COST IPLANTA CA15223 WG1 Meeting · sustainability and profitability of the viticulture industry...
Transcript of COST IPLANTA CA15223 WG1 Meeting · sustainability and profitability of the viticulture industry...
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, [email protected]
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: [email protected]
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 [email protected]
2 MICHEL RAVELONANDRO France [email protected]
3 EMELINE RICCIUTI France [email protected]
4 MATTHIEU CHABANNES France [email protected]
5 CHRISTOPHE RITZENTHALER France christophe.ritzenthaler@ibmp-
cnrs.unistra.fr;
6 MARIE-LINE CARUANA France [email protected]
7 LUC SWEVERS Greece [email protected]
8 FRANCESCA NEGRINI Italy [email protected]
9 ELENA BARALDI Italy [email protected]
10 CECILIA LIMERA Italy [email protected]
11 BRUNO MEZZETTI Italy [email protected]
12 SILVIA SABBADINI Italy [email protected]
13 TIZIANA PANDOLFINI Italy [email protected]
14 SONIA OSORIO Spain [email protected]
15 LORENZO BURGOS Spain [email protected]
16 EBRU KAFKAS Turkey [email protected]
17 SALÌH KAFKAS Turkey [email protected]
18 SAMUEL LEWIS UK [email protected]
19 JEREMY SWEET UK [email protected]
IN COLLABORATION WITH: