iPLANTA COST Action CA15223

3rd iPLANTA Conference

WHAT FUTURE FOR RNAi-BASED

PRODUCTS: RNAi MODIFIED PLANTS OR

SPRAY PRODUCTS

BOOK OF ABSTRACTS

POSTERS

ITQB NOVA (Oeiras)

FEBRUARY 27TH - MARCH 1ST, 2019

Grosshans H, Filipowicz W.Nature. 2008

3nd iPLANTA Conference

WHAT FUTURE FOR RNAi-BASED PRODUCTS: RNAi MODIFIED PLANTS OR SPRAY

PRODUCTS

AIMS AND THEMES

The 3rd iPlanta Conference and MC meeting, planned in Lisbon (PT), is the last major event to be organized for

Grant Period 3 of CA15223. The 3rd iPLANTA Conference will focus on defining the technical in planta and

topical applications of RNAi. The event will have the following organization:

1) Three full days of plenary conference, including max a 3 hours slot for the MC meeting and if needed

parallel small group meetings organized by the WGs.

2) The scientific program of the conference on RNAi technology (WG1, 2 and 3) will focus on the main

aspects of RNAi products and include novelties in RNAi stability of both SIGS and HIGS. About SIGS, the main

aspects related to the development of new RNAi based products should be included: dsRNA design for

specificity, testing activity, mobility, persistence, biosafety (food and environment), regulation. An invited

speaker from Brazil will be included in the program. About HIGS, priority will be given to novelties on in-planta

studies, efficiency and biosafety. An invited speaker from USDA –will be included in the program for presenting

results on food safety studies of RNAi products.

3) A session will be dedicated to socio-economic impacts (WG4) by inviting new stakeholders, thanks also

to the help of local organizers, and to present results from a survey started by WG4 Chair. An invited speaker

from USA can be expected.

4) For WG5 session will focus on the discussion on the state of preparation of scientific materials (videos,

position papers, reviews, book) to be used for a science-based communication program.

5) In the program about 3 hours will be dedicated to the Management Committee meeting (Thursday or

better Friday afternoon) mostly focused on the discussion and approval of GP4 program. WG and STSM Chairs

must be ready to present reports of GP3 activities and their plan of WG activities for GP4.

THE CONFERENCE WILL BE OPEN TO ALL PARTICIPANTS

BIOTECHNOLOGICAL SOLUTIONS FOR DELIVERY OF DSRNA TO CROP PESTS THAT DO

NOT INVOLVE TRANSGENIC ORGANISMS

Y.ZHAO1,2, F.REN2, Q.LU2, J.SUN2, L.SWEVERS1

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

2College of Animal Science, South China Agricultural University, Guangzhou, People’s Republic of China.

Pest control, dsRNA spray, cytoplasmic polyhedrosis virus, virus-like particle

A major issue in the applicability of the RNAi mechanism in pest control remains the efficient delivery of the dsRNA

trigger to the majority of crop pests. While transgenic plants expressing RNA hairpins have shown potential for control

of coleopteran and lepidopteran pests, strong societal and political opposition do not favour this approach within the EU.

The antagonism against genetically modified organisms (GMOs) likely is not restricted to transgenic crops but may be

extended to genetically engineered microorganisms (bacteria, yeasts, viruses) for which the capacity to deliver dsRNA

triggers is gaining more interest. On the other hand, it may be possible to design nanoparticles for the efficiently delivery

of dsRNA that are solely based on structural (capsid) proteins of dsRNA viruses and in vitro synthesized dsRNA. Since

viruses have evolved during long periods to achieve specific and efficient infections of host organisms, their viral capsids

must have acquired specialized properties for delivery of their genetic material. Such properties will be harnessed during

the production of viral-like particles (VLPs) in biotechnological platforms. VLPs loaded with dsRNA molecules therefore

could be developed as sophisticated nanoparticles that can be used in spray applications of pest control. Both capsid

proteins and incorporated dsRNA can be considered as “natural products” that are rapidly degraded in the environment

and therefore compatible with sustainable development and integrated pest management (IPM) approaches. We propose

that cytoplasmic polyhedrosis viruses that have a segmented dsRNA genome and of which the structure of the virion is

documented in great detail by cryo-electron microscopy, can function as the basis for the design of “inert” (i.e. no

amplification of genetic material) VLPs for specific and efficient delivery of RNAi effects in lepidopteran insects.

PHYTOREMEDIATION OF POLLUTED SOILS USING RNAi PLANTS

E. G. PAPAZOGLOU1, S. ARPAIA2

1Department of Crop Science, School of Agricultural Production, Infrastructure and Environment, Agricultural

University of Athens, Greece 2Division Bioenergy, Biorefinery and Green Chemistry, ENEA Research Centre Trisaia, Italy

GMO, gene silencing, trace elements, hyperaccumulation, hypertolerance

The pollution of the environment by trace elements (TE) has become a serious global problem. Recent estimates indicate

that in Europe there are roughly 640,000 ha of contaminated soil and 5 million of potentially contaminated ones.

