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The FTF ( Fusarium Transcription Factor ) gene family is composed of two members ( FTF1 and FTF2 ) with high-sequence homology that encode transcription factors involved in the modulation of virulence in the F. oxysporum species complex (FOSC). While FTF1 is a multicopy gene exclusive of highly virulent strains of FOSC and is located in the accessory genome, FTF2 is a single-copy gene, located in the core genome, and well-conserved in all filamentous ascomycete fungi, except yeast. The involvement of FTF1 in the colonization of the vascular system and regulation of the expression of SIX effectors has been stablished. To address the role of FTF2 , we generated and characterized mutants defective in FTF2 in a F. oxysporum f. sp. phaseoli weakly virulent strain and analyzed them together with the equivalent mutants formerly obtained in a highly virulent strain. The results obtained highlight a role for FTF2 as a negative regulator of the production of macroconidia and demonstrate that it is required for full virulence and the positive regulation of SIX effectors. In addition, gene expression analyses provided compelling evidence that FTF2 is involved in the regulation of hydrophobins likely required for plant colonization.

Nitric oxide regulates numerous physiological processes in species from all taxonomic groups. Here, its role in the early developmental stages of the fungal necrotroph Botrytis cinerea was investigated. Pharmacological analysis demonstrated that NO modulated germination, germ tube elongation and nuclear division rate. Experimental evidence indicates that exogenous NO exerts an immediate but transitory negative effect, slowing down germination-associated processes, and that this effect is largely dependent on the flavohemoglobin BCFHG1. The fungus exhibited a "biphasic response" to NO, being more sensitive to low and high concentrations than to intermediate levels of the NO donor. Global gene expression analysis in the wild-type and Δ Bcfhg 1 strains indicated a situation of strong nitrosative and oxidative stress determined by exogenous NO, which was much more intense in the mutant strain, that the cells tried to alleviate by upregulating several defense mechanisms, including the simultaneous upregulation of the genes encoding the flavohemoglobin BCFHG1, a nitronate monooxygenase (NMO) and a cyanide hydratase. Genetic evidence suggests the coordinated expression of Bcfhg 1 and the NMO coding gene, both adjacent and divergently arranged, in response to NO. Nitrate assimilation genes were upregulated upon exposure to NO, and BCFHG1 appeared to be the main enzymatic system involved in the generation of the signal triggering their induction. Comparative expression analysis also showed the influence of NO on other cellular processes, such as mitochondrial respiration or primary and secondary metabolism, whose response could have been mediated by NmrA-like domain proteins.

In planta gene expression analysis and GFP-based confocal microscopy are two powerful techniques that may be coupled to assess the extent and dynamics of plant colonization by a fungal pathogen. Here we describe methods to prepare common bean plants for inoculation with a highly virulent strain of Fusarium oxysporum f. sp. phaseoli, quantify the extent of colonization by RT-qPCR, and visualize the colonized tissues by confocal microscopy., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Botrytis cinerea is a necrotrophic plant pathogenic fungus with a wide host range. Its natural populations are phenotypically and genetically very diverse. A survey of B. cinerea isolates causing gray mold in the vineyards of Castilla y León, Spain, was carried out and as a result eight non-pathogenic natural variants were identified. Phenotypically these isolates belong to two groups. The first group consists of seven isolates displaying a characteristic mycelial morphotype, which do not sporulate and is unable to produce sclerotia. The second group includes one isolate, which sporulates profusely and does not produce sclerotia. All of them are unresponsive to light. Crosses between a representative mycelial non-pathogenic isolate and a highly aggressive field isolate revealed that the phenotypic differences regarding pathogenicity, sporulation and production of sclerotia cosegregated in the progeny and are determined by a single genetic locus. By applying a bulked segregant analysis strategy based on the comparison of the two parental genomes the locus was mapped to a 110 kb region in chromosome 4. Subcloning and transformation experiments revealed that the polymorphism is an SNP affecting gene Bcin04g03490 in the reference genome of B. cinerea . Genetic complementation analysis and sequencing of the Bcin04g03490 alleles demonstrated that the mutations in the mycelial isolates are allelic and informed about the nature of the alterations causing the phenotypes observed. Integration of the allele of the pathogenic isolate into the non-pathogenic isolate fully restored the ability to infect, to sporulate and to produce sclerotia. Therefore, it is concluded that a major effect gene controlling differentiation and developmental processes as well as pathogenicity has been identified in B. cinerea . It encodes a protein with a GAL4-like Zn(II)2Cys6 binuclear cluster DNA binding domain and an acetyltransferase domain, suggesting a role in regulation of gene expression through a mechanism involving acetylation of specific substrates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Acosta Morel, Anta Fernández, Baroncelli, Becerra, Thon, van Kan, Díaz-Mínguez and Benito.)

