COST IPLANTA CA15223

WG1 Meeting:

“Comparing siRNA and miRNA technology

and role for improving perennial plants”

Linked to “ISHS

International conference on Grape

Biotechnology”, Bordeaux (FR),

July 17-18, 2018.

Book of abstract

Grosshans H, Filipowicz W.Nature. 2008

iPLANTA WG1 Meeting:

“Comparing siRNA and miRNA technology and role for improving

perennial plants”

INRA – VILLENAVE D’ORNON - BORDEAUX

The WG1 meeting will focus siRNA and miRNA technology and role in regulating different

processes in plants and in their interaction with other organisms. A focus will be given in determining

which technology has been favourably used for annual and perennial plant species including the

rootstock-to-scion transfer of the silencing virus resistance signal. A scientific dissemination plan,

including the preparation of a review on RNAi technology, will be discussed.

The meeting will be organized with the following working program:

• July 17 (Morning): registered WG1 experts will attend the “ISHS international conference on Grape

Biotechnology”, at ENSEIRB, amphi Matmeca, 1 Avenue du Docteur Albert Schweitzer, 33402

Talence

• July 17 (Afternoon): iPLANTA WG1 meeting, registration at 2:00 pm, starting of the meeting 3:00

pm, at INRA- Bordeaux, 71Avenue Bourlaux, 33140 Villenave d’ornon.

• July 18 (morning): WG1 scientific session at INRA- Bordeaux, 71Avenue Bourlaux, 33140

Villenave d’ornon with selected oral presentations on major advances on RNAi technologies.

• July 18 (afternoon): WG1 discussion and planning of WG1 scientific and dissemination activities

at INRA- Bordeaux, 71 Avenue Bourlaux, 33140 Villenave d’ornon .

New specific topics will be introduced by identifying and invite 1 or 2 international speakers. All

presentations will be oral. As for all iPLANTA meetings will be reimbursed only experts selected for

abstract presentation as reported in the following program.

DAY 1 – Tuesday - July 17th, 2018, 9,00 – 13.00 – registered WG1 participants will

join the - XII INTERNATIONAL CONFERENCE ON GRAPEVINE BREEDING AND

GENETICS JOINT MEETING (ICGBG)

14:40 WG1 Meeting - Session 1 Chair Guy Smagghe and Jeremy Sweet

SMALL RNAs AS NATURAL AND ARTIFICIAL TOOLS TO CONTROL GENES IN

PERENNIALS

M. RAVELONANDRO

Institut National de la Recherches Agronomique Bordeaux, France

RNAi, miRNA, siRNA, perennials

Perennials are among relevant model plants exposed to the environment. Examining plant growth, these models are

potentially recipients wherever variable pests can invade via stem, leaves or roots. As like for a recipient, plant cells

resemble to a biological site where molecular interactions between biotic factors and plant genome may generate a

particular reaction in the occurrence of disease (susceptibility) or the set up of resistance. Based on those, plant

metabolism is enhancing the presence of disease symptoms when the susceptibility is occurring and interestingly the

symptomless character in resistant clones. Gene transcription competition indicated that plant-host is mechanistically

sharing for its functional genome with that of the incoming pathogen. Following to the study of the different proteins

encoded by the genome of either virus, bacteria or fungi that interact with those naturally produced by plant genome, the

basic step was the availability of tools for the control of mRNA regulation. Because tools for the control of regulation is

an essential prerequisite for investigating RNA machinery and particularly RNA interfering. The technology implies small

RNA from either messengers or precursor transcript. Disregard of targeted RNA, RNAi expressed as si- small interfering

RNA (siRNA) or mi-interfering RNA (miRNA) are two small fragments of RNA structured as either a double-stranded

(ds) RNA (siRNA) or a single-stranded (ss) RNA (miRNA). Both are involved in silencing machinery. In order to know

gene expression levels in grapevine, different putative miRNAs were investigated. As a reference for perennials, the

