New Taxon-Specific Heterobasidion PCR Primers Detect and Differentiate North American Heterobasidion spp. in Various Substrates and Led to the Discovery of Heterobasidion irregulare in British Columbia, Canada.

Heterobasidion annosum sensu lato is a species complex of pathogenic white-rot wood decay fungi which cause root and butt rot in conifer and hardwood species across the Northern hemisphere. Annual losses to forest managers are valued in the billions of dollars, due to tree mortality, reduction in timber yield, and wood decay. In North America, H. irregulare and H. occidentale have a partially overlapping host and geographic range, cause similar disease symptoms and produce similar fruiting bodies, making discrimination between the two of them often difficult. We developed two sets of primers that bind specifically to conserved, but species-specific portions of glyceraldehyde 3-phosphate dehydrogenase and elongation factor 1α alleles. The method is sensitive enough to detect either species from infected wood. Analysis of North American isolates has further clarified the distribution of both species on this continent, including the detection of H. irregulare for the first time on ponderosa pine (Pinus ponderosa) and eastern white pine (Pinus strobus) in British Columbia. This method has the potential to be a valuable tool for the detection of the pathogen in exported/imported wood products, as well as for the further identification and assessment of the distribution of North American Heterobasidion species.

Gene expression profiling of candidate virulence factors in the laminated root rot pathogen Phellinus sulphurascens.

BACKGROUND: Phellinus sulphurascens is a fungal pathogen that causes laminar root rot in conifers, one of the most damaging root diseases in western North America. Despite its importance as a forest pathogen, this fungus is still poorly studied at the genomic level. An understanding of the molecular events involved in establishment of the disease should help to develop new methods for control of this disease. RESULTS: We generated over 4600 expressed sequence tags from two cDNA libraries constructed using either mycelia grown on cellophane sheets and exposed to Douglas-fir roots or tissues from P. sulphurascens-infected Douglas-fir roots. A total of 890 unique genes were identified from the two libraries, and functional classification of 636 of these genes was possible using the Functional Catalogue (FunCat) annotation scheme. cDNAs were identified that encoded 79 potential virulence factors, including numerous genes implicated in virulence in a variety of phytopathogenic fungi. Many of these putative virulence factors were also among 82 genes identified as encoding putatively secreted proteins. The expression patterns of 86 selected fungal genes over 7 days of infection of Douglas-fir were examined using real-time PCR, and those significantly up-regulated included rhamnogalacturonan acetylesterase, 1,4-benzoquinone reductase, a cyclophilin, a glucoamylase, 3 hydrophobins, a lipase, a serine carboxypeptidase, a putative Ran-binding protein, and two unknown putatively secreted proteins called 1 J04 and 2 J12. Significantly down-regulated genes included a manganese-superoxide dismutase, two metalloproteases, and an unknown putatively secreted protein called Ps0058. CONCLUSIONS: This first collection of Phellinus sulphurascens EST sequences and its annotation provide an important resource for future research aimed at understanding key virulence factors of this forest pathogen. We examined the expression patterns of numerous fungal genes with potential roles in virulence, and found a collection of functionally diverse genes that are significantly up- or down-regulated during infection of Douglas-fir seedling roots by P. sulphurascens.

Comparative analysis of poxvirus orthologues of the vaccinia virus E3 protein: modulation of protein kinase R activity, cytokine responses, and virus pathogenicity.

Poxviruses are important human and animal pathogens that have evolved elaborate strategies for antagonizing host innate and adaptive immunity. The E3 protein of vaccinia virus, the prototypic member of the orthopoxviruses, functions as an inhibitor of innate immune signaling and is essential for vaccinia virus replication in vivo and in many human cell culture systems. However, the function of orthologues of E3 expressed by poxviruses of other genera with different host specificity remains largely unknown. In the present study, we characterized the E3 orthologues from sheeppox virus, yaba monkey tumor virus, swinepox virus, and myxoma virus for their ability to modulate protein kinase R (PKR) function, cytokine responses and virus pathogenicity. We found that the E3 orthologues of myxoma virus and swinepox virus could suppress PKR activation and interferon (IFN)-induced antiviral activities and restore the host range function of E3 in HeLa cells. In contrast, the E3 orthologues from sheeppox virus and yaba monkey tumor virus were unable to inhibit PKR activation. While the sheeppox orthologue was unable to restore the host range function of E3, the yaba monkey tumor virus orthologue partially restored E3-deficient vaccinia virus replication in HeLa cells, correlated with its ability to suppress IFN-induced antiviral activities. Moreover, poxvirus E3 orthologues show varying ability to inhibit the induction of antiviral and proinflammatory cytokines. Despite these in vitro results, none of the E3 orthologues tested was capable of restoring pathogenicity to E3-deficient vaccinia virus in vivo.

RNA species generated in vaccinia virus infected cells activate cell type-specific MDA5 or RIG-I dependent interferon gene transcription and PKR dependent apoptosis.

RNA species produced during virus replication are pathogen-associated molecular patterns (PAMPs) triggering cellular innate immune responses including induction of type I interferon expression and apoptosis. Pattern recognition receptors (PRRs) for these RNAs include the retinoic acid-inducible gene I (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation associated gene 5 (MDA5) and the dsRNA dependent protein kinase (PKR). Currently, poxvirus PAMPs and their associated PRRs are not well characterized. We report that RNA species generated in vaccinia infected cells can activate MDA5 or RIG-I dependent interferon-ß (IFN-ß) gene transcription in a cell type-specific manner. These RNA species also induce the activation of apoptosis in a PKR dependent, but MDA5 and RIG-I independent, manner. Collectively our results demonstrate that RNA species generated during vaccinia virus replication are major PAMPs activating apoptosis and IFN-ß gene transcription. Moreover, our results delineate the signaling pathways involved in the recognition of RNA-based poxvirus PAMPs.