The chestnut blight fungus, Cryphonectria parasitica, and associated virulence-attenuating hypoviruses have emerged as an important model system for studying molecular mechanisms underlying fungal-plant pathogenic interactions. As more gene sequence information becomes available as a result of C. parasitica express sequence tags (ESTs) and ongoing whole genome sequencing projects, the development of an efficient gene disruption system has become an urgent need for functional genomics studies of this important forestry pathogen. Here, we report the cloning of the C. parasitica gene cpku80 that encodes a key component of the nonhomologous end joining DNA repair pathway and the construction of a corresponding deletion mutant strain. The cpku80 mutant was indistinguishable from the parental wild-type strain EP155 in colony morphology, ability to support hypovirus replication, conidiation and virulence. As predicted, the Deltacpku80 strain did exhibit an increased sensitivity to the mutagen methyl methanesulfonate. A test with three selected genes resulted in a gene disruption efficiency of about 80% for the Deltacpku80 strain, a significant increase over the 2-5% levels of homologous recombination generally observed for the wild-type strain EP155. This efficient homologous recombination system provides a powerful tool for large-scale analysis of gene functions in C. parasitica.
Cryphonectria parasitica strain EP721 is infected with a strain of hypovirus CHV1, CHV1-EP721, and exhibits typical hypovirulence-associated traits such as reduced pigmentation and reduced asexual sporulation. However, the accumulation of the viral double-stranded RNA (dsRNA) in this hypovirus-infected C. parasitica strain is atypically low. We now report the complete nucleotide sequence and construction of a full-length infectious cDNA clone for hypovirus CHV1-EP721. The genome sequence of CHV1-EP721 was determined to be 12,724 bp in length and to share extensive homology with two other hypovirus strains, CHV1-Euro7 and CHV1-EP713, with an average of 99% and 90% identities at the nucleotide level and 99% and 92% identities at the amino acid level, respectively. CHV1-EP721 was successfully introduced into virus-free fungal host strain EP721(-v) by transfection with transcripts derived from a full-length viral cDNA. The transfected strain had a phenotype indistinguishable from that of EP721, and the accumulation of CHV1-EP721 dsRNA in the transfectant was lower than those transfected by CHV1-Euro7 and CHV1-EP713 transcripts. Through the construction of chimeric viruses by domain swapping using infectious cDNA clones of CHV1-EP721, CHV1-EP713, and CHV1-Euro7 hypoviruses, the determinant for the low level of viral dsRNA accumulation in CHV1-EP721 was mapped to the second of two CHV1-EP721 open reading frames (ORFs), ORF B. Further refined swapping of domains within ORF B identified a 2.5-kb coding region between p48 and the polymerase domain of CHV1-EP721 as being responsible for the low viral dsRNA accumulation. Evidence is also provided that low rates of hypovirus transmission through conidial spores correlates with low viral dsRNA accumulation.
Angiogenesis is an important mechanism that protects tissue against necrosis following acute ischemic injury. The aim of this study was to investigate whether the Toll-like receptor 2 (TLR2) signaling pathway is involved in angiogenesis following ischemic injury by cell migration and lymphocyte invasion assays in vitro, and a mouse model of hindlimb ischemia by ligation in vivo, respectively. To assess the potential role of TLR2 activation in endothelial cell permeability, HUVECs were pretreated with Pam3CSK4 and analyzed using wound repair and transwell assays. The results showed that the TLR2 agonist induced human umbilical vein endothelial cell (HUVEC) migration and increased the permeability of HUVECs to lymphocyte. The lymphocyte invasion of TLR2 knockout (TLR2-/-) mice was inhibited as compared to that of wild-type (WT) mice. In the mouse model of hindlimb ischemia by ligation, blood perfusion of operated limbs was significantly lower in TLR2-/- compared to WT mice, 7 and 14 days after ligation. TLR2-/- mice showed a decreased CD31 expression in ischemic gastrocnemius at 7 and 14 days after ligation, reduced interleukin-6 (IL-6) level and lowered tumor necrosis factor-α (TNF-α) levels. These findings demonstrated that TLR2 activation promotes cell migration, cell permeability and the lymphocyte invasion of endothelial cells. TLR2 activation promotes angiogenesis in vivo, which may be associated with the serum of TNF-α levels and IL-6 release.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.