BIOWINE web based resource has stimulated relevant analyses of miRNAs targeting specific genes expressed during the

phenological stages. Similar studies have been also expanded in Prunus domestica (plum). Attempts to use these miRNA

as an artificial regulatory molecule in plums were developed through genetic engineering approach. RNAi constructs

were designated to accumulate respectively both and mixed RNAi (mi and si-miRNA) in perennial host. Prior to PPV

challenging, plants were analysed for their RNAi content. Among these were first the detection of the primiRNA, then

second both mi and siRNA. Targeting the messenger RNA from the biotic agent like plum pox virus (PPV), we verified

that it has been specifically spliced via specific enzymes like dicer and slicer-like proteins. These studies permitted to

show that small RNA of the conservative size (21-24nt) can be used in a diversity of downstream applications including

detection, sequencing, and RNA interfering. Taking the proof-of-concept by inhibiting viral RNA replication with

artificial RNAi, stable and robust protection in these modified plums are valuable criteria to challenge disease pest in

perennials.

SOMATIC EMBRYOGENESIS IN GRAPEVINE: DEVELOPMENT OF AN

ALTERNATIVE EXPLANT FOR GENE SILENCING

C. LIMERA1, S. SABBADINI1, A. RICCI1, L. CAPRIOTTI1, S. DHEKNEY2, B. MEZZETTI1

1Department of agricultural, food and environmental sciences, Università Politecnica delle Marche, Ancona, italy.

2Sheridan Research and Extension Centre, University of Wyoming, 3401 Coffeen avenue, sheridan, wy 82801, usa

Somatic embryognesis, grapevine, rnai, nbt

Genetic improvement of grapevine for disease resistance and abiotic stress tolerance is of utmost importance, for the

sustainability and profitability of the viticulture industry worldwide. One of the most attractive features of cell cultures

is that, plants obtained are predominantly normal and devoid of any phenotypic or genotypic variation, as they are

derived from single cells hence, transformed somatic embryos are free of chimeras. This regeneration technique can be

utilised for genetic engineering using a suitable gene construct for the possible application of RNAi technique to control

grapevine diseases.

THE INVOLVEMENT OF FaMADS9 IN THE REGULATION OF STRAWBERRY FRUIT

RECEPTACLE DEVELOPMENT

JOSÉ G. VALLARINOa, CATHARINA MERCHANTEa, MARÍA ANGELS DE LUIS BALAGUERb, DELPHINE M.

POTTa, MARÍA T. ARIZAa, ANA CASAÑALa, DAVID POSEa, AMALIA VIOQUEa, LOTHAR WILLMITZERc,

ROSANGELA SOZZANIb,d, ALISDAIR R. FERNIEc, MIGUEL A. BOTELLAa, JAMES J. GIOVANNONIe,

VICTORIANO VALPUESTAa, SONIA OSORIOa

a Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”. Universidad de Málaga-Consejo Superior de

Investigaciones Científicas. Department of Molecular Biology and Biochemistry. Campus de Teatinos, 29071 Málaga,

Spain.

b Plant and Microbial Biology Department, North Carolina State University, Raleigh, NC 27695.

c Max-Planck-Institut für Molekulare Pflanzenphysiologie, 14476 Potsdam-Golm, Germany.

d Biomathematics Program, North Carolina State University, Raleigh, NC 27695.

e Boyce Thompson Institute for Plant Research and USDA-ARS, Robert W. Holley Center, Tower Road, Cornell

University Campus, Ithaca, NY 14853, USA.

