Mcchs1, a member of a chitin synthase gene family in Mucor circinelloides, is differentially expressed during dimorphism.

A complete chitin synthase gene and one chitin synthase gene fragment of the zygomycete Mucor circinelloides have been cloned and analyzed. Both genes encode zymogenic Class II chitin synthases. Hybridization analysis showed that there must exist at least another Class II chitin synthase gene in M. circinelloides highly homologous to the cloned Mcchs1 and Mcchs2. The expression of these genes during the dimorphic growing stages was analyzed. Northern hybridizations showed that Mcchs1 transcript accumulates only during the exponentially growing hyphal stage, while no expression could be detected in the yeast form. Expression of Mcchs2 could not be detected at any stage. Accumulation of Mcchs1 transcript was not influenced by visible light. The existence of a multigene chitin synthase family and the observation that Mcchs1 transcription depends upon the dimorphic stage indicate that various chitin synthase activities may have different roles in the dimorphic growth of M. circinelloides.

A bifunctional enzyme with lycopene cyclase and phytoene synthase activities is encoded by the carRP gene of Mucor circinelloides.

Using functional analyses in Escherichia coli and Mucor circinelloides, it has been shown that a single M. circinelloides gene (carRP) codes for a protein with two different enzymatic activities, lycopene cyclase and phytoene synthase, which are encoded by independent genes in organisms other than fungi. This gene was identified using complementation tests among different classes of carotenoid mutants of M. circinelloides. The carRP gene product contains two domains: the R domain is located at the N-terminus and determines lycopene cyclase activity; the P domain is located at the C-terminus and displays phytoene synthase activity. The R domain is functional even in the absence of the P domain, while the latter needs the proper R domain conformation to carry out its function. The carRP gene is closely linked to the phytoene dehydrogenase (carB) gene, and the promoter regions of both genes are located within only 446 bp. Northern analyses show a co-ordinated regulation of the expression of both genes by blue light. Several motifs found in this promoter region suggest a bi-directional mode of transcription control.

Blue-light regulation of phytoene dehydrogenase (carB) gene expression in Mucor circinelloides.

The carB gene, encoding the phytoene dehydrogenase of Mucor circinelloides, was isolated by heterologous hybridisation with a probe derived from the corresponding gene of Phycomyces blakesleeanus. The cDNA and genomic copies complemented phytoene dehydrogenase defects in Escherichia coli and in carB mutants of M. circinelloides, respectively. Fluence-response curves for transcript accumulation were constructed after different blue-light pulses. The level of carB mRNA accumulation reached values up to 150-fold higher than basal levels in darkness. Several elements in the promoter of this gene resemble a consensus sequence identified in Neurospora crassa (APE) which is essential for blue-light regulation. Comparison of the available phytoene dehydrogenase sequences from plants, fungi, algae and bacteria suggests that the two known types of phytoene dehydrogenase are more closely related to each other than previously thought.

[Pathogenicity factors in Botrytis cinerea]. / Factores de patogenicidad de Botrytis cinerea.

Botrytis cinereais an important plant pathogenic fungi with a wide host range, which can make use of different infection mechanisms. Although genetic variation for resistance to B. cinereahas been observed within some species, no gene-for-gene relationship has been found. The development of resistant genotypes is, therefore, complicated. Any attempt to develop control strategies makes it necessary a detailed knowledge of both the fungal infection mechanisms and the plant defence mechanisms. The application of different experimental approaches allows the analysis of the infection process in different hosts, the description of the elements that participate in each stage of the process and the identification of those pathogenicity factors which are essential for the establishment of the interaction. The characterisation of the latter will provide information about key elements of the infection process as the basis for the development of effective, long term and environmentally friendly control strategis.

Heterologous expression of the Phycomyces blakesleeanus phytoene dehydrogenase gene (carB) in Mucor circinelloides.

A phytoene dehydrogenase-deficient mutant of Mucor circinelloides accumulating only phytoene was transformed with the gene encoding the corresponding enzyme (carB gene) of Phycomyces blakesleeanus. Carotenoids derived from phytoene were detected in the transformants showing that the P. blakesleeanus carB gene complements the M. circinelloides carB mutation. These newly formed carotenoids accumulated in low quantities, indicating that functional complementation was poor. carB mRNA molecules correctly transcribed were detected in the transformants, but they represented a small proportion of the total population of carB-derived mRNAs, mostly constituted by truncated transcripts and by transcripts longer than the transcript that is functional in Phycomyces. These results showed that the P. blakesleeanus carB gene was expressed in M. circinelloides and suggested that the poor complementation observed was owing, at least in part, to the lack of specificity in the recognition of the transcription initiation and termination signals of the P. blakesleeanus carB gene by the M. circinelloides transcriptional machinery.

