The phoma-like dilemma.

Species of Didymellaceae have a cosmopolitan distribution and are geographically widespread, occurring in diverse ecosystems. The family includes several important plant pathogenic fungi associated with fruit, leaf, stem and root diseases on a wide variety of hosts, as well as endophytic, saprobic and clinically relevant species. The Didymellaceae was recently revised based on morphological and phylogenetic analyses of ex-type strains subjected to DNA sequencing of partial gene data of the LSU, ITS, rpb2 and tub2 loci. Several poly- and paraphyletic genera, including Ascochyta, Didymella and Phoma were redefined, along with the introduction of new genera. In the present study, a global collection of 1 124 Didymellaceae strains from 92 countries, 121 plant families and 55 other substrates, including air, coral, human tissues, house dust, fungi, insects, soil, and water were examined via multi-locus phylogenetic analyses and detailed morphological comparisons, representing the broadest sampling of Didymellaceae to date. Among these, 97 isolates representing seven new genera, 40 new species and 21 new combinations were newly introduced in Didymellaceae. In addition, six epitypes and six neotypes were designated to stabilise the taxonomy and use of older names. A robust, multi-locus reference phylogenetic tree of Didymellaceae was generated. In addition, rpb2 was revealed as the most effective locus for the identification of Didymellaceae at species level, and is proposed as a secondary DNA marker for the family.

Type 2A phosphoprotein phosphatase is required for asexual development and pathogenesis of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a necrotrophic, omnivorous plant pathogen with worldwide distribution. Sclerotia of S. sclerotiorum are pigmented, multihyphal structures that play a central role in the life and infection cycles of this pathogen. Plant infection depends on the formation of melanin-rich infection cushions, and secretion of hydrolytic enzymes and oxalic acid. Type 2A Ser/Thr phosphatases (PP2As) are involved in the regulation of a variety of cellular process. In the presence of cantharidin, a PP2A-specific inhibitor, hyphal elongation and sclerotia numbers were impaired whereas sclerotial size increased. We partially inactivated PP2A by antisense expression of the gene (pph1) encoding the PP2A catalytic subunit. When antisense expression was induced, almost complete cessation of fungal growth was observed, indicative of a crucial role for PP2A in fungal growth. RNAi-based gene silencing was employed to alter the expression of the 55-kDa R2 (B regulatory subunit). Isolates in which rgb1 RNA levels were decreased were slow growing, but viable. Melanin biosynthesis, infection-cushion production, and pathogenesis were significantly impaired in the rgb1 mutants, yet theses mutants were pathogenic on wounded leaves. Reduced ERK (extracellular signal-regulated kinases)-like mitogen-activated protein kinase (MAPK) function conferred a reduction in NADPH oxidase and PP2A activity levels, suggesting a functional link between MAPK, reactive oxygen species, and PP2A activity in S. sclerotiorum.

Calcineurin is required for sclerotial development and pathogenicity of Sclerotinia sclerotiorum in an oxalic acid-independent manner.

Sclerotinia sclerotiorum is a necrotrophic, omnivorous plant pathogen with worldwide distribution. Sclerotia of S. sclerotiorum are pigmented, multihyphal structures that play a central role in the life and infection cycles of this pathogen. Calcineurin, a Ser/Thr phosphatase linked to several signal-transduction pathways, plays a key role in the regulation of cation homeostasis, morphogenesis, cell-wall integrity, and pathogenesis in fungi. We demonstrate that calcineurin expression in S. sclerotiorum is altered in a phase-specific manner during sclerotial development. Inhibition of calcineurin by FK506, cysclosporin A, or inducible antisense calcineurin expression impaired sclerotial development at the prematuration phase and increased germination of preformed sclerotia. Induction of antisense calcineurin expression in S. sclerotiorum resulted in reduced pathogenesis on tomato and Arabidopsis. However, secretion of oxalic acid, a key virulence factor of S. sclerotiorum, was not altered. Inhibition of calcineurin conferred a reduction in cell wall beta-1,3-glucan content and increased sensitivity to cell-wall-degrading enzymes and to the glucan synthase inhibitor caspofungin. Thus, calcineurin plays a major role in both sclerotial development and pathogenesis of S. sclerotiorum and, most likely, other phytopathogens.

ppt-1, a Neurospora crassa PPT/PP5 subfamily serine/threonine protein phosphatase.