Strawberry, ripening, FaMADS9, RIN

FaMADS9 is the strawberry gene with highest homology to the tomato RIN. Transgenic lines were obtained with

FaMADS9 partially silenced. First analysis of four independent lines did not show significant differences in fruit firmness

and color, but presented lower values in Brix degrees. Transcriptional analysis of transgenic receptacle showed an

elevated number of significant differentially expressed genes. Categories enriched among the differentially expressed

genes identified as the most represented, at the white stage, the MapMan RNA.regulation of transcription category, that

included a number of transcription factors associated to hormones. At the red stage, transport, lipid metabolism, and cell

wall were the enriched categories. Metabolomic analysis of transgenic receptacle identified specific processes affected

after FaMADS9 silencing, such as disaccharides and proanthocyanidins metabolism at the green/white stage, and

anthocyanins and cuticular waxes at the red stage. These changes agreed with the transcriptional changes. The results

support a regulatory role of FaMADS9 at two stages of receptacle development. At the red stage, the analysis of the

strawberry fruit transcriptome of the ripening fruit allowed to infer a regulatory network with FaMADS9 as a main node,

directly connected to the FaERF61. Both genes share a high percentage of common targets, including the FaMYB1 gene.

NATURAL RESISTANCE OF THE DIPLOID MUSA BALBISIANA PISANG KLUTUK

WULUNG (PKW) BANANA PLANT TO INFECTIOUS ENDOGENOUS BANANA

STREAK VIRUS SEQUENCES IS DRIVEN BY TRANSCRIPTIONAL GENE SILENCING.

PIERRE-OLIVIER DUROY1,4, NATHALIE LABOUREAU1,2, JONATHAN SEGUIN3, RAJESWARAN

RAJENDRAN1,2, MIKHAIL POOGGIN1,2, MARIE-LINE ISKRA-CARUANA1,2 AND MATTHIEU

CHABANNES1,2.

1 CIRAD, UMR BGPI, F-34398 Montpellier Cedex 5.

2 BGPI, Univ Montpellier, CIRAD, INRA, Montpellier SupAgro, Montpellier, France.

3 FASTERIS SA, Ch. Du Pont-du-Centenaire 109, 1228 Plan-les-Ouates, Switzerland

4 Université de Lausanne, Institut de biotechnologie, EPFL FSB – LBTM, CH B1-391 (Bâtiment CH), Station 6, CH-

1015 Lausanne, Switzerland

Keywords: Banana, endogenous banana streak virus (eBSV), methylation, epigenetic

The genome of banana (Musa sp.) harbours multiple integrations of Banana streak virus (eBSV), whereas this badnavirus

does not require integration for the replication of its ds DNA genome. Some endogenous BSV sequences (eBSV), only

existing in the Musa balbisiana genome, are infectious by releasing a functional viral genome following stresses such as

those existing in in vitro culture and interspecific crosses context. The structure of these eBSV is much longer than a

single BSV genome, composed of viral fragments duplicated and more or less extensively rearranged.

Wild M. balbisiana diploid genotypes (BB) such as Pisang Klutuk Wulung (PKW) harbour such infectious eBSV

belonging to three widespread species of BSV (Goldfinger -BSGFV, Imové – BSIMV and Obino l’Ewai - BSOLV) but

are nevertheless resistant to any multiplication of BSV without any visible virus particles. Using deep sequencing of total

siRNAs of PKW we underlined the presence of virus-derived small RNA (vsRNA) from eBSOLV, eBSGFV and eBSIMV

by blasting sequences against the 3 BSV species genomes. Interestingly, we showed that hot and cold spots of vsRNA

generation do not target similar viral sequences from one eBSV species to the other but are directly correlated with the

structure of the integration. vsRNA are enriched in 24-nt class which represent about 75% of the total 21-24nt siRNA

matching eBSV. We also demonstrated that eBSV are highly methylated in the three different sequence contexts (CG,

CHH and CHG) throughout the whole sequence of eBSVs with no difference in methylation profile between siRNA

producing and non producing areas. Interestingly, methylation patterns of all three eBSV are similar whereas they are

located in different genomic context; eBSOLV being in a TE rich area whereas eBSIMV and eBSGFV are in genes rich

region. It seems that eBSV are controlled mainly by epigenetic mechanisms similar to those described for transposable

elements (TE). All together, our data indicate that eBSVs in PKW genome are likely silenced at the transcriptional level

and this is probably responsible for the natural resistance of this genotype to the activation of such infectious eBSV as

well as infection by external BSV particles.

