Related mobile pathogenicity chromosomes in Fusarium oxysporum determine host range on cucurbits

Fusarium oxysporum f. sp. radicis-cucumerinum (Forc) causes severe root rot and wilt in several cucurbit species, including cucumber, melon, and watermelon. Previously, a pathogenicity chromosome, chr^RC, was identified in Forc. Strains that were previously nonpathogenic could infect multiple cucurbit species after obtaining this chromosome via horizontal chromosome transfer (HCT). In contrast, F. oxysporum f. sp. melonis (Fom) can only cause disease on melon plants, even though Fom contains contigs that are largely syntenic with chr^RC. The aim of this study was to identify the genetic basis underlying the difference in host range between Fom and Forc. First, colonization of different cucurbit species between Forc and Fom strains showed that although Fom did not reach the upper part of cucumber or watermelon plants, it did enter the root xylem. Second, to select candidate genomic regions associated with differences in host range, high-quality genome assemblies of Fom001, Fom005, and Forc016 were compared. One of the Fom contigs that is largely syntenic and highly similar in sequence to chr^RC contains the effector gene SIX6. After HCT of the SIX6-containing chromosome from Fom strains to a nonpathogenic strain, the recipient (HCT) strains caused disease on melon plants, but not on cucumber or watermelon plants. These results provide strong evidence that the differences in host range between Fom and Forc are caused by differences between transferred chromosomes of Fom and chr^RC, thus narrowing down the search for genes allowing or preventing infection of cucumber and watermelon to genes located on these chromosomes.     

Role of cell wall-degrading enzymes in pathogenicity of Fusarium oxysporum

Fusarium oxysporum invades its host plants through the roots and colonizes the vascular system. It produces a great variety of cell-wall degrading enzymes (CWDE), such as cellulases, xylanases, pectinases and proteases. Our group has purified and characterized an endopolygalacturonase (PG1), two exopolygalacturonases (PG2 and PG3), an endoxylanase (XYL1) and an endo pectatelyase (PL1). We have isolated the following CWDE-encoding genes: pg1, pgx4, pg5, xyl2, xyl3, prt1 and pl1. Gene expression in different culture conditions has been determined by Northern analysis. The occurrence of these genes in different formae speciales has been analyzed by Southern analysis and PCR. All these genes are expressed during different stages of the interaction with the host plant indicating a possible role in pathogenesis. At present, targeted gene disruption is being carried out, in order to determine the role of each gene in the pathogenicity process.     

Dispensable chromosomes in Fusarium oxysporum f. sp. lycopersici

The genomes of many filamentous fungi consist of a ‘core’ part containing conserved genes essential for normal development as well as conditionally dispensable (CD) or lineage‐specific (LS) chromosomes. In the plant‐pathogenic fungus Fusarium oxysporum f. sp. lycopersici, one LS chromosome harbours effector genes that contribute to pathogenicity. We employed flow cytometry to select for events of spontaneous (partial) loss of either the two smallest LS chromosomes or two different core chromosomes. We determined the rate of spontaneous loss of the ‘effector’ LS chromosome in vitro at around 1 in 35 000 spores. In addition, a viable strain was obtained lacking chromosome 12, which is considered to be a part of the core genome. We also isolated strains carrying approximately 1‐Mb deletions in the LS chromosomes and in the dispensable core chromosome. The large core chromosome 1 was never observed to sustain deletions over 200 kb. Whole‐genome sequencing revealed that some of the sites at which the deletions occurred were the same in several independent strains obtained for the two chromosomes tested, indicating the existence of deletion hotspots. For the core chromosome, this deletion hotspot was the site of insertion of the marker used to select for loss events. Loss of the core chromosome did not affect pathogenicity, whereas loss of the effector chromosome led to a complete loss of pathogenicity.     

Codon bias and base composition are poor indicators of horizontally transferred genes

Horizontal gene transfer is now recognized as an important mechanism of evolution. Several methods to detect horizontally transferred genes have been suggested. These methods are based on either nucleotide composition or the failure to find a similar gene in closely related species. Genes that evolve vertically between closely related species can be divided into those that retain homologous chromosomal positions (positional orthologs) and those that do not. By comparing open reading frames in the Escherichia coli and Salmonella typhi genomes, we identified 2,728 positional orthologs since these species split 100 MYA. A group of 1,144 novel E. coli genes were unusually diverged from their S. typhi counterparts. These novel genes included those that had been horizontally transferred into E. coli, as well as members of gene pairs that had been rearranged or deleted. Positional orthologs were used to investigate compositional methods of identifying horizontally transferred genes. A large number of E. coli genes with normal nucleotide composition have no apparent ortholog in S. typhi, and many genes of atypical composition do, in fact, have positional orthologs. A phylogenetic approach was employed to confirm selected examples of horizontal transmission among the novel groups of genes. Our analysis of 80 E. coli genes determined that a number of genes previously classified as horizontally transferred based on base composition and codon bias were native, and genes previously classified as native appeared to be horizontally transferred. Hence, atypical nucleotide composition alone is not a reliable indicator of horizontal transmission.     