Phytoremediation is a new, non-invasive and publicly acceptable technology, which uses plants to remove pollutants from

the environment or to render them harmless by stabilization. Plant species thriving in polluted areas either avoid the

uptake of TE (heavy metals and metalloids) from the soil (excluders), or accumulate significantly high concentrations of

TE into their tissues, to levels which by far exceed the soil levels (hyperaccumulators). The growing application of

molecular-genetic technologies led to the well understanding of mechanisms of heavy metal(-loid)

tolerance/accumulation in plants, and subsequently many transgenic plants with increased resistance and uptake of TE

were developed for the purpose of phytoremediation [1]. However, the use of RNA interference to metal

hyperaccumulation or hypertolerance have been investigated to a limited extent so far. Research on the model crop

Arabidopsis halleri using RNA interference to downregulate HMA4 (Heavy Metal ATPase 4) expression, showed that

Zn hyperaccumulation and hypertolerance to Cd and Zn depend on the metal pump HMA4 [2,3]. The gene ACR2 with

moderate sequence homology to yeast arsenate reductase was identified in the Arabidopsis thaliana genome [4]. The

knockdown lines accumulated 10- to 16-fold more arsenic in shoots (350–500 ppm) and retained less arsenic in roots than

the wild type. Reducing expression of ACR2 homologs in plant species could play an important role in the

phytoremediation of environmental arsenic contamination. Other possible target genes in other plant species have been

identified; for instance, it is referred that OsNRAMP5 contributes to Mn, Fe and Cd transport in rice [5]. OsNRAMP5

RNAi (OsNRAMP5i) plants accumulated less Mn in the roots, and less Mn and Fe in shoots, and xylem sap. At the

contrary, the suppression of OsNRAMP5 promoted Cd translocation to shoots, indicating that rice RNAi plants could be

used for increased Cd extraction and for efficient phytoremediation of Cd from paddy fields [6], supporting an

environmentally friendly remediation approach.

References

[1] Visioli, G., Marmiroli, N. (2013). Journal of Proteomics 79, 133-145.[2] Hanikenne, M. et al. (2008) Nature 453:391–

395

[3] Memon, A.R., Schröder, P. (2009). Environmental Science and Pollution Research 16, 162-175.

[4] Dhankher, O. P. et al. (2006). Proceedings of National Academy of Sciences, 103(14), 5413-5418.

[5] Ishimaru, Y. et al. (2012). Scientific Reports 2: 286 | DOI: 10.1038/srep00286

[6] Takahashi, R. (2014). PLOS ONE 9, 1-7.

EVALUATION OF DIFFERENT FACTORS IN OPTIMIZING STABLE EXPRESSION OF

PPV-HAIRPIN CONSTRUCT IN HANSEN 536 (Prunus persica x Prunus amygdalus) PEACH

ROOTSTOCK

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

1Dipartimento di Scienze Agrarie, Alimentari ed Ambientali, Università Politecnica delle Marche, Ancona, IT

Genetic transformation, Prunus spp., rootstock, PPV, gene silencing

Sharka, caused by Plum Pox Virus (PPV), is the most devastating viral disease for Prunus spp.. Puting into consideration

the serious agronomic and economic damage caused worldwide, researches have been committed to introduce genetic

resistance to PPV in Prunus species, including peach varieties and rootstocks. A stimulating approach based on the

exploitation of a natural mechanism for gene expression regulation, known as post-transcriptional gene silencing (PTGS),

is being used. In order to induce PTGS against PPV in Prunus spp., it is essential to develop efficient and reproducible in

vitro protocols for regeneration and genetic transformation. For this reason, an in vitro regeneration protocol based on the

production of meristematic bulks (Sabbadini et al., 2015) was optimized for Hansen 536 (Prunus persica x Prunus

amygdalus), to enable insertion of a hairpin construct (ihp35s-PPV194) capable of inducing PPV resistance via PTGS. In

order to evaluate Hansen 536 transformation efficiency, meristematic bulks were used as starting explants for

Agrobacterium-mediated genetic transformation trials; nptii and eGFP genes were used as selection markers in

identifying putatively transformed events. In this study, two factors were evaluated to increase the transformation

efficiency in Hansen 536: i) acetosyringone added in the co-culture medium at different concentrations; ii) silver

thiosulfate (STS), an ethylene inhibitor, used during the co-culture medium and/or in regeneration/selection medium for

the first two weeks. Analysis of the results obtained was useful in the optimization of transformation protocol applied,

hence, obtainment of stably Hansen 536 cell lines (actively dividing calli) expressing eGFP.

MICRO RNAS IN TRANSGENIC AND NONTRANSGENIC TOBACCO PLANTS EXPOSED TO

ABIOTIC STRESS.

Z. GERSI1, M.ZIMOVA2,3 E.BOSZORADOVA3, I.MATUSIKOVA1, K. RAZNA4, J.MORAVCIKOVA3,5

1Department of Ecochemistry and Radioecology, University of Cyril and Methodius in Trnava, Slovak Republic. 2Department of Botany and Genetics, Faculty of Natural Sciences, The Constantine Philosopher University, Nitra, Slovak

Republic.

3Institute of Plant Genetics and Biotechnology, Plant Biology and Biodiversity Center Slovak Academy of Sciences,

Nitra, Slovak Republic. 4 Faculty of Agrobiology and Food Resources, Slovak University of Agriculture in Nitra, Slovak Republic.

5 Department of Biotechnologies, University of Cyril and Methodius in Trnava, Slovak Republic

abiotic stress, dehydrins, miRNA, transgenic tobacco plants

The miRNAs, being a class of short, endogenous, noncoding small RNAs that have been demonstrated to be involved in

many biological and metabolic processes and in a/biotic stresses. We studied the involvement of miRNAs (miR159,

miR319b, miR398, miR482d, miR156g and miR169q) in transgenic tobacco plants exposed to the selected types of abiotic

stresses. Trangenic plants were obtained via A. tumefaciens by introducing of the Arabidopsis dehydrin gene At3g50970.