Main Conclusion: ST1 and ST6 are possibly involved in primary and lateral root and symbiotic nodule development, but only ST6 participates in the interaction with hemibiotrophic fungi. Specific tissue (ST) proteins have been shown to be involved in several processes related to plant nutritional status, development, and responses to biotic agents. In particular, ST1 and ST6 are mainly expressed in roots throughout plant development. Here, we analyze where and how the expression of the genes encoding both proteins are modulated in the legume model plant Medicago truncatula in response to the plant developmental program, nodulation induced by a beneficial nitrogen-fixing bacterium (Sinorhizobium meliloti) and the defense response triggered by a pathogenic hemibiotrophic fungus (Fusarium oxysporum). Gene expression results show that ST1 and ST6 participate in the vasculature development of both primary and lateral roots, although only ST6 is related to meristem activity. ST1 and ST6 clearly display different roles in the biotic interactions analyzed, where ST1 is activated in response to a N 2 -fixing bacterium and ST6 is up-regulated after inoculation with F. oxysporum. The role of ST1 and ST6 in the nodulation process may be related to nodule organogenesis rather than to the establishment of the interaction itself, and an increase in ST6 correlates with the activation of the salicylic acid signaling pathway during the infection and colonization processes. These results further support the role of ST6 in response to hemibiotrophic fungi. This research contributes to the understanding of the complex network that controls root biology and strengthens the idea that ST proteins are involved in several processes such as primary and lateral root development, nodule organogenesis, and the plant-microbe interaction.

The FTF (Fusarium transcription factor) gene family comprises a single copy gene, FTF2, which is present in all the filamentous ascomycetes analysed, and several copies of a close relative, FTF1, which is exclusive to Fusarium oxysporum. An RNA-mediated gene silencing system was developed to target mRNA produced by all the FTF genes, and tested in two formae speciales: F. oxysporum f. sp. phaseoli (whose host is common bean) and F. oxysporum f. sp. lycopersici (whose host is tomato). Quantification of the mRNA levels showed knockdown of FTF1 and FTF2 in randomly isolated transformants of both formae speciales. The attenuation of FTF expression resulted in a marked reduction in virulence, a reduced expression of several SIX (Secreted In Xylem) genes, the best studied family of effectors in F. oxysporum, and lower levels of SGE1 (Six Gene Expression 1) mRNA, the presumptive regulator of SIX expression. Moreover, the knockdown mutants showed a pattern of colonization of the host plant similar to that displayed by strains devoid of FTF1 copies (weakly virulent strains). Gene knockout of FTF2 also resulted in a reduction in virulence, but to a lesser extent. These results demonstrate the role of the FTF gene expansion, mostly the FTF1 paralogues, as a regulator of virulence in F. oxysporum and suggest that the control of effector expression is the mechanism involved., (© 2016 The Authors Molecular Plant Pathology Published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Colletotrichum graminicola causes maize anthracnose, an agronomically important disease with a worldwide distribution. We have identified a fungalysin metalloprotease (Cgfl) with a role in virulence. Transcriptional profiling experiments and live cell imaging show that Cgfl is specifically expressed during the biotrophic stage of infection. To determine whether Cgfl has a role in virulence, we obtained null mutants lacking Cgfl and performed pathogenicity and live microscopy assays. The appressorium morphology of the null mutants is normal, but they exhibit delayed development during the infection process on maize leaves and roots, showing that Cgfl has a role in virulence. In vitro chitinase activity assays of leaves infected with wild-type and null mutant strains show that, in the absence of Cgfl, maize leaves exhibit increased chitinase activity. Phylogenetic analyses show that Cgfl is highly conserved in fungi. Similarity searches, phylogenetic analysis and transcriptional profiling show that C. graminicola encodes two LysM domain-containing homologues of Ecp6, suggesting that this fungus employs both Cgfl-mediated and LysM protein-mediated strategies to control chitin signalling., (© 2015 BSPP and John Wiley & Sons Ltd.)

