Abstract: Rapid progress in fungal genome sequencing presents many new opportunities for functional genomic analysis of fungal biology through the systematic mutagenesis of the genes identified through sequencing. However, the lack of efficient tools for targeted gene replacement is a limiting factor for fungal functional genomics, as it often necessitates the screening of a large number of transformants to identify the desired mutant. We developed an efficient method of gene replacement and evaluated factors affecting the efficiency of this method using two plant pathogenic fungi, Magnaporthe grisea and Fusarium oxysporum. This method is based on Agrobacterium tumefaciens-mediated transformation with a mutant allele of the target gene flanked by the herpes simplex virus thymidine kinase (HSVtk) gene as a conditional negative selection marker against ectopic transformants. The HSVtk gene product converts 5-fluoro-2-deoxyuridine to a compound toxic to diverse fungi. Because ectopic transformants express HSVtk, while gene replacement mutants lack HSVtk, growing transformants on a medium amended with 5-fluoro-2-deoxyuridine facilitates the identification of targeted mutants by counter-selecting against ectopic transformants. In addition to M. grisea and F. oxysporum, the method and associated vectors are likely to be applicable to manipulating genes in a broad spectrum of fungi, thus potentially serving as an efficient, universal functional genomic tool for harnessing the growing body of fungal genome sequence data to study fungal biology.
Abstract: The intergenic spacer (IGS) regions of the rDNA of several Fusarium spp. strains obtained from the collaborative researchers (Int. J. Food Microbiol. (2003)) were amplified by polymerase chain reaction (PCR), and an IGS–RFLP analysis was performed. Restriction digestion with AluI, MspI and PstI allowed differentiation between the related Fusarium poae and Fusarium kyushuense species. Fusarium langsethiae was also separated from Fusarium sporotrichioides (including var. minus) on the basis of the banding patterns after MspI digestion, while specific XhoI, AluI and MspI restriction patterns were found in the IGS amplicons of F. sporotrichioides var. minus. According to the phylogenetic analysis of IGS–RFLP patterns, F. langsethiae (except for one strain), F. sporotrichioides, F. poae and F. kyushuense strains formed four well-supported clades with high-bootstrap values. Based on the sequence differences in the IGS region, species-specific primers were designed for the F. langsethiae/F. sporotrichioides group and for F. poae. The specificity and sensitivity of the primers were tested on various Fusarium species and isolates, and on several other important fungal genera associated with cereals. The F. poae-specific primers, designed in this study, showed the same specificity as primers Fp82f/Fp82r developed previously. The two phylogenetic subgroups of F. langsethiae, found by IGS sequencing analysis, were separated on the basis of size differences of the amplification products with primers CNL12/PulvIGSr specific for the F. langsethiae/F. sporotrichioides group. RFLP analysis of the amplified IGS region is a useful molecular assay for characterisation and a phylogenetic study of several related Fusarium species—F. langsethiae, F. sporotrichioides, F. sporotrichioides var. minus, F. poae and F. kyushuense. The primers designed in this study were highly specific and allowed identification of F. poae and the F. langsethiae/F. sporotrichioides group.
Abstract: Genetic variation occurs at all levels across the genus Fusarium. In some cases such variation has been used to define species, and in others to describe populations or lineages. When amplified fragment length polymorphisms (AFLPs) are evaluated, strains in different species usually share at least 60% of the fragments and those in different species 40% of the fragments, or less, with isolates sharing between 40 and 60% of the fragments in an indeterminant situation. This gray area also is reflected in morphological characters, usually indistinguishable, and cross-fertility, usually some cross-fertility but often not as fertile as are strains that are more closely related. In terms of DNA sequence, the genes used for species diagnostics often have not been tested on large numbers of strains. For example, the TRI101 gene of F. graminearum contains at least 25 single nucleotide polymorphisms (SNPs) from 36 strains and yielded 17 alleles that have been proposed as a means to subdivide this species into at least nine. However these subdivisions fare poorly as more strains are analyzed, with the number of alleles increasing to > 40 when not, vert, similar 500 strains from Korea and South America are sequenced. Some of the newly identified alleles cannot be correctly assigned to one of the nine subdivisions based on the proposed diagnostic SNPs. Before SNPs are proposed as characters to define species, it is important to verify their specificity based on a sufficiently large sample and to evaluate the genetic variation present in terms of an independent measure of genetic relationships. Only in such a manner can names that are meaningful in the context of trade and quarantine regulations be developed.