We isolated a N. crassa cDNA clone encoding a novel-type serine/threonine phosphatase. The gene (mapped to LGVR), designated ppt-1, encodes a 479 amino acid putative polypeptide which contains a conserved tetratricopeptide repeat (TPR) motif. ppt-1 transcript levels are abundant in conidia and decrease during germination, indicating that ppt-1 is developmentally regulated.

pzl-1 encodes a novel protein phosphatase-Z-like Ser/Thr protein phosphatase in Neurospora crassa.

The gene and cDNA of a novel protein phosphatase were cloned from Neurospora crassa. The pzl-1 gene encompasses three introns and is localized to the left arm of chromosome I between cyt-21 and Fsr-12. It encodes a protein of 58.3 kDa containing a Ser/Pro rich N-terminal segment, and a C-terminal domain that is similar to the catalytic subunit of type 1 protein phosphatases. The first 51 amino acid residues, including a potential N-myristoylation site, as well as the C-terminal domain (about 300 residues) have a high level of sequence identity with yeast PPZ phosphatases. However, residues 52-208 do not share high similarity with other proteins. The mRNA of pzl-1 was detected in all phases of asexual development of the filamentous fungus.

Changes in Protein Kinase A Activity Accompany Sclerotial Development in Sclerotinia sclerotiorum.

ABSTRACT Sclerotia of Sclerotinia sclerotiorum are pigmented, multihyphal structures that play a central role in the life and infection cycles of this pathogen. Sclerotial formation has been shown to be affected by increased intracellular cAMP levels. Cyclic AMP (cAMP) is a key modulator of cAMP-dependent protein kinase A (PKA) and the latter may prove to play a significant role in sclerotial development. Therefore, we monitored changes in relative PKA activity levels during sclerotial development. To do so, we first developed conditions for near-synchronous sclerotial development in culture, based on hyphal maceration and filtering. Relative PKA activity levels increased during the white-sclerotium stage in the wild-type strain, while low levels were maintained in nonsclerotium-producing mutants. Furthermore, applying caffeine, an inducer of PKA activity, resulted in increased relative PKA activity levels and was correlated with the formation of sclerotial initial-like aggregates in cultures of the non-sclerotium-producing mutants. In addition, low PKA activities were found in an antisense smk1 strain, which exhibits low extracellular-signal-regulated kinase (ERK)-type mitogen-activated protein kinase (MAPK) activity, and does not produce sclerotia. The changes in PKA activity, as well as the abundance of phosphorylated MAPKs (ERK-like as well as p38-like) that accompany sclerotial development in a distinct developmental phase manner represent a potential target for antifungal intervention.

Fungal biology and agriculture: revisiting the field.

Plant pathology has made significant progress over the years, a process that involved overcoming a variety of conceptual and technological hurdles. Descriptive mycology and the advent of chemical plant-disease management have been followed by biochemical and physiological studies of fungi and their hosts. The later establishment of biochemical genetics along with the introduction of DNA-mediated transformation have set the stage for dissection of gene function and advances in our understanding of fungal cell biology and plant-fungus interactions. Currently, with the advent of high-throughput technologies, we have the capacity to acquire vast data sets that have direct relevance to the numerous subdisciplines within fungal biology and pathology. These data provide unique opportunities for basic research and for engineering solutions to important agricultural problems. However, we also are faced with the challenge of data organization and mining to analyze the relationships between fungal and plant genomes and to elucidate the physiological function of pertinent DNA sequences. We present our perspective of fungal biology and agriculture, including administrative and political challenges to plant protection research.

The chsA gene, encoding a class-I chitin synthase from Ampelomyces quisqualis.

Degenerate oligodeoxyribonucleotide primers, designed on the basis of conserved regions of the chitin synthase gene family, were used to amplify a fragment of the Ampelomyces quisqualis (Aq) chsA gene. Subsequently, the PCR product was used as a probe in order to identify and isolate genomic clones harboring the entire chsA gene. Aq chsA is 2786-nt long, has one intron and encodes a 910-amino-acid polypeptide belonging to the class-I chitin synthases. Low-stringency Southern hybridizations to Aq genomic DNA provided evidence for the presence of additional DNA fragments resembling chsA in the fungal genome, suggesting the presence of a multigene family of chitin synthases in Aq.

Photoregulation of cot-1, a Kinase-Encoding Gene Involved in Hyphal Growth in Neurospora crassa.

