A massive expansion of effector genes underlies gall-formation in the wheat pest Mayetiola destructor.

Gall-forming arthropods are highly specialized herbivores that, in combination with their hosts, produce extended phenotypes with unique morphologies [1]. Many are economically important, and others have improved our understanding of ecology and adaptive radiation [2]. However, the mechanisms that these arthropods use to induce plant galls are poorly understood. We sequenced the genome of the Hessian fly (Mayetiola destructor; Diptera: Cecidomyiidae), a plant parasitic gall midge and a pest of wheat (Triticum spp.), with the aim of identifying genic modifications that contribute to its plant-parasitic lifestyle. Among several adaptive modifications, we discovered an expansive reservoir of potential effector proteins. Nearly 5% of the 20,163 predicted gene models matched putative effector gene transcripts present in the M. destructor larval salivary gland. Another 466 putative effectors were discovered among the genes that have no sequence similarities in other organisms. The largest known arthropod gene family (family SSGP-71) was also discovered within the effector reservoir. SSGP-71 proteins lack sequence homologies to other proteins, but their structures resemble both ubiquitin E3 ligases in plants and E3-ligase-mimicking effectors in plant pathogenic bacteria. SSGP-71 proteins and wheat Skp proteins interact in vivo. Mutations in different SSGP-71 genes avoid the effector-triggered immunity that is directed by the wheat resistance genes H6 and H9. Results point to effectors as the agents responsible for arthropod-induced plant gall formation.

Evidence for stabilizing selection on codon usage in chromosomal rearrangements of Drosophila pseudoobscura.

There has been a renewed interest in investigating the role of stabilizing selection acting on genome-wide traits such as codon usage bias. Codon bias, when synonymous codons are used at unequal frequencies, occurs in a wide variety of taxa. Standard evolutionary models explain the maintenance of codon bias through a balance of genetic drift, mutation and weak purifying selection. The efficacy of selection is expected to be reduced in regions of suppressed recombination. Contrary to observations in Drosophila melanogaster, some recent studies have failed to detect a relationship between the recombination rate, intensity of selection acting at synonymous sites, and the magnitude of codon bias as predicted under these standard models. Here, we examined codon bias in 2798 protein coding loci on the third chromosome of D. pseudoobscura using whole-genome sequences of 47 individuals, representing five common third chromosome gene arrangements. Fine-scale recombination maps were constructed using more than 1 million segregating sites. As expected, recombination was demonstrated to be significantly suppressed between chromosome arrangements, allowing for a direct examination of the relationship between recombination, selection, and codon bias. As with other Drosophila species, we observe a strong mutational bias away from the most frequently used codons. We find the rate of synonymous and nonsynonymous polymorphism is variable between different amino acids. However, we do not observe a reduction in codon bias or the strength of selection in regions of suppressed recombination as expected. Instead, we find that the interaction between weak stabilizing selection and mutational bias likely plays a role in shaping the composition of synonymous codons across the third chromosome in D. pseudoobscura.

Comparative validation of the D. melanogaster modENCODE transcriptome annotation.

Accurate gene model annotation of reference genomes is critical for making them useful. The modENCODE project has improved the D. melanogaster genome annotation by using deep and diverse high-throughput data. Since transcriptional activity that has been evolutionarily conserved is likely to have an advantageous function, we have performed large-scale interspecific comparisons to increase confidence in predicted annotations. To support comparative genomics, we filled in divergence gaps in the Drosophila phylogeny by generating draft genomes for eight new species. For comparative transcriptome analysis, we generated mRNA expression profiles on 81 samples from multiple tissues and developmental stages of 15 Drosophila species, and we performed cap analysis of gene expression in D. melanogaster and D. pseudoobscura. We also describe conservation of four distinct core promoter structures composed of combinations of elements at three positions. Overall, each type of genomic feature shows a characteristic divergence rate relative to neutral models, highlighting the value of multispecies alignment in annotating a target genome that should prove useful in the annotation of other high priority genomes, especially human and other mammalian genomes that are rich in noncoding sequences. We report that the vast majority of elements in the annotation are evolutionarily conserved, indicating that the annotation will be an important springboard for functional genetic testing by the Drosophila community.

Natural variation in genome architecture among 205 Drosophila melanogaster Genetic Reference Panel lines.

