Peroxysomal carnitine acetyl transferase influences host colonization capacity in Sclerotinia sclerotiorum.

In lower eukaryotes, the glyoxylate cycle allows cells to utilize two-carbon compounds when simple sugars are not available. In filamentous fungi, glyoxylate metabolism is coupled with ß-oxidation of fatty acids, and both are localized to ubiquitous eukaryotic organelles called peroxisomes. Acetyl coenzyme A (acetyl-CoA) produced during ß-oxidation is transported via the cytosol into mitochondria for further metabolism. A peroxisomal-specific pathway for acetyl-CoA transport requiring peroxisomal carnitine acetyl transferase (CAT) activity has been identified in Magnaporthe grisea peroxisomes. Here, we report that a Sclerotinia sclerotiorum ortholog of the M. grisea peroxisomal CAT-encoding gene Pth2 (herein designated Ss-pth2) is required for virulence-associated host colonization. Null (ss-pth2) mutants, obtained by in vitro transposon mutagenesis, failed to utilize fatty acids, acetate, or glycerol as sole carbon sources for growth. Gene expression analysis of these mutants showed altered levels of transcript accumulation for glyoxylate cycle enzymes. Ss-pth2 disruption also affected sclerotial, apothecial, and appressorial development and morphology, as well as oxalic acid accumulation when cultured with acetate or oleic acid as sole carbon nutrient sources. Although mutants were able to penetrate and initially colonize host tissue, subsequent colonization was impaired. Genetic complementation with the wild-type Ss-pth2 restored wild-type virulence phenotypes. These findings suggest an essential role in S. sclerotiorum for the peroxisomal metabolic pathways for oxalic acid synthesis and host colonization.

Characterization of the glyoxalase I gene from the vascular wilt fungus Verticillium dahliae.

A glyoxalase I gene homologue (VdGLO1) was identified in the vascular wilt fungus Verticillium dahliae by sequence tag analysis of genes expressed during resting structure development. The results of the current study show that the gene encodes a putative 345 amino acid protein with high similarity to glyoxalase I, which produces S-D-lactoylglutathione from the toxic metabolic by-product methylglyoxal (MG). Disruption of the V. dahliae gene by Agrobacterium tumefaciens-mediated transformation resulted in enhanced sensitivity to MG. Mycelial growth of disruption mutants was severely reduced in the presence of 5 mmol/L MG. In contrast, spore production in liquid medium was abolished at 1 mmol/L MG, although not at physiologically relevant concentrations of

Sequence tag analysis of gene expression during pathogenic growth and microsclerotia development in the vascular wilt pathogen Verticillium dahliae.

Two cDNA libraries were constructed from cultures of the vascular wilt fungus Verticillium dahliae, grown either in simulated xylem fluid medium (SXM) or under conditions that induce near-synchronous development of microsclerotia. Expressed sequence tags (ESTs) were obtained for over 1000 clones from each library. Most sequences in the two EST collections were unique; nearly 55% of the translated ESTs had strong similarity to protein sequences in the NCBI nonredundant database. ESTs corresponding to melanin biosynthetic enzymes were exclusive to the developing microsclerotia (DMS) collection, and sequences corresponding to extracellular hydrolases (plant cell wall degrading enzymes) were more abundant in that collection. ESTs corresponding to proteins involved in transport and cell growth were more abundant in the SXM collection. The results of this preliminary analysis suggest that the in vitro growth conditions used here provide useful model systems that will facilitate studies of pathogenesis and microsclerotia development in V. dahliae.

Molecular Differentiation and Detection of Ginseng-Adapted Isolates of the Root Rot Fungus Cylindrocarpon destructans.

ABSTRACT The soilborne fungus Cylindrocarpon destructans (teleomorph: Neonectria radicicola) causes root rot in a wide range of plant hosts; the disease is of particular concern in ginseng production, and in conifer and fruit tree nurseries. beta-Tubulin gene and rRNA gene internal transcribed spacer (ITS) sequence data and pathogenicity assays were used to characterize isolates of C. destructans from ginseng and other hosts. The results of these studies demonstrated a high amount of sequence divergence among strains identified as C. destructans or N. radicicola, suggesting the existence of several phylogenetic species in this complex. Accordingly, we propose that the two varieties of N. radicicola be raised to species status. Certain highly aggressive ginseng isolates from Ontario, Korea, and Japan have identical ITS and beta-tubulin sequences, and form a monophyletic clade (designated "clade a"); these strains are identified as C. destructans f. sp. panacis. Other ginseng strains clustered in monophyletic groups with strains from angiosperm and conifers. A subtractive hybridization method was used to isolate genomic DNA sequences with diagnostic potential from the aggressive C. destructans Ontario ginseng isolate 1640. One of these sequences was similar to the rRNA gene intergenic spacer from a Fusarium oxysporum isolate from Pinus ponderosa, and hybridized to DNA from F. oxysporum and all C. destructans isolates tested. Primers were designed that could be used to amplify this sequence specifically from the highly aggressive, ginsengadapted C. destructans isolates from Ontario and Korea and other members of clade a.