Genetic diversity of Fusarium oxysporum strains from common bean fields in Spain.

Fusarium wilt is an endemic disease in El Barco de Avila (Castilla y León, west-central Spain), where high-quality common bean cultivars have been cultured for the last century. We used intergenic spacer (IGS) region polymorphism of ribosomal DNA, electrophoretic karyotype patterns, and vegetative compatibility and pathogenicity analyses to assess the genetic diversity within Fusarium oxysporum isolates recovered from common bean plants growing in fields around El Barco de Avila. Ninety-six vegetative compatibility groups (VCGs) were found among 128 isolates analyzed; most of these VCGs contained only a single isolate. The strains belonging to pathogenic VCGs and the most abundant nonpathogenic VCGs were further examined for polymorphisms in the IGS region and electrophoretic karyotype patterns. Isolates belonging to the same VCG exhibited the same IGS haplotype and very similar electrophoretic karyotype patterns. These findings are consistent with the hypothesis that VCGs represent clonal lineages that rarely, if ever, reproduce sexually. The F. oxysporum f. sp. phaseoli strains recovered had the same IGS haplotype and similar electrophoretic karyotype patterns, different from those found for F. oxysporum f. sp. phaseoli from the Americas, and were assigned to three new VCGs (VCGs 0166, 0167, and 0168). Based on our results, we do not consider the strains belonging to F. oxysporum f. sp. phaseoli to be a monophyletic group within F. oxysporum, as there is no correlation between pathogenicity and VCG, IGS restriction fragment length polymorphism, or electrophoretic karyotype.

Complementary mating types of Mucor circinelloides show electrophoretic karyotype heterogeneity.

Electrophoretic karyotypes of ten strains of Mucor circinelloides f. lusitanicus were generated by contour-clamped homogeneous electric field (CHEF) gel-electrophoresis. Most of the strains analyzed showed polymorphisms, but a different main karyotype pattern could be correlated with each mating type. Genome structure was further analyzed by gene assignment to the chromosome-sized DNAs. Nonradioactive hybridization techniques identified the chromosomal localization of seven cloned genes. The hybridization patterns confirmed the similarity between the mating-type (-) strains and showed some heterogeneity among the mating-type (+) strains. Linkage was found between genes pyrF and chs3 in all the strains, between the gene leuA and the rDNA in all mating-type (-) strains and ATCC 1216b (+), and between chs2 and the rDNA in CBS 969.68 (+). This is the first time that gene linkage in M. circinelloides has been reported, but in some cases the linkage relationships obtained are strain-dependent.

Interallelic complementation at the pyrF locus and the homodimeric nature of orotate phosphoribosyltransferase (OPRTase) in Mucor circinelloides.

Using 5-fluoroorotic acid (5-FOA) as a positive selection system we isolated mutants of Mucor circinelloides altered in the pyrimidine biosynthetic pathway. These mutants were found to be deficient either in orotidine-5'-monophosphate decarboxylase (OMPdecase), or in orotate phosphoribosyltransferase (OPRTase) activity. Complementation tests among mutants lacking OPRTase activity classified them into three groups, thus suggesting the possibility of interallelic complementation. To investigate this hypothesis a cDNA clone corresponding to the OPRTase-encoding gene of M. circinelloides was isolated by direct complementation of E. coli. The genomic copy transformed to prototrophy one member of each of the three classes of OPRTase-deficient mutants. We therefore concluded that they were all altered at the same locus, the pyrF locus. The corresponding alleles were cloned and sequenced. Comparisons of the amino acid sequence of M. circinelloides OPRTase with those of E. coli and S. typhimurium revealed a high degree of similarity in secondary and tertiary structure. As the two bacterial enzymes exist as dimers, a homodimeric quaternary structure of the M. circinelloides mature protein can be assumed. This would also explain the interallelic complementation between some pyrF mutants. The mutations found could affect either the active site or the structure of the dimer interface of the OPRTase.

Complementation analysis of carotenogenic mutants of Mucor circinelloides.

Mutants of the filamentous fungus Mucor circinelloides altered in the synthesis of beta-carotene (genotype car) have been isolated by direct inspection of the color shown by the colonies derived from mutagenized spores. The mutants were analyzed for the carotenoid content in darkness and light and studied with respect to complementation in heterokaryons made by spheroplast fusion. The results revealed the existence of at least four complementation groups. The carB mutants are white and accumulate phytoene. The carR mutants are red and accumulate lycopene. The carP mutants are most likely disturbed in the synthesis of phytoene: two of them are white and do not accumulate carotenoids; another carP mutant is yellowish, probably because it is leaky. There are three yellowish mutants which belong to one or more complementation groups different from carB, carR, and carP. Two white mutants obtained in a single mutagenization step failed to complement with the carR and the carP mutants. The wild-type and the carB and carR mutants tested for M. circinelloides showed similar photoinduction.

