Organization of ribosomal RNA genes in the fungus Cochliobolus heterostrophus.

The genes encoding the 17S, 5.8S and 25S ribosomal RNAs in the Ascomycete Cochliobolus heterostrophus were cloned and analyzed. They are arranged in tandemly repeated units (rDNA) either 9.0 or 9.15 kilobases in length, depending upon the strain. The 5S rRNA genes are not part of the tandemly repeated rDNA. Instead, many and perhaps all of the 5S genes are dispersed in the genome, as they are in the fungi Neurospora, Aspergillus and Schizosaccharomyces. Comparative restriction maps of the rDNA from C. heterostrophus and other filamentous fungi and yeasts are presented. A survey of rDNAs from twenty-three field isolates of C. heterostrophus collected worldwide demonstrated that each isolate has one or the other of two rDNA forms, which differ in length and in the presence or absence of at least three restriction enzyme sites. The differences are all located in spacer DNA outside the coding regions for the rRNA genes. Heterogeneity in the rDNA repeat was not observed within any single isolate. The copy number of the rRNA gene cluster in C. heterostrophus is approximately 130 per haploid genome.

Development of a fungal transformation system based on selection of sequences with promoter activity.

A novel strategy was used to develop a transformation system for the plant pathogenic fungus Cochliobolus heterostrophus. Sequences capable of driving the expression of a gene conferring resistance to the antibiotic hygromycin B in C. heterostrophus were selected from a library of genomic DNA fragments and used, with the selectable marker, as the basis for transformation. The library of random 0.5- to 2.0-kilobase-pair fragments of C. heterostrophus genomic DNA was inserted at the 5' end of a truncated, promoterless Escherichia coli hygromycin B phosphotransferase gene (hygB) whose product confers resistance to hygromycin B. C. heterostrophus protoplasts were transformed with the library and selected for resistance. Resistant colonies arose at low frequency. Each colony contained a transformation vector stably integrated into chromosomal DNA. When the transforming DNA was recovered from the genome and introduced into C. heterostrophus, resistant colonies appeared at higher frequency. We determined the sequences of two of the C. heterostrophus DNA fragments which had been inserted at the 5' end of hygB in the promoter library and found that both made translational fusions with hygB. One of the two fusions apparently adds 65 and the other at least 86 amino acids to the N-terminus of the hygB product. Plasmids containing hygB-C. heterostrophus promoter fusions can be used unaltered to drive hygB expression in several other filamentous ascomycetes. This approach to achieving transformation may have general utility, especially for organisms with relatively undeveloped genetics.

A cloned tryptophan-synthesis gene from the ascomycete Cochliobolus heterostrophus functions in Escherichia coli, yeast and Aspergillus nidulans.

A gene (TRP1) in the tryptophan biosynthetic pathway of the fungal plant pathogen Cochliobolus heterostrophus was isolated by complementation of an Escherichia coli trpF mutant which lacked phosphoribosylanthranilate isomerase (PRAI) activity. The cloned gene also complemented an E. coli trpC mutant lacking indoleglycerolphosphate synthase (IGPS) activity, a yeast trp1 mutant missing PRAI activity and an Aspergillus nidulans trpC mutant. It functioned in E. coli and A. nidulans without apparent rearrangement but in yeast only after the 5' end of the gene was deleted. The gene was subcloned on a 4.65-kb DNA fragment and the PRAI domain was localized to a 2.9-kb region. It showed homology to the A. nidulans trpC and Neurospora crassa trp-1 genes. There was one predominant transcript of C. heterostrophus TRP1, the same size (2.6-kb) as one of the two functional transcripts produced by A. nidulans trpC. The constitutive activity of the C. heterostrophus TRP1 gene was high whereas that of the A. nidulans trpC gene was low.

Mitochondrial DNA of the filamentous ascomycete Cochliobolus heterostrophus : Characterization of the mitochondrial chromosome and population genetics of a restriction enzyme polymorphism.

The mitochondrial chromosome of Cochliobolus heterostrophus is a circle approximately 115 kb in circumference, among the largest known from fungi. A physical map of C. heterostrophus mtDNA was constructed using the restriction enzymes BamHI, EcoRI, and PvulI by DNA-DNA hybridizations with cloned or purified mtDNA BamHI fragments. The following sequences were located on the mtDNA map: (1) the large and small mitochondrial ribosomal RNA genes (identified by heterologous hybridization to cloned Neurospora crassa rRNA genes); (2) the sequence homologous to a mitochondrial plasmid present in one field isolate of C. heterostrophus; and (3) a 1.05 kb EcoRI fragment that functions as an autonomously replicating sequence in Saccharomyces cerevisiae. An examination of mtDNA from 23 isolates of C. heterostrophus collected worldwide revealed polymorphisms in restriction enzyme sites. One such polymorphism, coupled with data on a polymorphism in nuclear rDNA, suggests that there are two genetically distinct but geographically overlapping mating populations of C. heterostrophus in the world.

