Abstract: We report further development of a novel recombinant protein expression system based on the Gram-negative bacterium, Ralstonia eutropha. In this study, we were able to express soluble, active, organophosphohydrolase (OPH), a protein that is prone to inclusion body formation in Escherichia coli, at titers greater than 10 g/L in high cell density fermentation. This represents a titer that is approximately 100-fold greater than titers previously reported in E. coli for this enzyme. R. eutropha strains expressing OPH were generated in two cloning steps. First, the T7 RNA polymerase gene was placed under the control of the strong, inducible phaP promoter and integrated into the phaP locus of R. eutropha NCIMB 40124. Second, a single copy of the oph gene under control of the T7 promoter was randomly integrated into the chromosome using a transposon cloning vector.
Abstract: This study is to investigate the role of lipid transfer protein (LTP1) gene of potato (Solanum tuberosum) in bacterial wilt (Ralstonia solanacearum) resistance. A novel cDNA clone encoding nsLTP was isolated from cultivated potato (Solanum tuberosum) infected with R. solanacearum by 5-rapid amplification of cDNA ends (RACE). The temporal and spatial expression of StLTPb1 was studied during the early stages of potato-R. solanacearum interaction by reverse transcriptase PCR (RT-PCR) and Northern blotting. The sequence analysis of the cloned cDNA, named StLTPb1, showed 691 bp which encoded a type 1 nsLTP of 91 amino acids. Construction of a phylogenic tree showed that StLTPb1 is well conserved in the coding region with high identity at the amino acid level with other Solanaceae nsLTPs. The temporal and spatial expression of StLTPb1 was studied during the early stages of potato-R. solanacearum interaction. StLTPb1 transcription is induced faster and transcripts accumulate to higher concentrations in resistant compared with susceptible genotypes by the pathogen. Dominant differences in the pathogen-induced gene expression pattern between the upper and lower leaves and stems were observed within the same genotypes. In situ hybridization results showed that the StLTPb1 mRNA was localized in phloem cells of vascular tissues in potato leaf and stem tissues after pathogen infection. Salicylic acid, methyl jasmonate and abscisic acid could induce StLTPb1 gene expression without significant difference between the upper and lower tissues. These abiotic elicitors could produce a long-lasting effect on the StLTPb1 during early stages of potato-R. solanacearum interaction. Differential expression of StLTPb1 gene between resistance and susceptible potato genotypes in response to R. solanacearum suggests that this gene plays a key role in plant defense mechanisms.
Abstract: E-lysis of Ralstonia eutropha H16, which belongs to the b-subclass, was undertaken to verify whether transmembrane tunnel formation is possible in bacteria which do not belong to the enterobacteriaceae. For this purpose, a new
gene E expression plasmid, pKG12, with two origins of replication, oriV and oriT, from plasmid pRP4, chloramphenicol and kanamycin resistance genes and a casette composed of lcI857 and lpR gene E was constructed.
Temperature upshift of R. eutropha H16 (pKG12) from 28 to 45°C during exponential growth resulted in lysis of the strain with features characteristic of E-mediated lysis of Escherichia coli. The cytoplasmic contents released can easily be separated from the still intact envelope fraction by centrifugation or filtration. As R. eutropha H16 represents an important industrial organism, E-mediated lysis could facilitate procedures for the recovery of intracellular mediators or products like polyhydroxyalkanoates.
Abstract: The reduced arbuscular mycorrhizal (AM) colonization mutant of tomato (rmc) is a single locus recessive mutation with different colonization phenotypes for different AM fungi. To test broader specificity and gain possible functional insights, we compared host status of rmc and near isogenic parent (Rio Grande 76R) for three root parasites. No significant colonization differences were found for bulb-and-potato aphid. However, root knot nematode (RKN) grew significantly better on rmc root cultures. Also, rmc was significantly more susceptible than 76R to Fusarium wilt. Our results indicate that the Rmc locus may have dual roles in symbiosis and parasitism.
Abstract: Crown rot (CR) of wheat, caused by Fusarium pseudograminearum (Fp) and other Fusarium species, is an important disease globally. To understand the host response to challenge by Fp, we examined gene expression changes in the wheat stem base following inoculation with macroconidia using the Affymetrix GeneChip Wheat Genome Array. Induced genes included mainly those with defensive functions such as genes encoding anti-microbial proteins as well as oxidative stress-related proteins, signalling molecules, and proteins involved in both primary and secondary metabolism. Comparison of genes induced by Fp and the biotrophic rust pathogen Puccinia triticina revealed substantial overlap in most functional classes of induced genes, except for oxidative stress-related genes which were specifically induced by the necrotroph, Fp. Differential expression of selected Fp-induced genes was confirmed and further analysed using real-time quantitative RT-PCR on an inoculation time-course of wheat cultivars Kennedy and Sunco. Interestingly, several genes were induced earlier, and to higher levels, in the partially CR-resistant cultivar Sunco than in susceptible Kennedy. Many Fp-induced genes were also activated by methyl jasmonate and benzothiadiazole, an analogue of salicylic acid, suggesting that these signalling molecules may be involved in activating defences during crown rot. Most of the genes identified here that were induced by Fp were also induced by deoxynivalenol (DON), a toxin produced by Fp during CR. In particular, DON induced several genes encoding glucosyltransferases that may be involved in DON detoxification. To initiate functional characterisation, one of these wheat glucosyltransferase genes was over-expressed in Arabidopsis thaliana, however this did not result in improved tolerance to DON. This study is the first comprehensive analysis of the wheat transcriptome during CR and provides new insights into the host processes potentially involved in plant defence against this pathogen.
