Fungicides are applied intensively to prevent downy mildew infections of grapevines (Vitis vinifera) with high impact on the environment. In order to develop alternative strategies we sequenced the genome of the oomycete pathogen Plasmopara viticola causing this disease. We show that it derives from a Phytophthora-like ancestor that switched to obligate biotrophy by losing genes involved in nitrogen metabolism and γ-Aminobutyric acid catabolism. By combining multiple omics approaches we characterized the pathosystem and identified a RxLR effector that trigger an immune response in the wild species V. riparia. This effector is an ideal marker to screen novel grape resistant varieties. Our study reveals an unprecedented bidirectional noncoding RNA-based mechanism that, in one direction might be fundamental for P. viticola to proficiently infect its host, and in the other might reduce the effects of the infection on the plant.

Apple trees require a long exposure to chilling temperature during winter to acquire competency to flower and grow in the following spring. Climate change or adverse meteorological conditions can impair release of dormancy and delay bud break, hence jeopardizing fruit production and causing substantial economic losses. In order to characterize the molecular mechanisms controlling bud dormancy in apple we focused our work on the MADS-box transcription factor gene MdDAM1. We show that MdDAM1 silencing is required for the release of dormancy and bud break in spring. MdDAM1 transcript levels are drastically reduced in the low-chill varieties 'Anna' and 'Dorsett Golden' compared to 'Golden Delicious' corroborating its role as a key genetic factor controlling the release of bud dormancy in Malus species. The functional characterization of MdDAM1 using RNA silencing resulted in trees unable to cease growth in winter and that displayed an evergrowing, or evergreen, phenotype several years after transgenesis. These trees lost their capacity to enter in dormancy and produced leaves and shoots regardless of the season. A transcriptome study revealed that apple evergrowing lines are a genocopy of 'Golden Delicious' trees at the onset of the bud break with the significant gene repression of the related MADS-box gene MdDAM4 as a major feature. We provide the first functional evidence that MADS-box transcriptional factors are key regulators of bud dormancy in pome fruit trees and demonstrate that their silencing results in a defect of growth cessation in autumn. Our findings will help producing low-chill apple variants from the elite commercial cultivars that will withstand climate change.

SUMMARYSo far, conventional hairpin RNA (hpRNA) constructs consisting of an inverted repeat (IR) of target promoters directly introduced into an expression cassette have been used to mediate de novo DNA methylation. Transcripts of such constructs resemble mRNA molecules, and are likely to be exported to the cytoplasm. The presence of hpRNAs in the cytoplasm and the nucleus may account for the simultaneous activation of posttranscriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM). We hypothesized that by retaining hpRNAs in the nucleus, efficient induction of only RdDM may be achieved. Thus, we introduced into tobacco a transgene containing an intron into which an IR of a target promoter was inserted. The intronic hpRNA initiated highly specific cis-and trans-methylation, but did not induce PTGS. No spreading of methylation into sequences flanking the region of homology between the hpRNA and the target DNA was detectable. The efficient methylation-directing activity of the intronic hpRNA may indicate a previously unrecognized role of introns, potentially regulating gene expression at the transcriptional level.

An efficient, simple, and small-scale procedure for isolating functional ribonucleic acid (RNA) was successfully applied to many different tissues of grape and apple. These woody plants are rich in polyphenolic compounds and polysaccharides that could impair the RNA extraction. The method chosen is based on the use of hot borate buffer at alkaline pH supplemented with several adjuvants and followed by selective precipitations. Starting with only 0.4 g of fresh tissue and working with small tubes (2 mL), we were able to obtain good yields of high-quality RNA suitable for further applications. The procedure can be proposed for many applications, and it is particularly highly recommended when isolating RNA from a large number of samples.

Edited by Tamas DalmayKeywords: RNA silencing Small RNA GFP DNA methylation Nicotiana benthamiana a b s t r a c tIn plants, transgenes are generally more sensitive against RNA silencing than endogenes are. In this study, we generated a transgene that structurally mimicks an endogene. It is composed of endogenous promoter, 5 0 -UTR, introns, 3 0 -UTR and terminator elements. Our data revealed that, in contrast to a conventional transgene, an endogene-resembling transgene was more stably expressed and poorly processed into small RNAs. In addition, although both constructs triggered methylation of homologous DNA sequences at similar levels, the endogene-resembling transgene exhibited significantly delayed onset of local and systemic silencing.