Evolution of chemosensory and detoxification gene families across herbivorous Drosophilidae.

Herbivorous insects are exceptionally diverse, accounting for a quarter of all known eukaryotic species, but the genomic basis of adaptations that enabled this dietary transition remains poorly understood. Many studies have suggested that expansions and contractions of chemosensory and detoxification gene families-genes directly mediating interactions with plant chemical defenses-underlie successful plant colonization. However, this hypothesis has been challenging to test because the origins of herbivory in many insect lineages are ancient (>150 million years ago (mya)), obscuring genomic evolutionary patterns. Here, we characterized chemosensory and detoxification gene family evolution across Scaptomyza, a genus nested within Drosophila that includes a recently derived (<15 mya) herbivore lineage of mustard (Brassicales) specialists and carnation (Caryophyllaceae) specialists, and several nonherbivorous species. Comparative genomic analyses revealed that herbivorous Scaptomyza has among the smallest chemosensory and detoxification gene repertoires across 12 drosophilid species surveyed. Rates of gene turnover averaged across the herbivore clade were significantly higher than background rates in over half of the surveyed gene families. However, gene turnover was more limited along the ancestral herbivore branch, with only gustatory receptors and odorant-binding proteins experiencing strong losses. The genes most significantly impacted by gene loss, duplication, or changes in selective constraint were those involved in detecting compounds associated with feeding on living plants (bitter or electrophilic phytotoxins) or their ancestral diet (fermenting plant volatiles). These results provide insight into the molecular and evolutionary mechanisms of plant-feeding adaptations and highlight gene candidates that have also been linked to other dietary transitions in Drosophila.

Linking discoveries, mechanisms, and technologies to develop a clearer perspective on plant long noncoding RNAs.

Long noncoding RNAs (lncRNAs) are a large and diverse class of genes in eukaryotic genomes that contribute to a variety of regulatory processes. Functionally characterized lncRNAs play critical roles in plants, ranging from regulating flowering to controlling lateral root formation. However, findings from the past decade have revealed that thousands of lncRNAs are present in plant transcriptomes, and characterization has lagged far behind identification. In this setting, distinguishing function from noise is challenging. However, the plant community has been at the forefront of discovery in lncRNA biology, providing many functional and mechanistic insights that have increased our understanding of this gene class. In this review, we examine the key discoveries and insights made in plant lncRNA biology over the past two and a half decades. We describe how discoveries made in the pregenomics era have informed efforts to identify and functionally characterize lncRNAs in the subsequent decades. We provide an overview of the functional archetypes into which characterized plant lncRNAs fit and speculate on new avenues of research that may uncover yet more archetypes. Finally, this review discusses the challenges facing the field and some exciting new molecular and computational approaches that may help inform lncRNA comparative and functional analyses.

High-Throughput Evolutionary Comparative Analysis of Long Intergenic Noncoding RNAs in Multiple Organisms.

Comparative genomic and transcriptomic analyses can help prioritize and facilitate the functional analysis of long noncoding RNAs (lncRNAs). Evolinc-II is a bioinformatic pipeline that automates comparative analyses, searching for sequence and structural conservation for thousands of lncRNAs at once. In addition, Evolinc-II takes a phylogenetic approach to infer key evolutionary events that may have occurred during the emergence of each query lncRNA. Here, we describe how to use command line or GUI (CyVerse's Discovery Environment) versions of Evolinc-II to identify lncRNA homologs and prioritize them for functional analysis.

Identification and functional annotation of long intergenic non-coding RNAs in Brassicaceae.

Long intergenic noncoding RNAs (lincRNAs) are a large yet enigmatic class of eukaryotic transcripts that can have critical biological functions. The wealth of RNA-sequencing (RNA-seq) data available for plants provides the opportunity to implement a harmonized identification and annotation effort for lincRNAs that enables cross-species functional and genomic comparisons as well as prioritization of functional candidates. In this study, we processed >24 Tera base pairs of RNA-seq data from >16,000 experiments to identify ∼130,000 lincRNAs in four Brassicaceae: Arabidopsis thaliana, Camelina sativa, Brassica rapa, and Eutrema salsugineum. We used nanopore RNA-seq, transcriptome-wide structural information, peptide data, and epigenomic data to characterize these lincRNAs and identify conserved motifs. We then used comparative genomic and transcriptomic approaches to highlight lincRNAs in our data set with sequence or transcriptional conservation. Finally, we used guilt-by-association analyses to assign putative functions to lincRNAs within our data set. We tested this approach on a subset of lincRNAs associated with germination and seed development, observing germination defects for Arabidopsis lines harboring T-DNA insertions at these loci. LincRNAs with Brassicaceae-conserved putative miRNA binding motifs, small open reading frames, or abiotic-stress modulated expression are a few of the annotations that will guide functional analyses into this cryptic portion of the transcriptome.

Identification and Characterization of the Heat-Induced Plastidial Stress Granules Reveal New Insight Into Arabidopsis Stress Response.