Botrytis cinerea is a widespread necrotrophic fungus which infects more than 200 plant species. In an attempt to characterize the physiological status of the fungus in planta and to identify genetic factors contributing to its ability to infect the host cells, a differential gene expression analysis during the interaction B. cinerea-tomato was carried out. Gene Bcmimp1 codes for a mRNA detected by differential display in the course of this analysis. During the interaction with the host, it shows a transient expression pattern with maximal expression levels during the colonization and maceration of the infected tissues. Bioinformatic analysis suggested that BCMIMP1 is an integral membrane protein located in the mitochondrial inner membrane. Co-localization experiments with a BCMIMP1-GFP fusion protein confirmed that the protein is targeted to the mitochondria. ΔBcmimp1 mutants do not show obvious phenotypic differences during saprophytic growth and their infection ability was unaltered as compared to the wild-type. Interestingly, the mutants produced increased levels of reactive oxygen species, likely as a consequence of disturbed mitochondrial function. Although Bcmimp1 expression is enhanced in planta it cannot be considered a pathogenicity factor.

Plant pathogens have the capacity to manipulate the host immune system through the secretion of effectors. We identified 27 putative effector proteins encoded in the genome of the maize anthracnose pathogen Colletotrichum graminicola that are likely to target the host's nucleus, as they simultaneously contain sequence signatures for secretion and nuclear localization. We functionally characterized one protein, identified as CgEP1. This protein is synthesized during the early stages of disease development and is necessary for anthracnose development in maize leaves, stems, and roots. Genetic, molecular, and biochemical studies confirmed that this effector targets the host's nucleus and defines a novel class of double-stranded DNA-binding protein. We show that CgEP1 arose from a gene duplication in an ancestor of a lineage of monocot-infecting Colletotrichum spp. and has undergone an intense evolution process, with evidence for episodes of positive selection. We detected CgEP1 homologs in several species of a grass-infecting lineage of Colletotrichum spp., suggesting that its function may be conserved across a large number of anthracnose pathogens. Our results demonstrate that effectors targeted to the host nucleus may be key elements for disease development and aid in the understanding of the genetic basis of anthracnose development in maize plants.

The dynamics of root and hypocotyl colonization, and the gene expression patterns of several fungal virulence factors and plant defense factors have been analyzed and compared in the interaction of two Fusarium oxysporum f. sp. phaseoli strains displaying clear differences in virulence, with a susceptible common bean cultivar. The growth of the two strains on the root surface and the colonization of the root was quantitatively similar although the highly virulent (HV) strain was more efficient reaching the central root cylinder. The main differences between both strains were found in the temporal and spatial dynamics of crown root and hypocotyl colonization. The increase of fungal biomass in the crown root was considerably larger for the HV strain, which, after an initial stage of global colonization of both the vascular cylinder and the parenchymal cells, restricted its growth to the newly differentiated xylem vessels. The weakly virulent (WV) strain was a much slower and less efficient colonizer of the xylem vessels, showing also growth in the intercellular spaces of the parenchyma. Most of the virulence genes analyzed showed similar expression patterns in both strains, except SIX1, SIX6 and the gene encoding the transcription factor FTF1, which were highly upregulated in root crown and hypocotyl. The response induced in the infected plant showed interesting differences for both strains. The WV strain induced an early and strong transcription of the PR1 gene, involved in SAR response, while the HV strain preferentially induced the early expression of the ethylene responsive factor ERF2.

Hemibiotrophic plant pathogens first establish a biotrophic interaction with the host plant and later switch to a destructive necrotrophic lifestyle. Studies of biotrophic pathogens have shown that they actively suppress plant defenses after an initial microbe-associated molecular pattern-triggered activation. In contrast, studies of the hemibiotrophs suggest that they do not suppress plant defenses during the biotrophic phase, indicating that while there are similarities between the biotrophic phase of hemibiotrophs and biotrophic pathogens, the two lifestyles are not analogous. We performed transcriptomic, histological, and biochemical studies of the early events during the infection of maize (Zea mays) with Colletotrichum graminicola, a model pathosystem for the study of hemibiotrophy. Time-course experiments revealed that mRNAs of several defense-related genes, reactive oxygen species, and antimicrobial compounds all begin to accumulate early in the infection process and continue to accumulate during the biotrophic stage. We also discovered the production of maize-derived vesicular bodies containing hydrogen peroxide targeting the fungal hyphae. We describe the fungal respiratory burst during host infection, paralleled by superoxide ion production in specific fungal cells during the transition from biotrophy to a necrotrophic lifestyle. We also identified several novel putative fungal effectors and studied their expression during anthracnose development in maize. Our results demonstrate a strong induction of defense mechanisms occurring in maize cells during C. graminicola infection, even during the biotrophic development of the pathogen. We hypothesize that the switch to necrotrophic growth enables the fungus to evade the effects of the plant immune system and allows for full fungal pathogenicity.

