[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.

Identification of plant-regulated genes in Ustilago maydis by enhancer-trapping mutagenesis.

To identify plant-induced genes in the maize pathogenic fungus Ustilago maydis we have developed a genetic screen that combines REMI (restriction enzyme mediated integration) mutagenesis with enhancer trapping using the gene for Green Fluorescent Protein (GFP) as vital reporter. Of 2,350 insertion mutants isolated, three were shown to express GFP only after the fungus had come into contact with the host maize plant. One of the genes tagged was mfa1, which encodes the pheromone precursor, while the second gene, pig2, codes for a product that showed similarity to protein disulfide isomerase. The third integration event had occurred in a locus which we designated the p -locus. This locus contains 11 genes in a 24-kb stretch. Of these, pig3, 4, 5, 6 and 7 show a plant-regulated expression pattern, while the other genes found at the locus (designated npi) do not. Of the plant-regulated genes only two were found to be similar to database entries: the pig4 product is related to membrane transporters of the major facilitator family, while the pig6 protein shows similarity to multidrug transporters. Detailed expression studies revealed that the five plant-regulated genes at the p -locus differ in their expression profiles. Mutants deleted for each of them showed no apparent phenotype, while the npi1 gene appeared to be essential. A viable deletion encompassing the entire p -locus could be generated when npi1 function was provided ectopically. This deletion mutant also showed no obvious alteration in virulence.

Identification of genes in the bW/bE regulatory cascade in Ustilago maydis.

In the phytopathogenic fungus Ustilago maydis, the switch to filamentous growth and pathogenic development is controlled by a heterodimeric transcription factor consisting of the bW and bE homeodomain proteins. To identify genes in the regulatory cascade triggered by the bW/bE heterodimer, we have constructed strains in which transcription of the b genes is inducible by either arabinose or nitrate. At different time-points after induction, genes that are switched on or off were identified through a modified, non-radioactive RNA fingerprint procedure. From 348 gene fragments isolated initially, 48 fragments representing 34 different genes were characterized in more detail. After eliminating known genes, false positives and genes influenced in their expression profile by media conditions, 10 new b-regulated genes were identified. Of these, five are upregulated and five are downregulated in presence of the b heterodimer. Two do not share significant similarity to database entries, whereas the other eight show similarity to disulphide isomerases, exochitinases, cation antiporters, plasma membrane (H+)-ATPases, acyl transferases, a capsular associated protein of Cryptococcus neoformans, DNA polymerases X, as well as to a potential protein of Neurospora crassa. We demonstrate that in one of the early upregulated genes, the promoter can be bound by a bW/bE fusion protein in vitro. Interestingly, three out of the four genes that are downregulated by the b heterodimer appear upregulated after pheromone stimulation, suggesting a connection to the mating process.

A split motor domain in a cytoplasmic dynein.

The heavy chain of dynein forms a globular motor domain that tightly couples the ATP-cleavage region and the microtubule-binding site to transform chemical energy into motion along the cytoskeleton. Here we show that, in the fungus Ustilago maydis, two genes, dyn1 and dyn2, encode the dynein heavy chain. The putative ATPase region is provided by dyn1, while dyn2 includes the predicted microtubule-binding site. Both genes are located on different chromosomes, are transcribed into independent mRNAs and are translated into separate polypeptides. Both Dyn1 and Dyn2 co-immunoprecipitated and co-localized within growing cells, and Dyn1-Dyn2 fusion proteins partially rescued mutant phenotypes, suggesting that both polypeptides interact to form a complex. In cell extracts the Dyn1-Dyn2 complex dissociated, and microtubule affinity purification indicated that Dyn1 or associated polypeptides bind microtubules independently of Dyn2. Both Dyn1 and Dyn2 were essential for cell survival, and conditional mutants revealed a common role in nuclear migration, cell morphogenesis and microtubule organization, indicating that the Dyn1-Dyn2 complex serves multiple cellular functions.

