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Cryphonectria parasitica is the causal agent of chestnut blight, a fungal disease that almost entirely eliminated mature American chestnut from North America over a 50-year period. Here, we formally report the genome of C. parasitica EP155 using a Sanger shotgun sequencing approach. After finishing and integration with simple-sequence repeat markers, the assembly was 43.8 Mb in 26 scaffolds (L50 = 5; N50 = 4.0Mb). Eight chromosomes are predicted: five scaffolds have two telomeres and six scaffolds have one telomere sequence. In total, 11,609 gene models were predicted, of which 85% show similarities to other proteins. This genome resource has already increased the utility of a fundamental plant pathogen experimental system through new understanding of the fungal vegetative incompatibility system, with significant implications for enhancing mycovirus-based biological control.

BackgroundA multicenter study of pectus excavatum was described previously. This report presents our final results.Study designPatients treated surgically at 11 centers were followed prospectively. Each underwent a preoperative evaluation with CT scan, pulmonary function tests, and body image survey. Data were collected about associated conditions, complications, and perioperative pain. One year after treatment, patients underwent repeat chest CT scan, pulmonary function tests, and body image survey. A subset of 50 underwent exercise pulmonary function testing.ResultsOf 327 patients, 284 underwent Nuss procedure and 43 underwent open procedure without mortality. Of 182 patients with complete follow-up (56%), 18% had late complications, similarly distributed, including substernal bar displacement in 7% and wound infection in 2%. Mean initial CT scan index of 4.4 improved to 3.0 post operation (severe >3.2, normal = 2.5). Computed tomography index improved at the deepest point (xiphoid) and also upper and middle sternum. Pulmonary function tests improved (forced vital capacity from 88% to 93%, forced expiratory volume in 1 second from 87% to 90%, and total lung capacity from 94% to 100% of predicted (p < 0.001 for each). VO2 max during peak exercise increased by 10.1% (p = 0.015) and O2 pulse by 19% (p = 0.007) in 20 subjects who completed both pre- and postoperative exercise tests.ConclusionsThere is significant improvement in lung function at rest and in VO2 max and O2 pulse after surgical correction of pectus excavatum, with CT index >3.2. Operative correction significantly reduces CT index and markedly improves the shape of the entire chest, and can be performed safely in a variety of centers.

Using an established spotted cDNA microarray platform, the nature of changes in the transcriptional profiles of 2200 unique genes from the chestnut blight fungus Cryphonectria parasitica in response to the absence of either the G[alpha] subunit CPG-1 or the G[beta] subunit CPGB-1 has been explored. It is reported that 216 transcripts were altered in accumulation in the [DELTA]cpg-1 strain and 163 in the [DELTA]cpgb- 1 strain, with a considerable overlap (100 genes) that were changed in both cases. Of note, these commonly altered transcripts were changed in the same direction in every instance, thus suggesting a considerable redundancy in pathway control or extensive crosstalk. To further knowledge of the potential impact on G-protein-signalling of infection by hypovirus CHV1-EP713, the accumulation of CPG-1 and CPGB-1 was also investigated by Western analysis. It was demonstrated that both signalling components were reduced in abundance to approximately 25% of wild-type levels, while their transcripts were slightly elevated. Comparison of a list of genes with altered expression in the presence of CHV1-EP713 to the data obtained in the absence of either G-protein subunit showed that more than one-half of all the transcripts changed by hypovirus infection were also changed in at least one G-protein mutant strain, with one-third being changed in both. Significantly, 95% of the co-changed genes were altered in the same direction. These data provide the first evidence for modulation of G[beta] protein levels as well as the G[beta][gamma]-signalling pathways by hypovirus infection, and support the hypothesis that modification of G-protein-signalling via both G[alpha] and G[beta][gamma] provides for a significant contribution to hypovirus-mediated phenotype.

Cryphonectria parasitica, the causative agent of chestnut blight, has proven to be a tractable experimental system for studying fungal pathogenesis. Moreover, the development of infectious cDNA clones of C. parasitica hypoviruses, capable of attenuating fungal virulence, has provided the opportunity to examine molecular aspects of fungal plant pathogenesis in the context of biological control. In order to establish a genomic base for future studies of C. parasitica, the authors have analysed a collection of expressed sequences. A mixed cDNA library was prepared from RNA isolated from wild-type (virus-free) and hypovirus-infected C. parasitica strains. Plasmid DNA was recovered from individual transformants and sequenced from the 5' end of the insert. Contig analysis of the collected sequences revealed that they represented approximately 2200 individual ORFs. An assessment of functional diversity present in this collection was achieved by using the BLAST software utilities and the NCBI protein database. Candidate genes were identified with significant potential relevance to C. parasitica growth, development, pathogenesis and vegetative incompatibility. Additional investigations of a 12.9 kbp genomic region revealed microsynteny between C. parasitica and both Neurospora crassa and Magnaporthe grisea, two closely related fungi. These data represent the largest collection of sequence information currently available for C. parasitica and are now forming the basis of further studies using microarray analyses to determine global changes in transcription that occur in response to hypovirus infection.