Fusarium oxysporum f. sp. phaseoli strains isolated from runner bean plants showing Fusarium wilt symptoms were characterized. The analysis of the genetic diversity of these strains and the comparison with strains formerly isolated from diseased common bean plants grown in the same region of Spain indicated a close genetic similarity among them. Pathogenicity assays carried out on runner bean plants showed virulence differences that allowed the classification of these strains into three groups: super virulent, highly virulent, and weakly virulent. However, all the analyzed strains behaved as highly virulent when inoculated on common bean plants, indicating that virulence is specific of the host-pathogen interaction. We also analyzed the number of copies and expression of the gene encoding the transcription factor ftf1, which has been shown to be specific of virulent F. oxysporum strains and highly up-regulated during plant infection. In planta real-time quantitative polymerase chain reaction expression analysis showed that expression of ftf1 was correlated with the degree of virulence. The comparative analysis of the polymorphic copies of ftf1 detected in the strains here characterized and those detected in the genome sequence of F. oxysporum f. sp. lycopersici strain 4287 indicates that some of the copies are likely nonfunctional.

We have identified a Fusarium oxysporum homolog of the Ste12 transcription factor that regulates mating and filamentation in Saccharomyces cerevisiae. The corresponding gene, fost12, from a highly virulent strain of F. oxysporum f. sp. phaseoli, was confirmed to share a high level of similarity and possessed the STE and C2H2 domains characteristic of the fungal Ste12 transcription factor family of proteins. Disruption of fost12 resulted in no visible alterations of colony morphology or in vitro growth characteristics. However, the disruption mutants showed a substantial reduction in virulence when inoculated in common bean seedlings. In planta transcription of fost12 is drastically increased between 12 and 24h after inoculation, as detected by real-time RT-PCR. The results of the transcriptional analyses carried out in several F. oxysporum strains during axenic growth suggest that the fost12 gene product is a virulence factor required to deal with the nutritional stress confronted by the pathogen during host plant colonization., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

The rhizobia-legume, root-nodule symbiosis provides the most efficient source of biologically fixed ammonia fertilizer for agricultural crops. Its development involves pathways of specificity, infectivity, and effectivity resulting from expressed traits of the bacterium and host plant. A key event of the infection process required for development of this root-nodule symbiosis is a highly localized, complete erosion of the plant cell wall through which the bacterial symbiont penetrates to establish a nitrogen-fixing, intracellular endosymbiotic state within the host. This process of wall degradation must be delicately balanced to avoid lysis and destruction of the host cell. Here, we describe the purification, biochemical characterization, molecular genetic analysis, biological activity, and symbiotic function of a cell-bound bacterial cellulase (CelC2) enzyme from Rhizobium leguminosarum bv. trifolii, the clover-nodulating endosymbiont. The purified enzyme can erode the noncrystalline tip of the white clover host root hair wall, making a localized hole of sufficient size to allow wild-type microsymbiont penetration. This CelC2 enzyme is not active on root hairs of the nonhost legume alfalfa. Microscopy analysis of the symbiotic phenotypes of the ANU843 wild type and CelC2 knockout mutant derivative revealed that this enzyme fulfils an essential role in the primary infection process required for development of the canonical nitrogen-fixing R. leguminosarum bv. trifolii-white clover symbiosis.

We report the isolation and analysis of the gene encoding ftf1 (Fusarium transcription factor 1), a previously undescribed putative transcription factor from highly virulent strains of Fusarium oxysporum f.sp. phaseoli that is transcribed specifically during early stages of infection of its host common bean (Phaseolus vulgaris L.). The predicted 1080 amino acid ftf1 protein contains a Zn(II)2-Cys6 binuclear cluster DNA-binding motif. ftf1 expression during axenic growth in culture was not detected by either Northern or RT-PCR. On the contrary, in planta transcription of ftf1 is increased about 24h after plant inoculation, as detected by real-time RT-PCR. This result suggests that ftf1 has a role in the establishment of the fungus within the plant and/or the progress of the disease. Multiple copies of ftf1 are present in highly virulent strains of F. oxysporum f.sp. phaseoli.

ABSTRACT We have characterized strains of Fusarium oxysporum from common bean fields in Spain that were nonpathogenic on common bean, as well as F. oxysporum strains (F. oxysporum f. sp. phaseoli) pathogenic to common bean by random amplified polymorphic DNA (RAPD) analysis. We identified a RAPD marker (RAPD 4.12) specific for the highly virulent pathogenic strains of the seven races of F. oxysporum f. sp. phaseoli. Sequence analysis of RAPD 4.12 allowed the design of oligonucleotides that amplify a 609-bp sequence characterized amplified region (SCAR) marker (SCAR-B310A280). Under controlled environmental and greenhouse conditions, detection of the pathogen by polymerase chain reaction was 100% successful in root samples of infected but still symptomless plants and in stem samples of plants with disease severity of >/=4 in the Centro Internacional de Agricultura Tropical (CIAT; Cali, Colombia) scale. The diagnostic procedure can be completed in 5 h and allows the detection of all known races of the pathogen in plant samples at early stages of the disease with no visible symptoms.

