Control of mating and development in Ustilago maydis.

In the fungus Ustilago maydis, the ability to distinguish between partners that are of the same or of different mating type is controlled by two mating-type loci. One locus allows extracellular recognition though a pheromone-based system. After cell fusion, the other mating-type locus, which exists in multiple alleles, determines intracellular recognition. Each allele encodes a pair of homeodomain proteins that are active only in pairwise combinations in which the two partners originate from different alleles of the locus. Recent discoveries suggest that the underlying molecular recognition mechanism is the ability to form heterodimers. Whereas the proteins in all different allelic combinations interact, it is a specific feature of proteins from the same allele not to interact. This suggests the existence of a code for protein-protein recognition.

Characterization of a Ustilago maydis gene specifically induced during the biotrophic phase: evidence for negative as well as positive regulation.

The phytopathogenic basidiomycete Ustilago maydis requires its host plant, maize, for completion of its sexual cycle. To investigate the molecular events during infection, we used differential display to identify plant-induced U. maydis genes. We describe the U. maydis gene mig1 (for "maize-induced gene"), which is not expressed during yeast-like growth of the fungus, is weakly expressed during filamentous growth in axenic culture, but is extensively upregulated during plant infection. mig1 encodes a small, highly charged protein of unknown function which contains a functional N-terminal secretion sequence and is not essential for pathogenic development. Adjacent to mig1 is a second gene (mdu1) related to mig1, which appears to result from a gene duplication. mig1 gene expression during the infection cycle was assessed by fusing the promoter to eGFP. Expression of mig1 was absent in hyphae growing on the leaf surface but was detected after penetration and remained high during subsequent proliferation of the fungus until teliospore formation. Successive deletions as well as certain internal deletions in the mig1 promoter conferred elevated levels of reporter gene expression during growth in axenic culture, indicative of negative regulation. During fungal growth in planta, sequence elements between positions -148 and -519 in the mig1 promoter were specifically required for high levels of induction, illustrating additional positive control. We discuss the potential applications of mig1 for the identification of inducing compounds and the respective regulatory genes.

Fungal gene expression during pathogenesis-related development and host plant colonization.

To successfully infect plants, pathogenic fungi must recognize and communicate with their host during different stages of the disease cycle. In past years, techniques such as insertional mutagenesis, sensitive GFP-based reporter systems and microarray techniques have been developed to analyze these processes at the molecular level, and now novel insights into this fascinating aspect of pathogen-plant communication are beginning to emerge. This is exemplified by a number of pathogenicity genes functioning in distinct stages of pathogenic development in Magnaporthe grisea.

Fungal-plant signalling in the Ustilago maydis-maize pathosystem.

The smut fungus Ustilago maydis needs the host plant maize for completion of its sexual life cycle. Recent experiments highlight the importance of cAMP and mitogen-activated protein (MAP) kinase signalling for cell fusion as well as for subsequent stages of plant colonisation and induction of disease symptoms. During these distinct developmental stages the same signalling cascades must be differentially regulated and accommodate multiple inputs and outputs.

Crystallization of the DNA-binding Escherichia coli protein FIS.

The specific DNA-binding protein FIS (factor for inversion stimulation), which stimulates site-specific DNA inversion by interaction with an enhancer sequence, was purified from an Escherichia coli strain overproducing the protein. FIS was crystallized at room temperature by microdialysis against 1.2 to 1.5 M-sodium/potassium phosphate containing 10 mM-Tris.HCl, 0.5 to 1 M-NaCl and 1 mM-NaN3 at pH 8.0 to 8.2. The crystals are stout prisms and suitable for X-ray diffraction study beyond 2.5 A resolution. They belong to the orthorhombic space group P2(1)2(1)2(1). The unit cell has dimensions a = 47.57(4) A, b = 51.13(4) A, c = 79.83(6) A and contains one FIS dimer in the asymmetric unit.

A MADS-box homologue in Ustilago maydis regulates the expression of pheromone-inducible genes but is nonessential.

Mating and pathogenic development in the smut fungus Ustilago maydis are controlled by a pheromone/receptor system and two homeodomain proteins, bEp and bWp, which form heterodimers in nonallelic combinations. We describe the isolation of a gene, umc1, encoding a MADS-box protein, which displays significant similarity to the Saccharomyces cerevisiae MCM1 gene. umc1 complemented the viability defect of yeast mcm1 mutants. In U. maydis, umc1 deletion mutants were viable and pathogenic development was unaffected. Nevertheless, the basal expression levels of several pheromone-inducible genes were significantly reduced leading to an attenuated mating reaction. In contrast to S. cerevisiae, where Mcm1p plays a crucial role in the cell-type specific expression of a- and alpha-specific genes, the U. maydis umc1 gene appears to have only a modulatory effect on the expression of mating type-specific genes.