Plants exhibit different physiological and molecular responses to adverse changes in their environment. One such molecular response is the sequestration of proteins, RNAs, and metabolites into cytoplasmic bodies called stress granules (cSGs). Here we report that, in addition to cSGs, heat stress also induces the formation of SG-like foci (cGs) in the chloroplasts of the model plant Arabidopsis thaliana. Similarly to the cSGs, (i) cpSG assemble rapidly in response to stress and disappear when the stress ceases, (ii) cpSG formation is inhibited by treatment with a translation inhibitor (lincomycin), and (iii) cpSG are composed of a stable core and a fluid outer shell. A previously published protocol for cSG extraction was successfully adapted to isolate cpSG, followed by protein, metabolite, and RNA analysis. Analogously to the cSGs, cpSG sequester proteins essential for SG formation, dynamics, and function, also including RNA-binding proteins with prion-like domain, ATPases and chaperones, and the amino acids proline and glutamic acid. However, the most intriguing observation relates to the cpSG localization of proteins, such as a complete magnesium chelatase complex, which is involved in photosynthetic acclimation to stress. These data suggest that cpSG have a role in plant stress tolerance.

The tomato cell death suppressor Adi3 is restricted to the endosomal system in response to the Pseudomonas syringae effector protein AvrPto.

The tomato (Solanum lycopersicum) AGC protein kinase Adi3 functions as a suppressor of cell death and was first identified as an interactor with the tomato resistance protein Pto and the Pseudomonas syringae effector protein AvrPto. Models predict that loss of Adi3 cell death suppression (CDS) activity during Pto/AvrPto interaction leads to the cell death associated with the resistance response initiated from this interaction. Nuclear localization is required for Adi3 CDS. Prevention of nuclear accumulation eliminates Adi3 CDS and induces cell death by localizing Adi3 to intracellular punctate membrane structures. Here we use several markers of the endomembrane system to show that the punctate membrane structures to which non-nuclear Adi3 is localized are endosomal in nature. Wild-type Adi3 also localizes in these punctate endosomal structures. This was confirmed by the use of endosomal trafficking inhibitors, which were capable of trapping wild-type Adi3 in endosomal-like structures similar to the non-nuclear Adi3. This suggests Adi3 may traffic through the cell using the endomembrane system. Additionally, Adi3 was no longer found in the nucleus but was visualized in these punctate endosomal-like membranes during the cell death induced by the Pto/AvrPto interaction. Therefore we propose that inhibiting nuclear import and constraining Adi3 to the endosomal system in response to AvrPto is a mechanism to initiate the cell death associated with resistance.

Evolution in an ancient detoxification pathway is coupled with a transition to herbivory in the drosophilidae.

Chemically defended plant tissues present formidable barriers to herbivores. Although mechanisms to resist plant defenses have been identified in ancient herbivorous lineages, adaptations to overcome plant defenses during transitions to herbivory remain relatively unexplored. The fly genus Scaptomyza is nested within the genus Drosophila and includes species that feed on the living tissue of mustard plants (Brassicaceae), yet this lineage is derived from microbe-feeding ancestors. We found that mustard-feeding Scaptomyza species and microbe-feeding Drosophila melanogaster detoxify mustard oils, the primary chemical defenses in the Brassicaceae, using the widely conserved mercapturic acid pathway. This detoxification strategy differs from other specialist herbivores of mustard plants, which possess derived mechanisms to obviate mustard oil formation. To investigate whether mustard feeding is coupled with evolution in the mercapturic acid pathway, we profiled functional and molecular evolutionary changes in the enzyme glutathione S-transferase D1 (GSTD1), which catalyzes the first step of the mercapturic acid pathway and is induced by mustard defense products in Scaptomyza. GSTD1 acquired elevated activity against mustard oils in one mustard-feeding Scaptomyza species in which GstD1 was duplicated. Structural analysis and mutagenesis revealed that substitutions at conserved residues within and near the substrate-binding cleft account for most of this increase in activity against mustard oils. Functional evolution of GSTD1 was coupled with signatures of episodic positive selection in GstD1 after the evolution of herbivory. Overall, we found that preexisting functions of generalized detoxification systems, and their refinement by natural selection, could play a central role in the evolution of herbivory.

Maintenance of genetic diversity through plant-herbivore interactions.

Identifying the factors governing the maintenance of genetic variation is a central challenge in evolutionary biology. New genomic data, methods and conceptual advances provide increasing evidence that balancing selection, mediated by antagonistic species interactions, maintains genome-wide functionally important genetic variation within species and natural populations. Because diverse interactions between plants and herbivorous insects dominate terrestrial communities, they provide excellent systems to address this hypothesis. Population genomic studies of Arabidopsis thaliana and its relatives suggest spatial variation in herbivory maintains adaptive genetic variation controlling defense phenotypes, both within and among populations. Conversely, inter-species variation in plant defenses promotes adaptive genetic variation in herbivores. Emerging genomic model herbivores of Arabidopsis could illuminate how genetic variation in herbivores and plants interact simultaneously.

Two Pdk1 phosphorylation sites on the plant cell death suppressor Adi3 contribute to substrate phosphorylation.

The tomato AGC kinase Adi3 is phosphorylated by Pdk1 for activation of its cell death suppression activity. The Pdk1 phosphorylation site for activation of Adi3 is at Ser539. However, there is at least one additional Pdk1 phosphorylation site on Adi3 that has an unknown function. Here we identify an Arabidopsis thaliana sequence homologue of Adi3 termed AGC1-3. Two Pdk1 phosphorylation sites were identified on AGC1-3, activation site Ser596 and Ser269, and by homology Ser212 on Adi3 was identified as a second Pdk1 phosphorylation site. While Ser212 is not required for Adi3 autophosphorylation, Ser212 was shown to be required for full phosphorylation of the Adi3 substrate Gal83.