Dynamic distribution of BIMG(PP1) in living hyphae of Aspergillus indicates a novel role in septum formation.

Mutation of bimG, the major protein phosphatase 1 gene in Aspergillus nidulans, causes multiple cell cycle and hyphal growth defects that are associated with overphosphorylation of subcellular components. We have used functional translational fusions with the green fluorescent protein (GFP) to show that BIMG has at least four discrete locations within growing hyphae. Three of these locations, the hyphal tip, the spindle pole body and the nucleus, correlate with previously known requirements for bimG(PP1) in mitosis and hyphal growth and are highly dynamic. BIMG-GFP in the hyphal tip seemed to be associated with the plasma membrane and formed a collar of fluorescence within the apical dome. The distribution of nuclear BIMG-GFP varied depending on nutritional conditions; on poor medium, it concentrated more in the nucleolus than in the nucleoplasm, whereas on rich medium, it was more evenly distributed between the two nuclear regions. The association of BIMG-GFP with developing septa was transient, and we present evidence that BIMG phosphatase plays a direct role in septum formation, distinct from its role in mitosis. We conclude that, by being physically present at several sites, the BIMG phosphatase has roles in multiple cellular processes.

A type 2A protein phosphatase gene from Aspergillus nidulans is involved in hyphal morphogenesis.

A PCR-based approach, using degenerate oligonucleotide primers, was used to isolate fragments of two genes encoding type 2A protein phosphatases from the filamentous fungus, Aspergillus nidulans. The complete genomic sequence of one of these genes, pphA, was isolated and characterised. The pphA gene was predicted to encode a 329-residue protein which is about 85% identical to mammalian protein phosphatase 2A. Ectopic expression of the wild-type pphA+ gene slightly inhibited growth in some transformants; but a mutant form of pphA, in which R259 was mutated to Q, led to slow growth, delayed germ tube emergence and mitotic defects at low temperature. These results indicate that the pphA+ gene plays an important role in hyphal growth.

The expression of D-cyclin genes defines distinct developmental zones in snapdragon apical meristems and is locally regulated by the Cycloidea gene.

Three D-cyclin genes are expressed in the apical meristems of snapdragon (Antirrhinum majus). The cyclin D1 and D3b genes are expressed throughout meristems, whereas cyclin D3a is restricted to the peripheral region of the meristem, especially the organ primordia. During floral development, cyclin D3b expression is: (a) locally modulated in the cells immediately surrounding the base of organ primordia, defining a zone between lateral organs that may act as a developmental boundary; (b) locally modulated in the ventral petals during petal folding; and (c) is specifically repressed in the dorsal stamen by the cycloidea gene. Expression of both cyclin D3 genes is reduced prior to the cessation of cell cycle activity, as judged by histone H4 expression. Expression of all three D-cyclin genes is modulated by factors that regulate plant growth, particularly sucrose and cytokinin. These observations may provide a molecular basis for understanding the local regulation of cell proliferation during plant growth and development.

sodVIC is an alpha-COP-related gene which is essential for establishing and maintaining polarized growth in Aspergillus nidulans.

Strains of Aspergillus nidulans carrying the conditional-lethal mutation sodVIC1 (stabilization of disomy) are defective in nuclear division and hyphal extension. The mutation affects both the establishment and maintenance of polar growth, since mutant spores do not germinate at restrictive temperature and preexisting hyphae stop growing upon upshift. The defect is reversible within the first 3-4 h at restrictive temperature but longer periods of incubation are lethal due to cell lysis and morphological abnormalities. There is no evidence for a specific cell cycle lesion, suggesting the existence of a feedback mechanism whereby hyphal extension is coordinated with nuclear partitioning. The sodVIC gene has been cloned from a chromosome VI-specific cosmid library and its product exhibits strong homology to the alpha-COP subunit of the coatomer complex involved in the secretory pathway in yeast and higher organisms. Molecular disruption of the gene is lethal, indicating that SodVIC is essential for growth in A. nidulans.