The genus Trichoderma includes biocontrol agents (BCAs) effective against soilborne plant pathogenic fungi. Several potentially useful strains for biological control are difficult to distinguish from other strains of Trichoderma found in the field. So, there is a need to find ways to monitor these strains when applied to natural pathosystems. We have used random amplified polymorphic DNA (RAPD) markers to estimate genetic variation among sixteen strains of the species T. asperellum, T. atroviride, T. harzianum, T. inhamatum and T. longibrachiatum previously selected as BCAs, and to obtain fingerprinting patterns. Analysis of these polymorphisms revealed four distinct groups, in agreement with previous studies. Some of the RAPD products generated were used to design specific primers. Diagnostic PCR performed using these primers specifically identify the strain T. atroviride 11, showing that DNA markers may be successfully used for identification purposes. This SCAR (sequence-characterised amplified region) marker can clearly distinguish strain 11 from other closely related Trichoderma strains.

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.

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.

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.

Fusarium oxysporum Schechtend.:Fr. f. sp. phaseoli J. B. Kendrick & W. C. Snyder (FOP) is the causal agent of the common bean (Phaseolus vulgaris L.) disease known as Fusarium wilt or Fusarium yellows. FOP has been reported from the Castilla y Leon region in Spain, where it is a serious problem on most commercial bean cultivars (1). Five FOP isolates from Spain (AB-6, AB-111, AB-112, AS-1, and AS-4) obtained from J. M. Díaz-Mínguez and the isolate FOP-CO1 (ATCC 90245) from Colorado were tested for pathogenicity on two American lines: Pinto U.I. 114, considered as a universal susceptible check, and Flor de Mayo, a Mexican landrace. Seedlings were root-clip inoculated and evaluated according to the CIAT 1 to 9 severity scale, in which 1 to 3 = resistant, 3.1 to 6 = intermediate, and 6.1 to 9 = susceptible. Three inoculum concentrations were tested: 10 4 , 10 5 , and 10 6 conidia/ml. AS-1 and AS-4 produced resistant reactions in both U.I. 114 (1.5 to 2.1 and 1.2 to 1.5) and Flor de Mayo (1.9 to 2.3 and 1.4), respectively, at all inoculum concentrations. Susceptible reactions to AB-6 (7.3 to 8.9), AB-111 (7.6 to 8.9), AB-112 (7.5 to 7.9), and FOP-CO1 (8.1 to 8.6) were observed in Pinto U.I. 114, regardless of inoculum concentration, although severity ratings increased as the inoculum concentration was increased. Flor de Mayo exhibited a susceptible reaction (6.7 to 7.8) to FOP-CO1 at all inoculum concentrations tested, and intermediate reactions to AB-6 (4.9), AB-111 (5.4), and AB-112 (5.0) at the lowest inoculum concentration. Susceptible reactions (7 to 8.2 for AB-6; 6.8 to 7.0 for AB-111, and 7.3 to 7.7 for AB-112) occurred with higher inoculum concentrations, and severity ratings increased as the inoculum concentration increased. Recently, FOP isolates from Greece and Italy were recognized as each belonging to different pathogenic races (2); consequently, more research on the Spanish isolates (AB-6, AB-111, and AB-112) is needed to determine if they are similar to the races reported from the Mediterranean Basin or should be classified as a new FOP race (s). References: (1) J. M. Díaz-Mínguez et al. Plant Dis. 80:600, 1996. (2) S. L. Woo et al. Phytopathology 86:966, 1996.

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.

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.

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.

Only eight genes are known to be involved in the phototropic response of Phycomyces (madA-H). Mutants affected in these genes have played a major role in the analysis of photosensory transduction processes in this system. A set of new mutants isolated by Alvarez et al. (1989) that are unable to bend towards dim unilateral blue light were studied by complementation and recombination. Two of these mutants have mutations in madE, one has a mutation in madF and one is a double madE madF mutant. The three remaining mutants tested did not complement each other and showed positive complementation with strains carrying mutations in the genes madA, madB, and madC, indicating that they carried mutations in a new gene designated madI. Recombination analysis showed that madI is unlinked to madA, madB and madC.