Activation of the cAMP pathway in Ustilago maydis reduces fungal proliferation and teliospore formation in plant tumors.

In the corn smut fungus Ustilago maydis, mating of two haploid sporidia is a prerequisite for subsequent colonization of the host. Cyclic AMP (cAMP) and pheromone signals have been implicated in this developmental program. The cAMP pathway is also needed for subsequent fungal development in planta, as null mutants in any component of the pathway fail to form tumors. Here we show that moderate activation of the pathway conferred either by mutation in the Galpha subunit or by mutation in the regulatory subunit of the protein kinase A influences tumor morphology. In the resulting tumors, the amount of fungal material is drastically reduced and fungal development is arrested at the stage of sporogenic hyphae. We conclude that tight regulation of the cAMP pathway is crucial for fungal development within the plant but does not interfere with the tumor induction process.

The sequence and mom-transactivation function of the C gene of bacteriophage Mu.

The mom gene of bacteriophage Mu encodes a DNA modification function. The gene is regulated on the transcriptional level by Dam-specific methylation and a trans-acting Mu function, and on a post-transcriptional level by the product of gene com. The gene encoding the transactivator has been cloned and mapped. By complementation analysis the activation function (also designated Dad) was shown to be the product of gene C. Transactivation of the mom promoter was shown in the following assay: the mom promoter and N-terminal part of com were fused in frame to lacZ. Cells containing such fusion plasmids were infected with M13 clones expressing C in the presence of IPTG and XGal. Successful transactivation results in the formation of blue plaques. Moreover, we have determined the sequence of gene C and found that it has a coding capacity of 140 amino acids. The promoter for C (pc) is likely to be located at least 0.5 kb upstream from the gene. A transcription terminator is found directly downstream from the C-coding region.

Post-transcriptional regulation of the bacteriophage Mu mom gene by the com gene product.

The mom gene of bacteriophage Mu encodes a DNA modification function, the expression of which is detrimental to the host cell. This may be reflected by the tight regulation of the mom gene at the level of transcription initiation by the Mu C gene product and the host Dam function. In addition, mom expression requires the positive regulatory function Com. The com and mom genes comprise the mom operon with the com coding region partially overlapping that of mom. The degree of overlap is defined by experiments reported here. We have tested Com for activity as an antiterminator of mom transcription. We show that in the absence of Com, premature termination affects at most 33% of the transcription across the mom operon. Although no premature termination is observed in the presence of Com, these results are inconsistent with a role for Com as an antiterminator. Northern blot analysis of Com+ and Com- Mu phage mRNA confirms this conclusion. Two models for the post-transcriptional regulation of mom gene expression by Com are presented.

The mom gene of bacteriophage mu: a unique regulatory scheme to control a lethal function.

The mom gene of bacteriophage Mu encodes a DNA modification function which converts adenine to acetamido adenine in a sequence-specific manner. The mom gene itself is subject to a complex regulation: gene expression requires methylation by the Escherichia coli Dam methylase of specific sites upstream of the mom promoter and transactivation of the promoter by a Mu gene product. The requirement for transactivation can be overcome when mom is transcribed from foreign promoters. When cloned into various sites in pBR322, the mom gene is always found in an orientation where transcription from vector promoters is excluded. The productive orientation is lethal to the cell. This effect is mediated by the concerted action of the mom gene product and the product of gene com (control of mom, previously termed ORF-x) whose coding region overlaps the 5-coding region of the mom gene. When mom is expressed from its own promoter, internal deletions in com completely abolish expression of the mom gene. Fragments lacking the 5' end of com can be cloned downstream of constitutive plasmid promoters. The com gene product itself is not lethal to the cell. The region encoding mom has been cloned in pL expression vectors. The mom gene product, a peptide of 27 kDal, has been visualized on gels. Efficient expression of Mom from pL requires gene com. A fusion between MS-2 polymerase and com has been generated. The fusion product is made in large amounts, whereas the mom gene product is not overproduced although the gene is present on the same transcriptional unit.(ABSTRACT TRUNCATED AT 250 WORDS)