Molecular Characterization of Vegetative Compatibility Group 4A and 4B Isolates of Verticillium dahliae Associated with Potato Early Dying.

Forty isolates of Verticillium dahliae, collected from potato seed tubers and potato plants from various regions in North America and previously assigned to vegetative compatibility groups (VCGs) 4A or 4B, were characterized using molecular markers. The VCG 4A isolates were previously shown to be a highly virulent pathotype of potato and to interact synergistically with the root-lesion nematode Pratylenchus penetrans to cause potato early dying. All but one of the VCG 4A isolates characterized in this study lacked the subspecies-specific repetitive DNA sequence E18 and could be differentiated from the remaining isolates by restriction fragment length polymorphisms (RFLPs) in the nuclear rDNA and Trp1 loci. The E18 RFLP patterns of several VCG 4B isolates from Maine and New York were highly similar to those of VCG 4B isolates previously collected from potato and tomato fields in Ontario. The data presented here suggest that the molecular markers will be useful for the detection and classification of isolates of V. dahliae associated with potato early dying.

Influences of Cropping Practices on Verticillium dahliae Populations in Commercial Processing Tomato Fields in Ontario.

ABSTRACT The abundance of Verticillium dahliae in the soil and the incidence of V. dahliae-infected plants were determined for 12 commercial processing tomato fields in Kent County, Ontario. Comparison of the data with those from a previous survey of fields in adjacent Essex County showed that soil inoculum levels and incidence of infection were generally lower in Kent County fields and that race 2 V. dahliae was not common in Kent County. From the two counties, 128 isolates were characterized by restriction fragment length polymorphism (RFLP) analysis, using the subspecies-specific repetitive DNA sequence E18. A subset of these isolates was also characterized by vegetative compatibility and DNA hybridization analysis with a second subspecies-specific DNA sequence. Isolates with E18 RFLP profiles highly similar to those of isolates previously collected from potato fields in North America were prevalent in Essex County tomato fields but not common in Kent County fields. The data are consistent with the hypothesis that the group I isolates were introduced into southwestern Ontario with potato and that the different cultural practices in Essex County and Kent County have contributed to the differences in the accumulation of these isolates in the two regions.

Treatment with 24-epibrassinolide, a brassinosteroid, increases the basic thermotolerance of Brassica napus and tomato seedlings.

Brassinosteroids are plant growth-promoting compounds that exhibit structural similarities to animal steroid hormones. Recent studies have indicated that brassinosteroids are essential for proper plant development. In addition to a role in development, several lines of evidence suggest that brassinosteroids exert anti-stress effects on plants. However, the mechanism by which they modulate plant stress responses is not understood. We show here that Brassica napus and tomato seedlings grown in the presence of 24-epibrassinolide (EBR) are significantly more tolerant to a lethal heat treatment than are control seedlings grown in the absence of the compound. Since a preconditioning treatment of seedlings was not required to observe this effect, we conclude that EBR treatment increases the basic thermotolerance of seedlings. An analysis of heat shock proteins (HSPs) in B. napus seedlings by western blot analysis indicated that the HSPs did not preferentially accumulate in EBR-treated seedlings at the control temperature. However, after heat stress, HSP accumulation was higher in EBR-treated than in untreated seedlings. The results of the present study provide the first direct evidence for EBR-induced expression of HSPs. The higher accumulation of HSPs in EBR-treated seedlings raises the possibility that HSPs contribute, at least in part, to thermotolerance in EBR-treated seedlings. A search for factors other than HSPs, which may directly or indirectly contribute to brassinosteroid-mediated increase in thermotolerance, is underway.

Production of an extracellular trypsin-like protease by the fungal plant pathogen Verticillium dahliae.

The plant pathogenic fungus Verticillium dahliae produced extracellular alkaline protease activity when grown in liquid medium supplemented with a protein source. A serine protease was purified 80-fold in a single step, using cation-exchange chromatography, from the filtrate of cultures grown with skim milk as a protein source. N-terminal amino acid sequence analysis of the 30-kDa protein (VDP30) that copurified with the serine protease activity suggested that VDP30 is a trypsin-like protein. The purified enzyme hydrolyzed the synthetic substrate N alpha-benzoyl-DL-arginine p-nitroanilide hydrochloride (BAPNA), and the activity on BAPNA was inhibited by leupeptin, further verifying the trypsin-like nature of the enzyme.

The ebb and flow of a fungal genome.

Transposable DNA elements have only recently been described in a few species of filamentous fungi, but may be more abundant than previously believed. Several different elements have been isolated from the rice blast pathogen Magnaporthe grisea. The distribution and amplification of these elements suggest a potential role in the evolution of the fungal genome.

Grasshopper, a long terminal repeat (LTR) retroelement in the phytopathogenic fungus Magnaporthe grisea.