Isolation of DNA from filamentous fungi and separation into nuclear, mitochondrial, ribosomal, and plasmid components.

A general procedure for purifying and efficiently separating four types of DNA from filamentous fungi has been developed. The protocol involves (i) disruption of mycelial cells by blending in liquid nitrogen followed by suspension of cell contents in buffer containing high concentrations of protease and EDTA; (ii) deproteinization with phenol; (iii) cesium chloride/bisbenzimide density gradient centrifugation to separate nuclear DNA, mitochondrial DNA, and ribosomal DNA; and (iv) agarose gel electrophoresis to identify and purify plasmid DNA, if present. All DNAs are suitable for digestion with restriction endonucleases, ligation, and cloning in Escherichia coli, and DNAs from step three are recovered in high-molecular-weight form. The procedure has been used successfully with several dozen isolates of the plant pathogenic fungus Cochliobolus heterostrophus (including both laboratory strains and isolates collected directly from the field), and has been found to be equally suitable for C. carbonum, Neurospora crassa, N. tetrasperma, and Nectria haematococca.

Elemental analysis of freeze-dried thin sections of Samanea motor organs: barriers to ion diffusion through the apoplast.

Leaflet movements in the legume Samanea saman are dependent upon massive redistribution of potassium (K), chloride (Cl), and other solutes between opposing (extensor and flexor) halves of the motor organ (pulvinus). Solutes are known to diffuse through the apoplast during redistribution. To test the possibility that solute diffusion might be restricted by apoplastic barriers, we analyzed elements in the apoplast in freeze-dried cryosections of pulvini using scanning electron microscopy/x-ray microanalysis. Large discontinuities in apoplastic K and Cl at the extensor-flexor interface provide evidence for a barrier to solute diffusion. The barrier extends from the epidermis on upper and lower sides of the pulvinus to cambial cells in the central vascular core. It is completed by hydrophobic regions between phloem and cambium, and between xylem rays and surrounding vascular tissue, as deduced by discontinuities in apoplastic solutes and by staining of fresh sections with lipid-soluble Sudan dyes. Thus, symplastic pathways are necessary for ion redistribution in the Samanea pulvinus during leaflet movement. In pulvini from leaflets in the closed state, all cells on the flexor side of the barrier have high internal as well as external K and Cl, whereas cells on the extensor side have barely detectable internal or external K or Cl. Approximately 60% of these ions are known to migrate to the extensor during opening; all return to the flexor during subsequent closure. We propose that solutes lost from shrinking cells in the outer cortex diffuse through the apoplast to plasmodesmata-rich cells of the inner cortex, collenchyma, and phloem; and that solutes cross the barrier by moving through plasmodesmata.

Apoplastic transport of ions in the motor organ of Samanea.

Turgor-mediated leaf movements of the legume Samanea saman are associated with the migration of K(+) and Cl(-) between opposing sides of the motor organs (pulvini). We have investigated the pathway of this ion migration by localizing K(+) and Cl(-) within the secondary pulvinus at various times during leaf movements. Ion distributions in freeze-dried cryosections of pulvini were determined by scanning electron microscopy/x-ray microanalysis. The results indicate that the apoplast is a major pathway for the migration of K(+) and Cl(-) within the pulvinus.

Vacuolar reorganization in the motor cells of Albizzia during leaf movement.

During the leaf movements of Albizzia julibrissin Durazzini, volume changes in the motor cells of the pulvinule (tertiary pulvinus) are closely correlated with a reversible reorganization of the vacuolar compartment. Motor cells have central vacuoles when expanded, but become multivacuolate during the time the cell volume decreases. The central vacuole reforms - apparently by fusion of small vacuoles - during motor-cell expansion. The volume changes of the vacuolar compartment account for all of the change in the size of the protoplast, while the cytoplasmic volume remains constant during the leaf movements.

The ultrastructure of mitosis in Plasmodiophora brassicae (Plasmodiophorales).

Mitosis was examined in plasmodia of Plasmodiophora brassicae within artifically inoculated cabbage roots, using light- and electron microscopy. Mitotic nuclear divisions are characterized by a persistent nucleolus, bipolar centrioles paired end-to-end, densely staining chromatin, and a complex array of membranes that surround and ramify through the spindle. Chromatin begins to condense in prophase, and is aligned at metaphase in a reticulate plate on the nuclear equator. The chromatin is not resolvable into distinct chromosomes at metaphase, and a chromosome count is not possible. Large amounts of membrane cisternae within the spindle are most clearly visible at metaphase, and apparently represent the remains of the nuclear envelope. The nuclear envelope is disrupted during prometaphase and may become entangled in the spindle when centriolar microtubules enter the nucleus. Several concentric sheets of perinuclear endoplasmic reticulum surround the spindle and give the mitotic nucleus the superficial appearance of having an intact nuclear envelope. This interpretation of the identity of nucleus-associated membranes differs from those previously reported for other protists, including members of the Plasmodiophorales.