INVESTIGATIONS ON A GRAPEVINE INVESTIGATIONS ON A GRAPEVINE VIRUS VIRUS- LIKE DISEASE: ENATIONS LIKE DISEASE: ENATIONS Chiumenti M. 1 , Giampetruzzi A. 2 , Pirolo C. 1 , Saldarelli P. 2 , Minafra A. 2 , Bottalico G. 1 , De Stradis A. 2 , Roseti V. 1 , Campanale A. 2 , Savino V. 1,2 and Martelli G.P. 1, 2 1. Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti (Di.S.S.P.A.), Università degli Studi di Bari, Aldo Moro“, Via Amendola, 165/A, 70126 Bari, Italy. 2. Istituto di Virologia Vegetale CNR, sezione di Bari, Via Amendola, 165/A, 70126 Bari, Italy. Corresponding author E-mail: p.saldarelli@ba.ivv.cnr.it ACKNOWLEDGEMENTS Work supported by the Projects CISIA “Valorizzazione delle risorse genetiche di colture mediterranee attraverso approcci di metagenomica, trascrittomica ed analisi funzionale per la caratterizzazione di germoplasma autoctono, endofiti, agenti di biocontrollo e fitopatogeni” (CNR, Italy) and ‘Applicazione di tecniche innovative di sequenziamento massale per lo studio di agenti patogeni di rilevante impatto economico per la viticoltura e l’agrumicoltura’ funded by Fondazione Cassa di Risparmio di Puglia ( University of Bari 2010-11) REFERENCES Al Rwahnih, M., Daubert, S., Golino, D., Rowhani, A., 2009.. Virology 387, 395–401. Giampetruzzi, A., et al. 2011. Journal of Plant Pathology (2011), 93 , S4.63-S4.89. Giampetruzzi, A.et al. 2012. Virus Res. 163:262-268. Graniti A., Martelli G.P.,, Lamberti F. 1966. Proc Int Conf Virus Vectors Per Hosts, Davis 293- 306 Manavella PA et al. 2012. PNAS doi: 10.1073/pnas.1200169109 Martelli, G.P. and Boudon-Padieu, E. 2006 Directory of Infectious Diseases of Grapevines. Options Méditerranéennes, Série B, 55: CIEHAM-IAM Bari. Moon J. and Hake S. 2011. Curr Opin Plant Biol 14: 24-30. Zerbino, D.R., Birney, E., 2008. Genome Res. 18, 821–829. INTRODUCTION Enations disease of the grapevine is an erratic disorder characterized by tissue outgrowths from the lower surface of the leaves, usually along the veins (Figure 1 and 2). The disease recalls the consequences of hormonal unbalance, whose appearance may depend on seasonal conditions (Martelli and Boudon Padieu, 2006). Even though graft- transmissibility of enations supports a viral aetiology of the disease, its putative agent(s) has not been identified. Initial histological observations described an altered developmental process in which affected leaves have an inverted abaxial/adaxial polarity in tissues showing enations (Graniti et al. 1965). In Arabidopsis thaliana the specification of organ symmetry and polarity is a tightly controlled process, which involves a network of microRNAs and trans acting RNAs (Rubio-Somoza and Weigel, 2011). Having the double objective of studying the “virome” and the small RNA profile of enations-affected vines we performed an analysis by deep sequencing (NGS) of small RNA fraction and /or dsRNA libraries (Al Rwanih et al., 2009) A B C Figure 1 – Enations symptoms on cv Panse precoce : A. Irregular fruit maturation; B. flowers with stamen necrosis; C. Enations on the underside of a leaf. MATERIALS AND METHODS Grapevine sources. Leaves collected in late spring from seven plants of cv. ‘Panse precoce’ with enations in a 10-years-old commercial vineyard (Figure 1, Trani, Southern Italy) were pooled for each NGS library. Libraries preparation and analysis. Purified dsRNAs and small sRNAs from leaf tissues of each accession were used to synthesize two cDNA libraries according to the Illumina protocol (Giampetruzzi et al., 2012). A 50 base-single read run was done on a HiScan SQ apparatus. Short sequences were processed with Fastx toolkit, de novo assembled with Velvet (Zerbino and Birney, 2008) and searched for homologies with viral sequences with BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) tools. Tissue preparation staining. Tissues were embedded in wax after formaldehyde fixation, ethanol dehydration and clarification. Trimmed sections mounted on poly-l-lysine slides, were de-waxed, rehydrated, stained with Toluidine blue 1% and observed on a light optical microscope. For electron microscopy, tissues were embedded in epoxy resin after glutharaldeide fixation, post-fixation in osmium tetroxide, dehydrated and included. Diamond-trimmed thin sections mounted on Ni grids were double stained with Pb citrate and Uranyl acetate. Small RNA hybridization. Membranes were hybridized with DNA oligonucleotide probes labelled with digoxigenin (Manavella et al. 2012). RESULTS The virome of tissues with enations: De novo assembly of sequences obtained by the dsRNA