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.

A homologue of the transcriptional repressor Ssn6p antagonizes cAMP signalling in Ustilago maydis.

In Ustilago maydis, cAMP signalling is crucial for successful infection of maize plants. Strains are non-pathogenic if mutated in any of the currently identified components of this signalling pathway, such as the alpha-subunit of a heterotrimeric G protein Gpa3, the adenylate cyclase Uac1 and the regulatory and catalytic subunit of protein kinase A Ubc1 and Adr1 respectively. Deletion of gpa3, uac1 or adr1 triggers filamentous growth, and certain point mutations in gpa3 and ubc1 that mimic a high cAMP level display a glossy colony phenotype. Screening an autonomously replicating U. maydis library in such a background (gpa3Q206L), we identified sql1 as a suppressor of the glossy colony phenotype. Interestingly, only alleles carrying C-terminal truncations of Sql1 were able to complement the mutant phenotype, suggesting a gain-of-function by these variants. Sql1 is a functional homologue of the yeast transcriptional repressor Ssn6p and contains 10 tetratricopeptide repeats (TPRs), of which the first six are important for suppressor function. Truncated sql1 alleles that suppress the glossy colony phenotype of gpa3Q206L strains induce filamentous growth when introduced in wild type. Filamentation of these strains is reversed in the presence of cAMP. We present a model in which Sql1 is part of an evolutionary conserved Sql1-Tup1 transcriptional repressor complex that antagonizes cAMP signalling by repressing cAMP-regulated 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.

A protein with similarity to the human retinoblastoma binding protein 2 acts specifically as a repressor for genes regulated by the b mating type locus in Ustilago maydis.

Pathogenic development in the corn smut fungus Ustilago maydis is controlled by a heterodimer of the two homeodomain proteins bE and bW which are encoded by the b mating type locus. The bE/bW heterodimer is thought to achieve its function as a transcriptional regulator of pathogenicity genes, either directly by binding to cis regulatory sequences or indirectly via a b-dependent regulatory cascade. In a screen for components of the b-dependent regulatory cascade we have isolated Rum1 (regulator U. maydis 1), a protein with similarities to the human retinoblastoma binding protein 2. Deletion of rum1 results in expression of several b regulated genes independently from their activation via the bE/bW heterodimer. rum1 mutant strains remain pathogenic, proliferate in planta, but fail to produce spores. The defect leads to an arrest in spore development at a defined stage before the spore wall is generated. Deduced from the highly conserved domain structure of Rum1 that includes a DNA-binding motif and a region known to facilitate the interaction with histone deacetylases, we propose that Rum1 functions as a transcriptional repressor through the modulation of chromatin structure.

Identification of a target gene for the bE-bW homeodomain protein complex in Ustilago maydis.

In the phytopathogenic fungus Ustilago maydis, sexual and pathogenic development are controlled by the multiallelic b mating type locus. The b locus encodes a pair of unrelated homeodomain proteins termed bE and bW that form a heterodimeric complex when both proteins originate from different alleles. The heterodimer is presumed to be the central regulator for pathogenicity genes. Here, we show that a translational fusion protein comprising specific domains of bE1 and bW2 remains biologically active and binds to a sequence motif in the promoter of lga2, a gene located in the a mating type locus. This b binding sequence 1 (bbs1) is also recognized by the native bE1-bW2 heterodimer in vivo and mediates the b-dependent regulation of the lga2 gene. Our data demonstrate that the bE-bW heterodimer can act as a positive transcriptional regulator.

A putative endosomal t-SNARE links exo- and endocytosis in the phytopathogenic fungus Ustilago maydis.