DIFFERENTIALLY EXPRESSED miRNAs IN THE INFLORESCENCE BUDS FROM ‘ON-

YEAR’AND ‘OFF-YEAR’ TREES IN PISTACHIO M. ZARIFIKHOSROSHAHI1, S. KAFKAS1 KAFKAS, E1., H. TOPCU1, H.KARCI1, M.GUNEY2, H. PAIZILA1

1University of Çukurova, Faculty of Agriculture, Department of Horticulture, 01330, Balcalı, Adana, TURKEY

2University of Bozok, Faculty of Agriculture, Department of Horticulture, Yozgat, TURKEY

Pistachio, Alternate bearing, miRNAs, Expression

Pistacia vera L. is the most economically important cultivated species of Pistacia genus that belongs to Anacardiaceae

family. Pistacia genus includes more than eleven species and Pistacia vera is the only economically important species

due to its edible nuts. Alternate bearing is one of the most economically important problems in pistachio, and the main

pistachio cultivars in Turkey have strong tendency to alternate bearing that causes fluctuations in the production and in

the market. The alternate bearing signal generated in the fruit, or in another organ determines buds fate-flowering or

vegetative growth by altering regulatory and metabolic pathways. Several factors may affect the balance between such

developmental phase-transition processes. Among them, miRNAs are gene-expression regulators that have been found to

be involved as key determinants in several physiological processes. In this study, the inflorescence buds from ‘ON-year’

and ‘OFF-year’ trees were sampled from early spring to mid-summer with seven sampling dates from Uzun cultivar. The

small RNA libraries were constructed from bud samples and sequenced by high-throughput Illumina sequencing.

Differentially expressed miRNAs from ‘ON-year’ and ‘OFF-year’ trees were detected at different developmental stages.

These miRNAs have been testing by expression analysis at various developmental stages and the results will be presented

in the meeting.

ENGINEERED FLOCK HOUSE VIRUS FOR VIRUS-INDUCED GENE SILENCING IN FRUIT

FLIES

CLAUVIS N. T. TANING, OLIVIER CHRISTIAENS AND GUY SMAGGHE

Department Of Plants And Crops, Faculty Of Bioscience Engineering, Ghent University, Ghent, Belgium

RNA interference (RNAi) is a powerful tool to study functional genomics in insects and the potential of using RNAi to

suppress crop pests has made outstanding progress. However, the delivery of dsRNA is a challenging step in the

development of RNAi bioassays. In this study, we investigated the ability of engineered flock house virus (FHV) to induce

targeted gene suppression through RNAi under in vitro and in vivo condition. As proxy for fruit flies of agricultural

importance, we worked with s2 cells derived from drosophila melanogaster embryos, and with adult stages of d.

Melanogaster. We found that the expression level for all of the targeted gene was reduced by more than 70% in both the

in vitro and in vivo bioassays. Furthermore, the cell viability and median survival time bioassays demonstrated that the

recombinant FHV expressing target gene sequence caused a significantly higher mortality than the wild type virus, in

both s2 cells and adult insects, respectively. This is the first report showing that a single stranded RNA insect virus such

as FHV, can be engineered as an effective in vitro and in vivo RNAi delivery system. Since FHV infects many insect

species, the described method could be exploited to improve the efficiency of dsRNA delivery for RNAi-related studies

in both FHV susceptible insect cell lines and live insects that are recalcitrant to the uptake of naked dsRNA.

VIRUS-INDUCED GENE SILENCING (VIGS) IN INSECTS

LUC SWEVERS1, CLAUVIS N T TANING2, ANNA KOLLIOPOULOU1, OLIVIER CHRISTIAENS2, AND GUY

SMAGGHE2

1Institute of Biosciences & Applications, NCSR “Demokritos”, Aghia Paraskevi, Athens, Greece

2Department Of Plants And Crops, Faculty Of Bioscience Engineering, Ghent University, Ghent, Belgium

While RNAi has been heralded as a specific and safe method of insect pest control, considerable obstacles still need to

be overcome for the development of economically viable products. One promising approach is the use of microorganisms,

such as bacteria, algae, symbionts and viruses as vehicles to increase the delivery of dsRNA silencing triggers. Here, a

perspective is presented regarding the engineering of viruses for the induction of gene silencing in insects.