Blue light plays a key role as an environmental signal in the regulation of growth and development of fungi and plants. Here we demonstrate that in Neurospora crassa hyphae branch more frequently in cultures grown in light. Previous studies had identified cot-1 as a gene that controls apical hyphal cell elongation. In the cot-1 mutant, cessation of elongation is accompanied by hyperbranching. Here we demonstrate that the cot-1 gene encodes two transcript species of about 2100 nt (cot-1 (s)) and about 2400 nt (cot-1 (l)) in length and that the ratio of both transcript species abundance is photoregulated. The origin of the difference between cot-1 (l) and cot-1 (s) was localized to the 5' end of the cot-1 transcripts, suggesting that two COT1 isoforms with different activities may be formed. Both light effects, on branching and on cot-1 expression, were dependent on functional wc-1 and wc-2 gene products. In addition to light, l-sorbose comprises another environmental cue that controls hyphal branching in N. crassa. In the presence of l-sorbose, photoregulation of cot-1 was blocked, suggesting the involvement of alternative and potentially interdependent signaling pathways for the regulation of hyphal elongation/branching. Copyright 1998 Academic Press.

The Mycoparasite Ampelomyces quisqualis Expresses exgA Encoding an exo-beta-1,3-Glucanase in Culture and During Mycoparasitism.

ABSTRACT Ampelomyces quisqualis, a mycoparasite of fungi causing powdery mildews, exhibited high levels of extracellular exo-beta-1,3-glucanase activity in culture compared with Neurospora crassa and Gliocladium roseum. A. quisqualis culture filtrates affected powdery mildew caused by Sphaerotheca fusca in a manner indicative of cell wall degradation, as determined by microscopic examination. A gene encoding an exo-beta-1,3-glucanase in A. quisqualis, designated exgA, was isolated and sequenced. The predicted polypeptide deduced from exgA had 46, 42, and 30% identity with amino acid sequences of Trichoderma harzianum exo-beta-1,3-glucanase and Cochliobolus carbonum EXG1 (both encoding exo-beta-1,3-glucanase) and T. harzianum bng13.1 (encoding an endo-beta-1,3-glucanase), respectively. The exgA gene had a predicted molecular mass of 84 kDa and a pI of 4.79. The gene was expressed during the late stages of growth in culture, and transcription was induced by fungal cell wall components. Transcript levels for exgA were present during late stages of hyperpar-asitism and were abundant along A. quisqualis mycelium and were slightly less abundant in A. quisqualis pycnidia.

Isolation and Characterization of a Cold-Tolerant Strain of Fusarium proliferatum, a Biocontrol Agent of Grape Downy Mildew.

ABSTRACT A cold-tolerant strain of the mycoparasite Fusarium proliferatum was isolated following UV mutagenesis of the G6 strain, which is a biocontrol agent of grape downy mildew. The isolated strain (designated 1505) exhibited radial growth two to threefold that of the parent strain when grown at 13 degrees C, which is generally suboptimal for growth of Fusarium spp., but desirable for its host, Plasmopara viticola. This rapid growth was correlated with improved biological control of P. viticola, determined by a detached-leaf assay. Even though radial growth of strain 1505 at higher temperatures was slower than that of G6 and the strain failed to conidiate, there was no reduction in biocontrol efficacy. Significantly higher levels of extracellular beta-glucosidase and endo-1,4-beta-glucanase activity were measured in the culture filtrate of strain 1505 relative to that of strain G6. A DNA-mediated transformation procedure that included the introduction of antibiotic resistance and a GUS reporter gene system was adapted for F. proliferatum. Using the GUS-engineered strains, we demonstrated that both G6 and 1505 exhibit the characteristic coiling and penetration of host structures.

Efficient gene replacement and direct hyphal transformation in Sclerotinia sclerotiorum.

Homologous recombination is required for gene-targeted procedures such as gene disruption and gene replacement. Ku80 is part of the non-homologous end-joining DNA repair mechanism in many organisms. We identified and disrupted the Ku80 homologue in Sclerotinia sclerotiorum and generated heterokaryon mutants enriched with Ku80-deficient nuclei (ssku80). Sclerotial formation and pathogenicity of ssku80 mutants were normal on tomato fruits. The frequencies of homologous recombination in these strains were much higher than those of the wild type when transformed with a cna1 (encoding calcineurin) replacement construct. We coupled the increase in homologous recombination with a direct BIM-LAB-mediated transformation procedure, which utilizes compressed air to assist the transforming DNA in penetrating fungal hyphae of S. sclerotiorum. We found this method to be efficient and reproducible, and it did not alter the fitness of the mutants. We also demonstrated the first case of direct transformation of sclerotia. Nourseothricin was introduced as a selectable marker in S. sclerotiorum. The tools and procedures described will improve our ability to study gene function in S. sclerotiorum and are most likely to be adaptable for use in other plant pathogens.

