In vivo targeted molecular magnetic resonance imaging of free radicals in diabetic cardiomyopathy within mice.

Free radicals contribute to the pathogenesis of diabetic cardiomyopathy. We present a method for in vivo observation of free radical events within murine diabetic cardiomyopathy. This study reports on in vivo imaging of protein/lipid radicals using molecular MRI (mMRI) and immuno-spin trapping (IST) in diabetic cardiac muscle. To detect free radicals in diabetic cardiomyopathy, streptozotocin (STZ)-exposed mice were given 5,5-dimethyl-pyrroline-N-oxide (DMPO) and administered an anti-DMPO probe (biotin-anti-DMPO antibody-albumin-Gd-DTPA). For controls, non-diabetic mice were given DMPO (non-disease control), and administered an anti-DMPO probe; or diabetic mice were given DMPO but administered a non-specific IgG contrast agent instead of the anti-DMPO probe. DMPO administration started at 7 weeks following STZ treatment for 5 days, and the anti-DMPO probe was administered at 8 weeks for MRI detection. MRI was used to detect a significant increase (p < 0.001) in MRI signal intensity (SI) from anti-DMPO nitrone adducts in diabetic murine left-ventricular (LV) cardiac tissue, compared to controls. Regional increases in MR SI in the LV were found in the apical and upper-left areas (p < 0.01 for both), compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised cardiac tissues, which indicated elevated fluorescence only in cardiac muscle of mice administered the anti-DMPO probe. Oxidized lipids and proteins were also found to be significantly elevated (p < 0.05 for both) in diabetic cardiac muscle compared to controls. It can be concluded that diabetic mice have more heterogeneously distributed radicals in cardiac tissue than non-diabetic mice.

Cercosporin-deficient mutants by plasmid tagging in the asexual fungus Cercospora nicotianae.

We have successfully adapted plasmid insertion and restriction enzyme-mediated integration (REMI) to produce cercosporin toxin-deficient mutants in the asexual phytopathogenic fungus Cercospora nicotianae. The use of pre-linearized plasmid or restriction enzymes in the transformation procedure significantly decreased the transformation frequency, but promoted a complicated and undefined mode of plasmid integration that leads to mutations in the C. nicotianae genome. Vector DNA generally integrated in multiple copies, and no increase in single-copy insertion was observed when enzymes were added to the transformation mixture. Out of 1873 transformants tested, 39 putative cercosporin toxin biosynthesis ( ctb) mutants were recovered that showed altered levels of cercosporin production. Seven ctb mutants were recovered using pre-linearized plasmids without the addition of enzymes, and these were considered to be non-REMI mutants. The correlation between a specific insertion and a mutant phenotype was confirmed using rescued plasmids as gene disruption vectors in the wild-type strain. Six out of fifteen rescued plasmids tested yielded cercosporin-deficient transformants when re-introduced into the wild-type strain, suggesting a link between the insertion site and the cercosporin-deficient phenotype. Sequence analysis of a fragment flanking the insert site recovered from one insertion mutant showed it to be disrupted in sequences with high homology to the acyl transferase domain of polyketide synthases from other fungi. Disruption of this polyketide synthase gene ( CTB1) using a rescued plasmid resulted in mutants that were defective in cercosporin production. Thus, we provide the first molecular evidence that cercosporin is synthesized via a polyketide pathway as previously hypothesized.

Isolation of PDX2, a second novel gene in the pyridoxine biosynthesis pathway of eukaryotes, archaebacteria, and a subset of eubacteria.

In this paper we describe the isolation of a second gene in the newly identified pyridoxine biosynthesis pathway of archaebacteria, some eubacteria, fungi, and plants. Although pyridoxine biosynthesis has been thoroughly examined in Escherichia coli, recent characterization of the Cercospora nicotianae biosynthesis gene PDX1 led to the discovery that most organisms contain a pyridoxine synthesis gene not found in E. coli. PDX2 was isolated by a degenerate primer strategy based on conserved sequences of a gene specific to PDX1-containing organisms. The role of PDX2 in pyridoxine biosynthesis was confirmed by complementation of two C. nicotianae pyridoxine auxotrophs not mutant in PDX1. Also, targeted gene replacement of PDX2 in C. nicotianae results in pyridoxine auxotrophy. Comparable to PDX1, PDX2 homologues are not found in any of the organisms with homologues to the E. coli pyridoxine genes, but are found in the same archaebacteria, eubacteria, fungi, and plants that contain PDX1 homologues. PDX2 proteins are less well conserved than their PDX1 counterparts but contain several protein motifs that are conserved throughout all PDX2 proteins.

Vitamin B6 (pyridoxine) and its derivatives are efficient singlet oxygen quenchers and potential fungal antioxidants.