and smallRNAs libraries showed a “virome” composed by ten viruses (Table 1) and two viroids: Grapevine yellow speckle viroid-1 (GYSVd-1) and Hop stunt viroid (HSVd) (not shown). Routine virus detection by RT-PCR assays (Table 1) confirmed the NGS detected viruses and revealed the first occurrence of Grapevine leafroll-associated virus 9 (GLRaV-9) in Italy (Giampetruzzi et al., 2011). In comparison a group of pot-grown vines originating from sanitized explants from vines of the same vineyard (vines named T in Table 1), did not show enations in a time span of six years. These latter vines lacked infections by ampelo- and closterovirus but were still infected by Grapevine fanleaf virus (GFLV), Grapevine Ruspestris stem pitting virus (GRSPaV), Grapevine Fleck virus (GFkV) and Grapevine virus A (GVA). CONCLUSIONS A comparative analysis of a group of plants from the same vineyard subjected to sanitation by heat therapy, which did not show any symptoms on a time span of three years, had a less severe sanitary status although maintaining GFLV infections. A similar situation was encountered in enation- affected vines of cv. Michele Palieri (unpublished data). These observations confirm the alleged lack of relationship between GFLV and enation disease but do not clarify the etiology of the disease that still remains undetermined. A survey of the current literature indicates that a complex network of small RNAs (microRNAs and trans acting smallRNAs) and mRNAs controls leaf morphogenesis (Figure 5C, Moon and Hake 2011), with the miR166 as a player. In this framework loss of abaxial identity observed in enations- affected leaves could rely on the disruption of these balances. Whether this unbalance exists during leaf development or is influenced by viral agents will be the object of future studies. Table 1 - Sanitary status of the grapevine field plants with enations (E-series). In light blue are reported all viruses detected by NGS analysis after assembly and BlastN homolgy search for each of the two libraries: dsRNA and small RNA. On the right, in green, RT-PCR results are shown. A group of partially sanitized plants (T-series), originating from the same vineyard and not showing enations disease was tested for comparison. dsRNA sRNA Healthy E1 E2 E3 E4 E5 E7 E8 T1 T2 T2 1 T2 2 T2 3 2040 355 Contigs/ Enation symptoms - + + + + + + + - - - - - 131 - GRSPaV - + + + + + + + + + + + + 1 16 GFLV - + + + + + + + + + - + + 65 91 GLRaV-1 - + + + + + + + - - - - - 80 12 GLRaV-2 - + + + + + + + - - - - - 132 - GLRaV-3 - + + + + + + + - - - - - 5 - GLRaV-5 - - - - - + - - - - - - - 40 - GLRaV-9 - + + - + + + - + - - - - 1 5 GFkV - + + + + + + + + + + + + 37 3 GVA - + + + + + + + + - - - - 3 - GVB - - - - + - - - - - - - - Figure 2 – Enations symptoms on cv Moscato D’Amburgo. Arrow points to a leaf-like outgrowth on the lower surface of the blade. Figure 4 – Transverse sections through leaves with enations (B, 100X) with the detail of epidermal outgrowth (C 400X). A (400X) grapevine normal leaf blade. A B C upper surface veins Figure 5 – Transverse sections through leaves with enations showing the presence of palisade tissue on the lower surface of the leaf (400x). Details of the palisade (B 2500X) and the ectopic localization of spongy tissues (A 2500X) are showed by transmission electrographs. C: specification of adaxial/abaxial polarity in the development of A. thaliana leaf. A B The small RNA profile of tissues with enations: Expression profile of known Vitis vinifera micro RNAs (vvi-miRNAs) in enation-showing leaf tissues showed an increase of miR166 which controls leaf morphogenesis by targeting transcription factors of the Class III HD-Zip gene family members in Arabidopsis thaliana (Moon and Hake 2011). To verify this finding the small RNA fractions from sectors with enations and corresponding leaf blade were probed by Dig-hybridization (Fig 3). Results confirmed the increased vvimiR166 expression in the outgrowth sections of leaves. 1 2 3 4 Figure 3 – VvimiR166 hybridization of small RNAs fractions from enations (1,3) and leaf blade sectors (2, 4) of affected leaves. Equal loading of small RNAs is showed on the left image. Histological observations: Transverse sections of leaves with enations outgrowths showed profoundly altered tissue organization (Figures 4B-C and 5). Development of palisade tissues on the abaxial face of the leaf (Figure 4B and 5) is a typical alteration followed by an excess of epidermal tissues (Figure 4C) and the ectopic localization of spongy tissue (Figure 5A). C Moon and Hake 2011