Ustilago maydis Mating Hyphae Orient Their Growth toward Pheromone Sources

Snetselaar, K. M., Bolker, M., and Kahmann, R. 1996. Ustilago maydis mating hyphae orient their growth toward pheromone sources. Fungal Genetics and Biology 20, 299-312. When small drops of Ustilago maydis sporidia were placed 100-200 µm apart on agar surfaces and covered with paraffin oil, sporidia from one drop formed thin hyphae that grew in a zig-zag fashion toward the other drop if it contained sporidia making the appropriate pheromone. For example, a2b2 mating hyphae grew toward a1b1 and a1b2 mating hyphae, and the filaments eventually fused tip to tip. Time-lapse photography indicated that the mating hyphae can rapidly change orientation in response to nearby compatible sporidia. When exposed to pheromone produced by cells in an adjacent drop, haploid sporidia with the a2 allele began elongating before sporidia with the a1 allele. Sporidia without functional pheromone genes responded to pheromone although they did not induce a response, and sporidia without pheromone receptors induced formation of mating hyphae although they did not form mating hyphae. Diploid sporidia heterozygous at b but not at a formed straight, rigid, aerial filaments when exposed to pheromone produced by the appropriate haploid sporidia. Again, the a2a2b1b2 strain formed filaments more quickly than the a1a1b1b2 strain. Taken together, these results suggest that the a2 pheromone diffuses less readily or is degraded more quickly than the a1 pheromone.

Lumbosacral kyphosis, tethered cord, and diplomyelia. A unique spinal dysraphic condition.

Four patients are described with a triad of neuroectodermal abnormalities consisting of lumbosacral kyphosis, tethered cord, and dyplomyelia. Of utmost importance is the recognition of the association between this type of kyphosis and the underlying spinal cord pathology and the progressive nature of the deformity. The patient presenting with lumbosacral kyphos and the presence of sacral hypoplasia should have a neuroradiographic evaluation (magnetic resonance imaging scanning or enhanced computed tomography) to rule out the presence of a tethered cord or other neural abnormalities. Early surgical intervention for release of the tether and fusion should be carried out to prevent neurologic deterioration and curve progression.

In vivo facet joint loading of the canine lumbar spine.

This study describes a technique to measure in vivo loads and the resultant load-contact locations in the facet joint of the canine lumbar spine. The technique is a modification of a previously described in vitro method that used calibrated surface strains of the lateral aspect of the right L3 cranial articular process. In the present study, strains were measured during various in vivo static and dynamic activities 3 days after strain gage implantation. The in vivo recording technique and its errors, which depend on the location of the applied facet loads, is described. The results of applying the technique to five dogs gave the following results. Relative resultant contact load locations on the facet tended to be in the central and caudal portion of the facet in extension activities, central and cranial in standing, and cranial and ventral in flexion or right-turning activities. Right-turning contact locations were ventral and cranial to left-turning locations. Resultant load locations at peak loading during walking were in the central region of the facet, whereas resultant load locations at minimum loading during walking were relatively craniad. This resultant load-contact location during a walk gait cycle typically migrated in an arc with a displacement of 4 mm from minimum to maximum loading. Static tests resulted in a range of facet loads of 0 N in flexion and lying to 185 N for two-legged standing erect, and stand resulted in facet loads of 26 +/- 15 N (mean +/- standard deviation [SD]). Dynamic tests resulted in peak facet loads ranging from 55 N while walking erect to 170 N for climbing up stairs. Maximum walk facet loads were 107 +/- 27 N. The technique is applicable to in vivo studies of a canine facet joint osteoarthritis model and may be useful for establishing an understanding of the biomechanics of low-back pain.

Platelet function in adolescent idiopathic scoliosis.

Recent studies have reported abnormal platelet morphology and function in patients with adolescent idiopathic scoliosis. These abnormalities include increased platelet size and dense body numbers, abnormal aggregation, thromboxane A2 synthesis, serotonin release to adenosine diphosphate and epinephrine stimulus, and decreased myosin-adenosine-triphosphatase-specific activity. It was postulated that a membrane-specific defect in calcium transport may be partially responsible for the abnormalities found. In response to a suggestion in the literature that platelet screening could be clinically useful in scoliosis evaluation as well as in basic research of its pathophysiology, a study was performed to evaluate platelet morphology, biochemistry, and function in patients with adolescent idiopathic scoliosis. Platelets from nine volunteers with adolescent idiopathic scoliosis were compared with cells from a control group of nine patients. No significant differences in measured platelet parameters were noted between adolescent idiopathic scoliosis patients and control groups. Platelets from both groups demonstrated normal aggregation and release patterns with all agents except for a mild decreased aggregation and secretion response to epinephrine. No significant differences were noted in serotonin or adenine nucleotide levels. No significant ultrastructural differences were noted. Earlier findings of an abnormal aggregation and secretion response to adenosine diphosphate, increased numbers of dense bodies, or increased intracellular calcium could not be confirmed. On the contrary, we found normal, if not slightly decreased, numbers of dense bodies per platelet and calcium levels that were not different from controls.