Abstract: The Gibberella fujikuroi complex includes many plant pathogens of agricultural crops and trees, all of which have anamorphs assigned to the genus Fusarium. In this study, an interspecific hybrid cross between Gibberella circinata and Gibberella subglutinans was used to compile a genetic linkage map. A framework map was constructed using a total of 578 AFLP markers together with the mating type (MAT-1 and MAT-2) genes and the histone (H3) gene. Twelve major linkage groups were identified (n = 12). Fifty percent of the markers showed significant deviation from the expected 1:1 transmission ratio in a haploid F1 cross (P < 0.05). The transmission of the markers on the linkage map was biased towards alleles of the G. subglutinans parent, with an estimated 60% of the genome of F1 individuals contributed by this parent. This map will serve as a powerful tool to study the genetic architecture of interspecific differentiation and pathogenicity in the two parental genomes.
Abstract: With the increase of sequenced fungal genomes, high-throughput methods for functional analyses of genes are needed. We assessed the potential of a new transposon mutagenesis tool deploying a Fusarium oxysporum miniature inverted-repeat transposable element mimp1, mobilized by the transposase of impala, a Tc1-like transposon, to obtain knock-out mutants in Fusarium graminearum. We localized 91 mimp1 insertions which showed good distribution over the entire genome. The main exception was a major hotspot on chromosome 2 where independent insertions occurred at exactly the same nucleotide position. Furthermore insertions in promoter regions were over-represented. Screening 331 mutants for sexual development, radial growth and pathogenicity on wheat resulted in 19 mutants (5.7%) with altered phenotypes. Complementation with the original gene restored the wild-type phenotype in two selected mutants demonstrating the high tagging efficiency. This is the first report of a MITE transposon tagging system as an efficient mutagenesis tool in F. graminearum.
Abstract:Fusarium species are among the most studied plant-pathogenic fungi, with several species causing diseases on maize, wheat, barley, and other food and feed grains. Decreased yield, as well as diminished quality and value of the grain, results in significant worldwide economic losses. Additionally, Fusarium species produce a chemically diverse array of mycotoxins such as diacetoxyscirpenol, deoxynivalenol, nivalenol, T-2 toxin, zearalenone, fumonisins, fusarin C, beauvericin, moniliformin, and fusaproliferin. The dominant Fusarium species associated with feed grain that produce these mycotoxins are reviewed with emphasis on their current taxonomy, phylogenetic relationships, and general biology. Ecological and environmental factors associated with plant–fungal interactions and potential mycotoxin contamination of feed also are discussed with primary emphasis on two main diseases: head blight of small grains and ear rot of maize. The past quarter-century has provided much detail on the morphology, physiology, genetics and genomics of Fusarium species. Such data are critical for understanding these fungi and for managing their impact on the safety, value, and yield of quality grain.
Abstract: Recently the genome sequences of several filamentous fungi have become available, providing the opportunity for large-scale functional analysis including genome-wide expression analysis. We report the design and validation of the first Affymetrix GeneChip microarray based on the entire genome of a filamentous fungus, the ascomycetous plant pathogen Fusarium graminearum. To maximize the likelihood of representing all putative genes (~14,000) on the array, two distinct sets of automatically predicted gene calls were used and integrated into the online F. graminearum Genome DataBase. From these gene sets, a subset of calls was manually annotated and a non-redundant extract of all calls together with additional EST sequences and controls were submitted for GeneChip design. Experiments were conducted to test the performance of the F. graminearum GeneChip. Hybridization experiments using genomic DNA demonstrated the usefulness of the array for experimentation with F. graminearum and at least four additional pathogenic Fusarium species. Differential transcript accumulation was detected using the F. graminearum GeneChip with treatments derived from the fungus grown in culture under three nutritional regimes and in comparison with fungal growth in infected barley. The ability to detect fungal genes in planta is surprisingly sensitive even without efforts to enrich for fungal transcripts. The Plant Expression Database (PLEXdb, http://www.plexdb.org) will be used as a public repository for raw and normalized expression data from the F. graminearum GeneChip. The F. graminearum GeneChip will help to accelerate exploration of the pathogen–host pathways that may involve interactions between pathogenicity genes in the fungus and disease response in the plant.
Abstract:Gibberella zeae, the causal agent of Fusarium head blight, is a devastating pathogen of small grains worldwide. The sexual cycle is a crucial component of head blight epidemiology, as forcibly discharged ascospores serve as the primary inoculum. The recent development of an Affymetrix GeneChip containing probesets representative of all predicted genes of G. zeae has opened the door to studies of differential gene expression during sexual development. Using GeneChips, a developmental time course was performed in culture, from vegetative hyphae to mature perithecia with multiseptate ascospores. Time-points represent the development of the major cell types comprising the mature perithecium. The majority of the 17,830 G. zeae probesets, 78%, were expressed during at least one of the developmental stages; 12% of these appear to be specific to sexual development. Analysis of the 162 predicted ion transporter genes is reported in detail, due to their association with perithecium function. Expression patterns of the MirA-type siderophores, chloride channels, P-type ATPases and potassium transporters show some specialization in regard to developmental stage. This is the first whole-genome analysis of differential transcript accumulation during sexual development in a filamentous fungus.
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