Different reverse genetics systems are already available for production of recombinant RNA viruses such as nodaviruses,

tetraviruses, dicistroviruses, tymoviruses, reoviruses, alphaviruses, flaviviruses and rhabdoviruses and these can be tested

and eventually promoted as platforms for VIGS in insects. Production systems for DNA viruses such as baculoviruses

also exist but their use as VIGS vectors may not be as straightforward as for RNA viruses. While many viruses are insect-

specific, it is also noted that some plant viruses are vectored by insects in a persistent and circulative manner and therefore

could represent a natural delivery system.

The use of viruses for VIGS poses important challenges that need to be overcome including robust production systems,

efficiency of triggering RNAi in the targeted insects and safety aspects. It requires a better understanding of the infection

process in the gut, the replication capacity of the viral genome, the antiviral immune response and the viral host range.

THE EVOLUTION AND FUNCTION OF PIRNAS ACROSS ARTHROPODS

SAMUEL LEWIS

Department Of Genetics, University Of Cambridge, Downing Street, Cambridge, Cb2 3eh, Uk, Sl782@Cam.Ac.Uk

The small RNA pathway is an evolutionarily ancient mechanism that uses small RNA molecules bound to argonaute

proteins to regulate gene expression, combat viruses and suppress transposable elements (TEs). Due to their potent and

highly-specific regulatory effects, small RNAs are of considerable interest in the development of novel pesticides, but the

evolution of these pathways across different species is still relatively unexplored. In animals, small RNAs termed piwi-

interacting RNAs (piRNAs) silence TEs in the germline, preventing genomic instability and mutation. piRNAs have been

detected in the soma in a few animals, but these are believed to be specific adaptations of individual species. To investigate

the evolution of small RNA pathways across the arthropods, we sampled somatic and germline tissue from 20 species

including horseshoe crabs, woodlice, bees and locusts. We sequenced small RNAs and transcriptomes from each tissue,

and find that somatic piRNAs are common across the arthropods. By mapping this data onto the arthropod phylogeny,

we show that somatic piRNAs were likely present in the ancestral arthropod more than 500 million years ago, and have

been lost on multiple independent occasions. We also find that somatic piRNAs target TEs in all species, but genes and

viruses are targeted in only a subset of lineages. Our results call into question the view that the ancestral role of the piRNA

pathway was to protect the germline, and demonstrate that small RNA silencing pathways have been repurposed for both

somatic and germline functions throughout arthropod evolution.

DAY 2 – Wednesday, JULY 18th AT INRA WG1 Meeting at INRA VILLENAVE D’ORNON – BORDEAUX

9.00 WG1 Meeting - Session 1 Chair Michel Ravelonandro and Bruno Mezzetti

INTEGRATED SEQUENCES OF BANANA STREAK VIRUS (BSV) IN BANANA

PLANTAIN: HOW DOES GENE SILENCING-BASED PLANT DEFENSE SYSTEM

WORK?

EMELINE RICCIUTI1, DELPHINE MASSÉ2, BRUNO HOESTACHY2, MARIE-LINE ISKRA-CARUANA1

1 CIRAD, UMR BGPI, F-34398 Montpellier, France. BGPI, Univ Montpellier, CIRAD, INRA, Montpellier SupAgro,

Montpellier, France.

