iPLANTA COST Action CA15223

4th iPLANTA Conference

CONTRIBUTION OF RNAI TO

SUSTAINABLE AGRICULTURE, FOOD

SAFETY AND SECURITY

iPLANTA MC meeting

ATHENS (President Hotel)

FEBRUARY 26-28TH, 2020

Grosshans H, Filipowicz W.Nature. 2008

4th iPLANTA Conference

CONTRIBUTION OF RNAI TO SUSTAINABLE AGRICULTURE, FOOD SAFETY AND

SECURITY

AIMS AND THEMES

The 4rd iPLANTA Conference will focus on RNAi future developments.

The MC will summarize the activities of GP4 and plan the strategy and detailed plant for GP5.

The Wg5 Meeting will build the communication plan, and outline our activities to reach out to all our stakeholder

groups

THE CONFERENCE WILL BE OPEN TO ALL PARTICIPANTS

DAY 1 – WEDNESDAY, FEBRUARY 26TH, 2020

RNA-BASED BIOCONTROL COMPOUNDS: CURRENT STATUS AND PERSPECTIVES

TO REACH THE MARKET

C. N. T. TANING1, S. ARPAIA2, O. CHRISTIAENS1, A. DIETZ-PFEILSTETTER3, H. JONES4, B.

MEZZETTI5, S. SABBADINI5, H. SORTEBERG6, J. SWEET7, V. VENTURA8, G. SMAGGHE1

1Ghent University, Ghent, Belgium; 2Italian National Agency for New Technologies, Energy and Sustainable

Economic Development, Rotondella, Italy; 3Julius Kühn-Institut, Braunschweig, Germany; 4Aberystwyth University,

Wales, UK; 5Università Politecnica delle Marche, Ancona, Italy; 6Norwegian University of Life Sciences, Norway;

7JT Environmental Consultants Ltd, Cambridge, UK; 8Università degli Studi di Milano, Milan, Italy

RNA interference, biocontrol, sprayable, dsRNA, regulatory

Concerns over the impact of chemical pesticides on the environment and health during the past years

have stimulated the search for alternative and environmentally friendly solutions. RNA interference

(RNAi) has been identified as a very promising new approach to complement the arsenal of chemical

and biological pest control agents in a sustainable way. RNA-based active ingredients (AIs) possess

a unique mode of action and can be implemented via both exogenous and genetic modification (GM)

approaches. While the exploitation of RNAi as a tool in agriculture is still limited to a few GM crops,

and only adopted in restricted parts of the world, scientists and industry are already seeking

innovations in leveraging and exploiting the potential of RNAi in the form of sprayable RNA-based

products. RNA-based AIs promise to deliver the selectivity and sustainability desired in future crop

protection agents. This is due to their utilization of a natural process to exert control and their high

level of selectivity, which leads to reduced risk for non-target organisms (NTOs). This study

highlights the expanding research and development pipeline, commercial landscape and regulatory

environment surrounding the pursuit of sprayable RNA-based products with improved environmental

profiles. These RNA-based products are expected to become valuable new tools complementing the

current arsenal of crop-protection solutions.

ROLE OF MIRNA444C AND ITS TARGET GENE MADS27 TRANSCRIPTION FACTOR

IN BARLEY ROOT RESPONSE TO NITROGEN EXCESS STRESS AND GRAIN

PRODUCTION CONTROL.

A. SMOCZYNSKA1, A. GRABOWSKA1, A. PACAK1, A. JARMOLOWSKI1 AND Z. SZWEYKOWSKA–

KULINSKA1

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

Mickiewicz University in Poznan, Poland

Hordeum vulgare, RNA silencing, miRNA444c, MADS27 transcription factor, nitrogen excess stress

Barley is one of the most important crops used in the production of food and in brewing industry.

Cereal grain quality is influenced by many factors, also fertilization.