Deyhdrins that are rich of histidin regions are thought to have a potential to bind free metals; therefore, they could

contribute to increased tolerance to heavy metals. The miRNA analyses were performed on roots and leaves of transgenic

and non-transgenic (control) tobacco plants that were exposed to 100 μM CdCl2, 200 μM ZnCl2, 100 μM CuCl2 or 150

mM NaCl for 24 hour. Our data showed that among the analysed miRNAs, miR319b, miR156g and miR169q were

significantly down regulated in the Cd2+ treated leaves and/or roots of transgenic tobacco plants suggesting that the

expression of their target genes was turned on in response to the100 μM CdCl2. We did not observed decreased expression

of these miRNAs in the leaves and roots of Cd2+ treated nontransgenic (control) plants.

This work was supported by the project VEGA 2/0035/17

SIMPLE WORKFLOW FOR RNAI-INDUCING CONSTRUCT USING GOLDENBRAID

CLONING SYSTEM

K. MULLER1, Z. VONDRAKOVA1, R. FILEPOVA1, T. MORAVEC1, J. PETRASEK1

1Institute of Experimental Botany, Czech Academy of Sciences, Prague, Czech Republic

gene silencing, tobacco, cell culture, auxin oxidase, GoldenBraid cloning

Auxin is important signaling molecule involved in regulation of many processes during plant life cycle. Activity of auxin

action is regulated on several levels: transport, biosynthesis and metabolism. Oxidation of the indoleacetic acid, the most

common natural auxin, has been recently linked with the activity of auxin oxidase (DAO1) enzyme. Since DAO1 is not

member of any large gene family, it has important role in auxin metabolism and its activity is easy to follow, it presents

good target for transcriptional regulation by RNAi.

Preparation of RNAi inducing construct can be challenging cloning process. Here we report simple strategy, where single

PCR amplification of the target sequence is followed by several Golden Gate assembly steps enabling the choice of

modulation of dsRNA transcription. GoldenBraid is a tool utilizing Golden Gate assembly for construction of complex

DNA assemblies in Agrobacterium compatible binary plasmids. It offers choice of constitutive, tissue specific or inducible

promoters that can be easily incorporated into the cloning strategy. Following GoldenBraid standard we have prepared

several new DNA parts and verified their effectiveness for the transcriptional silencing of NtDAO1 in tobacco cell

cultures.

GRAPEVINE-LEAFROLL ASSOCIATED VIRUS AND ITS CURE VIA RNAI

N.SADOT, O.SHOSEYOV

The Robert H. Smith Faculty of Agriculture, Food and Environment

The Hebrew University of Jerusalem, Israel

GLRaV, Grapevine, RNAi

Grapevine leafroll disease (GLD) is among the most severe grapevine (Vitis Venifera) viral diseases known to man. Its

damaging effects cause massive economic losses on the wine market, affecting not only the crop yield, but also the vines’

health and the quality of the grapes. The main pathogens of GLD are the Grapevine leafroll-associated viruses (GLRaV)

1-9, which inhabit the phloem after infection. GLRaV-1 and GLRaV-3 lead to very strong symptoms of the disease, in

contrast to GLRaV-7 that causes very little to no symptoms. GLRaV-3 occurs worldwide; it’s an ssRNA virus from the

Ampelovirus genus and is by far the most notable pathogen causing this disease. GLRaV viruses are transmitted by the

vine mealybug (Planococcus ficus) at a very high rate. Due to its strong symptoms of the disease GLRaV-3 is the main

interest of this study [1]. Symptoms of the disease may vary, causing the detection of GLD to be very difficult. The

symptoms may change due to season, weather and grape cultivar. On a phenotypic level, at the beginning of the growing

season, in red cultivars, increased redness of the interveinal areas is seen, while the veins remain green. In white cultivars

chlorosis appears in the interveinal areas. As the growing season progresses, leaves in both red and white cultivars will

show leaf-rolling downwards. In some cases, the vine doesn’t show any symptoms at all, making the detection of the

virus without genetic testing impossible. We wish to address these issues using RNAi solutions.

References:

1. Maree, H. J. et al. Grapevine leafroll-associated virus 3. Front. Microbiol. 4, 1–21 (2013).

HOST-INDUCED GENE SILENCING IN WHEAT POWDERY MILDEW

L. SCHAEFER1, S. BOURRAS2, B. KELLER1

1Department of Plant and Microbial Biology, University of Zurich, Zurich, Switzerland. 2Division of Plant Pathology, Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural

Sciences, Uppsala, Sweden.

Wheat powdery mildew, genetic transformation, HIGS, gene silencing

Wheat is the most important staple crop for human consumption. Wheat powdery mildew is one of the top fungal diseases

reducing yield globally. Breeding for mildew resistance traditionally focuses on major resistance traits, which have proven

unsustainable, as resistance is typically broken within a few years. Therefore, research efforts increasingly focus on minor

resistance traits and pathogen-informed strategies, as these are expected to be more durable. In the effort to advance

understanding of the pathogen side of the infection, the principle of host induced gene silencing (HIGS) has been used to

identify several barley powdery mildew effectors that have a role in virulence. In wheat powdery mildew the effector

suppressor of avirulence SvrPm3a1/f1 is so far the only known wheat powdery mildew virulence factor.

Here, we transformed the spring wheat Bobwhite SH 98 26 with a SvrPm3-RNAi construct. The resulting transgenic HIGS

wheat lines were characterized for powdery mildew resistance in the lab and under semi-field conditions.