Facet loads in the canine lumbar spine before and after disc alteration.

The effect of externally applied spinal loads on facet mechanics before and after facet capsulectomy and disc alteration was investigated. Four control and four chymopapain-treated specimens were tested. Facet load and resultant load location were determined in the unaltered state, after facet capsulectomy, and after discectomy using a newly developed, noninvasive measurement technique. Resultant contact load locations shifted in a predictable manner. Caudal shift in contact locations occurred with chymopapain treatment or discectomy but was variable with capsulectomy. Facet load changed with applied load magnitude and direction; no significant change was noted after intradiscal chymopapain, discectomy, or capsulectomy. The significance of the lack of force change with disc narrowing is unknown. Perhaps, observed facet degeneration results from a change in the joint motion and contact location rather than change in facet load.

[Limits to arthroscopic treatment of degenerative triangular fibrocartilage complex lesions depending on the ulnar variance]. / Grenzen der arthroskopischen Behandlung degenerativer Läsionen des ulnokarpalen Komplexes in Abhängigkeit der Ulnavarianz.

PURPOSE: The present prospective study investigated the influence of the static ulnar variance on the success of arthroscopic debridement of degenerative TFCC lesions. PATIENTS AND METHODS: 10 patients with an ulnar positive variance ("Ulna+") and 12 patients with ulnar neutral or ulnar negative variance ("Ulna-/0") were examined preoperatively (U0), as well as at 2 (U2) and 6 (U6) months after arthroscopic debridement of degenerative TFCC lesions and compared with each other. After the U2 investigation due to persistent complaints in 9 of 10 patients with an ulnar positive variance there was a need for further surgery, consisting of ulnar shortening osteotomy (USO). The following parameters were recorded in each case: pain at rest and with load, the summed wrist range of motion - consisting of extension and flexion, radial and ulnar deviation, pronation and supination - compared to the contralateral side, the strength of the affected hand compared to the contralateral side, the Mayo modified wrist score (MMWS), the Krimmer score and the DASH score. Preoperatively there were no significant differences between the 2 cohorts "Ulna+" and "Ulna-/0" except for the characteristic "pain at rest". RESULTS: At 2 months postoperatively (U2), the results in the cohort "Ulna+" remained at a significantly or tendentially poorer level compared to the cohort "Ulna-/0". The subsequent surgical treatment of the subgroup "Ulna+" with USO led to almost complete approximation of the results at 6 months postoperatively (U6). In addition to this, with time (U6) within each subgroup there were tendential or significant improvements of all characteristics compared to the preoperative situation (U0). At U6 four of 22 patients were -unable to work. CONCLUSION: Degenerative lesions of the TFCC can be treated successfully by arthroscopic debridement in cases of ulnar negative and ulnar neutral variance. Patients with ulnar positive variance and persistent complaints after debridement of the TFCC can be treated successfully with a secondary ulnar shortening osteotomy.

A H2O2-producing glyoxal oxidase is required for filamentous growth and pathogenicity in Ustilago maydis.

In the phytopathogenic fungus Ustilago maydis the mating-type loci control the transition from yeast-like to filamentous growth required for pathogenic development. In a large REMI (restriction enzyme mediated integration) screen, non-pathogenic mutants were isolated in a haploid strain that had been engineered to be pathogenic. In one of these mutants, which showed a specific morphological phenotype, the tagged gene, glo1 , was found to encode a product that is highly homologous to a glyoxal oxidase gene from the wood-rot fungus Phanerochaete chrysosporium. Glyoxal oxidase homologues are found in human, plant pathogenic fungi and in plants, but not in other mammals or yeasts. To confirm the function of the glo1 gene, null mutations were generated in compatible haploid U. maydis strains. In crosses null mutants were unable to generate filamentous dikaryons, and were completely non-pathogenic. Using a Glo1-overproducing strain we demonstrated that Glo1 is membrane bound, oxidizes a series of small aldehydes (< C4) and produces H2O2. The enzyme needs to be activated, presumably by auto-oxidation, to show full activity. A potential role for Glo1 during filamentous growth and pathogenic development of U. maydis is proposed.