In the Department of Gene Expression three members of MIR444 family were discovered. This family

is exclusively found in monocots and is characterized by the presence of intron separating miRNA

and miRNA* within each MIR444 gene. Target genes for miRNAs from this family are encoded on

the opposite DNA strand and are MADS-box transcription factors. MADS27 transcription factor is a

target gene for miRNA444c and is expressed mainly in barely roots. In the promotors of MIR444c

and MADS27 genes we identified regulatory motifs controlling nitrogen excess response. Analysis

of the plant response to nitrogen excess stress is particularly important in the context of over-

fertilization in agriculture. Nitrogen that is not utilised by the plants travels into deeper layers of the

soil and is toxic for animals and humans when present in groundwater. Also over-fertilization causes

excessive emissions of ammonia and nitrous oxide into the atmosphere, which significantly

contributes to the greenhouse effect. MiRNA444c is highly induced in nitrogen excess stress in barley

roots while MADS27 is down-regulated. Analysis of mutants with knock down of MADS27

(KDMADS27) and overexpression of MADS27 revealed differences in root system architecture and

anatomy of the roots. Plants overexpressing MADS27 produce longer roots with additional

protoxylem vessels in the root stele which results in more than two fold increase of the grain

productivity of these plants in comparison to WT. Analysis of ABA sensitivity of MADS27 mutants

revealed differences in response to ABA stress and prompted us to analyse connection between

nitrogen and ABA signalling. Presence of high nitrogen concentration in the rhizosphere causes the

activation of β-glucosidase that releases biologically functional ABA. KDMADS27 plants are not

sensitive to ABA while opposite effect was observed for plants overexpressing MADS27. We

identified motifs recognized by MADSbox TF family in promotors of several genes involved in this

pathway.

Our results paint interesting landscape of the role of miRNA444c and its target gene MADS27 in

barley response to nitrogen excess stress and their effect on grain productivity.

VALIDATION OF TWO HAIRPIN-BASED GENE CONSTRUCTS IN VITIS VINIFERA

CULTIVARS TO INDUCE RESISTANCE AGAINST DOWNY MILDEW

CAPRIOTTI L.1, GEBREMICHAEL D.2, BARALDI E.2, MEZZETTI B.1, SABBADINI S.1

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

2University of Bologna, DISTAL (Department of Food and Agricultural Science), Italy.

Vitis vinifera, Host-induced-gene-silencing, downy mildew, genetic transformation

Among the other species in the Vitis genus, Vitis vinifera is the most susceptible to different biotic

stress, like diseases caused by fungi and oomycetes. Plasmopara viticola, the causal agent of downy

mildew, is responsible for severe economic losses for grapevine cultivation during every growing

season, which makes the massive use of fungicide treatments one of the main preventive strategy

applied.

Genes for resistance to many fungal diseases have been partially transferred to V. vinifera cultivars

by traditional breeding techniques but commercial value of interspecific hybrids can be very limited.

As alternative strategy, we are validating two different RNAi-based gene constructs, introduced in

different grapevine cultivars by genetic transformation, in order to hinder the aforementioned

pathogen.

In collaboration with UniBO, two genes codifying for dicer-like proteins of Plasmopara viticola (Pv)

were identified to be targeted by two hairpin gene constructs: i) Pv-DCL441 (hairpin sequence which

targets 258 bp of Pv-DCL 1 gene) and Pv-DCL441+DCL331 (constituted by a RNAi chimeric

sequence silencing both 258 bp of PvDCL 1 and 257 bp of PvDCL 2) under the control of 35S

promoter; both these hairpin constructs express GFP as reporter gene and NPTII as selective marker

gene. The silencing of DCL 1 and/or 2 proteins, known to be involved in the production of sRNAs in

other fungal pathogens (i.e Botrytis cinerea (Wang et al., 2016)), should block and/or reduce

Plasmopara viticola virulence.

To validate both these hairpin gene constructs (i.e Pv-DCL441 and Pv-DCL441+DCL331),

meristematic bulk slices of the cultivar Thompson Seedless (Sabbadini et al., 2019), and somatic

embryos of the Italian cultivar Ancellotta were used as starting explants for Agrobacterium-mediated

genetic transformation trials. The obtained independent transgenic lines of both Thompson Seedless

and Ancellotta cultivars, will be molecularly characterized and evaluated for their resistance against

P. viticola.

DOUBLE-STRANDED RNAS (DSRNAS) AGAINST GREY MOLD (BOTRYTIS CINEREA):

A PROMISING SUSTAINABLE TOOL FOR GRAPEVINE PROTECTION

L. NERVA1,2, M. SANDRINI1,3, G. GAMBINO2, W. CHITARRA1,2

1Research Centre for Viticulture and Enology, Council for Agricultural Research and Economics (CREA-VE), Via