The phytoene dehydrogenase gene of Phycomyces: regulation of its expression by blue light and vitamin A.

By using a polymerase chain reaction based cloning strategy we isolated the gene (carB) encoding the enzyme phytoene dehydrogenase from Phycomyces blakesleeanus. The deduced protein, a 583 residue polypeptide, showed great similarity to carotenoid dehydrogenases from other fungi and bacteria, especially in the amino-terminal region. The main conserved regions found in other phytoene dehydrogenases, which are thought to be essential for the enzymatic activity, are present in the sequence from Phycomyces. Heterologous expression of the Phycomyces gene in Escherichia coli showed that, as in other fungi and bacteria, a single polypeptide catalyzes the four dehydrogenations that convert phytoene to lycopene. RNA measurements indicated that the level of expression of the phytoene dehydrogenase gene in wild-type mycelia increased in response to blue light. The kinetics of this increase in transcription of the gene after blue light induction (0.1 and 0.4 W/m2) exhibit a two-step (biphasic) dependence on fluence rate, suggesting that there could be two separate components involved in the reception of the low and high blue light signal. The presence of vitamin A in the medium stimulated transcript accumulation in the wild type and in some carotenogenic mutant strains. Diphenylamine, a phytoene dehydrogenase inhibitor, did not affect the level of transcription of this gene.

Purification and characterization of an extracellular aspartate protease from Phycomyces blakesleeanus.

An acid protease has been found in the culture broth of Phycomyces blakesleeanus growing under standard conditions. It has been induced up to 70-fold with several complex growth media and the enzyme has been purified to homogeneity and characterized. The molecular mass of the native enzyme was estimated by gel filtration to be 40 kDa. The acid protease of Phycomyces migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to a molecular mass of 35 kDa. The glycoprotein nature for the acid protease was deduced from its binding to a concanavalin A-Sepharose 4B column. The carbohydrate moiety is composed of mannose and rhamnose. Its amino acid composition was determined, and its isoelectric point was estimated to be 4.2, the optimum pH was 2.5 to 3, and the optimum temperature was 70 degrees C, using hemoglobin as a substrate. The enzyme showed thermal stability between 37 and 50 degrees C. The thermodynamic parameters for hemoglobin hydrolysis and thermal inactivation were calculated. With Lys-Pro-Ile-Glu-Phe-Phe(4-N02)-Arg-Leu as the substrate, the Km, kcat, and Vmax values were 8.78 microM, 1.25 s(-1), and 2.12 mumol min(-1) mg(-1), respectively. The protease was insensitive to phenylmethylsulfonyl fluoride, O-phenanthroline, N-ethylmaleimide, iodoacetamide, ethylenediaminetetraacetate, [ethyl-enebis(oxyethylenenitrilo)]tetraacetic acid, and trypsin inhibitor. However, pepstatin A established a strong competitive inhibition against it, with a K(i) value of 1.33 nM. The data suggest that this protease has properties of an aspartate-type proteinase.

Blue-light receptor requirement for gravitropism, autochemotropism and ethylene response in Phycomyces.

Light, gravity and ethylene represent for plants and fungi important environment cues for spatial orientation and growth regulation. Coordination of the frequently conflicting stimuli requires signal-integration sites, which, however, remain largely unidentified. The genetic and physiological basis for signal integration was investigated with a set of phototropism mutants (genotype mad) of the UV- and blue-light-sensitive fungus Phycomyces blakesleeanus, which responds also to gravity, ethylene and nearby obstacles (autochemotropism or avoidance response). Both, class 1 and class 2 mutants display a reduced sensitivity to visible light. Class 1 mutants with defects in genes madA, B, C, I have preserved their sensitivity to gravity and ethylene, whereas class 2 mutants with defects in genes madD,E,F,G,J have lost it. We found that the phototropic sensitivity of class 1 mutants is affected roughly to the same extent in far UV and blue light. In contrast, the sensitivity loss of class 2 mutants is restricted mainly to the near-UV and the blue-light region, whereas the sensitivity to far UV is only mildly affected. This behavior of the class 2 mutants indicates that different photoreceptors mediate phototropism in far-UV and in near-UV/ blue light. The photogravitropic action spectra for two class 2 mutants with defects in genes madF and madJ display distortions between 342 and 530 nm and a bathochromic shift relative to the action spectrum of the wild type. These features indicate that the madF and madJ mutants are affected at the level of the blue-light photoreceptor system. As an implication we infer that an intact near-UV/blue-light photoreceptor system is required even in darkness for negative gravitropism, the ethylene response and autochemotropism. In Phycomyces, signal integration occurs, at least in part, at the level of the near-UV/blue-light photoreceptor system.