Distinct classes of cdc2-related genes are differentially expressed during the cell division cycle in plants.

cdc2 and several related genes encode the catalytic subunits of cyclin-dependent kinases, which have been implicated in a number of cellular processes, including control of cell division. As a first step in exploring their function in plants, we isolated four cdc2-related genes from Antirrhinum. Two genes, cdc2a and cdc2b, encode proteins that contain a perfectly conserved PSTAIRE motif characteristic of cdc2 homologs, whereas the products of the two remaining genes, cdc2c and cdc2d, appear to represent a new subclass of proteins that have so far only been identified in plants. Transcripts of these novel genes were localized in isolated cells dispersed throughout actively dividing regions of the inflorescence. This localization is consistent with accumulation that is specific to particular phases of the cell cycle. Correlating cell labeling with nuclear condensation and double-labeling experiments using cdc2 and histone H4 as probes indicated that cdc2c transcripts accumulate during S phase as well as during the G2 and M transition, whereas cdc2d expression was specific to the G2 and M phases. All cells labeled with cdc2d also contained cdc2c label, Indicating that expression of cdc2d completely overlapped with that of cdc2c. Transcripts of cdc2a and cdc2b were detected in all cells within actively dividing regions, but at levels that were only slightly higher than those observed in nondividing areas. These transcripts did not appear to accumulate in a cell cycle-specific fashion. The genes cdc2a and cdc2b were able to partially complement a yeast cdc2 mutation, although all four genes appeared to interfere with the sizing mechanism of yeast cells. We propose that plants contain at least two classes of cdc2-related genes that differ in structure, expression, and perhaps function.

A temperature-sensitive splicing mutation in the bimG gene of Aspergillus produces an N-terminal fragment which interferes with type 1 protein phosphatase function.

Progression through anaphase requires high levels of type 1 protein phosphatase (PP1) activity in a variety of eukaryotes, including Aspergillus nidulans. A conditional lethal, temperature-sensitive mutant in one of the Aspergillus PP1 genes, bimG, prevents the normal completion of anaphase when cells are grown at restrictive temperature and this has been shown to be due to a reduction in type 1 phosphatase activity. We show that the bimG11 allele is recessive to the wild-type allele in heterozygous diploids, implying that the mutation is due to loss of function at restrictive temperature, but molecular disruption of the wild-type bimG gene shows that the gene is not essential and has no discernable phenotype under laboratory conditions. Sequence comparison of wild-type and mutant alleles reveals a single base pair difference between the two genes, within the 5' splicing site of the second intron. We demonstrate that the conditional lethal phenotype of bimG11 strains is due to impaired splicing of the mutant mRNA and that this leads to the production of a truncated protein comprising an intact N-subdomain and a modified C-terminus. Over-expression of this truncated form of PP1 in a wild-type haploid produces a lethal phenotype and reduced PP1 activity, supporting the idea that a toxic interfering protein is produced. PP1, therefore, may have at least two spatially separated sites, both of which are required for function. Temperature-sensitive splicing mutations may provide a novel means of engineering conditional versions of other proteins, particularly other phosphatases.

Fungal pathogens of oat roots and tomato leaves employ closely related enzymes to detoxify different host plant saponins.

Antifungal saponins are produced by many plants and have been implicated as preformed determinants of resistance to fungal attack. The importance of saponin detoxification in fungal pathogenesis has recently been demonstrated for the fungus Gaeumannomyces graminis var. avenae, which produces the enzyme avenacinase. Avenacinase detoxifies the triterpenoid oat root saponin avenacin A-1, and is essential for pathogenicity of G. graminis var.avenae to oats. Here we demonstrate an unexpected relatedness between avenacinase and the tomatinase enzyme produced by Septoria lycopersici (a tomato leaf-infecting fungus), which acts on the steroidal glycoalkaloid alpha-tomatine. The two enzymes share common physicochemical properties and are immunologically cross-reactive; however, there are critical differences in their substrate specificities which reflect the host preferences of the fungi from which the enzymes were purified. The DNA encoding tomatinase was isolated from a S. lycopersici cDNA library using avenacinase DNA as a probe. Comparison of the predicted amino acid sequences of avenacinase and tomatinase revealed that the enzymes are clearly similar.

A novel Arabidopsis type 1 protein phosphatase is highly expressed in male and female tissues and functionally complements a conditional cell cycle mutant of Aspergillus.

