The \u3ci\u3ePseudomonas syringae\u3c/i\u3e HopPtoV Protein Is Secreted in Culture and Translocated into Plant Cells via the Type III Protein Secretion System in a Manner Dependent on the ShcV Type III Chaperone

The bacterial plant pathogen Pseudomonas syringae depends on a type III protein secretion system and the effector proteins that it translocates into plant cells to cause disease and to elicit the defense-associated hypersensitive response on resistant plants. The availability of the P. syringae pv. tomato DC3000 genome sequence has resulted in the identification of many novel effectors. We identified the hopPtoV effector gene on the basis of its location next to a candidate type III chaperone (TTC) gene, shcV, and within a pathogenicity island in the DC3000 chromosome. A DC3000 mutant lacking ShcV was unable to secrete detectable amounts of HopPtoV into culture supernatants or translocate HopPtoV into plant cells, based on an assay that tested whether HopPtoV-AvrRpt2 fusions were delivered into plant cells. Coimmunoprecipitation and Saccharomyces cerevisiae two-hybrid experiments showed that ShcV and HopPtoV interact directly with each other. The ShcV binding site was delimited to an N-terminal region of HopPtoV between amino acids 76 and 125 of the 391-residue full-length protein. Our results demonstrate that ShcV is a TTC for the HopPtoV effector. DC3000 overexpressing ShcV and HopPtoV and DC3000 mutants lacking either HopPtoV or both ShcV and HopPtoV were not significantly impaired in disease symptoms or bacterial multiplication in planta, suggesting that HopPtoV plays a subtle role in pathogenesis or that other effectors effectively mask the contribution of HopPtoV in plant pathogenesis

A Conserved Long Noncoding RNA Affects Sleep Behavior in Drosophila

Metazoan genomes encode an abundant collection of mRNA-like, long noncoding (lnc)RNAs. Although lncRNAs greatly expand the transcriptional repertoire, we have a limited understanding of how these RNAs contribute to developmental regulation. Here, we investigate the function of the Drosophila lncRNA called yellow-achaete intergenic RNA (yar). Comparative sequence analyses show that the yar gene is conserved in Drosophila species representing 40–60 million years of evolution, with one of the conserved sequence motifs encompassing the yar promoter. Further, the timing of yar expression in Drosophila virilis parallels that in D. melanogaster, suggesting that transcriptional regulation of yar is conserved. The function of yar was defined by generating null alleles. Flies lacking yar RNAs are viable and show no overt morphological defects, consistent with maintained transcriptional regulation of the adjacent yellow (y) and achaete (ac) genes. The location of yar within a neural gene cluster led to the investigation of effects of yar in behavioral assays. These studies demonstrated that loss of yar alters sleep regulation in the context of a normal circadian rhythm. Nighttime sleep was reduced and fragmented, with yar mutants displaying diminished sleep rebound following sleep deprivation. Importantly, these defects were rescued by a yar transgene. These data provide the first example of a lncRNA gene involved in Drosophila sleep regulation. We find that yar is a cytoplasmic lncRNA, suggesting that yar may regulate sleep by affecting stabilization or translational regulation of mRNAs. Such functions of lncRNAs may extend to vertebrates, as lncRNAs are abundant in neural tissues

Context Differences Reveal Insulator and Activator Functions of a Su(Hw) Binding Region

Insulators are DNA elements that divide chromosomes into independent transcriptional domains. The Drosophila genome contains hundreds of binding sites for the Suppressor of Hairy-wing [Su(Hw)] insulator protein, corresponding to locations of the retroviral gypsy insulator and non-gypsy binding regions (BRs). The first non-gypsy BR identified, 1A-2, resides in cytological region 1A. Using a quantitative transgene system, we show that 1A-2 is a composite insulator containing enhancer blocking and facilitator elements. We discovered that 1A-2 separates the yellow (y) gene from a previously unannotated, non-coding RNA gene, named yar for y-achaete (ac) intergenic RNA. The role of 1A-2 was elucidated using homologous recombination to excise these sequences from the natural location, representing the first deletion of any Su(Hw) BR in the genome. Loss of 1A-2 reduced yar RNA accumulation, without affecting mRNA levels from the neighboring y and ac genes. These data indicate that within the 1A region, 1A-2 acts an activator of yar transcription. Taken together, these studies reveal that the properties of 1A-2 are context-dependent, as this element has both insulator and enhancer activities. These findings imply that the function of non-gypsy Su(Hw) BRs depends on the genomic environment, predicting that Su(Hw) BRs represent a diverse collection of genomic regulatory elements