Carotenoid biosynthesis in wild type and mutant strains of Mucor circinelloides.

Carotenoid biosynthesis in wild type Mucor circinelloides has been investigated and the biochemical characterisation of the MS1 and MS9 mutant strains, impaired in carotenoid formation, carried out. In liquid cultures, all strains produced carotenoids (mainly beta-carotene, but also xi-carotene, lycopene and gamma-carotene) at the onset of stationary phase of growth. Carotenogenesis was light dependent. In liquid cultures carotenoid formation in wild type was affected by diphenylamine, which prevented desaturation, nicotine, resulting in reduced carotenoid levels, but CPTA caused an increase in the total carotenoid content but a reduced beta-carotene level, with the accumulation of lycopene and gamma-carotene. The mutant strains MS1 and MS9 contained only 5.0 and 11.5% of wild type carotenoid levels, respectively. Cell extracts of light-grown mycelia, incubated with 3(R)-[2-14C] mevalonic acid, produced beta-carotene, but incorporations into carotenoids were substantially reduced in the cell extracts of MS1 and MS9. Analysis of prenyl diphosphate intermediates indicated that, compared to wild type, geranylgeranyl diphosphate accumulated in MS1. MS9 extracts produced a larger amount of prenyl phosphates and a more even distribution of radioactivity from mevalonic acid into farnesyl and geranylgeranyl diphosphates. Squalene and long chain prenyl phosphates were formed by the cell extracts of all strains. It is proposed that the MS1 strain possesses a mutation in a gene responsible for phytoene formation, whilst a regulatory mutation, affecting prenyl transferase activities has occurred in MS9.

Isolation, characterization and transformation, by autonomous replication, of Mucor circinelloides OMPdecase-deficient mutants.

Pyrimidine auxotrophs of Mucor circinelloides were isolated after mutagenesis with nitrosoguanidine and selected for resistance to 5-fluoroorotate. These mutants were genetically and biochemically characterized and found to be deficient either in orotidine-5'-monophosphate decarboxylase (OMPdecase) activity or in orotate phosphoribosyl transferase (OPRTase) activity. Different circular DNA molecules containing the homologous pyrG gene were used to transform a representative OMPdecase-deficient strain to uracil prototrophy. Southern analysis, as well as mitotic stability analysis of the transformants, showed that the transforming DNA is always maintained extrachromosomally. The smallest fragment tested that retained both the capacity to complement the pyrG4 mutation and the ability to be maintained extrachromosomally when cloned in a suitable vector is a 1.85 kb M. circinelloides genomic DNA fragment. This fragment consists of the pyrG coding region flanked by 606 nucleotides at the 5' and 330 nucleotides at the 3' ends, respectively. Sequence analysis reveals that it does not share any element in common with another M. circinelloides genomic DNA fragment which also promotes autonomous replication in this organism, except those related to transcription. Furthermore, it differs from elements which have been shown to be involved in autonomous replication in other fungal systems. An equivalent plasmid harbouring the heterologous Phycomyces blakesleeanus pyrG gene yielded lower transformation rates, but the transforming DNA was also maintained extrachromosomally. Our results suggest that autonomous replication in M. circinelloides may be driven by elements normally present in nuclear coding genes.

Genetic characterization of two phototropism mutants of Phycomyces with defects in the genes madI and madJ.

Two Phycomyces genes, madI and madJ, which are involved in phototropism, were characterized by recombination and complementation analyses. The madI gene was located on linkage group IV of the genetic map of Phycomyces, 27 map units away from the gene carA. Complementation and recombination studies involving the genes madD, madE, madF, and madG, in combination with previous genetic studies, show that the recently isolated mad-407 mutation defines a novel behavioural gene, madJ, of Phycomyces. A regulatory role of the madJ gene product in the light-sensory transduction pathway is suggested.

Isolation and characterization of phototropism mutants of Phycomyces insensitive to ultraviolet light.