The Drosophila melanogaster Genetic Reference Panel (DGRP) is a community resource of 205 sequenced inbred lines, derived to improve our understanding of the effects of naturally occurring genetic variation on molecular and organismal phenotypes. We used an integrated genotyping strategy to identify 4,853,802 single nucleotide polymorphisms (SNPs) and 1,296,080 non-SNP variants. Our molecular population genomic analyses show higher deletion than insertion mutation rates and stronger purifying selection on deletions. Weaker selection on insertions than deletions is consistent with our observed distribution of genome size determined by flow cytometry, which is skewed toward larger genomes. Insertion/deletion and single nucleotide polymorphisms are positively correlated with each other and with local recombination, suggesting that their nonrandom distributions are due to hitchhiking and background selection. Our cytogenetic analysis identified 16 polymorphic inversions in the DGRP. Common inverted and standard karyotypes are genetically divergent and account for most of the variation in relatedness among the DGRP lines. Intriguingly, variation in genome size and many quantitative traits are significantly associated with inversions. Approximately 50% of the DGRP lines are infected with Wolbachia, and four lines have germline insertions of Wolbachia sequences, but effects of Wolbachia infection on quantitative traits are rarely significant. The DGRP complements ongoing efforts to functionally annotate the Drosophila genome. Indeed, 15% of all D. melanogaster genes segregate for potentially damaged proteins in the DGRP, and genome-wide analyses of quantitative traits identify novel candidate genes. The DGRP lines, sequence data, genotypes, quality scores, phenotypes, and analysis and visualization tools are publicly available.

Epistasis dominates the genetic architecture of Drosophila quantitative traits.

Epistasis-nonlinear genetic interactions between polymorphic loci-is the genetic basis of canalization and speciation, and epistatic interactions can be used to infer genetic networks affecting quantitative traits. However, the role that epistasis plays in the genetic architecture of quantitative traits is controversial. Here, we compared the genetic architecture of three Drosophila life history traits in the sequenced inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and a large outbred, advanced intercross population derived from 40 DGRP lines (Flyland). We assessed allele frequency changes between pools of individuals at the extremes of the distribution for each trait in the Flyland population by deep DNA sequencing. The genetic architecture of all traits was highly polygenic in both analyses. Surprisingly, none of the SNPs associated with the traits in Flyland replicated in the DGRP and vice versa. However, the majority of these SNPs participated in at least one epistatic interaction in the DGRP. Despite apparent additive effects at largely distinct loci in the two populations, the epistatic interactions perturbed common, biologically plausible, and highly connected genetic networks. Our analysis underscores the importance of epistasis as a principal factor that determines variation for quantitative traits and provides a means to uncover genetic networks affecting these traits. Knowledge of epistatic networks will contribute to our understanding of the genetic basis of evolutionarily and clinically important traits and enhance predictive ability at an individualized level in medicine and agriculture.

Preferential DNA cleavage under anaerobic conditions by a DNA-binding ruthenium dimer.

In the absence of dioxygen, the cationic complex [(phen)2Ru(tatpp)Ru(phen)2]4+ (P4+) undergoes in situ reduction by glutathione (GSH) to form a species that induces DNA cleavage. Exposure to air strongly attenuates the cleavage activity, even in the presence of a large excess of reducing agent (e.g., 40 equiv of GSH per P4+), suggesting that the complex may be useful in targeting cells with a low-oxygen microenvironment (hypoxia) for destruction via DNA cleavage. The active species is identified as the doubly reduced, doubly protonated complex H2P4+, and a carbon-based radical species is implicated in the cleavage action. We postulate that the dioxygen concentration regulates the degree to which the carbon radical forms and thus regulates the DNA cleavage activity.

Influence of pH on the photochemical and electrochemical reduction of the dinuclear ruthenium complex, [(phen)2Ru(tatpp)Ru(phen)2)Cl4, in water: proton-coupled sequential and concerted multi-electron reduction.

The dinuclear ruthenium complex [(phen)2Ru(tatpp)Ru(phen)2]4+ (P; in which phen is 1,10-phenanthroline and tatpp is 9,11,20,22-tetraaza tetrapyrido[3,2-a:2'3'-c:3'',2''-l:2''',3''']-pentacene) undergoes a photodriven two-electron reduction in aqueous solution, thus storing light energy as chemical potential within its structure. The mechanism of this reduction is strongly influenced by the pH, in that basic conditions favor a sequential process involving two one-electron reductions and neutral or slightly acidic conditions favor a proton-coupled, bielectronic process. In this complex, the central tatpp ligand is the site of electron storage and protonation of the central aza nitrogen atoms in the reduced products is observed as a function of the solution pH. The reduction mechanism and characterization of the rich array of products were determined by using a combination of cyclic and AC voltammetry along with UV-visible reflectance spectroelectrochemistry experiments. Both the reduction and protonation state of P could be followed as a function of pH and potential. From these data, estimates of the various reduced species' pKa values were obtained and the mechanism to form the doubly reduced, doubly protonated complex, [(phen)2Ru(H2tatpp)Ru(phen)2]4+ (H2P) at low pH (< or =7) could be shown to be a two-proton, two-electron process. Importantly, H2P is also formed in the photochemical reaction with sacrificial reducing agents, albeit at reduced yields relative to those at higher pH.