Chitin synthase 1 plays a major role in cell wall biogenesis in Neurospora crassa.

In filamentous fungi, chitin is a structural component of morphologically distinct structures assembled during various phases of growth and development. To investigate the role of chitin synthase in cell wall biogenesis in Neurospora crassa, we cloned a chitin synthase structural gene and examined the consequences of its inactivation. Using degenerate oligonucleotide mixtures designed on the basis of conserved sequences of the Saccharomyces cerevisiae CHS1 and CHS2 polypeptides, a DNA fragment encoding a similar predicted amino acid sequence was amplified from N. crassa genomic DNA. This product was used to probe N. crassa libraries for a gene homologous to one of the yeast genes. Full-length genomic and partial cDNA clones were identified, isolated, and sequenced. The amino acid sequence deduced from a cloned 3.4-kb gene [designated chitin synthase 1 (chs-1)] was very similar to that of the S. cerevisiae CHS1 and CHS2 and the Candida albicans CHS1 polypeptides. Inactivation of the N. crassa chs-1 gene by repeat-induced point mutation produced slow-growing progeny that formed hyphae with morphologic abnormalities. The chs-1RIP phenotype was correlated with a significant reduction in chitin synthase activity. Calcofluor staining of the chs-1RIP strain cross-walls, residual chitin synthase activity, and the increased sensitivity of the chs-1RIP strain to Nikkomycin Z suggest that N. crassa produces additional chitin synthase that can participate in cell wall formation.

Inactivation of a single-2A phosphoprotein phosphatase is lethal in Neurospora crassa.

A PCR approach, employing the use of degenerate oligonucleotide mixtures, was used to isolate pph-1, a type-2A protein phosphatase (catalytic subunit)-encoding gene, from Neurospora crassa. The isolated single copy gene is 1327 nucleotides in length, contains four putative introns and encodes a 310 amino-acid polypeptide. pph-1 is located between pdx-1 and col-4 on the right arm of N.crassa linkage group IV. pph-1 transcript levels are highest during the first hours of conidial germination. Failure to obtain viable progeny in which pph-1 had been inactivated via the repeat-induced point (RIP) mutation process, and evidence that nuclei harboring a disrupted pph-1 gene could only be maintained in a heterokaryon, indicated that a functional pph-1 gene is essential for fungal growth. This is the first report providing evidence that inactivation of a single-type-2A protein phosphatase gene results in a lethal phenotype in fungi.

The B regulatory subunit of protein phosphatase 2A is required for completion of macroconidiation and other developmental processes in Neurospora crassa.

rgb-1, encoding the tentative B regulatory subunit of the type 2A Ser/Thr phosphatase in Neurospora crassa, was isolated from cDNA and genomic libraries. Based on analysis of cDNA and genomic clones, rgb-1 is 3387 nucleotides in length, contains seven putative introns and encodes a 461-amino-acid polypeptide. Intron I, which is 5' to the presumed translation initiation codon, contains a uORF encoding 34 amino acids. Intron VI undergoes alternative splicing. Inactivation of rgb-1 by the repeat-induced point (RIP) mutation procedure produced progeny that grow slowly, have abnormal hyphal morphology, are female sterile and produce abundant amounts of arthroconidia. The rgb-1RIP strain does not produce major constriction chains or mature macroconidia. Minor constriction chains are formed, yet the growth process reverts to hyphal elongation. Microscopic and genetic analyses indicate that rgb-1 is a regulator of the budding subroutine of the macroconidiation process and that arthroconidiation, which shares common early and late events with macroconidiation, is induced as a default mechanism for asexual reproduction in this fungus.

The Neurospora crassa chs-2 gene encodes a non-essential chitin synthase.

Chitin is a structural component of morphologically distinct structures assembled during various phases of growth and development in filamentous fungi. In Neurospora crassa, at least three different DNA fragments related to chitin synthase have been identified. In this study we cloned, sequenced and characterized the chitin synthase 2 structural gene (designated chs-2). The amino acid sequence deduced from the cloned chs-2 genomic DNA fragments is very similar to that of chitin synthase genes isolated from other fungi. Inactivation of the N. crassa chs-2 gene by repeat-induced point (RIP) mutation produced progeny which under standard growth conditions were indistinguishable from the wild-type. However, a significant reduction in chitin synthase activity and increased sensitivity to the phosphatidylcholine biosynthesis inhibitor edifenphos are characteristic of the chs-2RIP strain.

