Toll/interleukin receptor (TIR) domain-containing proteins encoded in the Arabidopsis (Arabidopsis thaliana) genome include the TIR-nucleotide binding site (TN) and TIR-unknown site/domain (TX) families. We investigated the function of these proteins. Transient overexpression of five TX and TN genes in tobacco (Nicotiana benthamiana) induced chlorosis. This induced chlorosis was dependent on ENHANCED DISEASE RESISTANCE1, a dependency conserved in both tobacco and Arabidopsis. Stable overexpression transgenic lines of TX and TN genes in Arabidopsis produced a variety of phenotypes associated with basal innate immune responses; these were correlated with elevated levels of salicylic acid. The TN protein AtTN10 interacted with the chloroplastic protein phosphoglycerate dehydrogenase in a yeast (Saccharomyces cerevisiae) two-hybrid screen; other TX and TN proteins interacted with nucleotide binding-leucine-rich repeat proteins and effector proteins, suggesting that TN proteins might act in guard complexes monitoring pathogen effectors.
Epoxide hydrolase (EH) is an enzyme in the ?/?-hydrolase fold superfamily that uses a water molecule to transform an epoxide to its corresponding diol. In insects, EHs metabolize among other things critical developmental hormones called juvenile hormones (JHs). EHs also play roles in the detoxification of toxic compounds that are found in the insects diet or environment. In this study, a full-length cDNA encoding an epoxide hydrolase, Hovi-mEH1, was obtained from the xylem-feeding insect Homalodisca vitripennis. H. vitripennis, commonly known as the glassy-winged sharpshooter, is an economically important vector of plant pathogenic bacteria such as Xylella fastidiosa. Hovi-mEH1 hydrolyzed the general EH substrates cis-stilbene oxide and trans-diphenylpropene oxide with specific activities of 47.5 ± 6.2 and 1.3 ± 0.5 nmol of diol formed min?¹ mg?¹, respectively. Hovi-mEH1 metabolized JH III with a Vmax of 29.3 ± 1.6 nmol min?¹ mg?¹, kcat of 0.03 s?¹, and KM of 13.8 ± 2.0 ?M. These Vmax and kcat values are similar to those of known JH metabolizing EHs from lepidopteran and coleopteran insects. Hovi-mEH1 showed 99.1% identity to one of three predicted EH-encoding sequences that were identified in the transcriptome of H. vitripennis. Of these three sequences only Hovi-mEH1 clustered with known JH metabolizing EHs. On the basis of biochemical, phylogenetic, and transcriptome analyses, we hypothesize that Hovi-mEH1 is a biologically relevant JH-metabolizing enzyme in H. vitripennis.
Next generation sequence analyses were used to assess virus-derived small RNA (vsRNA) profiles for Homalodisca coagulata virus-1 (HoCV-1), family Dicistroviridae, and Homalodisca vitripennis reovirus (HoVRV), family Reoviridae, from virus-infected H. vitripennis, the glassy-winged sharpshooter. The vsRNA reads were mapped against the monopartite genome of HoCV-1 and all 12 genome segments of HoVRV, and 21nt vsRNAs were most common. However, strikingly contrasting patterns for the HoCV-1 and HoVRV genomic RNAs were observed. The majority of HoCV-1 vsRNAs mapped to the genomic positive-strand RNA and, although minor hotspots were observed, vsRNAs mapped across the entire genomic RNA. In contrast, HoVRV vsRNAs mapped to both positive and negative-sense strands for all genome segments, but different genomic segments showed distinct hotspots. The HoVRV vsRNAs were more common for 5 and 3 regions of HoVRV regions of all segments. These data suggest that taxonomically different viruses in the same host offer different targets for RNA-antiviral defense.
The glassy-winged sharpshooter Homalodisca vitripennis (Hemiptera: Cicadellidae), is a xylem-feeding leafhopper and important vector of the bacterium Xylella fastidiosa; the causal agent of Pierces disease of grapevines. The functional complexity of the transcriptome of H. vitripennis has not been elucidated thus far. It is a necessary blueprint for an understanding of the development of H. vitripennis and for designing efficient biorational control strategies including those based on RNA interference.
The technological advances in DNA sequencing over the past five years have changed our approaches to gene expression analysis, fundamentally altering the basic methods used and in most cases driving a shift from hybridization-based approaches to sequencing-based approaches. Quantitative, tag-based studies of gene expression were one of the earliest applications of these next-generation technologies, but the tremendous depth of sequencing facilitates de novo transcript discovery, which replaces traditional expressed sequence tag (EST) sequencing. In addition, these technologies have created new opportunities for understanding the generation, stability, and decay of RNA and the impacts of chromatin differences on gene expression. As we review the impact of these methods on plant biology, we also mention published studies from animal systems when the methods are broadly applicable. We can anticipate that the published work over the past few years is a harbinger of much broader studies that are yet to be published and are sure to further advance our understanding of plant genomes in a field changing at a dizzying pace.
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