Vitamin B6 (pyridoxine, 1) and its derivatives: pyridoxal (2), pyridoxal 5-phosphate (3) and pyridoxamine (4) are important natural compounds involved in numerous biological functions. Pyridoxine appears to play a role in the resistance of the filamentous fungus Cercospora nicotianae to its own abundantly produced strong photosensitizer of singlet molecular oxygen (1O2), cercosporin. We measured the rate constants (kq) for the quenching of 1O2 phosphorescence by 1-4 in D2O. The respective total (physical and chemical quenching) kq values are: 5.5 x 10(7) M-1 s-1 for 1; 7.5 x 10(7) M-1 s-1 for 2, 6.2 x 10(7) M-1 s-1 for 3 and 7.5 x 10(7) M-1 s-1 for 4, all measured at pD 6.2. The quenching efficacy increased up to five times in alkaline solutions and decreased approximately 10 times in ethanol. Significant contribution to total quenching by chemical reaction(s) is suggested by the degradation of all the vitamin derivatives by 1O2, which was observed as declining absorption of the pyridoxine moiety upon aerobic irradiation of RB used to photosensitize 1O2. This photodegradation was completely stopped by azide, a known physical quencher of 1O2. The pyridoxine moiety can also function as a redox quencher for excited cercosporin by forming the cercosporin radical anion, as observed by electron paramagnetic resonance. All B6 vitamers fluoresce upon UV excitation. Compounds 1 and 4 emit fluorescence at 400 nm, compound 2 at 450 nm and compound 3 at 550 nm. The fluorescence intensity of 3 increased approximately 10 times in organic solvents such as ethanol and 1,2-propanediol compared to aqueous solutions, suggesting that fluorescence may be used to image the distribution of 1-4 in Cercospora to understand better the interactions of pyridoxine and 1O2 in the living fungus.

CFP, the putative cercosporin transporter of Cercospora kikuchii, is required for wild type cercosporin production, resistance, and virulence on soybean.

Many species of the fungal genus Cercospora, including the soybean pathogen C. kikuchii, produce the phytotoxic polyketide cercosporin. Cercosporin production is induced by light. Previously, we identified several cDNA clones of mRNA transcripts that exhibited light-enhanced accumulation in C. kikuchii. Targeted disruption of the genomic copy of one of these, now designated CFP (cercosporin facilitator protein), results in a drastic reduction in cercosporin production, greatly reduced virulence of the fungus to soybean, and increased sensitivity to exogenous cercosporin. Sequence analysis of CFP reveals an 1,821-bp open reading frame encoding a 65.4-kDa protein similar to several members of the major facilitator superfamily (MFS) of integral membrane transporter proteins known to confer resistance to various antibiotics and toxins in fungi and bacteria. We propose that CFP encodes a cercosporin transporter that contributes resistance to cercosporin by actively exporting cercosporin, thus maintaining low cellular concentrations of the toxin.

A novel gene required for cercosporin toxin resistance in the fungus Cercospora nicotianae.

Cercosporin, a photosensitizing perylenequinone toxin produced by the plant pathogenic Cercospora fungi, generates the highly toxic singlet oxygen (1O2) upon exposure to light. Cercosporin shows broad toxicity against a wide range of organisms, including bacteria, fungi, plants, and animals; however, Cercospora fungi are resistant to its effects. A novel gene, crg1 (cercosporin-resistance gene) was isolated from a wild-type strain of C. nicotianae by genetic complementation of a C. nicotianae mutant (CS10) which is cercosporin sensitive and down-regulated in cercosporin production. Sequence analysis indicated that crg1 encodes a putative protein of 550 amino acids with four putative transmembrane helical regions, however CRG1 shows no strong similarity to any other protein in sequence databases. Northern analysis identified two transcripts (4.5 and 2.6 kb) that are unaffected by the presence of light or cercosporin. Southern analysis demonstrated that crg1 is present in a single copy in the C. nicotianae genome and can be detected only in Cercospora species. Targeted disruption of crg1 resulted in mutants that, like CS10, are sensitive to cercosporin. However, unlike CS10, crg1 disruption mutants are not down-regulated in toxin production. Both CS10 and the crg1 disruption mutants are unaffected in their response to other 1O2-generating photosensitizers, suggesting that CRG1 functions specifically against cercosporin, rather than against 1O2.

A highly conserved sequence is a novel gene involved in de novo vitamin B6 biosynthesis.