PRELIMINARY RESULTS ON ROOTSTOCK-TO-SCION TRANSFER OF TRANSGENE-

DERIVED SMALL INTERFERING RNAS AND THEIR EFFECT ON SHARKA RESISTANCE IN

NON-TRANSGENIC FRUIT TREES

NURIA ALBURQUERQUE, PEDRO DÍAZ-VIVANCOS, LYDIA FAIZE, JOSÉ ANTONIO HERNÁNDEZ,

LORENZO BURGOS

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

30.100 Murcia (Spain)

Email de contacto: burgos@cebas.csic.es

Genetic engineering is a breeding technique that allows the introduction of genes or genomic sequences, which are not

available within the species gene pool, to induce desired characteristics in plants. Although these technologies are

undoubtedly useful, unfortunately, transgenic products are been rejected in Europe and other parts of the world due to the

negative publicity given to them. Therefore, few or none new transgenic plants are being commercialize in Europe.

Sharka, caused by plum pox virus, is probably one of the most devastating diseases affecting stone fruit trees. In an

attempt to introduce resistance to this disease we produced transgenic plum trees with a construct designed to silence

virus genes inducing a “pathogen-derived resistance” by interfering RNA. We demonstrated that some of these lines were

highly resistant to the virus.

Commercialization of these plants would be much easier if they could be used as rootstocks so that they will never flower

and therefore transgene dispersion through pollen would not be possible.

In order to test if the small-interfering-RNAs-mediated resistance found in these plants is transmitted to other Prunus

grafted onto them, we designed an experiment where four of these lines were grafted with wild apricots heavily infected

by sharka. The highly resistant St5’-1 and St5’-9 lines, the intermediate resistant St5’-6 line (the virus was found during

initial evaluation but disappeared later), and the susceptible St5’-7 line were bud-grafted with infected apricot.

After artificial winter in cold chamber and sprouting of buds in the greenhouse plants were evaluated for virus presence

by ELISA and confirmed by RT-PCR. Virus was transmitted to all susceptible St5’-7 rootstocks grafted with infected

buds and, obviously, was also found in all graftings. In the intermediate St5’-6 line the virus was transmitted to half of

the grafted rootstock plants and was found in all graftings. Finally, both resistant lines St5’-1 and St5’-9 behaved similarly.

The virus was never transmitted to the rootstock plants and was not found in half of the scions sprouted from heavily

infected apricot buds.

Although results are promising (especially considering the high viral inoculum density in this experimental approach)

more plants need to be grafted in order to have more consistent results. Graftings will also be examined after subsequent

artificial winters to see if the silencing mechanism works on the long term, eliminating viral particles from the apricot

grafted onto resistant plums rootstocks.

GENE SILENCING IN TRIBOLIUM CASTANEUM AS A TOOL FOR THE TARGETED

IDENTIFICATION OF CANDIDATE RNAI TARGETS IN CROP PESTS

KNORR E1, FISHILEVICH E2, TENBUSCH L1, FREY MLF2, RANGASAMY M2, BILLION A1, WORDEN SE2,

GANDRA P2, ARORA K2, LO W2, SCHULENBERG G2, VALVERDE-GARCIA P2, VILCINSKAS A1,3, NARVA

KE2

1Fraunhofer Institute for Molecular Biology and Applied Ecology, Department of Bioresources, Giessen, Germany

2Dow AgroSciences, Indianapolis, USA

3Institute for Insect Biotechnology, Giessen, Germany

Pest management, Meligethes aeneus, Tribolium castaneum, Diabrotica virgifera, GM crops

Agricultural crops are exposed to a variety of insect pests, causing tremendous annual losses worldwide. Current control

strategies like pest resistant crops or insecticides, are facing efficacy challenges as species evolve resistances. Alternative

pest management technologies are therefore urgently needed and one promising approach is the RNA interference

(RNAi). RNAi is a post-transcriptional gene silencing mechanism triggered by double-stranded RNA, which can be

utilized for pest control by disrupting the expression of essential genes. The number of potent RNAi targets that cause

robust lethality in pest insects is limited and the identification of new target genes requires high-throughput screening

approaches, which may not be possible in every pest species. We used 50 lethal RNAi targets from the model organism

Tribolium castaneum to demonstrate efficacy of orthologous targets in severe pests western corn rootworm (Diabrotica

virgifera virgifera) and European pollen beetle (Meligethes aeneus). Injection and oral application of dsRNA led to the

identification of highly active RNAi targets in M. aeneus as well as D. v. virgifera. Maize expressing RNA hairpins of

the prioritized candidates Rop, dre4, or RpII140 exhibited root protection against D. v. virgifera larvae. Additionally,

silencing of the orthologous genes in M. aeneus was also lethal, both in injection and feeding assays. These data show the

feasibility of using high throughput RNAi screens in model organisms for the identification of effective new RNAi targets

for non-model insect pests.

THE CHOICE OF HOMOLOGY REGION IN THE TOMATO SPOTTED WILT VIRUS GENOME

IS CRUCIAL TO INDUCE RESISTANCE USING EXOGENOUSLY APPLIED DOUBLE-

STRANDED RNAs

TABEIN S1, BEHJATNIA SAA1, ACCOTTO GP2, NORIS E2, VAIRA AM2, MIOZZI L2

1Plant Virology Research Center, College of Agriculture, Shiraz University, Shiraz, Iran 2Institute for Sustainable Plant Protection, National Research Council of Italy, Torino, Italy

TSWV, RNAi-based vaccination, gene silencing

Tomato spotted wilt virus, (TSWV), a negative-sense RNA virus belonging to the Bunyaviridae family, is a devastating

plant pathogen, causing huge crop losses worldwide. Indeed, due to its wide host range and the emergence of resistance

breaking strains, its management is challenging. Up to now, resistance to TSWV infection based on RNA interference

(RNAi) has been achieved in transgenic plants expressing parts of the viral genome or artificial miRNAs targeting it.

