Analysis of the Phlebiopsis gigantea genome, transcriptome and secretome provides insight into its pioneer colonization strategies of wood.

Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on fresh-cut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea's extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.

Transcriptome Analysis of a Ustilago maydis ust1 Deletion Mutant Uncovers Involvement of Laccase and Polyketide Synthase Genes in Spore Development.

Ustilago maydis, causal agent of corn smut disease, is a dimorphic fungus alternating between a saprobic budding haploid and an obligate pathogenic filamentous dikaryon. Maize responds to U. maydis colonization by producing tumorous structures, and only within these does the fungus sporulate, producing melanized sexual teliospores. Previously we identified Ust1, an APSES (Asm1p, Phd1p, Sok2p, Efg1p, and StuAp) transcription factor, whose deletion led to filamentous haploid growth and the production of highly pigmented teliospore-like structures in culture. In this study, we analyzed the transcriptome of a ust1 deletion mutant and functionally characterized two highly upregulated genes with potential roles in melanin biosynthesis: um05361, encoding a putative laccase (lac1), and um06414, encoding a polyketide synthase (pks1). The Δlac1 mutant strains showed dramatically reduced virulence on maize seedlings and fewer, less-pigmented teliospores in adult plants. The Δpks1 mutant was unaffected in seedling virulence but adult plant tumors generated hyaline, nonmelanized teliospores. Thus, whereas pks1 appeared to be restricted to the synthesis of melanin, lac1 showed a broader role in virulence. In conclusion, the ust1 deletion mutant provided an in vitro model for sporulation in U. maydis, and functional analysis supports the efficacy of this in vitro mutant analysis for identification of genes involved in in planta teliosporogenesis.

A single amino acid alteration in PGR5 confers resistance to antimycin A in cyclic electron transport around PSI.

In Arabidopsis thaliana, the main route of cyclic electron transport around PSI is sensitive to antimycin A, but the site of inhibition has not been clarified. We discovered that ferredoxin-dependent plastoquinone reduction in ruptured chloroplasts was less sensitive to antimycin A in Arabidopsis that overaccumulated PGR5 (PROTON GRADIENT REGULATION 5) originating from Pinus taeda (PtPGR5) than that in the wild type. Consistent with this in vitro observation, infiltration of antimycin A reduced PSII yields and the non-photochemical quenching (NPQ) of Chl fluorescence in wild-type leaves but not in leaves accumulating PtPGR5. There are eight amino acid differences between PGR5 of Arabidopsis (AtPGR5) and PtPGR5 in their mature forms. To determine the site conferring antimycin A resistance, a series of AtPGR5 and PtPGR5 variants was introduced into the Arabidopsis pgr5 mutant. We determined that the presence of lysine rather than valine at the third amino acid position was necessary and sufficient for resistance to antimycin A. High levels of resistance to antimycin A required overaccumulation of PtPGR5 in ruptured chloroplasts, suggesting that PtPGR5 is partly resistant to antimycin A. In contrast, PSII yield was almost fully resistant to antimycin A in leaves accumulating endogenous levels of PtPGR5 or AtPGR5 V3K that had lysine instead of valine at the third position. NPQ was also dramatically recovered in leaves of these lines. These results imply that partial recovery of PSI cyclic electron transport is sufficient for maintaining redox homeostasis in photosynthesis. Our discovery suggests that antimycin A inhibits the function of PGR5 or proteins localized close to PGR5.

Two members of the Ustilago maydis velvet family influence teliospore development and virulence on maize seedlings.