We identified a temperature-sensitive mutant of the plant pathogenic fungus Ustilago maydis that is defective in the polar distribution of cell wall components and shows abnormal morphology. The affected gene, yup1, was cloned by complementation. It encodes a putative target soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (t-SNARE), suggesting a function in membrane fusion. A Yup1-GFP fusion protein localized to vesicles that showed rapid saltatory motion along microtubules. These vesicles are part of the endocytic pathway and accumulate at sites of active growth, thereby supporting the expansion of the hyphal tip. In yup1(ts) cells, endocytosis is impaired and accumulation of Yup1-carrying endosomes at cell poles is abolished, resulting in apolar distribution of wall components and morphological alterations. This suggests that a membrane recycling process via early endosomes supports polar growth of U. maydis.

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.

The MAP kinase kpp2 regulates mating and pathogenic development in Ustilago maydis.

In the phytopathogenic fungus Ustilago maydis, fusion of compatible haploid cells is a prerequisite for infection. This process is genetically controlled by the biallelic a locus, encoding pheromone precursors and receptors. These are presumed to be coupled to a heterotrimeric G protein and a MAP kinase cascade, leading to activation of the HMG domain transcription factor Prf1. Here, we have demonstrated that putative MAP kinase sites in Prf1 are required for its activity during mating. In addition, we have identified a gene, kpp2, which encodes a putative MAP kinase related to Pmk1 of Magnaporthe grisea and Fus3p of Saccharomyces cerevisiae. kpp2 deletion mutants are attenuated in several steps of development: cell fusion, induction of pheromone-responsive genes and pathogenicity. Epistasis analysis shows that kpp2 does not affect pheromone gene expression through the cAMP signalling cascade. Pathogenicity of kpp2 mutants can be partially restored by overexpressing the b genes, indicating a regulation of Prf1 by Kpp2. These data support the hypothesis that the MAP kinase Kpp2 transmits the pheromone signal.

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.

Environmental signals controlling sexual development of the corn Smut fungus Ustilago maydis through the transcriptional regulator Prf1.

Environmental signals induce and coordinate discrete morphological transitions during sexual development of Ustilago maydis. In this fungus, mating of two compatible haploid sporidia is a prerequisite for plant infection. Cell fusion is governed by the action of pheromones and receptors, whereas the subsequent pathogenicity program is controlled by the combinatorial interaction of homeodomain proteins. The U. maydis pheromone response factor (Prf1) is a central regulator of both processes. We have analyzed the regulation of the prf1 gene and demonstrate that pheromone and cAMP signaling regulate prf1 post-transcriptionally. Transcriptional activation of prf1 was observed in the presence of carbon sources, such as glucose and fructose, allowing us to define the cis-acting element in the prf1 promoter that mediates these effects. The same element provides for negative control of prf1 gene transcription at high cAMP levels. A protein that specifically binds to this element was purified and analyzed for its role in prf1 gene regulation. On the basis of these results, we present a model in which prf1 integrates different environmental signals to control development in U. maydis.

Crosstalk between cAMP and pheromone signalling pathways in Ustilago maydis.

In the phytopathogenic basidiomycete Ustilago maydis mating and dikaryon formation are controlled by a pheromone/receptor system and the multiallelic b locus. Recently, a gene encoding a G protein alpha subunit, gpa3, was isolated and has subsequently been implicated in pheromone signal transduction. Mutants deleted for gpa3 are sterile and nonpathogenic, and exhibit a morphology that is similar to that of mutants with defects in the adenylate cyclase gene uac1. We have found that the sterility and mutant morphology of gpa3 deletion strains can be rescued by exogenous cAMP. In these mutants and in the corresponding wild-type strains, exogenous cAMP stimulates pheromone gene expression to a level comparable to that seen in the pheromone-stimulated state. In addition, we demonstrate that uac1 is epistatic to gpa3. We conclude that Gpa3 controls the cAMP signalling pathway in U. maydis and discuss how this pathway feeds into the pheromone response.

Kinesin from the plant pathogenic fungus Ustilago maydis is involved in vacuole formation and cytoplasmic migration.