Transcript and activity levels of different Pleurotus ostreatus peroxidases are differentially affected by Mn2+.

The white-rot fungus Pleurotus ostreatus produces both manganese-dependent peroxidase (MnP) and versatile peroxidase (VP) in non-manganese-amended peptone medium (PM). We studied the effect of Mn2+ supplementation on MnPs and VPs in P. ostreatus by analysing the enzymatic and transcript abundance profiles of the peroxidases, as well as the lignin mineralization rate. The fungus was grown in PM under solid-state conditions using perlite as an inert solid support. Mn2+ amendment resulted in a 1.7-fold increase in [14C]-lignin mineralization relative to unamended medium. Anion-exchange chromatography was used to resolve the fungal peroxidase's enzymatic activity profile. Five peaks (P1-P5) of VP and one peak (P6) of MnP activity were detected in unamended medium. In Mn2+-amended medium, a reduction in the activity of the VPs was observed. On the other hand, a sharp increase in the MnP activity level of peak P6 was detected. The P6 isoenzyme was purified and showed manganese-dependent peroxidation of phenolic substrates. Internal sequence analysis of the purified enzyme revealed 100% identity with the deduced amino acid sequence of P. ostreatus MnP3 (GenBank AB016519). The effect of Mn2+ on the relative abundance of gene transcripts of three VPs and one MnP from P. ostreatus was monitored using reverse transcription-polymerase chain reaction (RT-PCR) with oligonucleotide primer sets synthesized on the basis of non-conserved sequences of the different peroxidases. The reduction in VP gene transcript abundance and the increase in mnp3 transcript level were collinear with the changes observed in the enzyme activity profiles. These results indicate that the activity of peroxidases is regulated at the transcriptional level. We suggest that the expression of MnP and VP may be differentially regulated by the presence of Mn2+.

Serine/threonine protein kinases and phosphatases in filamentious fungi.

Protein phosphorylation and dephosphorylation are one of the central currencies by which living cells perceive and respond to environmental cues. A number of fundamental processes in fungi such as the cell cycle, transcription, and mating have been shown to require protein phosphorylation. The analysis of protein kinases and phosphatases in filamentous fungi is in its infancy; however, it has already become clear that kinases and phosphatases are likely to be important mediators of fungal proliferation and development as well as signal transduction and infection-related morphogenesis. In this review, we describe, summarize, and consider the rapidly expanding field of protein phosphorylation/dephosphorylation in various aspects of filamentous fungal growth and development.

A dominant selectable marker that is meiotically stable in Neurospora crassa: the amdS gene of Aspergillus nidulans.

When Neurospora crassa is transformed using a Neurospora gene as the selectable marker, the vegetatively stable transformants obtained cannot be used successfully in a cross because the selectable marker will be inactivated by the process of RIP (repeat-induced point mutation). Introduction of the acetamidase-encoding gene amdS of Aspergillus nidulans into N. crassa by transformation yielded transformants that would grow in minimal medium containing acetamide as a sole nitrogen source. In mitotically stable transformants containing a single copy of the amdS gene, the capacity to utilize acetamide as a sole nitrogen source was maintained in the progeny of a sexual cross. Therefore, the A. nidulans amdS gene is an appropriate dominant selectable marker for use in transformation analyses with N. crassa in which sexual crosses will be subsequently performed.

The involvement of polyphenols and peroxidase activities in heavy-metal accumulation by epidermal glands of the waterlily (Nymphaeaceae).

Co-localization of polyphenols and peroxidase activity was demonstrated in epidermal glands of the waterlily (Nymphaea) by histochemistry. Total phenols, tannins and peroxidase activity were determined quantitatively in plant extracts. Polyphenols were partially identified and were found to consist mainly of hydrolyzable tannins, gallic and tannic acid derivatives. Nymphaea polyphenols were shown to chelate Cr, Hg, and Pb in vitro, and Cd-binding by polymerized polyphenols was demonstrated in leaves exposed to Cd in vivo. Both polyphenols and peroxidases were found at very high constitutive levels, which were not induced or altered by external conditions, such as light and heavy-metal stress. It is suggested that the polymerization of polyphenols by peroxidases, enhanced after heavy-metal uptake and detoxification, is responsible for the binding of heavy metals in Nymphaea epidermal glands.