Reduced accumulation of the GTP-binding protein [G.sub.i][Alpha] subunit CPG-1, due either to hypovirus infection or transgenic cosuppression, correlates with virulence attenuation of the chestnut blight fungus, Cryphonectria parasitica. The role of G protein-mediated signal transduction in fungal virulence was further examined by targeted disruption of the gene cpg-1, encoding CPG-1, and a second G[Alpha] gene, cpg-2, encoding the subunit CPG-2. Disruption of cpg-1 resulted in a set of phenotypic changes similar to, but more severe than, those associated with hypovirus infection. Changes included a marked reduction in fungal growth rate and loss of virulence, asexual sporulation, female fertility, and transcriptional induction of the gene lac-1, encoding the enzyme laccase. In contrast, cpg-2 disruption resulted in only slight reductions in growth rate and asexual sporulation and no significant reduction in virulence, female fertility, or lac-1 mRNA inducibility. These results provide definitive confirmation of previous correlative evidence that suggested a requirement of CPG-1-linked signaling for a number of fungal processes, including virulence and reproduction, while demonstrating that a second G[Alpha], CPG-2, is dispensable for these processes. They also significantly strengthen support for the apparent linkage between hypovirus-mediated disruption of G protein signal transduction and attenuation of fungal virulence.

The gene enpg-1 from the chestnut blight fungus, Cryphonectria parasitica that codes the main extracellular endopolygalacturonase (endoPG) does not contribute to fungal virulence. The gene product, ENPG-1 has a molecular weight of 34.5 kDa and a pI of 7.2, and is 66% similar to endoPG from the maize pathogen Cochliobolus carbonum. Isoelectric focusing of the polygalacturonase activity indicates that infected tissues have low ENGP-1 activity and disruption mutant-infected tissues have no ENGP-1.

Analysis of the chestnut blight fungus Cryphonectria parasitica strains carrying RNA viruses of the Hypovirus genus reveals reduced levels of a heterotrimeric GTP-binding protein (G protein) alpha subunit. These strains exhibit hypovirulence. Transgenic suppression diminishes this alpha subunit levels in virus-free fungal strains which also exhibit hypovirulence. Hence G-protein-linked signal transmission contributes to fungal pathogenesis.

A natural form of biological control was developed as an alternative to the environmentally hazardous chemical means of control of microbial pests. The virulence of the chestnut blight fungus, Cryphonectaria parasitica, was attentuated by the manipulation of an endogenous viral RNA genetic element. Further studies had been planned in the use of the genetically engineered transmissible hypovirulence for a more effective management of pathogenic fungi.

Targeted disruption of G[Alpha] and G[Beta] genes has established the requirement of an intact G protein signaling pathway for optimal execution of several important physiological processes, including pathogenesis, in the chestnut blight fungus Cryphonectria parasitica. We now report the identification of a G protein signal transduction component, beta disruption mimic factor-1, BDM-1. Disruption of the corresponding gene, bdm-1, resulted in a phenotype indistinguishable from that previously observed after disruption of the G[Beta] subunit gene, cpgb-1. The BDM-1 deduced amino acid sequence contained several significant clusters of identity with mammalian phosducin, including a domain corresponding to a highly conserved 11-amino acid stretch that has been implicated in binding to the G[Beta][Gamma], dimer and two regions of defined G[Beta]/ phosducin contact points. Unlike the negative regulatory function proposed for mammalian phosducin, the genetic data presented in this report suggest that BDM-1 is required for or facilitates G[Beta] function. Moreover, disruption of either bdm-1 or cpgb-1 resulted in a significant, posttranscriptional reduction in the accumulation of CPG-1, a key G[Alpha] subunit required for a range of vital physiological processes. Cryphonectria parasitica | fungal pathogenesis | phosducin

Transmission of mycoviruses that attenuate virulence (hypovirulence) of pathogenic fungi is restricted by allorecognition systems operating in their fungal hosts. We report the use of systematic molecular gene disruption and classical genetics for engineering fungal hosts with superior virus transmission capabilities. Four of five diallelic virus-restricting allorecognition [vegetative incompatibility (vic)] loci were disrupted in the chestnut blight fungus Cryphonectria parasitica using an adapted Cre-loxP recombination system that allowed excision and recycling of selectable marker genes (SMGs). SMG-free, quadruple vic mutant strains representing both allelic backgrounds of the remaining vic locus were then produced through mating. In combination, these super donor strains were able to transmit hypoviruses to strains that were heteroallelic at one or all of the virus-restricting vic loci. These results demonstrate the feasibility of modulating allorecognition to engineer pathogenic fungi for more efficient transmission of virulence-attenuating mycoviruses and enhanced biological control potential.a [ABSTRACT FROM AUTHOR]