationally sorting reads in segregating progeny based on γ-decalactone presence eliminated transcripts not directly relevant to the volatile, revealing transcripts possibly imparting quantitative contributions. One candidate encodes an omega-6 fatty acid desaturase, an enzyme known to participate in lactone production in fungi, noted here as FaFAD1. This candidate was induced by ripening, was detected in certain harvests, and correlated with γ-decalactone presence. The FaFAD1 gene is present in every genotype where γ-decalactone has been detected, and it was invariably missing in non-producers. A functional, PCR-based molecular marker was developed that cosegregates with the phenotype in F1 and BC1 populations, as well as in many other cultivars and wild Fragaria accessions.ConclusionsGenetic, genomic and analytical chemistry techniques were combined to identify FaFAD1, a gene likely controlling a key flavor volatile in strawberry. The same data may now be re-sorted based on presence/absence of any other volatile to identify other flavor-affecting candidates, leading to rapid generation of gene-specific markers.

In this report, we describe the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecET proteins encoded by the lambda and Rac bacteriophages of Escherichia coli. The ability of the pseudomonad-encoded proteins to promote recombination was tested in P. syringae pv. tomato DC3000 using a quantitative assay based on recombination frequency. The results show that the Pseudomonas RecT homolog is sufficient to promote recombination of single-stranded DNA oligonucleotides and that efficient recombination of doublestranded DNA requires the expression of both the RecT and RecE homologs. Additionally, we illustrate the utility of this recombineering system to make targeted gene disruptions in the P. syringae chromosome.There are currently more than 1,500 completed or draft bacterial genome sequences available for public access. This data resource continues to grow rapidly and provides potential insights into the roles of individual genes and regulons. However, testing hypotheses based on sequence data requires direct experimental manipulation of each genome. While many established methods for modifying bacterial DNA can assist in genetic analysis of these organisms, they are often time-consuming and limited with respect to the types of changes that can be directed.New advances in recombineering (genetic engineering by recombination) offer powerful alternative strategies for sitedirected mutagenesis of genomic loci and provide methods for rapid and precise functional genomic analysis in some organisms (9, 29, 36-38, 41, 43). In these cases, recombineering is very efficient when phage-encoded recombinases are supplied, such that in vivo expression of these proteins enables direct genetic engineering of chromosomal and episomal replicons. These proteins catalyze RecA-independent recombination (21) of linear DNA substrates with homologous genomic target loci. The phage recombination functions typically involve the coordinated action of a 5Ј-to-3Ј exonuclease (i.e., RecE or lambda Exo) and a single-stranded DNA (ssDNA)-annealing and strand invasion protein (i.e., RecT or lambda Beta), which we shall refer to as recombinases for brevity. The recombinase binds to 3Ј ssDNA ends that are exposed by the action of the exonuclease, forming a protein-DNA filament, which protects the substrate DNA and promotes annealing with the homologous genomic sequence (4,17,19,24). The recombinases are sufficient to facilitate recombination of ssDNA oligonucleotides, presumably because the oligonucleotides resemble the 5Ј-end-resected double-stranded DNA (dsDNA) substrate (11). Most of the recombinase proteins that have been shown to facilitate recombination are located in operons and are adjacent to the exonuclease-encoding genes, although there are cases where functional recombinase proteins have been identified without an...

HighlightThis work in strawberry fruit predicts new genes relevant to a key metabolic pathway using correlation analysis, and then demonstrates their function in a rapid and effective transient assay.

No abstract