Members of the fungal-specific velvet protein family regulate sexual and asexual spore production in the Ascomycota. We predicted, therefore, that velvet homologs in the basidiomycetous plant pathogen Ustilago maydis would regulate sexual spore development, which is also associated with plant disease progression in this fungus. To test this hypothesis, we studied the function of three U. maydis velvet genes, umv1, umv2 and umv3. Using a gene replacement strategy, deletion mutants were made in all three genes in compatible haploid strains, and additionally for umv1 and umv2 in the solopathogenic strain, SG200. None of the mutants showed novel morphological phenotypes during yeast-like, in vitro growth. However, the Δumv1 mutants failed to induce galls or teliospores in maize. Chlorazol black E staining of leaves infected with Δumv1 dikaryons revealed that the Δumv1 hyphae did not proliferate normally and were blocked developmentally before teliospore formation. The Δumv2 mutants were able to induce galls and teliospores in maize, but were slow to do so and thus reduced in virulence. The Δumv3 mutants were not affected in teliospore formation or disease progression. Complementation of the Δumv1 and Δumv2 mutations in the SG200 background produced disease indices similar to those of SG200. These results indicate that two U. maydis velvet family members, umv1 and umv2, are important for normal teliospore development and disease progression in maize seedlings.

One fungus, one name: defining the genus Fusarium in a scientifically robust way that preserves longstanding use.

In this letter, we advocate recognizing the genus Fusarium as the sole name for a group that includes virtually all Fusarium species of importance in plant pathology, mycotoxicology, medicine, and basic research. This phylogenetically guided circumscription will free scientists from any obligation to use other genus names, including teleomorphs, for species nested within this clade, and preserve the application of the name Fusarium in the way it has been used for almost a century. Due to recent changes in the International Code of Nomenclature for algae, fungi, and plants, this is an urgent matter that requires community attention. The alternative is to break the longstanding concept of Fusarium into nine or more genera, and remove important taxa such as those in the F. solani species complex from the genus, a move we believe is unnecessary. Here we present taxonomic and nomenclatural proposals that will preserve established research connections and facilitate communication within and between research communities, and at the same time support strong scientific principles and good taxonomic practice.

The genome of Nectria haematococca: contribution of supernumerary chromosomes to gene expansion.

The ascomycetous fungus Nectria haematococca, (asexual name Fusarium solani), is a member of a group of >50 species known as the "Fusarium solani species complex". Members of this complex have diverse biological properties including the ability to cause disease on >100 genera of plants and opportunistic infections in humans. The current research analyzed the most extensively studied member of this complex, N. haematococca mating population VI (MPVI). Several genes controlling the ability of individual isolates of this species to colonize specific habitats are located on supernumerary chromosomes. Optical mapping revealed that the sequenced isolate has 17 chromosomes ranging from 530 kb to 6.52 Mb and that the physical size of the genome, 54.43 Mb, and the number of predicted genes, 15,707, are among the largest reported for ascomycetes. Two classes of genes have contributed to gene expansion: specific genes that are not found in other fungi including its closest sequenced relative, Fusarium graminearum; and genes that commonly occur as single copies in other fungi but are present as multiple copies in N. haematococca MPVI. Some of these additional genes appear to have resulted from gene duplication events, while others may have been acquired through horizontal gene transfer. The supernumerary nature of three chromosomes, 14, 15, and 17, was confirmed by their absence in pulsed field gel electrophoresis experiments of some isolates and by demonstrating that these isolates lacked chromosome-specific sequences found on the ends of these chromosomes. These supernumerary chromosomes contain more repeat sequences, are enriched in unique and duplicated genes, and have a lower G+C content in comparison to the other chromosomes. Although the origin(s) of the extra genes and the supernumerary chromosomes is not known, the gene expansion and its large genome size are consistent with this species' diverse range of habitats. Furthermore, the presence of unique genes on supernumerary chromosomes might account for individual isolates having different environmental niches.

Adenosine A2A receptor gene polymorphisms (ADORA2A) are associated with maximal concentric contraction pain.