XXVIII Aprile 26, 31015 Conegliano (Italy);

2Institute for Sustainable Plant Protection, National Research Council (IPSP-CNR), Strada delle Cacce 73, 10135 Torino

(Italy);

3Dipartimento di Scienze Agroalimentari, Ambientali e Animali, Università degli Studi di Udine, Via delle Scienze 206,

33100 Udine (Italy)

dsRNA, spray induced gene silencing, plant protection, sustainability, grapevine, Vitis vinifera,

Botrytis cinerea

Grapevine is one of the most important and globally widespread fruit species, with a high impact on

the economy of many countries but with an intense environmental effect. Therefore, new

environmental-friendly defence strategies against fungal pathogens are needed for a more sustainable

agriculture. A novel emerging approach is the spray-induced gene silencing (SIGS), which concern

in the exogenous application of double-stranded RNA (dsRNA) inducing an enhanced plant

resistance against fungal pathogens. We decide to test the ability of SIGS to prevent and counteract

infection of Botrytis cinerea, one of the most economic impacting pathogens of grapevine. In

particular, we tested three independent approaches for dsRNA delivery into plants: i) high pressure

spraying of leaves; ii) petiole adsorption of dsRNAss; iii) post-harvest spraying of bunches. We

demonstrated that, independently form the method of application, SIGS is able to reduce virulence of

the fungus. Moreover, we also observed three different level of efficacy depending on the method of

application. Thus, the present data provide crucial information on the possibility to exploit SIGS as

an alternative sustainable and eco-friendly strategy for grapevine pre- and post-harvest protection.

BIOLOGICAL FUNCTION OF PUTATIVE PROTEINS OR LNCRNAS ENCODED BY PRI-

MIRNAS DERIVED FROM MIR444 FAMILY GENES (HORDEUM VULGARE)

A. GRABOWSKA1, A. SMOCZYŃSKA1, A. JARMOŁOWSKI1 AND Z. SZWEYKOWSKA-KULIŃSKA1

1Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam

Mickiewicz University in Poznań, Poland

microRNA, pri-miRNA, ORF derived from pri-miRNA, barley, abiotic stresses

MicroRNAs from MIR444 family are present exclusively in monocots. The characteristic feature of

these genes is the presence of an intron that separates two halves of microRNA precursor stem and

loop structure. We analysed promoters of all three genes and in all of them we identified regulatory

elements for nitrogen, copper and light stress. Based on small RNA sequencing and microRNA

sequence homology we deduced there are three MIR444 genes in barley genome. RACE as well as

RT-PCR experiments proved that all three pri-miRNAs undergo alternative splicing generating

multiple pri-miRNA444 isoforms that can be divided in two classes: functional and non-functional.

Bioinformatics analysis revealed the presence of short open reading frames (ORFs) in all three pri-

miRNAs444. Previous studies have shown that plant plant pri-microRNAs encode peptides that

stimulate the biogenesis of their corresponding microRNAs [Lauressergues et al., 2015]. To verify

whether pri-miRNAs from the MIR444 family can be translated we carried out polysome profiling.

We demonstrated that some isoforms of pri-miRNAs444 are associated with ribosomes in root under

conditions of nitrogen excess and consequently, they may be translated. We confirmed the synthesis

of the peptide of 168 amino acids encoded by the longest ORF in Escherichia coli. Nitrogen is a major

essential element for plant growth and development which significantly increases and enhances the

yield and grain quality by playing a crucial role in biochemical and physiological functions of plant.

Further experiments are required to reveal the function of proteins encoded by MIR444 genes in

barley which are involved in the nitrogen excess response in barley.

RNAi FOR ENHANCING ESSENTIAL OIL FEEDSTOCKS AND HIGH-VALUE

PRODUCTS FROM MENTHA SPECIES

LORENZ FUCHS, MORGAN JENKINS, JOHN PICKETT AND SIMON SCOFIELD

Cardiff University, Wales.