2 Laboratoire de la santé des végétaux, Station de la Réunion, Pôle de Protection des Plantes, Bât. CIRAD, Ligne Paradis,

7 chemin de l'IRAT, 97410 SAINT PIERRE

Banana streak virus (BSV), plantain, integrated sequences, gene silencing, epidemiologic risk

The majority of cultivated bananas are intra and interspecific hybrids of the two main species Musa acuminata (denoted

genome A) and Musa balbisiana (denoted genome B). The Banana streak virus (BSV) is responsible of the banana streak

mosaic disease and causes severe damage on the Musa acuminata hybrids, which are extremely susceptible. The virus is

mealybugs- vector transmitted from plant to plant. Curiously, BSV also exists as endogenous sequences named (eBSV)

within all B genomes and can give rise active viruses leading to a systemic infection in banana plants.

The banana plantain is a natural triploid interspecific hybrid AAB and harbors viral sequences for two BSV species within

its B genome. It spontaneously develops BSV infections following abiotic stresses by releasing active viral genome from

eBSVs. However, no epidemic has been reported so far, whereas several plantains regularly show infections in the fields.

We suspected that the banana plantain regulates such endogenous viral infections by a gene silencing-based plant defense

mechanism resulting from a co-evolution process.

Our project aims understanding how this pathosystem works by following after an endogenous infection the kinetic of

the virus distribution in the plant via molecular (IC-PCR, qPCR) and histologic (serological detection) markers as well as

the small viral RNA profiles involved in gene silencing (TGS, PTGS) via Northern blots, NGS and LNA probes.

CHARACTERIZATION OF THE ROLE OF A RAPID ALKALINIZATION FACTOR (RALF)

GENE IN THE SUSCEPTIBILITY OF STRAWBERRY FRUITS TO COLLETOTRICHUM

ACUTATUM

M. GUIDARELLI1, M.C. MERINO2, F. NEGRINI, D. DE BIASE3, A. PESSION3, E. BARALDI1

1Università degli Studi di Bologna, Dipartimento di Scienze Agrarie (DipSA), Viale Fanin 46, 40127, Bologna, Italy. 2 Consejo

Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de Córdoba, Argentina. 3Department

of Pharmacy and Biotechnology (FaBiT), University of Bologna, Bologna, Italy. E-mail: elena.baraldi@unibo.it

Strawberry fruit; Rapid Alkalinization factor; Antrachnose susceptibility; gene expression

The fungal pathogen Colletotrichum acutatum, the causal agent of strawberry (Fragaria × ananassa) anthracnose, can infect

strawberry fruit host both at unripe and ripe stages causing anthracnose symptoms only on red ripe fruit. In order to understand

the molecular basis of the high susceptibility of red ripe strawberry fruits, the role of a gene encoding for a Rapid

Alkalinizaztion Factor (RALF), a secreted peptide causing a rapid apoplastic alkalinization, has been investigated.

The variation of the expression of RALF was monitored by using qRT-PCR at the early time points (8-16-20-24 hours post

inoculum) of the interaction in both white and red strawberry fruits inoculated with C. acutatum. The expression of RALF

was found to increase exclusively in ripe susceptible inoculated fruits starting from 20 to 24 hpi suggesting a role for this gene

in modulating the susceptibility of red strawberries. For this reason, an Agrobacterium- mediated transient transformation was

used for silencing and overexpressing the RALF gene in ripe and unripe strawberry fruits respectively. RALF-silenced ripe

fruits did not show any decrease in the susceptibility with respect to control fruits. The overexpression of RALF in white

unripe inoculated strawberries determined a slight decrease in the resistance of these fruits, but further investigation are needed

to clearly confirm a role for RALF expression in fruit susceptibility, independently from the use of agrobacterium and/or

agroinfiltration procedure.

DIFFERENTIALLY EXPRESSED miRNAs DURING MALE AND FEMALE INFLORESCENCE

BUD DEVELOPMENT IN PISTACHIO

S. KAFKAS, H. TOPCU, H. KARCI

University of Çukurova, Faculty of Agriculture, Department of Horticulture, 01330, Balcalı, Adana, TURKEY

Pistachio, sex expression, dioecious, miRNA

The genus Pistacia belongs to Anacardiacdeae family and includes 11 species. P. vera (pistachio) is only one cultivated

species and it has been widely grown in the Middle East, Mediterranean regions of Europe, North Africa and USA.

