www.nature.com/scientificreports OPEN A multi-omics study of the grapevine-downy mildew (Plasmopara viticola) pathosystem Received: 8 June 2017 Accepted: 15 December 2017 unveils a complex protein coding- Published: xx xx xxxx and noncoding-based arms race during infection Matteo Brilli 1,2, Elisa Asquini1, Mirko Moser 1, Pier Luigi Bianchedi1, Michele Perazzolli 1 & Azeddine Si-Ammour 1 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 identifed a RxLR efector that trigger an immune response in the wild species V. riparia. This efector 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 profciently infect its host, and in the other might reduce the efects of the infection on the plant. Grapevine (Vitis vinifera L.) is an important commodity and comprises varieties for wine production and table grape for human consumption1. Wine production is a very lucrative activity and the world wine trade is worth almost US $30 billion. France, Italy and Spain are the largest European wine producing countries representing altogether half of the world production (http://www.oiv.int/). Grapevines can be infected by a myriad of plant pathogens at all growth stages and in order to secure harvest large quantities of agrochemicals are used to control their spread2. Te treatments against powdery and downy mildews, including the oomycete Plasmopara viticola, requires almost two thirds of all synthetic fungicides sprayed in the European Union with adverse efects on the environment3. Plasmopara viticola (Berk. and Curt.) Berl. and de Toni belongs to the group of oomycetes (Order: Peronosporales, Family: Peronosporaceae) that comprises the most devastating plant pathogens such as Phytophthora infestans responsible for the Irish potato famine in the 19th century4. Unlike the hemibiotroph Phytophthora species, P. viticola is an obligate biotroph and therefore relies entirely on grape as a host to complete its life cycle. P. viticola is endemic in North America where the Vitis species such as V. riparia are naturally resist- ant, likely as a consequence of a long co-evolutionary process5. Conversely, P. viticola was introduced uninten- tionally in Europe in the 1870s and immediately spread on V. vinifera cultures causing pandemics all over Europe in the following decades6. Soon afer, the extensive use of copper formulations known as the “Bordeaux mixture” restricted the disease spread and later on paved the path to excessive usage of synthetic agrochemicals. As a 1Department of Genomics and Biology of Fruit Crops, Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010, San Michele all’Adige (TN), Italy. 2Present address: Department of Agronomy, Food, Natural Resources, Animals and Environment (DAFNAE), University of Padova, Agripolis, V.le dell’Università, 16, 35020, Legnaro (PD), Italy. Correspondence and requests for materials should be addressed to A.S.-A. (email: azeddine. [email protected]) SCIENTIFIC REPORTS | (2018) 8:757 | DOI:10.1038/s41598-018-19158-8 1 www.nature.com/scientificreports/ PvitFEM01 INRA-PV221a JL-7–2b Natural Vitis host V. vinifera V. vinifera V. riparia Geographical origin Italy France China Genome assembly Sequencing platform Illumina Illumina Illumina + PacBio Number of scafolds 57,890 1,883 2,165 N50c (kb) 4.645 180.6 172.3 Assembly size (Mb) 83.54 74.74 101.3 Gene annotation Protein coding genes (predicted) 38,298 nd 17,014 Validated transcripts (RNA-Seq) 18,335 nd 11,670 Orthologous groups in Oomycetesd 6,552 nd nd Genome completeness Oomycete core genes presente 81% na na CEGMA/BUSCOf na/87.2% 95%/na 97%/90% Table 1. Characteristics of the three available P. viticola genomes. aData retrieved from Dussert et al.21. nd: not determined in the study. na: not applicable. bData retrieved from Yin et al.22. nd: not determined in the study. na: not applicable. cN50 is defned as the scafold size such that 50% of the assembled nucleotides resides on contigs at least this length. dNumber of orthologous groups found in oomycetes with sequenced genomes and containing at least one protein from P. viticola. eIn total, 1,299 genes shared by all oomycetes were found, of which 1,054 were present in P. viticola. fIndicated CEGMA and BUSCO numbers include the counting of partial matches. consequence P. viticola became recently resistant to many fungicides7. Terefore, research on grape resistance mechanisms and the development of alternative strategies to control P. viticola infections are urgently needed