Exogenous application of double-stranded RNAs (dsRNAs) for inducing virus resistance in plants, namely RNAi-based

vaccination, represents a very attractive and promising alternative, already shown to effectively protect plants against

different positive-sense RNA viruses and viroids. In the present study, the efficacy of external application of dsRNAs

against the TSWV nucleocapsid (N) or the movement protein (NSm) coding genes in protecting plants against TSWV

infection was evaluated. DsRNA molecules corresponding to portions of the viral genome were synthesized in vitro using

a PCR approach followed by T7 RNA polymerase transcription. Almost all Nicotiana benthamiana plants (~90%) treated

with TSWV N-derived dsRNAs remained asymptomatic and free of virus until 40 days post inoculation (dpi), while NSm-

derived dsRNAs showed a lower efficacy (~30%). Preliminary data suggest that movement and stability of dsRNAs may

be important factors for effectively induce resistance and that the different dynamic in dsRNA systemic spread, possibly

linked to their primary sequence, could be responsible for the different efficacy in protecting plants. These results indicate

that the choice of the viral target sequence in designing dsRNA-based vaccines against plant viruses is crucial to obtain

virus resistance through exogenous application of dsRNAs.

AN ENDOGENOUS SUPPRESSOR OF RNA SILENCING IN ARABIDOPSIS THALIANA

JELENA GERASSIMENKO, LENNE NIGUL, ERKKI TRUVE, CECILIA SARMIENTO

Department of Chemistry and Biotechnology, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn,

Estonia.

RNA silencing, suppressor, Arabidopsis, AtRLI2, CRISPR/Cas9

ATP-binding cassette sub-family E member 1 (ABCE1) is a highly conserved protein among eukaryotes and archaea.

First identified as RNase L inhibitor, ABCE1 is currently recognized as an essential translation factor involved in several

stages of eukaryotic translation and ribosome biogenesis. We have demonstrated that AtRLI2, the homolog of ABCE1 in

Arabidopsis thaliana, is an endogenous suppressor of RNA silencing. Mutational analysis of AtRLI2 shows that ATP

binding and hydrolysis by AtRLI2 is necessary for its suppressor function. In addition, the N-terminal domain with two

iron-sulfur clusters is important, probably playing a role in the interaction with partners.

Interestingly, ABCE1 is crucial for the viability of several organisms (knockouts in yeast, C. elegans and Trypanosoma

brucei are lethal). We have determined that AtRLI2 is ubiquitously expressed, showing an increased expression in flowers

and siliques. At the moment, we are characterizing one Arabidopsis thaliana T-DNA insertional line of AtRLI2 and have

not been able to obtain homozygous plants containing the disrupted gene. Therefore, we are phenotyping heterozygous

plants paying special attention to ovules and siliques. We have observed that the amount of fertilized ovules in the siliques

from heterozygous plants is reduced. Moreover, in some developed flowers the ovules display abnormal central cells. As

endogenous RNAi pathway is important for the development of plants, we are trying to find a link between the observed

phenotype and AtRLI2 suppressor function. In addition, Arabidopsis thaliana, as some other species including Oryza

sativa, encodes two paralogues of ABCE. We are currently using CRISPR/Cas9 to explore the knockout of both AtRLI2

and AtRLI1 genes.

INNOVATIVE BIOTECHNOLOGICAL STRATEGIES AGAINST WDV INFECTION IN WHEAT

AND BARLEY

T. VLCKO1, K.S. SINGH2,, P. CEJNAR2,3, J.KUMAR2, L. OHNOUTKOVA1

1Institute of Experimental Botany AS CR & Palacký University, Laboratory of Growth Regulators, Centre of the Region

Hana for Biotechnological and Agricultural Research, Šlechtitelů 27, 78371 Olomouc, Czech Republic 2Crop Research Institute, Division of Crop Protection and Plant Health, Drnovská 507/73, 16106 Prague 6, Czech

Republic 3Department of Plant Protection, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences,

Kamýcká 129, 16500 Prague 6, Czech Republic

Wheat dwarf virus, PDR, RNAi, CRISPR/Cas9

Wheat dwarf virus (WDV) is a viral pathogen attacking exclusively barley and wheat. Losses in yield can reach up to 90

%. Currently, there is not a natural source of resistance against WDV infection. Biotechnological progress provided

alternative approaches to deal with viral infection via various ways of induction of resistance. Initially, pathogen-derived

resistance (PDR) was expected to induce resistance by introducing partial viral sequence into plant genome that results

in interference with virus affecting its replication. Then, an advanced approach utilizing naturally occurring process of

regulation of transcription - RNA interference (RNAi) can be exploited. Mechanism of resistance is based on activity of

RISC complex that recognize viral sequence, which is degraded in similar way as regular transcripts. The latest innovation

is utilization of CRISPR/Cas9 technology, which was originally described as an adaptive bacterial immune system against

viral infection, analogously in plants. WDV genome consists of genes for replication (Rep/RepA), systemic spread in

plant tissues (CP, MP), and intergenic regions LIR and SIR that are required for replication. Three guide RNA’s were

designed to target distinct parts of WDV genome. Particularly, sequences for Rep, CP, and MP proteins. Plants carrying

Cas9 transgene and guide RNA are in development and will be tested for resistance against WDV.