The Cercospora nicotianae SOR1 (singlet oxygen resistance) gene was identified previously as a gene involved in resistance of this fungus to singlet-oxygen-generating phototoxins. Although homologues to SOR1 occur in organisms in four kingdoms and encode one of the most highly conserved proteins yet identified, the precise function of this protein has, until now, remained unknown. We show that SOR1 is essential in pyridoxine (vitamin B6) synthesis in C. nicotianae and Aspergillus flavus, although it shows no homology to previously identified pyridoxine synthesis genes identified in Escherichia coli. Sequence database analysis demonstrated that organisms encode either SOR1 or E. coli pyridoxine biosynthesis genes, but not both, suggesting that there are two divergent pathways for de novo pyridoxine biosynthesis in nature. Pathway divergence appears to have occurred during the evolution of the eubacteria. We also present data showing that pyridoxine quenches singlet oxygen at a rate comparable to that of vitamins C and E, two of the most highly efficient biological antioxidants, suggesting a previously unknown role for pyridoxine in active oxygen resistance.

Functional characterization of SOR1, a gene required for resistance to photosensitizing toxins in the fungus Cercospora nicotianae.

The Cercospora nicotianae SOR1 gene is required for resistance to singlet oxygen-generating photosensitizers. SOR1 was characterized in the wild-type and in five photosensitizer-sensitive mutant strains which are complemented to photosensitizer resistance by transformation with SOR1. Sequence analysis determined that three of the mutants contain SOR1 copies with mutations encoding substitutions in the protein-coding sequence; however, two other mutants had wild-type SOR1 protein and promoter sequences. All five mutants accumulate SOR1 mRNA at levels comparable to that of the wild-type strain. In the wild-type strain, SOR1 accumulation is enhanced two-fold by light, but is unaffected by the presence of cercosporin, the photosensitizer synthesized by C. nicotianae. Southern analysis indicates that SOR1 is present in other fungi that synthesize structurally related perylenequinone photosensitizers.

Roles for mannitol and mannitol dehydrogenase in active oxygen-mediated plant defense.

Reactive oxygen species (ROS) are both signal molecules and direct participants in plant defense against pathogens. Many fungi synthesize mannitol, a potent quencher of ROS, and there is growing evidence that at least some phytopathogenic fungi use mannitol to suppress ROS-mediated plant defenses. Here we show induction of mannitol production and secretion in the phytopathogenic fungus Alternaria alternata in the presence of host-plant extracts. Conversely, we show that the catabolic enzyme mannitol dehydrogenase is induced in a non-mannitol-producing plant in response to both fungal infection and specific inducers of plant defense responses. This provides a mechanism whereby the plant can counteract fungal suppression of ROS-mediated defenses by catabolizing mannitol of fungal origin.

SOR1, a gene required for photosensitizer and singlet oxygen resistance in Cercospora fungi, is highly conserved in divergent organisms.

Filamentous Cercospora fungi are resistant to photosensitizing compounds that generate singlet oxygen. C. nicotianae photosensitizer-sensitive mutants were restored to full resistance by transformation with SOR1 (Singlet Oxygen Resistance 1), a gene recovered from a wild-type genomic library. SOR1 null mutants generated via targeted gene replacement confirmed the requirement for SOR1 in photosensitizer resistance. SOR1 RNA is present throughout the growth cycle. Although resistance to singlet oxygen is rare in biological systems, SOR1, a gene with demonstrated activity against singlet-oxygen-generating photosensitizers, is highly conserved in organisms from widely diverse taxa. The characterization of SOR1 provides an additional phenotype to this large group of evolutionarily conserved genes.

Isolation, sequence, and characterization of the Cercospora nicotianae phytoene dehydrogenase gene.

We have cloned and sequenced the Cercospora nicotianae gene for the carotenoid biosynthetic enzyme phytoene dehydrogenase. Analysis of the derived amino acid sequence revealed it has greater than 50% identity with its counterpart in Neurospora crassa and approximately 30% identity with prokaryotic phytoene dehydrogenases and is related, but more distantly, to phytoene dehydrogenases from plants and cyanobacteria. Our analysis confirms that phytoene dehydrogenase proteins fall into two groups: those from plants and cyanobacteria and those from eukaryotic and noncyanobacter prokaryotic microbes. Southern analysis indicated that the C. nicotianae phytoene dehydrogenase gene is present in a single copy. Extraction of beta-carotene, the sole carotenoid accumulated by C. nicotianae, showed that both light- and dark-grown cultures synthesize carotenoids, but higher levels accumulate in the light. Northern (RNA) analysis of poly(A)+ RNA, however, showed no differential accumulation of phytoene dehydrogenase mRNA between light- and dark-grown fungal cultures.

Mutants of Cercospora kikuchii Altered in Cercosporin Synthesis and Pathogenicity.