Pistachio is a dioecious species and staminate and pistillate flowers are apetalous. It is known in pistachio that whorls

organs of both sexes initiate development and ultimately become unisexual by the developmental arrest of the organs of

the opposite sex. The development of staminate and pistillate flowers was studied by scanning electron microscopy

to detect arresting period of the opposite sex organ development to reveal differentially expressed miRNAs at

that stages in this study. The male and female inflorescence buds were sampled from early spring to mid summer as

well as before bud burst in the spring with 13 sampling dates from Siirt and Kaska pistachio female and male cultivars,

respectively. Small RNA sequencing was performed in all samples and differentially expressed miRNAs in male and

female infloresences were detected at different developmental stages. These miRNAs have been testing by expression

analysis at various developmental stages of buds of different male and female pistachio cultivars, and the results

will be presented in the meeting.

DOUBLE-STRANDED RNA: UNIVERSAL HALLMARK OF INFECTION OF

CELLULAR ORGANISMS BY VIRUSES

CHRISTOPHE RITZENTHALER1

1 Institut de Biologie Moléculaire des Plantes CNRS-UPR 2357, associée à l’Université de Strasbourg, 12 rue du

Général Zimmer, 67084 Strasbourg, France

Double-stranded (ds)RNA that results from the pairing in cis or in trans of two complementary RNA strands has been

postulated to be the earliest form of life. Long dsRNA is also regarded as a universal hallmark of infection of cellular

organisms by viruses. dsRNA plays essential functions in many biological processes, including the activation of innate

immune responses and RNA interference.

Methods for detecting dsRNA rely essentially on immunological approaches and their use is often limited to in vitro

applications, although recent developments have allowed the visualization of dsRNA in vivo. We recently reported about

the sensitive and rapid detection of long dsRNA both in vitro and in vivo using the dsRNA binding domain of the B2

protein from Flock house virus (Monsion et al., 2018). In vitro, we adapted the system for the detection of dsRNA either

enzymatically by northwestern blotting or by direct fluorescence labeling on fixed samples. In vivo, we produced stable

transgenic Nicotiana benthamiana and Arabodopsis lines allowing the visualization of viral replication complexes by

fluorescence microscopy. Using these techniques, we were able to discriminate healthy and positive-sense single-

stranded RNA virus-infected material in plants and insect cells. In N. benthamiana and Arabodopsis, our system proved

to be very potent for the spatio-temporal visualization of replicative RNA intermediates of a broad range of positive-sense

RNA viruses, including high- vs. low-copy number viruses. Finally, dsRNA capture procedures were developed that

allowed (i) the identification of host factors associated to virus replication complex and also the identification of a novel

virus isolates using NGS approaches.

Reference

Monsion, B., Incarbone, M., Hleibieh, K., Poignavent, V., Ghannam, A., Dunoyer, P., Daeffler, L., Tilsner, J., and

Ritzenthaler, C. (2018). Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain

from FHV B2 Protein. Frontiers in Plant Science 9.

TRANSFORMATION OF EUROPEAN PLUM (PRUNUS DOMESTICA) WITH HAIRPIN

RNA AND WITH AMIRNA TO INDUCE RESISTANCE TO PLUM POX VIRUS

L. BURGOS

Grupo de Biotecnología de Frutales. Departamento de Mejora. CEBAS-CSIC. Campus Universitario de Espinardo, Edif.

Nº 25, 30.100 Murcia (Spain)

hpRNA, amiRNA, sharka

We have transformed European plums with a hairpin construction that was designed by Dr. V. Ilardi’s group aimed to

silence a virus gene close to the 5’ end. Additionally, transgenic plums were produced transformed with amiRNA

constructions designed to silence two different virus genes simultaneously by Dr. J.A. García’s group.

Although it is not easy, given that different genes have been targeted and that strategies are quite different, both are

compared in terms of efficiency to generate plants resistant to the virus infection. Also a reflection on the facility that the

virus may have to avoid the resistant mechanism by mutations and generate resistant strains is done.