A gene encoding the heavy chain of conventional kinesin (kin2) has recently been identified in the dimorphic fungus Ustilago maydis (Lehmler et al., 1997). From the phenotype of kin2 null-mutants it was concluded that Kin2 might be involved in vesicle traffic towards the tip. However, this model did not explain why kin2-null mutant hyphae were unable to create empty cell compartments that are normally left behind the growing tip cell. Here we present a re-investigation of the function of Kin2 in hyphae and sporidia. We provide evidence that suggests a different and unexpected role of this kinesin motor in hyphal growth of Ustilago maydis. In addition, Kin2 was partially purified from U. maydis and in vitro properties were investigated. Isolated kinesin supported in vitro microtubule gliding at speeds of up to 1.8 micron/second, and showed motility properties and hydrodynamic behavior similar to those described for kinesin from N. crassa. It appears to be the product of the kin2 gene. Compared with wild-type sporidia, the kin2-null mutant sporidia grew normally but were defective in accumulation of Lucifer Yellow in their vacuoles, which were smaller than normal and often misplaced. The dikaryotic hyphae, produced by the fusion of two kin2-null sporidia, showed tip growth, but unlike wild-type hyphae, these structures lacked the large, basal vacuole and contain significantly more 200-400 nm vesicles scattered over the hole hypha. This defect was accompanied by a failure to generate regular empty cell compartments that are left behind in wild-type tip cells as the hyphae grow longer. These results suggest that Kin2 is a microtubule-dependent motor enzyme which is involved in the formation of vacuoles. The accumulation of these vacuoles at the basal end of the tip cell might be crucial for the formation of the empty sections and supports cytoplasmic migration during the growth of dikaryotic hyphae.

Regulation of crp transcription by oscillation between distinct nucleoprotein complexes.

FIS belongs to the group of small abundant DNA-binding proteins of Escherichia coli. We recently demonstrated that, in vivo, FIS regulates the expression of several genes needed for catabolism of sugars and nucleic acids, a majority of which are also transcriptionally regulated by cAMP-cAMP-receptor protein (CRP) complex. Here we provide evidence that FIS represses transcription of the crp gene both in vivo and in vitro. Employing crp promoter-lacZ fusions, we demonstrate that both FIS and cAMP-CRP are required to keep the crp promoter in a repressed state. We have identified in the crp promoter other transcription initiation sites which are located 73, 79 and 80 bp downstream from the previously mapped start site. Two CRP- and several FIS-binding sites with different affinities are located in the crp promoter region, one of them overlapping the downstream transcription initiation sites. We show that initiation of transcription at the crp promoter is affected by the composition of nucleoprotein complexes resulting from the outcome of competition between proteins for overlapping binding sites. Our results suggest that the control of crp transcription is achieved by oscillation in the composition of these regulatory nucleoprotein complexes in response to the physiological state of the cell.

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.

Stimulation of DNA inversion by FIS: evidence for enhancer-independent contacts with the Gin-gix complex.

Efficient DNA inversion catalysed by the invertase Gin requires the cis-acting recombinational enhancer and the Escherichia coliFIS protein. Binding of FIS bends the enhancer DNA and, on a negatively supercoiled DNA inversion substrate, facilitates the formation of a synaptic complex with specific topology. Previous studies have indicated that FIS-independent Gin mutants can be isolated which have lost the topological constraints imposed on the inversion reaction yet remain sensitive to the stimulatory effect of FIS. Whether the effect of FIS is purely architectural, or whether in addition direct protein contacts between Gin and FIS are required for efficient catalysis has remained an unresolved question. Here we show that FIS mutants impaired in DNA binding are capable of either positively or negatively affecting the inversion reaction both in vivo and in vitro. We further demonstrate that the mutant protein FIS K25E/V66A/M67T dramatically enhances the cleavage of recombination sites by FIS-independent Gin in an enhancer-independent manner. Our observations suggest that FIS plays a dual role in the inversion reaction and stimulates both the assembly of the synaptic complex as well as DNA strand cleavage.