Large-scale gene discovery in the septoria tritici blotch fungus Mycosphaerella graminicola with a focus on in planta expression.

The foliar disease septoria tritici blotch, caused by the fungus Mycosphaerella graminicola, is currently the most important wheat disease in Europe. Gene expression was examined under highly different conditions, using 10 expressed sequence tag libraries generated from M. graminicola isolate IPO323 using seven in vitro and three in planta growth conditions. To identify fungal clones in the interaction libraries, we developed a selection method based on hybridization with the entire genomic DNA of M. graminicola, to selectively enrich these libraries for fungal genes. Assembly of the 27,007 expressed sequence tags resulted in 9,190 unigenes, representing 5.2 Mb of the estimated 39-Mb genome size of M. graminicola. All libraries contributed significantly to the number of unigenes, especially the in planta libraries representing different stages of pathogenesis, which covered 15% of the library-specific unigenes. Even under presymptomatic conditions (5 days postinoculation), when fungal biomass is less than 5%, this method enabled us to efficiently capture fungal genes expressed during pathogenesis. Many of these genes were uniquely expressed in planta, indicating that in planta gene expression significantly differed from in vitro expression. Examples of gene discovery included a number of cell wall-degrading enzymes, a broad set of genes involved in signal transduction (n=11) and a range of ATP-binding cassette (n=20) and major facilitator superfamily transporter genes (n=12) potentially involved in protection against antifungal compounds or the secretion of pathogenicity factors. In addition, evidence is provided for a mycovirus in M. graminicola that is highly expressed under various stress conditions, in particular, under nitrogen starvation. Our analyses provide a unique window on in vitro and in planta gene expression of M. graminicola.

In silico identification of putative promoter motifs of White Spot Syndrome Virus.

BACKGROUND: White Spot Syndrome Virus, a member of the virus family Nimaviridae, is a large dsDNA virus infecting shrimp and other crustacean species. Although limited information is available on the mode of transcription, previous data suggest that WSSV gene expression occurs in a coordinated and cascaded fashion. To search in silico for conserved promoter motifs (i) the abundance of all 4 through 8 nucleotide motifs in the upstream sequences of WSSV genes relative to the complete genome was determined, and (ii) a MEME search was performed in the upstream sequences of either early or late WSSV genes, as assigned by microarray analysis. Both methods were validated by alignments of empirically determined 5' ends of various WSSV mRNAs. RESULTS: The collective information shows that the upstream region of early WSSV genes, containing a TATA box and an initiator, is similar to Drosophila RNA polymerase II core promoter sequences, suggesting utilization of the cellular transcription machinery for generating early transcripts. The alignment of the 5' ends of known well-established late genes, including all major structural protein genes, identified a degenerate motif (ATNAC) which could be involved in WSSV late transcription. For these genes, only one contained a functional TATA box. However, almost half of the WSSV late genes, as previously assigned by microarray analysis, did contain a TATA box in their upstream region. CONCLUSION: The data may suggest the presence of two separate classes of late WSSV genes, one exploiting the cellular RNA polymerase II system for mRNA synthesis and the other generating messengers by a new virus-induced transcription mechanism.

Complete genome sequence of Lactobacillus plantarum WCFS1.

The 3,308,274-bp sequence of the chromosome of Lactobacillus plantarum strain WCFS1, a single colony isolate of strain NCIMB8826 that was originally isolated from human saliva, has been determined, and contains 3,052 predicted protein-encoding genes. Putative biological functions could be assigned to 2,120 (70%) of the predicted proteins. Consistent with the classification of L. plantarum as a facultative heterofermentative lactic acid bacterium, the genome encodes all enzymes required for the glycolysis and phosphoketolase pathways, all of which appear to belong to the class of potentially highly expressed genes in this organism, as was evident from the codon-adaptation index of individual genes. Moreover, L. plantarum encodes a large pyruvate-dissipating potential, leading to various end-products of fermentation. L. plantarum is a species that is encountered in many different environmental niches, and this flexible and adaptive behavior is reflected by the relatively large number of regulatory and transport functions, including 25 complete PTS sugar transport systems. Moreover, the chromosome encodes >200 extracellular proteins, many of which are predicted to be bound to the cell envelope. A large proportion of the genes encoding sugar transport and utilization, as well as genes encoding extracellular functions, appear to be clustered in a 600-kb region near the origin of replication. Many of these genes display deviation of nucleotide composition, consistent with a foreign origin. These findings suggest that these genes, which provide an important part of the interaction of L. plantarum with its environment, form a lifestyle adaptation region in the chromosome.

The sequence of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus genome.

The nucleotide sequence of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) DNA genome was determined and analysed. The circular genome encompasses 131,403 bp, has a G+C content of 39.1 mol% and contains five homologous regions with a unique pattern of repeats. Computer-assisted analysis revealed 135 putative ORFs of 150 nt or larger; 100 ORFs have homologues in Autographa californica multicapsid NPV (AcMNPV) and a further 15 ORFs have homologues in other baculoviruses such as Lymantria dispar MNPV (LdMNPV), Spodoptera exigua MNPV (SeMNPV) and Xestia c-nigrum granulovirus (XcGV). Twenty ORFs are unique to HaSNPV without homologues in GenBank. Among the six previously sequenced baculoviruses, AcMNPV, Bombyx mori NPV (BmNPV), Orgyia pseudotsugata MNPV (OpMNPV), SeMNPV, LdMNPV and XcGV, 65 ORFs are conserved and hence are considered as core baculovirus genes. The mean overall amino acid identity of HaSNPV ORFs was the highest with SeMNPV and LdMNPV homologues. Other than three 'baculovirus repeat ORFs' (bro) and two 'inhibitor of apoptosis' (iap) genes, no duplicated ORFs were found. A putative ORF showing similarity to poly(ADP-ribose) glycohydrolases (parg) was newly identified. The HaSNPV genome lacks a homologue of the major budded virus (BV) glycoprotein gene, gp64, of AcMNPV, BmNPV and OpMNPV. Instead, a homologue of SeMNPV ORF8, encoding the major BV envelope protein, has been identified. GeneParityPlot analysis suggests that HaSNPV, SeMNPV and LdMNPV (group II) have structural genomic features in common and are distinct from the group I NPVs and from the granuloviruses. Cluster alignment between group I and group II baculoviruses suggests that they have a common ancestor.