Phototropism mutants of the zygomycete fungus Phycomyces blakesleeanus were isolated on the basis of their loss of responsivity to UV light. Four of these mutants had retained a partial sensitivity to near-UV and to blue light. Gravitropism and the avoidance response were unaffected in these mutants. One mutant, A909, had lost most of its sensitivity to near-UV and blue light while the sensitivity to far-UV light was only slightly affected. Additionally, the gravitropic and the avoidance responses were significantly reduced in A909. A complementation analysis of the five strains of Phycomyces with known phototropism mutants indicated that strains A896, A897, and A898 were defective in the madA gene, and that A905 was affected in the madC gene. In strain A909 the input, as well as the output, of the transduction chain is affected.

Isolation, characterization and mapping of pyrimidine auxotrophs of Phycomyces blakesleeanus.

A total of seven pyrimidine auxotrophs of Phycomyces were isolated from among 5-fluoroorotate acid (5-FOA)-resistant mutants. They were classified by complementation into two groups. A representative mutant strain belonging to one group was deficient in orotate phosphoribosyl transferase (OPRTase; EC 2.4.2.10) activity; the mutant strain belonging to the second group was deficient in orotidine-5'-monophosphate decarboxylase (OMPdecase; EC 4.1.1.23). These mutants are defective in the genes pyrF and pyrG respectively. The results from random spore analysis, tetrad analysis, and gene-centromere distances showed that these two markers are located in linkage group VI, with pyrG being a proximal marker and pyrF a distal one.

Cloning and sequence analysis of the Mucor circinelloides pyrG gene encoding orotidine-5'-monophosphate decarboxylase: use of pyrG for homologous transformation.

A 3.2-kb BamHI genomic DNA fragment containing the pyrG gene of Mucor circinelloides was isolated by heterologous hybridization using a pyrG cDNA clone of Phycomyces blakesleeanus as the probe. The complete nucleotide sequence of the M. circinelloides pyrG gene encoding orotidine-5'-monophosphate decarboxylase (OMPD) was determined and the transcription start points (tsp) were mapped by primer extension analysis. The predicted amino acid sequence showed homology with the OMPD sequences reported from other filamentous fungi, with 96% similarity with the OMPD of P. blakesleeanus. Analysis of the sequence revealed the presence of two short introns whose length and location were confirmed by sequencing a cDNA clone and comparing this with its genomic counterpart. The intron splice sites and the 5'- and 3'-noncoding flanking regions show general features of fungal genes. Northern-blot hybridization revealed the pyrG transcript to be approx. 1.0 kb. The M. circinelloides pyrG cDNA clone was able to complement the pyrF::Mu-1 mutation of Escherichia coli when inserted between bacterial expression signals. Additionally, the genomic clone complemented the M. circinelloides pyrG4 mutation. When an M. circinelloides autonomous replication sequence was included in the transforming plasmid, the average transformation frequency obtained was 600 to 800 transformants per micrograms DNA and per 10(6) viable protoplasts.

Heterologous transformation of Mucor circinelloides with the Phycomyces blakesleeanus leu1 gene.

The leu1 gene of Phycomyces blakesleeanus was isolated within a HindIII-HindIII genomic DNA fragment by heterologous hybridization screening of a cosmid library, making use of the Mucor circinelloides leuA gene as a probe. The complete nucleotide sequence of this fragment reveals a single 2070 bp ORF with no introns, which presents at least 68% homology with that of the leuA gene. The P. blakesleeanus leu1 gene has also been expressed in the M. circinelloides mutant R7B (leu-), which was used to isolate the leuA gene by complementation. The homology with other known sequences shows that the leu1 gene encodes the P. blakesleeanus alpha-IPM (isopropylmalate) isomerase.

Isolation and molecular analysis of the orotidine-5'-phosphate decarboxylase gene (pyrG) of Phycomyces blakesleeanus.

The pyrG gene of Phycomyces was isolated from a Phycomyces genomic library, constructed in the cosmid pHS255, by hybridization with a 170 bp fragment of the pyrG gene of Aspergillus niger. This fragment includes a consensus sequence found in almost all species in which the orotidine-5'-phosphate decarboxylase (OMPdecase) gene has been sequenced. The complete nucleotide sequence of the cloned pyrG gene from Phycomyces was determined and the transcription start sites mapped. In the predicted amino acid sequence there are regions of strong homology to the equivalent genes of Saccharomyces cerevisiae, A. niger, Schizophyllum commune and Homo sapiens. Analysis of the sequence revealed the presence of two introns. The precise length and location of these introns was determined by sequencing the pyrG cDNA and comparing it with the genomic clone. Non-coding flanking regions showed obvious homology to the consensus TATA and CAAT boxes, and the polyadenylation signal "AATAAA". The pyrG gene is the second Phycomyces gene that has been cloned and analysed. This is the first time that introns have been reported in Phycomyces.