Abstract: The sti35 gene of the vascular wilt fungus Fusarium oxysporum was originally identified based on induced expression under stress conditions. In this study, the transcriptional regulation and biological function of sti35 were examined in the tomato pathogen F. oxysporum f.sp. lycopersici. Expression of sti35 was repressed by thiamine and induced by high temperatures. Sti35 transcripts were detected both during early and late stages of infection of tomato plants by F. oxysporum. Heterologous expression of the sti35 cDNA restored thiamine prototrophy in a Saccharomyces cerevisiae thi4 mutant and increased UV tolerance in a uvr− mutant of Escherichia coli. Targeted Δsti35 knockout mutants of F. oxysporum exhibited a thiamine auxotrophic phenotype and reduced tolerance to the superoxide-generating agent menadione, indicating that Sti35 has a dual role in thiamine biosynthesis and oxidative stress response. RT-PCR analysis revealed the presence of differential RNA splicing of the second 5-UTR intron, suggesting that thiamine may regulate sti35 expression via a post-transcriptional mechanism. F. oxysporum transformants carrying a transcriptional fusion of the sti35 promoter to the lacZ reporter gene produced high levels of β-galactosidase activity when grown in the absence, but not in the presence of thiamine. Thus, the sti35 promoter represents a useful tool for the controlled expression of genes of interest in F. oxysporum.
Abstract: To assess potential effects of genetically modified (GM) potatoes on the abundance and diversity of rhizobacteria with in vitro antagonistic activity in relation to natural variability among cultivars, two GM potato lines accumulating the carotenoid zeaxanthin in their tubers, the parental cultivar and four additional commercial cultivars were planted at two field sites in Germany. Rhizosphere samples were taken at three developmental stages of the plants. A total of 3,985 bacteria isolated from the rhizosphere were screened for their in vitro antagonistic activity towards Rhizoctonia solani, Verticillium dahliae and Phytophthora infestans using a dual-culture assay. Genotypic characterisation, 16S rRNA gene sequencing and antifungal metabolite analysis was performed to characterize the 595 antagonists obtained. The 16S rRNA gene-based identification of in vitro antagonists revealed strong site-dependent differences in their taxonomic composition. This study showed that the site was the overriding factor determining the proportion and diversity of antagonists from the rhizosphere of potato while the effect of the genetic modification on the proportion of antagonists obtained did not exceed natural variability among the five commercial cultivars tested.
Abstract: We have increased the gene dosage of the poly(3-hydroxybutyrate) biosynthesis operon in Ralstonia eutropha (formerly Alcaligenes eutrophus) to test whether PHB synthesis rates may be increased by recombinant methods. The native R. eutropha phbCAB operon was inserted into the broad-host-range vector pKT230. This PHB operon-containing plasmid, and a control plasmid containing the identical broad-host-range replicon but not the PHB genes, were transferred to R. eutropha H16. Analysis of whole-cell lysates indicated that the strain harboring the operon-containing plasmid possessed β-ketothiolase and acetoacetyl-CoA reductase specific activities that were 6.0 and 6.2 times elevated, respectively, as compared to the control strain with a single operon. After growth on fructose, PHB synthesis rates were sharply dependent on the type of carbon source offered during the PHB accumulation phase under nitrogen limitation. In the case of the strain harboring the control plasmid, and in comparison to fructose as carbon source, PHB accumulation was 2.15, 2.83, and 2.60 times faster when resuspended in nitrogen-free medium with lactate, acetate, or 3-hydroxybutyrate, respectively. The strain harboring the PHB operon-containing plasmid synthesized PHB at a lower specific rate in each case. During exponential growth on fructose, the strain harboring the control plasmid was again more efficient at forming PHB. These results suggest that increasing the intracellular concentration of PHB precursors may be a superior alternative to raising the levels of PHB enzymes for enhancing PHB productivity in R. eutropha.