The Pseudomonas syringae HopPtoV Protein Is Secreted in Culture and Translocated into Plant Cells via the Type III Protein Secretion System in a Manner Dependent on the ShcV Type III Chaperone

The bacterial plant pathogen Pseudomonas syringae depends on a type III protein secretion system and the effector proteins that it translocates into plant cells to cause disease and to elicit the defense-associated hypersensitive response on resistant plants. The availability of the P. syringae pv. tomato DC3000 genome sequence has resulted in the identification of many novel effectors. We identified the hopPtoV effector gene on the basis of its location next to a candidate type III chaperone (TTC) gene, shcV, and within a pathogenicity island in the DC3000 chromosome. A DC3000 mutant lacking ShcV was unable to secrete detectable amounts of HopPtoV into culture supernatants or translocate HopPtoV into plant cells, based on an assay that tested whether HopPtoV-AvrRpt2 fusions were delivered into plant cells. Coimmunoprecipitation and Saccharomyces cerevisiae two-hybrid experiments showed that ShcV and HopPtoV interact directly with each other. The ShcV binding site was delimited to an N-terminal region of HopPtoV between amino acids 76 and 125 of the 391-residue full-length protein. Our results demonstrate that ShcV is a TTC for the HopPtoV effector. DC3000 overexpressing ShcV and HopPtoV and DC3000 mutants lacking either HopPtoV or both ShcV and HopPtoV were not significantly impaired in disease symptoms or bacterial multiplication in planta, suggesting that HopPtoV plays a subtle role in pathogenesis or that other effectors effectively mask the contribution of HopPtoV in plant pathogenesis

Whole-Genome Expression Profiling Defines the HrpL Regulon of \u3ci\u3ePseudomonas syringae\u3c/i\u3e pv. \u3ci\u3etomato\u3c/i\u3e DC3000, Allows de novo Reconstruction of the Hrp \u3ci\u3ecis\u3c/i\u3e Element, and Identifies Novel Coregulated Genes

Pseudomonas syringae pv. tomato DC3000 is a model pathogen of tomato and Arabidopsis that uses a hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS) to deliver virulence effector proteins into host cells. Expression of the Hrp system and many effector genes is activated by the HrpL alternative sigma factor. Here, an open reading frame-specific whole-genome microarray was constructed for DC3000 and used to comprehensively identify genes that are differentially expressed in wild-type and ΔhrpL strains. Among the genes whose differential regulation was statistically significant, 119 were upregulated and 76 were downregulated in the wild-type compared with the ΔhrpL strain. Hierarchical clustering revealed a subset of eight genes that were upregulated particularly rapidly. Gibbs sampling of regions upstream of HrpL-activated operons revealed the Hrp promoter as the only identifiable regulatory motif and supported an iterative refinement involving real-time polymerase chain reaction testing of additional HrpL-activated genes and refinements in a hidden Markov model that can be used to predict Hrp promoters in P. syringae strains. This iterative bioinformatic-experimental approach to a comprehensive analysis of the HrpL regulon revealed a mix of genes controlled by HrpL, including those encoding most type III effectors, twin-arginine transport (TAT) substrates, other regulatory proteins, and proteins involved in the synthesis or metabolism of phytohormones, phytotoxins, and myo-inositol. This analysis provides an extensively verified, robust method for predicting Hrp promoters in P. syringae genomes, and it supports subsequent identification of effectors and other factors that likely are important to the host-specific virulence of P. syringae