ASSESSING THE EFFECTS OF A LARGE SIZE OF A VIRUS TRANSGENE

TRANSCRIPT ON VIRUS REPLICATION IN PLUMS.

M. RAVELONANDRO1, P. BRIARD1, R. SCORZA2

1INSTITUT NATIONAL DE LA RECHERCHES AGRONOMIQUE BORDEAUX, FRANCE

2 APPALACHIAN FRUIT RESEARCH STATION USDA-ARS, KEARNEYSVILLE, WV, USA

prunus domestica, rnai, resistance, plum pox virus

The quarantine plum pox virus (ppv) infects prunus genera resulting in yield losses and a restrictive

control for exchanging plant materials. As an alternate to the classical breeding technique, genetic

engineering of rnai allows to tackle the virus genome replication and confers resistance to disease.

Ppv rna is a virus genome within 9800 nt including both a 145nt of leader and 220nt of 3’untranslated

sequences. Attempting to challenging both virus genome replication and translation was our goal. In

these studies, two constructs harbouring a 2.5kb transcript including the p1 (5’gene) fused to the cp

(3’gene) of ppv genome were designated. The first is an encoding construct within the p1-cp flanked

with the 5’leader and the 3’ untranslated sequences. However the second was build with the same

gene constructs fused in a tandem inverted repeat split by an intron (intron hairpin rna construct) into

the phellsgate plant transformation vector. Following to plum (prunus domestica) transformation,

resistance studies were assessed with plants graft-inoculated with ppv in greenhouse conditions. If

only two resistant clones were identified with the first gene construct, high proportion of plums

transformed with the second engineered with an ihrna have provided resistant plants. Of the 2

transgene constructs we have proven the criteria widely shown by the scientific community that sirna

accumulated in resistant plants confers virus resistance. With regard to the 5’leader and the

3’untranslated sequences that were remoted from the 2nd gene construct, the p1 fused to the cp

sequences comparably reproduced the same resistance phenotype. These findings led us to

hypothesize as interestingly efficient tackling for virus rna replication. In conclusion, these studies

aim at strengthening the use of sirna accumulated in perennials. Picking up information about these

results, significant benefits from plant biotechnology are promising to support the use of a variable

source of sirna for conferring a stable and durable resistance to ppv.

PARTICIPANTS

N NAME SURNAME COUNTRY EMAIL

1 GUY SMAGGHE Belgium guy.smagghe@ugent.be

2 MICHEL RAVELONANDRO France Michel.ravelonandro@inra.fr

3 EMELINE RICCIUTI France emeline.ricciuti@cirad.fr

4 MATTHIEU CHABANNES France matthieu.chabannes@cirad.fr

5 CHRISTOPHE RITZENTHALER France christophe.ritzenthaler@ibmp-

cnrs.unistra.fr;

6 MARIE-LINE CARUANA France Marie-line.caruana@cirad.fr

7 LUC SWEVERS Greece swevers@bio.demokritos.gr

8 FRANCESCA NEGRINI Italy francesca.negrini6@unibo.it

9 ELENA BARALDI Italy elena.baraldi@unibo.it

10 CECILIA LIMERA Italy cnlimera1983@hotmail.com

11 BRUNO MEZZETTI Italy b.mezzetti@univpm.it

12 SILVIA SABBADINI Italy s.sabbadini@univpm.it

13 TIZIANA PANDOLFINI Italy tiziana.pandolfini@univr.it

14 SONIA OSORIO Spain sosorio@uma.es

15 LORENZO BURGOS Spain burgos@cebas.csic.es

16 EBRU KAFKAS Turkey ebruyasakafkas@gmail.com

17 SALÌH KAFKAS Turkey skafkas@cu.edu.tr

18 SAMUEL LEWIS UK sl782@cam.ac.uk

19 JEREMY SWEET UK jeremysweet303@aol.com

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