RELATION BETWEEN KNOWLEDGE AND PERCEPTION OF GMO

M. TENGLEROVA1, B. KLCOVA2, F. ZNEBEJANEK3, L. OHNOUTKOVA2

1Department of Botany, Faculty of Science, Palacký University in Olomouc, Šlechtitelů 27, 78371 Olomouc, Czech

Republic 2Institute of Experimental Botany AS CR & Palacký University, Laboratory of Growth Regulators, Centre of the Region

Hana for Biotechnological and Agricultural Research, Šlechtitelů 27, 78371 Olomouc, Czech Republic 3 Faculty of Arts, The Department of Sociology, Andragogy and Cultural Anthropology, Svobody 26, 77900, Olomouc,

Czech Republic

GMO, public, legislation, questionnaire survey

Genetically modified organisms (GMOs) are negatively perceived by the public in European Union (EU), despite their

wide ranging applicability; they are used in many areas such as biology research, pharmacology, and industry. Notably,

strongly negative opinion to food and feedstuff made of GM products persists among European consumers. Lack of public

awareness about these new technologies might lead to poor judgements and negative beliefs. The purpose of this work is

to detect through questionnaires, whether students awareness might have an impact on their relation to GMO. The research

part is based on a quantitative questionnaire survey carried out on a sample of 100 students of Palacky University in

Olomouc from different faculties and departments recruited through Facebook social network. The data, which was

collected by knowledge and relationship issues, was subsequently statistically evaluated. The results revealed that the

respondents with high level of awareness have very positive relation to GMO whereas the results of respondents with

lower level of awareness rather refer to negative relation.

PATHOGEN DERIVED RESISTANCE TO WHEAT DWARF VIRUS IN BARLEY AND WHEAT

M. TRAJEROVA1, P. CEJNAR2,3, T. VLCKO1, A. PACAK4, P. Z.SZWEYKOWSKA-KULIŃSKA4, J.KUMAR3,

L.OHNOUTKOVA1

1Institute of Experimental Botany AS CR & Palacký University, Laboratory of Growth Regulators, Centre of the Region

Hana for Biotechnological and Agricultural Research, Šlechtitelů 27, 78371 Olomouc, Czech Republic 2Department of Plant Protection, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences,

Kamýcká 129, 16500 Prague 6, Czech Republic 3Crop Research Institute, Division of Crop Protection and Plant Health, Drnovská 507/73, 16106 Prague 6, Czech

Republic

4Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam

Mickiewicz University, Umultowska 89, 61-614 Poznan, Poland

Wheat dwarf virus, PDR, wheat, barley Agrobacterium-mediated transformation

The principle of pathogen-derived resistance (PDR) by transforming host plants with viral genes or sequences with the

purpose to block a specific step during virus multiplication in the plant is one of strategy how to obtain resistance to virus

diseases. Wheat dwarf virus (WDV) is one of the most economically serious viral disease in cereals in the different part

in the word. The virus affects economically important species of cereals, mainly winter wheat and winter barley. The

strategy of PDR-WDV was designed and applied to obtain resistant plant of wheat and barley. Expression vector

pIPKb002 was prepared with a selected target sequence of WDV Rep, labeled J1, and with a selected target sequence of

WDV Rep with several point mutations, labeled K5 for wheat and barley and AC4 and O2 for barley and also potentially

for wheat. Agrobacterium tumefaciens-mediated transformation was used for transforming of immature zygotic embryos

of the spring barley cv. Golden Promise and for the spring wheat of cv. Fielder. Barley and wheat regenerated plants T0

generation were evaluated by PCR, the efficiency of transformation was determined. Three different phenotypes of

transgenic plants with pIPKb002:J1 were identified in the barley. The dihaploid transgenic plants of T3DH2 generation

were evaluated by using Northern Blot to detect J1 mRNA transcript. Verification of resistance in transformed plants T1

and T2 barley and wheat are tested in CRI in Prague.

RISK ASSESSMENT OF NBT CROPS FOR THE SOIL ENVIRONMENT

P.H. KROGH, J. JENSEN

Department of Bioscience, Aarhus University, Vejlsøvej 25, 8600 Silkeborg, Denmark

Ecological risk assessment (ERA), soil invertebrates, unintended effects, baseline scenarios, problem

formulation

All crops developed using NBT and RNAi can trigger an updating, specification and evaluation of the ecological risk

assessment (ERA) as described in the recommended EFSA GMO ERA Guidance (EFSA Journal 2010, 8(11):1879). Such

an update is the recent scientific opinion on pesticide ERA (EFSA Journal 2017, 15(2):4690) by clarifying specific

protection goals for soil biodiversity (SPG) and soil functions in terms of ecosystem services (ES). Soil biodiversity is

considered to deliver a range of ESs and are therefore service-providing units (SPU).

Hitherto, direct effects of a new intended property of an NBT plant could rarely trigger testing at the tier 1 testing level,

while the unintended effects with unknown consequences at the plant phenotypic level always raises concerns. In this

case, the problem formulation will need to provide the rationale for selection of a range of representative focal soil

invertebrate species, in order to increase the chance of protecting all relevant SPGs and SPUs within the soil ecosystem.

A minimum requirement of focal species would typically consist of an earthworm, a collembolan and a mite species.

Baseline soil invertebrate biodiversity data available from international databases such as www.edaphobase.org can aid

in determining site and land-use specific protection goals and ESs. However, the estimation of ESs is not particularly

developed for soil fauna and there is a need to convert biodiversity information into ecosystem services, when it is not

feasible to quantify the functions directly. Such proxies could be developed for ES like water infiltration capacity based

on earthworm abundance and species composition. Similar relationships can be tested for ES such as fungal decease

suppression.