The fungal phytopathogen Magnaporthe grisea parasitizes a wide variety of gramineous hosts. In the course of investigating the genetic relationship between pathogen genotype and host specificity we identified a retroelement that is present in some strains of M. grisea that infect finger millet and goosegrass (members of the plant genus Eleusine). The element, designated grasshopper (grh), is present in multiple copies and dispersed throughout the genome. DNA sequence analysis showed that grasshopper contains 198 base pair direct, long terminal repeats (LTRs) with features characteristic of retroviral and retrotransposon LTRs. Within the element we identified an open reading frame with sequences homologous to the reverse transcriptase, RNaseH, and integrase domains of retroelement pol genes. Comparison of the open reading frame with sequences from other retroelements showed that grh is related to the gypsy family of retrotransposons. Comparisons of the distribution of the grasshopper element with other dispersed repeated DNA sequences in M. grisea indicated that grasshopper was present in a broadly dispersed subgroup of Eleusine pathogens, suggesting that the element was acquired subsequent to the evolution of this host-specific form. We present arguments that the amplification of different retroelements within populations of M. grisea is a consequence of the clonal organization of the fungal populations.

A protein required for RNA processing and splicing in Neurospora mitochondria is related to gene products involved in cell cycle protein phosphatase functions.

The Neurospora crassa cyt-4 mutants have pleiotropic defects in mitochondrial RNA splicing, 5' and 3' end processing, and RNA turnover. Here, we show that the cyt-4+ gene encodes a 120-kDa protein with significant similarity to the SSD1/SRK1 protein of Saccharomyces cerevisiae and the DIS3 protein of Schizosaccharomyces pombe, which have been implicated in protein phosphatase functions that regulate cell cycle and mitotic chromosome segregation. The CYT-4 protein is present in mitochondria and is truncated or deficient in two cyt-4 mutants. Assuming that the CYT-4 protein functions in a manner similar to the SSD1/SRK1 and DIS3 proteins, we infer that the mitochondrial RNA splicing and processing reactions defective in the cyt-4 mutants are regulated by protein phosphorylation and that the defects in the cyt-4 mutants result from failure to normally regulate this process. Our results provide evidence that RNA splicing and processing reactions may be regulated by protein phosphorylation.

Mutations in nuclear gene cyt-4 of Neurospora crassa result in pleiotropic defects in processing and splicing of mitochondrial RNAs.

The nuclear cyt-4 mutants of Neurospora crassa have been shown previously to be defective in splicing the group I intron in the mitochondrial large rRNA gene and in 3' end synthesis of the mitochondrial large rRNA. Here, Northern hybridization experiments show that the cyt-4-1 mutant has alterations in a number of mitochondrial RNA processing pathways, including those for cob, coI, coII and ATPase 6 mRNAs, as well as mitochondrial tRNAs. Defects in these pathways include inhibition of 5' and 3' end processing, accumulation of aberrant RNA species, and inhibition of splicing of both group I introns in the cob gene. The various defects in mitochondrial RNA synthesis in the cyt-4-1 mutant cannot be accounted for by deficiency of mitochondrial protein synthesis or energy metabolism, and they suggest that the cyt-4-1 mutant is defective in a component or components required for processing and/or turnover of a number of different mitochondrial RNAs. Defective splicing of the mitochondrial large rRNA intron in the cyt-4-1 mutant may be a secondary effect of failure to synthesize pre-rRNAs having the correct 3' end. However, a similar explanation cannot be invoked to account for defective splicing of the cob pre-mRNA introns, and the cyt-4-1 mutation may directly affect splicing of these introns.

Effect of the delta subunit of Bacillus subtilis RNA polymerase on initiation of RNA synthesis at two bacteriophage phi 29 promoters.

Initiation of RNA synthesis by Bacillus subtilis RNA polymerase (sigma-43) has been examined at two early promoters of phage phi 29: the A2 promoter, which is a weak promoter, and the G2 promoter, which is a strong promoter. The delta subunit of the polymerase inhibits the rate of initiation at A2, but not G2. In addition, formation of stable complexes by the polymerase at A2, but not at G2, requires the presence of the first two nucleotides of the A2 transcript.

Nucleotide sequence and transcription of a bacteriophage 29 early promoter.

We have studied the in vitro and in vivo transcription of a promoter for Bacillus subtilis RNA polymerase on bacteriophage phi 29 DNA. The promoter is identified as an early promoter as it is transcribed in vitro by uninfected B. subtilis sigma 55-containing RNA polymerase; is transcribed in vivo at both 7 min after infection and in the presence of chloramphenicol; and is transcribed right to left on the standard phi 29 map. The nucleotide sequence of the promoter and the initiation site for RNA synthesis are reported. We have also examined the kinetics of RNA synthesis initiation using a single round run-off transcription assay. The overall rate of initiation was found to be 1.6 X 10(6) M-1 s-1 while the rate of conversion of unstable to stable polymerase-promoter complexes was 0.049 s-1. These values are comparable to those for similar promoters for Escherichia coli RNA polymerase.