Menthol, Metabolic Engineering, Mentha, Monoterpenes, Uganda

Mints are perennial herbs cultivated for their aromatic oils, which have uses in the food flavour,

cosmetic and pharmaceutical industries. This GCRF-funded project seeks to develop novel mint

varieties to maximise the production and usage of the essential oil compound menthol. It involves

partnerships between Cardiff University and Makerere University that will enable local rural

Ugandan farming communities to cultivate and extract mint essential oils to develop new products

for commercial exploitation and the benefit of the local communities through sustainable

enhancement of their agricultural production. The monoterpenoid essential oil biosynthetic pathway

in peppermint has been well characterised, facilitating metabolic engineering strategies to increase

essential oil yield and improve composition. Increasing plastidic carbon flow into the monoterpenoid

biosynthetic pathway through overexpression of key genes has led to increased essential oil yields.

Furthermore, the co-expression of multiple biosynthetic enzymes has had a strong additive effect

resulting in increased monoterpenoid oil accumulation in these transgenic lines. RNAi technology

has led to a reduced level of the unwanted side product menthofuran through antisense-mediated

silencing of menthofuran synthase gene expression. The first stage of the project is to overexpress the

geranyl diphosphate synthase and limonene synthase genes in peppermint to direct carbon flow into

the monoterpenoid pathway toward menthol production. The second stage is to employ an improved

RNAi approach that uses an intron-containing hairpin RNA to target two genes, menthofuran

synthase, a cytochrome p450 enzyme which catalyses the conversion of pulegone to menthofuran and

YABBY5, a transcription factor is a repressor of secondary metabolism and whose suppression in

spearmint resulted in an increased level of terpene accumulation. These four genes will be targeted

both individually and in combination in natural mint varieties with high levels of menthol that have

been found to grow well in Uganda, facilitating the commercialisation of Mentha spp. for the benefit

local Ugandan communities.

GMO-FREE RNAi IN PLANTS.

ATHANASIOS DALAKOURAS1,2, MICHAEL WASSENEGGER3, ELENA DADAMI1, IOANNIS

GANOPOULOS2, MARIA PAPPAS4 AND KALLIOPE PAPADOPOULOU1

1University of Thessaly, Department of Biochemistry & Biotechnology, Larissa, Greece

2Institute of Plant Breeding and Genetic Resources ELGO-DEMETER, Thessaloniki, Greece

3RLP AgroScience GmbH, AlPlanta Institute for Plant Research, Neustadt, Germany

4Democritus University of Thrace, Department of Agricultural Development, Orestiada, Greece

RNAi, siRNAs, dsRNAs, high pressure spraying, trunk injection, petiole uptake.

Since its discovery more than 20 years ago, RNA interference (RNAi) has been extensively used in

crop improvement and protection platforms. So far, RNAi approaches have been conventionally

based on the use of transgenic plants expressing double stranded RNAs (dsRNAs) against selected

targets. However, the use of transgenes and genetically modified organisms (GMOs) has raised

considerable scientific and public concerns. Hence emerged the need for alternative approaches that

avoid the use of transgenes and resort instead to direct exogenous application of RNA molecules that

have the potential to trigger RNAi. Herewith, we describe methodologies based on high pressure

spraying, petiole uptake and trunk injection for exogenous delivery of RNA molecules with the

capacity to trigger RNAi in both herbaceous and woody plants (Dalakouras et al., 2020). Importantly,

depending on the method of application, the RNA molecules are delivered in the symplast or the

apoplast, greatly influencing the efficiency of RNAi.

References

Dalakouras A, Wassenegger M, Dadami E, Ganopoulos I, Pappas M, Papadopoulou KK (2020). Genetically Modified

Organism-Free RNA Interference: Exogenous Application of RNA Molecules in Plants. Plant Physiol 182(1): 38-50.

TERMINATOR-LESS TRANSGENE CONSTRUCTS TRIGGER RNAI PATHWAY

M.A.AKBUDAK1, V. SRIVASTAVA2

1 Department of Agricultural Biotechnology, Akdeniz University, Antalya 07059, Turkey

2Department of Crop, Soil & Environmental Science, 115 Plant Science Building, University of Arkansas, Fayetteville

72701, AR, USA

Arabidopsis, Rice, Down-regulation, RNAi pathway, Terminator-less construct

Transgene-mediated gene silencing is an important biotechnological and research tool. There are

several RNAi-mediated techniques available for silencing genes in plants. The basis of all these

techniques is to generate double-stranded RNA precursors in the cell, which are recognized by the

cellular surveillance system, and marked for degradation by the Dicer family RNases into siRNAs.