Objectives: To test associations of adenosine A2A receptor gene (ADORA2A) polymorphisms with maximal concentric contraction pain, caffeine-induced perceptual changes, and responses to mild eccentric injury. Design: Targeted candidate gene association study. Methods: ADORA2A (rs5751876) was genotyped in 26 adults. Pain of a maximal elbow flexor contraction was assessed. Participants performed 18 eccentric actions of the elbow flexors. Arm volume was measured before and 24- and 48-h post-injury. At 24- and 48-h post-injury, caffeine or placebo capsules were given using a randomized cross-over design and effort and pain were assessed. Results: Maximal concentric contraction pain was higher for the CC compared to the CT/TT group (effect size d = 0.89) and lower for the TT compared to the CT/CC group (d = 0.62). There were moderate sized differences between CC and TT/CT groups for post-injury exertion and arm volume 24-h post injury. Conclusions: ADORA2A polymorphisms are moderately associated with muscle pain during maximal contractions in uninjured muscle.

Investigation of an Outbreak of Fusarium Foot and Fruit Rot of Pumpkin Within the United States.

Isolates of two biologically and phylogenetically distinct species, referred to as Fusarium solani f. sp. cucurbitae race 1 (Fsc-1 = Nectria haematococca mating population I [MPI]) and F. solani f. sp. cucurbitae race 2 (Fsc-2 = N. haematococca mating population V [MPV]), were suspected of causing an outbreak of Fusarium foot and fruit rot of pumpkin during 2001 to 2003 in Connecticut, New York, Ohio, and Missouri. Both species affect the fruit, but Fsc-1 also affects the crown and causes a stem rot. In this study, 156 isolates from affected plants and from soil under diseased fruit that tentatively were identified morphologically as members of the F. solani species complex were assayed for pathogenicity on pumpkin seedlings and mature fruit. Results of the pathogenicity assay indicated that 81 of the isolates were Fsc-1. The remaining 74 isolates were either nonpathogenic or only weakly pathogenic on the fruit. A subset of 53 test isolates from soil and plants, plus reference isolates of Fsc-1 and Fsc-2 and an isolate from wheat reported to cause a seedling rot on cucurbits, were characterized phylogenetically by sequencing a portion of the translation elongation factor 1-α gene. A BLAST query of the FUSARIUM-ID database at Pennsylvania State University indicated that 42 of the 53 test isolates were Fsc-1, whereas none were typed as Fsc-2. A polymerase chain reaction assay for mating-type (MAT) idiomorph revealed that all of the Fsc-1 isolates were MAT-1-2, suggesting that the pathogen may be strictly clonal in the affected fields. These findings provide convincing evidence that the Fusarium foot and fruit rot outbreaks were incited by Fsc-1.

Conserved role of proton gradient regulation 5 in the regulation of PSI cyclic electron transport.

There are at least two photosynthetic cyclic electron transport (CET) pathways in most C(3) plants: the NAD(P)H dehydrogenase (NDH)-dependent pathway and a pathway dependent upon putative ferredoxin:plastoquinone oxidoreductase (FQR) activity. While the NDH complex has been identified, and shown to play a role in photosynthesis, especially under stress conditions, less is known about the machinery of FQR-dependent CET. Recent studies indicate that FQR-dependent CET is dependent upon PGR5, a small protein of unknown function. In a previous study we found that overexpression of PGR5 causes alterations in growth and development associated with decreased chloroplast development and a transient increase in nonphotochemical quenching (NPQ) after the shift from dark to light. In the current study we examine the spatiotemporal expression pattern of PGR5, and the effects of overexpression of PGR5 in Arabidopsis under a host of light and stress conditions. To investigate the conserved function of PGR5, we cloned PGR5 from a species which apparently lacks NDH, loblolly pine, and overexpressed it in Arabidopsis. Although greening of cotyledons was severely delayed in overexpressing lines under low light, mature plants survived exposure to high light and drought stress better than wild-type. In addition, PSI was more resistant to high light in the PGR5 overexpressors than in wild-type plants, while PSII was more sensitive to this stress. These complex responses corresponded to alterations in linear and cyclic electron transfer, suggesting that over-accumulation of PGR5 induces pleiotropic effects, probably via elevated CET. We conclude that PGR5 has a developmentally-regulated, conserved role in mediating CET.

Genetics of morphogenesis and pathogenic development of Ustilago maydis.