to control the environmental burden of grapevine cultures. Molecular mechanisms occurring during the compatible interaction between P. viticola and V. vinifera are largely unknown. Most of our knowledge about oomycete pathogenicity factors derives from studies focused on Phytophthora species8. During infection oomycetes secrete cytoplasmic and apoplastic efector proteins that usually suppress plant immunity by triggering susceptibility (efector-triggered susceptibility, ETS)9. Certain oomycete efectors with a special protein motif RxLR are recognized by the plant resistance genes and this inter- action triggers immunity (efector-triggered immunity, ETI) resulting in localized cell death or hypersensitive response (HR)10. RxLR efectors are found abundantly in all sequenced oomycete genomes and are rapidly evolv- ing11. Tey even acquired novel functions such as suppressing plant RNA silencing mechanisms12. Teir role during the grape downy mildew infection is unclear and their identifcation requires multi-omics approaches. To this end, we sequenced the DNA extracted from a P. viticola strain isolated in Northern Italy and present its assembled draf genome. By using comparative genomics we discovered the missing metabolic feature in the P. viticola genome that could explain its biotrophic mode of life. We complemented our genome sequencing eforts with genome-wide diferential gene expression analyses during the infection process and we identifed a protein efector of the RxLR type triggering ETI in the resistant grapevine V. riparia. Small RNA sequencing (sRNA-Seq) combined to a genome-wide degradome study13 revealed a comprehensive interaction network of small RNAs (sRNAs) that target genes during infection allowing us to uncover a potential bi-directional RNA silencing strat- egy between the pathogen and its host despite species barriers. Results Nuclear and mitochondrial genome assemblies, gene annotation and phylogenetic analysis. Assembling the draf genome for P. viticola was a prerequisite for gene and sRNA expression profling studies during the infection process of V. vinifera. We therefore sequenced the genome of a downy mildew strain, named ‘PvitFEM01’, isolated from a local vineyard in the Trentino region in Italy. DNA extracted from asexual sporan- gia from in vitro infected plants was used to construct Illumina paired-end libraries (2 × 100 bp) and further sequenced. Although P. viticola genetic material was extracted under sterile conditions, potential contamination with DNA originating from other sources such as the host (grapevine), potential grape endophytic or epiphytic bacteria were addressed. 83 million reads remained for assembly, afer fltering against V. vinifera and bacterial sequences (Supplementary Fig. S2 and Supplementary note). In total, 57,890 scafolds were obtained correspond- ing to a N50 of 4,645 bp (Table 1, Supplementary Figs S1–S6 and Supplementary note). Te total length of the assembly was 83.54 Mb corresponding to 74% of the genome size previously determined by Feulgen staining14. Contigs corresponding to the mitochondrial genome were also identifed and provided the frst P. viticola mito- chondrial reconstruction estimated to be ca. 39.2 kb (Supplementary Fig. S7). P. viticola mitochondrial proteins identity ranged from 80 to 97% with respect to their P. infestans counterparts. A a single amino acid change from Glycine to Alanine at position 143 (G143A) in the mitochondrial apocytochrome b protein suggests that ‘PvitFEM01’ is resistant to Quinone outside Inhibitors (QoI) fungicides (Supplementary Fig. S8). Several poly- morphic sites were identifed on the P. viticola mitochondrial genome suggesting that our draf assembly might condense several haplotypes (Supplementary Fig. S9). SCIENTIFIC REPORTS | (2018) 8:757 | DOI:10.1038/s41598-018-19158-8 2 www.nature.com/scientificreports/ Figure 1. Phylogenetic relationship between Plasmopara viticola and other oomycetes and abundance of their efectors. Te maximum-likelihood phylogenetic tree was built using 312 concatenated proteins selected from single copy genes belonging to the oomycete core genome. Te abundance of each class of cytoplasmic and apoplastic efectors efector in biotroph (B), hemibiotroph (H) or necrotroph (N) oomycete species is indicated by a number and a color code. Out of the 87 YxSLK efectors identifed in our study only 25 contained a signal peptide and reported in this fgure. Darker colors indicate higher