Unique properties of eukaryote-type actin and profilin horizontally transferred to cyanobacteria

A eukaryote-type actin and its binding protein profilin encoded on a genomic island in the cyanobacterium Microcystis aeruginosa PCC 7806 co-localize to form a hollow, spherical enclosure occupying a considerable intracellular space as shown by in vivo fluorescence microscopy. Biochemical and biophysical characterization reveals key differences between these proteins and their eukaryotic homologs. Small-angle X-ray scattering shows that the actin assembles into elongated, filamentous polymers which can be visualized microscopically with fluorescent phalloidin. Whereas rabbit actin forms thin cylindrical filaments about 100 μm in length, cyanobacterial actin polymers resemble a ribbon, arrest polymerization at 5-10 μm and tend to form irregular multi-strand assemblies. While eukaryotic profilin is a specific actin monomer binding protein, cyanobacterial profilin shows the unprecedented property of decorating actin filaments. Electron micrographs show that cyanobacterial profilin stimulates actin filament bundling and stabilizes their lateral alignment into heteropolymeric sheets from which the observed hollow enclosure may be formed. We hypothesize that adaptation to the confined space of a bacterial cell devoid of binding proteins usually regulating actin polymerization in eukaryotes has driven the co-evolution of cyanobacterial actin and profilin, giving rise to an intracellular entity.     

Identification and categorization of horizontally transferred genes in prokaryotic genomes

Horizontal gene transfer （HGT）, a process through which genomes acquire genetic materials from distantly related organisms, is believed to be one of the major forces in prokaryotic genome evolution.However, systematic investigation is still scarce to clarify two basic issues about HGT： （1） what types of genes are transferred; and （2） what influence HGT events over the organization and evolution of biological pathways. Genome-scale investigations of these two issues will advance the systematical understanding of HGT in the context of prokaryotic genome evolution. Having investigated 82 genomes, we constructed an HGT database across broad evolutionary timescales. We identified four function categories containing a high proportion of horizontally transferred genes： cell envelope, energy metabolism, regulatory functions, and transport/binding proteins. Such biased function distribution indicates that HGT is not completely random;instead, it is under high selective pressure, required by function restraints in organisms. Furthermore, we mapped the transferred genes onto the connectivity structure map of organism-specific pathways listed in Kyoto Encyclopedia of Genes and Genomes （KEGG）. Our results suggest that recruitment of transferred genes into pathways is also selectively constrained because of the tuned interaction between original pathway members. Pathway organization structures still conserve well through evolution even with the recruitment of horizontally transferred genes. Interestingly, in pathways whose organization were significantly affected by HGT events, the operon-like arrangement of transferred genes was found to be prevalent. Such results suggest that operon plays an essential and directional role in the integration of alien genes into pathways.     

Fusarium oxysporum hijacks COI1-mediated jasmonate signaling to promote disease development in Arabidopsis

Although defense responses mediated by the plant oxylipin jasmonic acid (JA) are often necessary for resistance against pathogens with necrotrophic lifestyles, in this report we demonstrate that jasmonate signaling mediated through COI1 in Arabidopsis thaliana is responsible for susceptibility to wilt disease caused by the root-infecting fungal pathogen Fusarium oxysporum . Despite compromised JA-dependent defense responses, the JA perception mutant coronatine insensitive 1 ( coi1 ), but not JA biosynthesis mutants, exhibited a high level of resistance to wilt disease caused by F. oxysporum . This response was independent from salicylic acid-dependent defenses, as coi1/NahG plants showed similar disease resistance to coi1 plants. Inoculation of reciprocal grafts made between coi1 and wild-type plants revealed that coi1 -mediated resistance occurred primarily through the coi1 rootstock tissues. Furthermore, microscopy and quantification of fungal DNA during infection indicated that coi1 -mediated resistance was not associated with reduced fungal penetration and colonization until a late stage of infection, when leaf necrosis was highly developed in wild-type plants. In contrast to wild-type leaves, coi1 leaves showed no necrosis following the application of F. oxysporum culture filtrate, and showed reduced expression of senescence-associated genes during disease development, suggesting that coi1 resistance is most likely achieved through the inhibition of F. oxysporum -incited lesion development and plant senescence. Together, our results indicate that F. oxysporum hijacks non-defensive aspects of the JA-signaling pathway to cause wilt-disease symptoms that lead to plant death in Arabidopsis.     