A proper prediction of the state of soil-related ESs needs agronomic information, as the individual ES depends on the

entire farming system and farming operations. Hence, agronomic metadata must be included along with biodiversity

observations uploaded to baseline databases. Such a system level approach is paramount for evaluation of NBT, as

potential changes in farming practice driven by the use of NBT GMO’s may result in environmental impact, while the

direct effect of the NBT plant may be absent or neglectable. Such a multidisciplinary and holistic approach is already

implicit in the EFSA GMO ERA Guidance and is further supported by the need to ensure compliance with the UN goal

of farming system sustainability. This will include assessment of additional soil ESs such as carbon sequestration, water

holding capacity and retention of plant nutrients.

IN SILICO EVIDENCE OF MICRORNA-MEDIATED CROSS-KINGDOM REGULATION OF

GENES INVOLVED IN THE CELLULAR RESPONSE TO VIRUSES IN MEDICAGO

TRUNCATULA AND HUMANS

C.GUALTIERI1, D.DE MARCHI2, M.BELLATO2, E.SAUTA2, L.PASOTTI2, P.MAGNI2, A.BALESTRAZZI1,

A.MACOVEI1

1Plant Biotechnology Lab, Department of Biology and Biotechnology, University of Pavia, Italy 2Laboratory of Bioinformatics, Mathematical Modelling and Synthetic Biology, Department of Electrical, Computer and

Biomedical Engineering - Centre for Health Technology, University of Pavia, Italy

Medicago truncatula, microRNA, cross-kingdom, virus, human

MicroRNAs, highly-conserved small RNAs, act as key regulators of many biological functions in both plants and animals

by post-transcriptionally regulating gene expression through interactions with their target mRNAs. The microRNA

research is a dynamic field, in which new and unconventional aspects are emerging alongside with the well-established

roles in development and stress adaptation. A recent hypothesis states that miRNAs can be transferred from one species

to another and potentially target genes across distant species. Here, we propose to look into the cross-kingdom potential

of miRNAs as a tool to unravel conserved pathways between plants and animals. Hence, a pipeline to investigate the

cross-kingdom interactions between plant miRNA and human targets was developed. Starting from a collection of

Medicago truncatula microRNAs deposited in miRBase, we retrieved public plant/human datasets (genome, proteome,

transcriptome and/or 3'UTR-ome, depending on the organism) and use them to predict candidate targets. Subsequently,

GeneMania was used to generate gene interaction/co-expression networks in both organisms. The topology of the

networks was analysed by using Cytoscape applications to identify clusters of genes to be individually studied. The

subnets were then processed through enrichment analysis via the Cytoscape ClueGO application. Finally, the derived

networks were compared to find common features, e.g., microRNAs targeting similar biological processes. Among these,

the most represented GO terms were ‘cellular response to virus’ and ‘modulation by virus of host morphology or

physiology’, common to both plants and humans. Therefore, this study provides a list of miRNAs and target genes that

can be used for RNAi approaches to develop plants resistant to biotic stresses. Additionally, their cross-kingdom potential

could be useful for interesting advances in therapeutics via daily diet.

THE ROLE OF miRNAS POST-TRANSCRIPTIONAL REGULATION DURING

THE ACQUISITION OF EMBRYOGENIC COMPETENCE IN TAMARILLO

(Solanum betaceum CAV.)

CORDEIRO, D.1, RITO, M.1, CORREIA, S.1,2 AND CANHOTO, J.1,2

1Centre for Functional Ecology, University of Coimbra, Calçada Martim de Freitas, 3000-456 Coimbra,

Portugal 2Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal

post-transcriptional regulation, small RNAs, somatic embryogenesis;

Somatic embryogenesis (SE) is the expression of totipotency of plant cells. The ability to induce embryogenic

cells has been widely used in biotechnology as an efficient tool for plant regeneration and as a model system

for studying plant embryogenesis. Recently, the generation of genomewide profiles of microRNAs

(miRNAs)/small interfering RNAs (siRNAs) and their target genes in several developmental processes have

pointed out several of these molecules as key factors controlling many developmental processes, including SE.

Based on very effective protocols of SE induction in the fruit tree tamarillo (Solanum betaceum Cav.), we have

been working with the objective of evaluating how miRNAs are involved in this system, highlighting the

mechanism of somatic embryo formation and development, to better manipulate the conditions of plant cloning.

High-throughput sequencing (HTS) of small RNAs libraries and functional analysis tools are being used to

validate selected miRNA/target genes and their involvement on SE efficiency. These results will develop new

insights to understand the competency of embryogenesis and regenerability which is very important for the

improvement of several crops.

EVALUATION OF HIGS AND SIGS AS CONTROL STRATEGY FOR GRAPEVINE DOWNY

MILDEW

DANIEL GEBREMICHAEL1, LIJING MA1, LUCA CAPRIOTTI2, SILVIA SABBADINI, BRUNO MEZZETTI2, ELENA

BARALDI1

1University of Bologna, DISTAL (Department of Food and Agricultural Science), Viale Fanin 46, 40127, Bologna, Italy. 2Polytechnic University of Marche, D3A (Department of Agricultural, Food and Environmental Sciences), Ancona, Italy

HIGS; SIGS; dsRNA; Plasmopara viticola; grapevine

Fungal pathogens deliver small RNAs into plant cells to silence host immunity genes, which are mostly produced by

Dicer-like proteins. The host induced gene silencing (HIGS) and spray-induced gene silencing (SIGS) strategies of

disease control can be an efficient tool to protect plants, but more information is needed about the specificity and efficacy

of dsRNAs molecules. HIGS and SIGS targeting fungal Dicer-like genes (necessary for fungal sRNA generation) have

already been effectively applied to control pathogenic fungi during fruit infection.