Improperly terminated, unpolyadenylated RNA are precursors of double-stranded RNA, and,

therefore, can serve as silencing triggers in plants. Such transcripts can easily be synthesized from

transgene constructs lacking transcription-terminator signals (terminator-less constructs).

Our studies in Arabidopsis determined the silencing efficiency of terminator-less constructs on six

different genes: Phytochrome A (PHYA), Brassinosteroid Insensitive 1 (BRI1), Variegated 2

(VAR2), Constans (CO), Apetala 1 (AP1) and Transparent Testa Glabra 1 (TTG1). Expression of

terminator-less gene fragments of PHYA, AP1, and VAR2 resulted in a ~90 % decline, and those of

BRI1 and CO resulted in a ~70 % decline, in the steady state level of the respective transcript in

transgenic lines compared to the wild-type.

Our studies in rice, indicated at least 36-87% transcript reduction in HCT lines, 75-94% in CCR lines,

and 14-85% in CAD lines. Of the nine down-regulated lines (three lines from each genes) analyzed

for lignin, total lignin content was significantly reduced in seven lines (HCT-4, HCT-7, CAD-1,

CAD-7, CCR-3, CCR-7, and CCR-12) with lignin reduction ranged from 4.6% to 10.8%. The results

from our studies indicated that the simple binary vector without termination sequence can be used for

down-regulation of genes in plants.

TOWARDS THE MANAGEMENT OF VECTORS OF PLANT PATHOGENS USING RNA

INTERFERENCE

MURAD GHANIM, SAPTARSHI GHOSH, SVETLANA KONTSEDALOV, GALINA LEBEDEV, OLA

JASSAR, , POULAMI SARKAR, SURAPATHRUDU KANAKALA

Department of Entomology, Volcani Center, Rishon LeZion, Israel

whitefly, psyllid, virus, bacteria, transmission

Many plant pathogens such as viruses and bacteria are vector-borne and depend on arthropod vectors

for their transmission. Examples of such pathogens include Tomato yellow leaf curl virus (TYLCV)

which is transmitted by the whitefly Bemisia tabaci in a persistent-circulative manner, and bacteria

of the genus Liberibacter which are transmitted by psyllid vectors. The most important of this group

is Candidatus Liberibacter asiaticus, which in recent years is devastating the citrus industry by

causing the citrus greening disease and transmitted by the citrus psyllid Diaphorina citri. The closely-

related bacterium Candidatus Liberibacter solanacearum, which is associated with vegetative

disorders in carrots and the zebra chips disease in potatoes, is transmitted by other psyllid species

including Bactericera trigonica in carrots and B. ckockerelli in potatoes. While chemical sprays are

currently the prevailing method for managing these diseases by limiting psyllid populations; they are

harmful and limited in their effectiveness. RNA interference has been proposed as an alternative and

an environment-freindly method for pest control or the prevention of disease transmission by

interferring with the pest development or with the pathogen transmission. Research in our lab has

identified several proteins in the whitefly-TYLCV system (Heat shock Protein 70, Cyclophilin and

others) and the psyllid-Liberibacter system (Endoplasmic Reticulum Associated Degradation ERAD

proteins). When the genes coding for those proteins were targeted by RNAi using delivery methods

(artificial feeding or expression in plants), the insect development or the transmission of the respective

pathogen were significantly affected. Examples from those pathosystems will be presented and the

results and their implications will be discussed.

RNA-NANOVECTORS AS A TOOL TO STUDY GENE FUNCTION AND MODIFY THE

EXPRESSION OF TARGET GENES IN TOMATO FLOWER BUDS

PENNISI F, CRESSONI C, SPEGHINI A, PANDOLFINI T, MOLESINI B

University of Verona, Department of Biotechnology, Strada le Grazie 15, 37134-Verona, Italy

Exogenous dsRNA, nanovectors, flower buds, fruit set, RNA silencing

RNA silencing is a molecular mechanism activated by double-stranded RNA (dsRNA) which results

in the degradation of target transcripts. RNA silencing of target genes can be obtained by exogenous

application of dsRNAs. In plants, coupling dsRNAs to layered double hydroxides clay nanosheets

(nanoLDHs) has been proved to favour RNA delivery and long-term silencing effects. The aim of our

research activity was the development of a system for the delivery of dsRNA-LDHs in very young

tomato ovaries to trigger the silencing of target genes. We used as molecular model the parthenocarpy

in tomato, consisting in the development of fruit in the absence of fertilization. It has been

demonstrated in stably transformed tomato plants that the downregulation by RNA silencing of ovary

growth repressors, activates parthenocarpic fruit set (Wang et al., 2005; Molesini et al., 2009).