The SNARE protein FolVam7 mediates intracellular trafficking to regulate conidiogenesis and pathogenicity in Fusarium oxysporum f. sp. lycopersici

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are conserved in fungi, plants and animals. The Vam7 gene encodes a v-SNARE protein that involved in vesicle trafficking in fungi. Here, we identified and characterized the function of FolVam7, a homologue of the yeast SNARE protein Vam7p in Fusarium oxysporum f. sp. lycopersici (Fol), a fungal pathogen of tomato. FolVam7 contains SNARE and PX (Phox homology) domains that are indispensable for normal localization and function of FolVam7. Targeted gene deletion showed that FolVam7-mediated vesicle trafficking is important for vegetative growth, asexual development, conidial morphology and plant infection. Further cytological examinations revealed that FolVam7 is localized to vesicles and vacuole membranes in the hyphae stage. Moreover, the ΔFolvam7 mutant is insensitive to salt and osmotic stresses and hypersensitive to cell wall stressors. Taken together, our results suggested that FolVam7-mediated vesicle trafficking promotes vegetative growth, conidiogenesis and pathogenicity of Fol.     

Abnormal glycogen in astrocytes is sufficient to cause adult polyglucosan body disease

Background

A 45-year old woman of Cambodian ethnic background presented with fatal respiratory failure due to a severe diaphragmatic dysfunction. Two years before, she had developed early onset of urinary symptoms.

Methods and results

Neuroimaging showed atrophy of the spine and medulla as well as a leukodystrophy affecting both supra- and infra-tentorial regions. At autopsy, polyglucosan bodies (PB) were seen in several peripheral tissues, including the diaphragm, and nervous tissues such as peripheral nerves, cerebral white matter, basal ganglia, hippocampus, brainstem and cerebellum. Immunohistochemistry and electron microscopy of the brain revealed an exclusive astrocytic localization of the PB. The diagnosis of adult polyglucosan body disease (APBD) was confirmed by enzymatic and molecular studies.

Conclusion

Storage of abnormal glycogen in astrocytes is sufficient to cause the leukodystrophy of APBD. Since brain glycogen is almost exclusively metabolized in astrocytes, this observation sheds light on the pathophysiology of APBD. In addition, this is the first report of an APBD patient presenting with a subacute diaphragmatic failure.     

Expression and role of CYP505A1 in pathogenicity of Fusarium oxysporum f. sp. lactucae

BackgroundCytochrome P450 enzymes (CYPs) are monooxygenases present in every domain of life. In fungi CYPs are involved in virulence. Fusarium wilt of lettuce, caused by F. oxysporum f. sp. lactucae, is the most serious disease of lettuce. F. oxysporum f.sp. lactucae MSA35 is an antagonistic fungus. Pathogenic formae specialis of F. oxysporum possess a CYP belonging to the new family CYP505. This enzyme hydroxylates saturated fatty acids that play a role in plant defence.MethodsMolecular tools were adopted to search for cyp505 gene in MSA35 genome. cyp505 gene expression analysis in pathogenic and antagonistic Fusarium was performed. The enzyme was expressed in its recombinant form and used for catalytic reactions with fatty acids, the products of which were characterized by mass spectrometry analysis.ResultsA novel MSA35 self-sufficient CYP505 is differentially expressed in antagonistic and pathogenic F. oxysporum. Its expression is induced by the host plant lettuce in both pathogenesis and antagonism during the early phase of the interaction, while it is silenced during the late phase only in antagonistic Fusarium. Mass-spectrometry investigations proved that CYP505A1 mono-hydroxylates lauric, palmitic and stearic acids.ConclusionsThe ability of CYP505A1 to oxidize fatty acids present in the cortical cell membranes together with its differential expression in its Fusarium antagonistic form point out to the possibility that this enzyme is associated with Fusarium pathogenicity in lettuce.General significanceThe CYP505 clan is present in pathogenic fungal phyla, making CYP505A1 enzyme a putative candidate as a new target for the development of novel antifungal molecules.     

Cross pathogenicity studies with isolates of Fusarium oxysporum from either cucumber or watermelon pathogenic to both crop species*

Isolates of Fusarium oxysporum from Abaco, the Bahamas, whether obtained from wilted plants of cucumber (Cucumis sativus) or watermelon (Citrullus lanatus), were pathogenic to cucumber, watermelon, and cantaloupe (Cucumis melo var. reticulatus). The West Indian gherkin (Cucumis anguria), pumpkin (Cucurbita pepo), and three cultivars of summer squash (C. pepo var. melopepo) were not susceptible. Strains of F. oxysporum from wilted cucumber or watermelon plants from Florida were highly pathogenic only to their original host species and are regarded as different formae speciales.     