Abstract: The self-transmissible megaplasmid pHG1 carries essential genetic information for the facultatively lithoautotrophic and facultatively anaerobic lifestyles of its host, the Gram-negative soil bacterium Ralstonia eutropha H16. We have determined the complete nucleotide sequence of pHG1. This megaplasmid is 452,156 bp in size and carries 429 potential genes. Groups of functionally related genes form loose clusters flanked by mobile elements. The largest functional group consists of lithoautotrophy-related genes. These include a set of 41 genes for the biosynthesis of the three previously identified hydrogenases and of a fourth, novel hydrogenase. Another large cluster carries the genetic information for denitrification. In addition to a dissimilatory nitrate reductase, both specific and global regulators were identified. Also located in the denitrification region is a set of genes for cytochrome c biosynthesis. Determinants for several enzymes involved in the mineralization of aromatic compounds were found. The genes for conjugative plasmid transfer predict that R. eutropha forms two types of pili. One of them is related to the type IV pili of pathogenic enterobacteria. pHG1 also carries an extensive junkyard region encompassing 17 remnants of mobile elements and 22 partial or intact genes for phage-type integrase. Among the mobile elements is a novel member of the IS5 family, in which the transposase gene is interrupted by a group II intron.
Abstract: Sequence analysis of the genomic region of Phytophthora sojae close to the Avr4/6 locus specifying virulence on soybean identified a Ty1/Copia-like retrotransposon that we have named Phytophthora sojae Copia-like retrotransposon (PSCR). Twelve near-complete homologs of PSCR were found in the published P. sojae genome sequence, none of which encoded a full-length polyprotein characteristic of Copia-like retrotransposons, or appears to exhibit transcriptional activity or show evidence of recent movement, suggesting they are non-functional and unlikely to have caused pathogenic variability. However, reconstructed consensus PSCR sequence encoding a full-length polyprotein resembles a functional, ancestral retroelement within P. sojae. Homologs were also found in sequence databases of other Phytophthora species. Database searches found other families of Copia-like elements in genomes of P. sojae, P. ramorum and P. infestans that were different from members of the PSCR family and from Copia-like elements reported in other organisms. It is possible that the various families of Copia-like retroelements identified in this study represent introgressions into the genome of ancient ancestor(s) of current Phytophthora species, where they have evolved and diverged considerably during the speciation. Some Copia-like families are transcriptionally active with the potential to transpose and contribute to pathogenic variation in current populations of P. sojae.
Abstract: The mitochondrial genome of an isolate of Phytophthora ramorum from Europe (EU) was sequenced and compared to the previously published genome sequence of an isolate from California (NA). The EU mitochondrial genome had the identical gene order and encoded for the same suite of genes as the NA mitochondrial genome, but had 13 single nucleotide polymorphisms (SNPs) and at 39,494 bp was 180 bp longer. This length difference was due to an increase in the size of the spacer region between the nad5 and nad6 genes caused by a chimeric region containing duplication of the spacer sequence and additional sequences from the flanking genes. Recombination between the 1,150 bp-inverted repeats (IR) generated orientational isomers where the gene order was reversed between the IR. A total of seven primer pairs were developed for amplification of regions where the SNPs were located and two other regions where additional SNPs were encountered when a larger number of isolates were examined. Sequence data for a total of 5,743 bp for 40 isolates collected from a range of geographic areas was compared and 28 loci were found to be polymorphic. The combination of these polymorphisms revealed a total of 4 mitochondrial haplotypes; the traditional EU (haplotype I), the traditional NA (haplotype IIa), the third nuclear lineage of the pathogen recovered from a nursery in Washington State (haplotype III) and a new haplotype representing a subgroup of NA isolates from an Oregon forest (haplotype IIb). Phylogenetic analysis using the sequences generated from the haplotype analysis supported a high affinity for haplotypes IIa and IIb, both of which were distinct from haplotype I, with haplotype I basal to these and haplotype III representing the ancestral state.
Abstract: Analysis of gene expression using real-time reverse transcription polymerase chain reaction (RT-PCR) requires reference genes to normalize expression values between samples. We have developed a novel reference for real-time RT-PCR using an arbitrary primer to amplify a random set of genes. The arbitrary primer amplifies over 30 genes, whose cumulative expression as measured by real-time RT-PCR closely follows that of UBQ 11, an Arabidopsis thaliana gene that is used as a reference on microarrays. The expression of arbitrary genes is also compared with potato (Solanum tuberosum spp. tubersosum) housekeeping genes and was shown to be stable during Phytophthora infestans infection.
Abstract