Type 1 protein phosphatases are very highly conserved throughout eukaryotes where they regulate a number of key metabolic and morphogenetic processes. A cDNA, AtPP1bg, representing a new member of the type 1 protein phosphatase gene family in Arabidopsis has been isolated on the basis of hybridization with the Aspergillus bimG protein phosphatase gene. The AtPP1bg gene potentially encodes a 37 kDa protein very closely related to PP1 but with divergent N- and C-termini. The predicted amino acid sequence shows 71% identity to the ORF of the bimG gene. When expressed in Aspergillus under the alcA promoter, this phosphatase complements the temperature-sensitive bimG11 mutation allowing nearly normal vegetative growth at 37 degrees C (but not at 42 degrees C). Notably, the plant PP1 does not support morphogenesis (conidiation) at 37 degrees C. This may indicate that conidophore formation has particular phosphatase requirement(s) which the plant PP1 cannot supply. The pattern of expression of the AtPP1bg transcript has been studied during development of the plant. In situ hybridization of Arabidopsis with antisense probes shows that this phosphatase gene is expressed at a low level throughout the plant, but is strongly upregulated within developing flowers, especially in the tapetum, the developing and mature pollen and in the ovaries. This implies that the AtPP1bg either has a specialized role in the formation of these organs, or that there is an increased requirement for protein phosphatase 1 at these stages. It was found that the level of AtPP1bg transcript, as judged by the relative intensity of staining in different cells within the floral meristems, did not vary during the cell cycle.

Host range of a plant pathogenic fungus determined by a saponin detoxifying enzyme.

Antifungal saponins occur in many plant species and may provide a preformed chemical barrier to attack by phytopathogenic fungi. Some fungal pathogens can enzymatically detoxify host plant saponins, which suggests that saponin detoxification may determine the host range of these fungi. A gene encoding a saponin detoxifying enzyme was cloned from the cereal-infecting fungus Gaeumannomyces graminis. Fungal mutants generated by targeted gene disruption were no longer able to infect the saponin-containing host oats but retained full pathogenicity to wheat (which does not contain saponins). Thus, the ability of a phytopathogenic fungus to detoxify a plant saponin can determine its host range.

Analysis of the genetic variability of maize streak virus.

The nucleotide sequences of the small intergenic region (SIR) and the gene encoding the coat protein of 12 maize streak virus (MSV) isolates from different geographic locations have been determined. These have been used to assess the variability of the virus and to construct evolutionary dendrograms. For the viruses analyzed, the maximum levels of sequence divergence were found to be 10.9% and 2.0% at the nucleotide and amino acid levels, respectively. A genetically distinct strain of MSV was collected from islands in the Indian ocean. The significance of these findings for detection of the virus in epidemiological studies and breeding of resistant plant varieties is discussed.

Rapid production of full-length, infectious geminivirus clones by abutting primer PCR (AbP-PCR).

The application of the polymerase chain reaction (PCR) method of DNA amplification for the isolation of full-length, infectious clones of geminiviruses is described. Non-overlapping, abutting 20-mer oligonucleotide primers were used to produce a linear product from the circular geminivirus genomic template. Clones of African cassava mosaic virus (ACMV) DNA A, obtained by this method, were infectious following mechanical inoculation (in the presence of ACMV DNA B) onto Nicotiana benthamiana. Normal ACMV symptoms resulted and typical geminate viral particles were detected by electron microscopy. The use of PCR for the detection and production of full-length, infectious geminivirus clones is discussed.

The nucleotide sequence of an infectious insect-transmissible clone of the geminivirus Panicum streak virus.

The infectious genome of a Kenyan isolate of Panicum streak virus (PSV) has been cloned and sequenced. Infection of host plants was done using an Agrobacterium binary vector containing a partial repeat of the genome. Progeny virus from resultant infections proved to be transmissible by the leafhopper Cicadulina mbila (Naude). Comparisons of the amino acid sequences of PSV DNA-encoded proteins with those of previously characterized geminiviruses infecting monocotyledonous plants, including maize streak virus, revealed high levels of identity. The evolutionary relationship between PSV and other geminiviruses infecting monocotyledons is discussed.