The specific genes for lantibiotic mutacin II biosynthesis in Streptococcus mutans T8 are clustered and can be transferred en bloc

Mutacin II is a ribosomally synthesized peptide lantibiotic produced by group II Streptococcus mutans. DNA sequencing has revealed that the mutacin II biosynthetic gene cluster consists of seven specific open reading frames: a regulator (mutR), the prepromutacin structural gene (mutA), a modifying protein (mutM), an ABC transporter (mutT), and an immunity cluster (mutFEG). Transformations of a non-mutacin-producing strain, S. mutans UA159, and a mutacin I-producing strain, S. mutans UA140, with chromosomal DNA from S. mutans T8 with an aphIII marker inserted upstream of the mutacin II structural gene yielded transformants producing mutacin II and mutacins I and II, respectively.     

Mutation of an arginine biosynthesis gene causes reduced pathogenicity in Fusarium oxysporum f. sp. melonis

Restriction enzyme-mediated integration (REMI) mutagenesis was used to tag genes required for pathogenicity of Fusarium oxysporum f. sp. melonis. Of the 1,129 REMI transformants tested, 13 showed reduced pathogenicity on susceptible melon cultivars. One of the mutants, FMMP95-1, was an arginine auxotroph. Structural analysis of the tagged site in FMMP95-1 identified a gene, designated ARG1, which possibly encodes argininosuccinate lyase, catalyzing the last step for arginine biosynthesis. Complementation of FMMP95-1 with the ARG1 gene caused a recovery in pathogenicity, indicating that arginine auxotrophic mutation causes reduced pathogenicity in this pathogen.     

Studies on sterol-ester hydrolase from Fusarium oxysporum. I. Partial purification and properties.

1. A search for a long chain fatty acyl sterol-ester hydrolase in microorganisms led to the isolation from soil of five strains belonging to Fusarium sp. which produced strong activity in the culture medium. 2. The cholesterol esterase from Fusarium oxysporum IGH-2 was purified about 270-fold by means of CaCl2 precipitation and Sephadex G-75 column chromatography. 3. The cholesterol esterase was activated by adekatol and Triton X-100. It was inhibited by lecithin and lysolecithin, and completely inactivated by heat treatment (60 degrees C for 30 min, at pH 7.0). 4. The optimum pH of the enzyme was found to be around 7.0. 5. Among various cholesterol esters tested, cholesterol linoleate was the most suitable substrate. 6. Cholesterol esters in serum were also hydrolyzed by this enzyme.     

Only a small subset of the horizontally transferred chromosomal genes in Escherichia coli are translated into proteins

Horizontally transferred genes are believed to play a critical role in the divergence of bacterial strains from a common ancestor, but whether all of these genes express functional proteins in the cell remains unknown. Here, we used an integrated LC-based protein identification technology to analyze the proteome of Escherichia coli strain K12 (JM109) and identified 1,480 expressed proteins, which are equivalent to approximately 35% of the total open reading frames predicted in the genome. This subset contained proteins with cellular abundance of several dozens to hundreds of thousands of copies, and included nearly all types of proteins in terms of chemical characteristics, subcellular distribution, and function. Interestingly, the subset also contained 138 of 164 gene products that are currently known to be essential for bacterial viability (84% coverage). However, the subset contained only a very small population (10%) of protein products from genes mapped within K-loops, which are "hot spots" for the integration of foreign DNAs within the K12 genome. On the other hand, these genes in K-loops appeared to be transcribed to RNAs almost as efficiently as the native genes in the bacterial cell as monitored by DNA microarray analysis, raising the possibility that most of the recently acquired foreign genes are inadequate for the translational machinery for the native genes and do not generate functional proteins within the cell.     

Escherichia coli can produce recombinant chitinase in the soil to control the pathogenesis by Fusarium oxysporum without colonization

Fusarium wilt of cucumbers was effectively controlled by Escherichia coli expressing an endochitinase gene (chiA), and the rate was as effective (60.0%) as the wildtype strain S. proteamaculans 3095 (55.0%) where the gene was cloned. However, live cells of soil inoculated E. coli host harboring the chiA gene did not proliferate but declined 100-fold from 108 CFU during the first week and showed less than 10 cells after day 14, suggesting that E. coli was able to express and produce the chitinase enzyme to the soil even as the population was gradually decreasing. Because the majority of the strains was alive for only a short period of time and the Fusarium-affected seedlings showed symptoms of wilting within 7-10 days, it seems that the pathogen control was decided early after the introduction of the biocontrol agent, eliminating the survival of the antagonist. These results indicated that soil inoculated E. coli could sufficiently express and produce the recombinant protein to control the pathogen, and root or soil colonization of the antagonist might not be a significant factor in determining the efficacy of biological control.