We have obtained spontaneous and UV-induced stable mutants, altered in the synthesis of cercosporin, of the fungal soybean pathogen Cercospora kikuchii. The mutants were isolated on the basis of colony color on minimal medium. The UV-induced mutants accumulated, at most, 2% of wild-type cercosporin levels on all media tested. In contrast, cercosporin accumulation by the spontaneous mutants was strongly medium regulated, occurring only on potato dextrose medium but at concentrations comparable to those produced by the wild-type strain. UV-induced mutants unable to synthesize cercosporin on any medium were unable to incite lesions when inoculated onto the soybean host. Cercosporin was reproducibly isolated from all inoculated leaves showing lesions. Although cercosporin involvement in disease has been indirectly suggested by many previous studies, this is the first report in which mutants blocked in cercosporin synthesis have been used to demonstrate that cercosporin is a crucial pathogenicity factor for this fungal genus.

Genetic Transformation System for the Fungal Soybean Pathogen Cercospora kikuchii.

An altered beta-tubulin gene that confers resistance to the fungicide benomyl was isolated from a genomic library of a UV-induced mutant of Cercospora kikuchii and used as a selectable marker for transformation. The level of benomyl resistance conferred to the transformants was at least 150-fold greater than the intrinsic resistance of the C. kikuchii recipient protoplasts. In the majority of cases, the tubulin fragment was integrated at the native beta-tubulin locus, apparently by gene replacement or gene conversion. The frequency of transformation ranged from 0.2 to 6 transformants per mug of DNA, depending on the recipient strain. Transformation with linearized plasmid resulted in a higher frequency, without changing the type of integration event. Transformants were phenotypically stable after eight consecutive transfers on medium without benomyl. This is the first report of a genetic transformation system for a Cercospora species.

Isolation of Light-Enhanced cDNAs of Cercospora kikuchii.

Cercospora kikuchii is a fungal pathogen of soybeans which produces a photosensitizing phytotoxic polyketide metabolite, cercosporin. Cercosporin synthesis in culture is modulated by several environmental factors. In addition to the light requirement for toxin action, cercosporin biosynthesis is also highly light regulated. As a first step towards identifying genes involved in cercosporin regulation and biosynthesis, we have used subtractive hybridization to isolate light-enhanced cDNA clones. Six distinct cDNA clones representing genes from a wild-type C. kikuchii strain for which transcript accumulation is positively regulated by light were isolated. To assess the relationship of these light-enhanced cDNAs to cercosporin biosynthesis, we compared corresponding steady-state RNA levels in the wild type and in three mutant strains altered in toxin biosynthesis. Two of the mutant C. kikuchii strains which fail to accumulate cercosporin in response to light also fail to exhibit light-enhanced accumulation of transcripts corresponding to all six light-enhanced cDNAs. Cercosporin accumulation in the third mutant strain, S2, is regulated by medium composition as well as light. S2 fails to accumulate cercosporin in complete medium, a medium which allows significant cercosporin accumulation by the wild-type strain. When cultured in complete medium, this mutant strain also fails to show the wild-type, light-enhanced accumulation of transcripts corresponding to five of the six light-enhanced cDNAs. Kinetic analysis demonstrated that transcript accumulation for two of the six light-enhanced cDNAs strongly paralleled cercosporin accumulation in light-grown wild-type culture.

Respiration and mitochondrial biogenesis in germinating embryos of maize.

Function of the cyanide-sensitive mitochondrial electron transport system was required for germination of the Zea mays embryo. Respiration of the standard electron transport system (rather than the alternate oxidase) began immediately upon initiation of imbibition. This respiration depended upon cytochrome c oxidase and ATPase that were conserved in an active form in the quiescent embryo rather than upon newly synthesized or assembled enzyme complexes. Immunoprecipitation of radiolabeled subunits of these enzymes showed that the initiation of mitochondrial biogenetic activities, including de novo synthesis of nuclear- and mitochondrial-encoded enzyme subunit peptides, was strongly induced after 6 hours of embryo germination. Undetectable or very low levels of transcripts for subunits 1 and 2 of the F(1)-ATPase and subunit 2 of cytochrome c oxidase were present in the quiescent embryo; these transcripts accumulated rapidly between 6 and 12 hours of germination and their translation products were rapidly synthesized between 6 and 24 hours. An exception was the gene for subunit 9 of the ATPase; transcripts of this mitochondrial gene were abundant in the dry embryo and rapidly accumulated further upon initiation of imbibition; they were translated actively during the first 6 hours. We isolated and sequenced a near full-length cDNA for subunit 2 (beta) of the F(1)-ATPase, and we compared the deduced protein sequence with related sequences of other organisms.

Construction of a cosmid clone library of Pseudomonas syringae pv. phaseolicola and isolation of genes by functional complementation.

A genomic library constructed from a wild-type strain of Pseudomonas syringae pv. phaseolicola in the broad-host-range cosmid vector pVK102 was used to isolate wild-type genes by complementation of Tn5-induced auxotrophic mutants. Selection pressure was required for maintenance of the vector and members of the library in strains of P. syringae.