Ustilago maydis has emerged as an important model system for the study of fungi. Like many fungi, U. maydis undergoes remarkable morphological transitions throughout its life cycle. Fusion of compatible, budding, haploid cells leads to the production of a filamentous dikaryon that penetrates and colonizes the plant, culminating in the production of diploid teliospores within fungal-induced plant galls or tumors. These dramatic morphological transitions are controlled by components of various signaling pathways, including the pheromone-responsive MAP kinase and cAMP/PKA (cyclic AMP/protein kinase A) pathways, which coregulate the dimorphic switch and sexual development of U. maydis. These signaling pathways must somehow cooperate with the regulation of the cytoskeletal and cell cycle machinery. In this chapter, we provide an overview of these processes from pheromone perception and mating to gall production and sporulation in planta. Emphasis is placed on the genetic determinants of morphogenesis and pathogenic development of U. maydis and on the fungus-host interaction. Additionally, we review advances in the development of tools to study U. maydis, including the recently available genome sequence. We conclude with a brief assessment of current challenges and future directions for the genetic study of U. maydis.

A balanced PGR5 level is required for chloroplast development and optimum operation of cyclic electron transport around photosystem I.

PSI cyclic electron transport contributes markedly to photosynthesis and photoprotection in flowering plants. Although the thylakoid protein PGR5 (Proton Gradient Regulation 5) has been shown to be essential for the main route of PSI cyclic electron transport, its exact function remains unclear. In transgenic Arabidopsis plants overaccumulating PGR5 in the thylakoid membrane, chloroplast development was delayed, especially in the cotyledons. Although photosynthetic electron transport was not affected during steady-state photosynthesis, a high level of non-photochemical quenching (NPQ) was transiently induced after a shift of light conditions. This phenotype was explained by elevated activity of PSI cyclic electron transport, which was monitored in an in vitro system using ruptured chloroplasts, and also in leaves. The effect of overaccumulation of PGR5 was specific to the antimycin A-sensitive pathway of PSI cyclic electron transport but not to the NAD(P)H dehydrogenase (NDH) pathway. We propose that a balanced PGR5 level is required for efficient regulation of the rate of antimycin A-sensitive PSI cyclic electron transport, although the rate of PSI cyclic electron transport is probably also regulated by other factors during steady-state photosynthesis.

FsFKS1, the 1,3-beta-glucan synthase from the caspofungin-resistant fungus Fusarium solani.

The cell wall, a mesh of carbohydrates and proteins, shapes and protects the fungal cell. The enzyme responsible for the synthesis of one of the main components of the fungal wall, 1,3-beta-glucan synthase, is targeted by the antifungal caspofungin acetate (CFA). Clinical isolates of Candida albicans and Aspergillus fumigatus are much more sensitive to CFA than clinical isolates of Fusarium species. To better understand CFA resistance in Fusarium species, we cloned and sequenced FsFKS1, which encodes the Fusarium solani f. sp. pisi beta(1,3)-D-glucan synthase, used RNA interference to reduce its expression and complemented deletion of the essential fks gene of the CFA-sensitive fungus A. fumigatus with FsFKS1. Reduction of the FsFKS1 message in F. solani f. sp. pisi reduced spore viability and caused lysis of spores and hyphae, consistent with cell wall defects. Compensating for the loss of A. fumigatus fks1 with FsFKS1 caused only a modest increase in the tolerance of A. fumigatus for CFA. Our results suggest that FsFKS1 is required for the proper construction of F. solani cell walls and that the resistance of F. solani to CFA is at best only partially due to resistance of the FsFKS1 enzyme to this antifungal agent.

Differential expression of pine and Cronartium quercuum f. sp. fusiforme genes in fusiform rust galls.