Downy mildew caused by the Oomycete Plasmopara viticola, is a major pathogen of grapevine and several agrochemical

applications are need to control this disease raising social concern and pressure to develop alternative protection strategies.

In order to assess HIGS and SIGS to control P. viticola we set out to produce dsRNA targeting dicer like genes of this

pathogens. Silencing plasmid constructs with intron-spaced partial sense and antisense DNA sequences, encoding self-

complementary ‘hairpin’ RNA, specific for dicer-like genes of P. viticola, were obtained. Agroinfiltration of Nicotiana

benthmiana leaves with these constructs is performed to produce dsRNA molecules that will then be assessed as new

control means against P. viticola infecting grape leaves. Furthermore, Agrobacterium-mediated genetic tranformation

trials are performed to validate these gene constructs by using meristematic bulks of the grapevine cultivar Thompson

Seedless as starting in vitro explant. Molecular analysis will be performed to confirm the transgenic state of the selected

lines and their resistance against P.viticola will be assessed in vivo.

DIFFERENTIALLY EXPRESSED miRNAs DURING MALE AND FEMALE INFLORESCENCE

BUD DEVELOPMENT IN PISTACHIO

H. TOPCU, L. GUNDUZ, H. KARCI, A.PAIZILA AND S. KAFKAS,

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. Differentially expressed miRNAs were tested

and the results will be presented in the meeting.

THE INFLUENCE OF LEAD AND ACYRTHOSIPHON PISUM ON GENERATION OF SIGNALING

MOLECULES AND EXPRESSION LEVELS OF SELECTED GENES INVOLVED IN MOLECULE

BIOSYNTHESIS

A. WOŹNIAK1, D. NAROŻNA2, W. NOWAK3, I. MORKUNAS1

1Department of Plant Physiology, Poznań University of Life Sciences, Wołyńska 35, 60-637 Poznań, Poland; 2Department of Biochemistry and Biotechnology, Poznań University of Life Sciences, Dojazd 11, 60-632 Poznań, Poland;

Acyrthosiphon pisum, lead, the enzymes involved in molecul biosynthesis; signaling molecules, Pisum sativum

Plants are exposed to a wide range of pollutants found in the environment at varying concentrations and toxicity. In this

study, we focus on the impact of heavy metal at hormetic and toxic doses on plant defense responses and how their

presence in the surrounding environment affects the response of plants to biotic stressors such as aphids playing a definite

role in the trophic chain. Among a variety of herbivores that feed on legumes, the pea aphid Acyrthosiphon pisum Harris

(Homoptera: Aphididae) is of special importance. It is a well-known almost worldwide oligophagous species that infests

leguminous plants and transmits over 30 virus diseases. The primary objective of the study is to identify the regulation

of biosynthesis of signal molecules in plant response to abiotic (heavy metal-lead) and biotic (A. pisum) stress factors.

The first objective was to investigate the effect of lead on the level of generation of signal molecules such as

phytohormones and hydrogen peroxide, and next to determined how cross-interactions of both stress factors, regulated

the level of signaling molecules. In parallel, we determined expression levels of selected genes involved in the

biosynthesis of these molecules by Real-Time qPCR. A significant accumulation of total salicylic acid (SA), abscisic acid

(ABA), jasmonates (JA/MeJA) and hydrogen peroxide (H2O2) was recorded in the roots and leaves of pea seedlings

growing on lead-supplemented medium and next during A. pisum infestation. The results of this study indicate that the

response of pea seedlings to the heavy metal and aphid differed greatly at the hormetic and toxic lead doses and was

dependent on the organ. These results will not only contribute new knowledge to contemporary plant biology but in the

future may be used by breeders of legumes to generate cultivars resistant to the above mentioned stressors.

Acknowledgements:

This work was supported by National Science Centre, Poland grant number 2017/25/N/NZ9/00704

FAST-FORWARD IDENTIFICATION OF HIGHLY EFFECTIVE ARTIFICIAL SMALL RNAS

AGAINST PLANT VIRUSES AND VIROIDS

A.CARBONELL1, P.LISÓN1, C.LÓPEZ2, J.A.DARÒS1

1Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas-Universitat

Politècnica de València, Valencia, Spain 2Instituto de Conservación y Mejora de la Agrodiversidad Valenciana, Universitat Politècnica de València, Valencia,

Spain

amiRNA, syn-tasiRNA, antiviral resistance, TSWV, viroid

Artificial small RNAs (sRNAs), including artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering

RNAs (syn-tasiRNAs), are used to silence viral RNAs and confer antiviral resistance in plants. However, the generation

of resistant lines is still a long, tedious and unpredictable process in which multiple factors such as target site accessibility

or stability (among others) may limit the effectiveness of the transformed artificial sRNA construct. Therefore, there is a

need to develop strategies for identifying effective antiviral sRNA sequences prior to the time-consuming generation of

stably transformed plants.

Here, we describe a systematic methodology for the simple and rapid design, generation and functional analysis of large

numbers of artificial sRNA constructs to induce high levels of antiviral resistance in plants. This methodology uses the

P-SAMS web-tool and B/c vectors for the automated design and one-step cloning, respectively, of highly specific artificial

sRNAs, together with a resistance transient assay in Nicotiana benthamiana. Following this methodology, we have

identified highly effective artificial sRNAs against Potato spindle tuber viroid and Tomato spotted wilt virus (TSWV).

Most effective anti-TSWV sRNAs, particularly syn-tasiRNAs, induced high levels of antiviral resistance when expressed

in transformed tomatoes. All together, these results highlight the usefulness of this high-throughput methodology for the

fast-forward identification of artificial sRNAs with high antiviral activity prior to plant transformation.

IN COLLABORATION WITH: