contributed equally to this work Calcium-dependent protein kinases (CDPKs) comprise a large family of serine/threonine kinases in plants and protozoans. We isolated two related CDPK cDNAs (NtCDPK2 and NtCDPK3) from Nicotiana tabacum. These CDPK transcripts are elevated after race-speci®c defence elicitation and hypo-osmotic stress. Transiently expressed myc-epitope-tagged NtCDPK2 in Nicotiana benthamiana and N.tabacum leaves showed a rapid transient interconversion to an activated form after elicitation and hypo-osmotic stress. The Avr9 race-speci®c elicitor caused a more pronounced and sustained response. This transition is due to phosphorylation of the CDPK. Immunocomplex kinase assays with epitope-tagged NtCDPK2 showed that stress-induced phosphorylation and interconversion of NtCDPK2 correlates with an increase in enzymatic activity. The function of NtCDPK2 in plant defence was investigated by employing virus-induced gene silencing (VIGS) in N.benthamiana. CDPKsilenced plants showed a reduced and delayed hypersensitive response after race-speci®c elicitation in a gene-for-gene interaction, and lacked an accompanying wilting phenotype. Silencing correlated with loss of CDPK mRNA, whereas mRNA accumulation of mitogen-activated protein kinase WIPK remained unaltered.

Since the initial annotation of miRNAs from cloned short RNAs by the Ambros, Tuschl, and Bartel groups in 2001, more than a hundred studies have sought to identify additional miRNAs in various species. We report here a meta-analysis of short RNA data from Drosophila melanogaster, aggregating published libraries with 76 data sets that we generated for the modENCODE project. In total, we began with more than 1 billion raw reads from 187 libraries comprising diverse developmental stages, specific tissue- and cell-types, mutant conditions, and/or Argonaute immunoprecipitations. We elucidated several features of known miRNA loci, including multiple phased byproducts of cropping and dicing, abundant alternative 5′ termini of certain miRNAs, frequent 3′ untemplated additions, and potential editing events. We also identified 49 novel genomic locations of miRNA production, and 61 additional candidate loci with limited evidence for miRNA biogenesis. Although these loci broaden the Drosophila miRNA catalog, this work supports the notion that a restricted set of cellular transcripts is competent to be specifically processed by the Drosha/Dicer-1 pathway. Unexpectedly, we detected miRNA production from coding and untranslated regions of mRNAs and found the phenomenon of miRNA production from the antisense strand of known loci to be common. Altogether, this study lays a comprehensive foundation for the study of miRNA diversity and evolution in a complex animal model.

Drosophila utilizes two small-RNA systems to restrict transposon activity in the germline (mostly via piRNAs) and in the soma (mostly via siRNAs).

We conclude that VPEgamma is a caspase-like enzyme that has been recruited in plants to regulate vacuole-mediated cell dismantling during cell death, a process that has significant influence in the outcome of a diverse set of plant-pathogen interactions.