Cronartium quercuum f. sp. fusiforme is the causative agent of fusiform rust disease of southern pines in the United States. This disease is characterized by the formation of woody branch and stem galls. Differential display was used to identify pine genes whose expression is altered by C. quercuum f. sp. fusiforme infection and to identify C. quercuum f. sp. fusiforme genes that are expressed in fusiform rust galls. Six pine cDNAs that appeared to be differentially expressed in galled and healthy stems and 13 C. quercuum f. sp. fusiforme cDNAs expressed in galled tissues were identified. A probe that hybridizes specifically to C. quercuum f. sp. fusiforme 18S rRNA was used to estimate that 14% of the total RNA in fusiform rust galls was from C. quercuum f. sp. fusiforme. This finding was used to calibrate gene expression levels in galls when comparing them to expression levels in uninfected pines or in isolated C. quercuum f. sp. fusiforme cultures. According to Northern analysis and reverse transcriptase PCR analysis, all six of the pine clones were expressed at lower levels in galls than in healthy tissues. Seven of the nine C. quercuum f. sp. fusiforme clones that were assayed were expressed at higher levels in galls than in axenic culture. A number of the cDNAs encode proteins that are similar to those that play roles in plant development, plant defense, or fungal stress responses.

Nht2, a copia LTR retrotransposon from a conditionally dispensable chromosome in Nectria haematococca.

In the plant pathogenic ascomycete Nectria haematococca mating population (MP) VI, the conditionally dispensable chromosomes are unstable during sexual reproduction. During mapping of such a chromosome, three dispersed repeats were identified. Nht2, one of these repeated elements, is a long terminal repeat (LTR) retrotransposon that is 5.9 kb in length. Its deduced amino acid sequence is homologous to the four enzymatic domains characteristic of copia retrotransposons, but it contains multiple stop codons and probably is no longer able to transpose autonomously. Nht2's LTRs differ at ten positions and the characteristics of these differences resemble the changes induced by repeat-induced point mutation (RIP) in Neurospora crassa. The likelihood that Nectria haematococca MP VI has a RIP-like process, however, is reduced by the fact that a multi-copy transposon cloned from the same ascospore isolate as Nht2 encodes an intact open reading frame. Nht2 is broadly distributed among isolates collected from a variety of host plants. A limited survey of three field isolates suggests that Nht2 is on only one or a few chromosomes in every genome. Nht2's degeneracy and its widespread distribution within the species both suggest that it is an ancient element within N. haematococca MP VI.

Physical mapping of plasmid and cosmid clones in filamentous fungi by fiber-FISH.

Fluorescence in situ hybridization to extended DNA fibers (fiber-FISH) serves as a powerful tool for direct physical mapping in plants and animals. Here, we show that fiber-FISH is useful for contig mapping as well as for estimating the physical distance between genetic markers in fungi. A five-cosmid contig from a chromosome of Nectria haematococca and four cloned genetic markers from a linkage map of Cochliobolus heterostrophus were chosen as models for the application of this technology. In N. haematococca, overlapping and non-overlapping clones were visually mapped on individual DNA fibers, confirming the results from conventional physical mapping perfectly. Fiber-FISH concomitantly indicated the gap size or the extent of overlap between two clones. In C. heterostrophus, the physical distance between the two pairs of genetic markers could be estimated from the microscopic measurements of the intervals. Chromosomal DNA isolated from a pulsed field gel was suitable for preparing the DNA fibers.

Pine genes regulated by the necrotrophic pathogen Fusarium circinatum.

A targeted genomics approach was used to construct a cDNA array of potential pathogen-regulated genes for investigating host-pathogen interactions in pine trees (Pinus species). This array, containing a nonredundant set of 311 cDNAs, was assembled by combining smaller sets of cDNAs generated by differential display or suppression subtraction hybridization using a variety of pathogen treatments and elicitors. The array was probed to identify host genes regulated by Fusarium circinatum, a necrotrophic fungus that incited pitch canker disease on pine stems. A set of 29 cDNAs were induced during the disease state. Notably, cDNAs on the array that were derived from experiments with fusiform rust, incited by Cronartium quercuum f. sp. fusiforme (a biotrophic fungus) were unregulated by Fusarium. the results imply distinct genetic responses in pine to diseases incited by necrotrophs and biotrophs. This cDNA collection expands the genomics toolkit for understanding interactions between conifers and their microbial associates in forest ecosystems.