Variations in hypovirus interactions with the fungal-host RNA-silencing antiviral-defense response.

Hypoviruses Cryphonectria hypovirus 1 (CHV-1)/EP713, CHV-1/Euro7, and CHV-1/EP721, which infect the chestnut blight fungus Cryphonectria parasitica, differ in their degrees of virulence attenuation (hypovirulence), symptom expression, and viral RNA accumulation, even though they share between 90% and 99% amino acid sequence identity. In this report we examine whether this variability is influenced by interactions with the C. parasitica Dicer gene dcl2-dependent RNA-silencing antiviral defense response. The mild symptoms exhibited by strains infected with CHV-1/Euro7 and CHV-1/EP721 relative to those with severe hypovirus CHV-1/EP713 did not correlate with a higher induction of the RNA-silencing pathway. Rather, dcl2 transcripts accumulated to a higher level (∼8-fold) following infection by CHV-1/EP713 than following infection by CHV-1/Euro7 (1.2-fold) or CHV-1/EP721 (1.4-fold). The differences in dcl2 transcript accumulation in response to CHV-1/EP713 and CHV-1/EP721 were unrelated to the suppressor of RNA silencing, p29, encoded by the two viruses. Moreover, the coding strand viral RNA levels increased by 33-, 32-, and 16-fold for CHV-1/EP713, CHV-1/Euro7, and CHV-1/EP721, respectively, in Δdcl2 mutant strains. This indicates that a very robust antiviral RNA-silencing response was induced against all three viruses, even though significant differences in the levels of dcl2 transcript accumulation were observed. Unexpectedly, the severe debilitation previously reported for CHV-1/EP713-infected Δdcl2 mutant strains, and observed here for the CHV-1/Euro7-infected Δdcl2 mutant strains, was not observed with infection by CHV-1/EP721. By constructing chimeric viruses containing portions of CHV-1/EP713 and CHV-1/EP721, it was possible to map the region that is associated with the severe debilitation of the Δdcl2 mutant hosts to a 4.1-kb coding domain located in the central part of the CHV-1/EP713 genome.

Resolving arthropod phylogeny: exploring phylogenetic signal within 41 kb of protein-coding nuclear gene sequence.

This study attempts to resolve relationships among and within the four basal arthropod lineages (Pancrustacea, Myriapoda, Euchelicerata, Pycnogonida) and to assess the widespread expectation that remaining phylogenetic problems will yield to increasing amounts of sequence data. Sixty-eight regions of 62 protein-coding nuclear genes (approximately 41 kilobases (kb)/taxon) were sequenced for 12 taxonomically diverse arthropod taxa and a tardigrade outgroup. Parsimony, likelihood, and Bayesian analyses of total nucleotide data generally strongly supported the monophyly of each of the basal lineages represented by more than one species. Other relationships within the Arthropoda were also supported, with support levels depending on method of analysis and inclusion/exclusion of synonymous changes. Removing third codon positions, where the assumption of base compositional homogeneity was rejected, altered the results. Removing the final class of synonymous mutations--first codon positions encoding leucine and arginine, which were also compositionally heterogeneous--yielded a data set that was consistent with a hypothesis of base compositional homogeneity. Furthermore, under such a data-exclusion regime, all 68 gene regions individually were consistent with base compositional homogeneity. Restricting likelihood analyses to nonsynonymous change recovered trees with strong support for the basal lineages but not for other groups that were variably supported with more inclusive data sets. In a further effort to increase phylogenetic signal, three types of data exploration were undertaken. (1) Individual genes were ranked by their average rate of nonsynonymous change, and three rate categories were assigned--fast, intermediate, and slow. Then, bootstrap analysis of each gene was performed separately to see which taxonomic groups received strong support. Five taxonomic groups were strongly supported independently by two or more genes, and these genes mostly belonged to the slow or intermediate categories, whereas groups supported only by a single gene region tended to be from genes of the fast category, arguing that fast genes provide a less consistent signal. (2) A sensitivity analysis was performed in which increasing numbers of genes were excluded, beginning with the fastest. The number of strongly supported nodes increased up to a point and then decreased slightly. Recovery of Hexapoda required removal of fast genes. Support for Mandibulata (Pancrustacea + Myriapoda) also increased, at times to "strong" levels, with removal of the fastest genes. (3) Concordance selection was evaluated by clustering genes according to their ability to recover Pancrustacea, Euchelicerata, or Myriapoda and analyzing the three clusters separately. All clusters of genes recovered the three concordance clades but were at times inconsistent in the relationships recovered among and within these clades, a result that indicates that the a priori concordance criteria may bias phylogenetic signal in unexpected ways. In a further attempt to increase support of taxonomic relationships, sequence data from 49 additional taxa for three slow genes (i.e., EF-1 alpha, EF-2, and Pol II) were combined with the various 13-taxon data sets. The 62-taxon analyses supported the results of the 13-taxon analyses and provided increased support for additional pancrustacean clades found in an earlier analysis including only EF-1 alpha, EF-2, and Pol II.