Differently from wild-type flower buds, which are unable to grow in vitro if collected before

fertilization, unfertilised flower buds from parthenocarpic plants are able to produce fruits (Pandolfini

et al., 2009). We used exogenous dsRNA targeting SlIAA9, which codes for a negative regulator of

fruit set and growth (Wang et al., 2005), to obtain fruit growth from in vitro cultivated wild-type

flower buds. To increase the stability and delivery of SlIAA9-dsRNAs, we loaded in vitro synthesised

dsRNAs to nanoLDHs after determining the optimal dsRNA–LDH mass ratio. SlIAA9-

dsRNAs/nanoLDHs were applied to the cut pedicel of emasculated flower buds; as negative control

we used nanoLDHs alone. To facilitate the entry of the effector molecules, the tissue has been

subjected to air stress in a laminar airflow hood. Ovary/fruit weight has been measured 3 weeks after

SlIAA9-dsRNAs/nanoLDHs application. Differently from flower buds treated with nanoLDHs alone,

that senesced after few days or remained in a static condition, many of the flower buds treated with

SlIAA9-dsRNAs/nanoLDHs displayed ovary growth. To evaluate the silencing of SlIAA9 gene,

reverse-transcription quantitative PCR has been performed. Although the method will require further

technical optimizations and additional analyses on dsRNA distribution within these tissues, our

preliminary data suggest that this in vitro system could represent a very fast strategy to screen new

candidate genes playing a role in fruit set and growth.

MODIFIED RNAI CONSTRUCTS CARRYING THE GENE TO BE SILENCED FLANKED

BY TWO 35S-PROMOTERS

MATTHIAS FLADUNG

Thünen-Institute of Forest Genetics, D-22927 Grosshansdorf, Sieker Landstr. 2, Germany

RNA interference, promoter, spacer

RNAi involves the silencing of a target gene by introduction of dsRNA corresponding to the

sequences within the target gene to be silenced. To make dsRNA, one needs to transcribe both sense

and antisense strands of RNA from a cDNA, separated by a spacer, and allow them to anneal. By

using the GUS reporter gene was as a test system, functioning of a set of such RNAi vectors in poplar

was confirmed by Meyer et al. (2004).

A novel RNAi approach without spacer but with two promoters flanking the gene to be silenced has

been proposed by DNA Cloning Service (Hamburg, Germany; http://www.dna-cloning.com). The

advantage of this approach is that no cloning of sense AND antisense sequences of the gene to be

silenced is needed. The approach was first tested in transgenic poplar constitutively expressing the

GUS gene under the cauliflower mosaic virus 35S promoter.

A modified RNAi construct carrying the GUS gene flanked by two 35S-promoters was transferred to

stably GUS-expressing poplar. Silencing of the GUS gene was validated in GUS-staining

experiments of chlorophyll-less leaf discs harvested from different independent GUS:RNAi-

35::GUS-transgenic poplar lines. The GUS-stains ranged from slightly decreased blue to nearly

complete white leaf discs

A similar construct system was used for RNAi of the Chl a/b Light harvesting complex (LHC). As

result, green, pale-green, and white poplar regenerants were obtained.

HIGHLY EFFICACIOUS ANTIVIRAL PROTECTION OF PLANTS BY SMALL

INTERFERING RNAS IDENTIFIED IN VITRO

SELMA GAGO-ZACHERT1,3, JANA SCHUCK1, CLAUS WEINHOLDT2, MARIE KNOBLICH1,

VITANTONIO PANTALEO4, IVO GROSSE2, TORSTEN GURSINSKY1 AND SVEN-ERIK BEHRENS1

1Institute of Biotechnology,

2Institute of Computer Science, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany.

3Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Halle/Saale, Germany.

4Institute for Sustainable Plant Protection-Consiglio Nazionale delle Ricerche, Research Unit of Bari, Italy.

RNAi, plant protection, siRNA, Argonaute, DCL

In response to a viral infection, the plant’s RNA silencing machinery processes viral RNAs into a

huge number of small interfering RNAs (siRNAs). However, very few of these siRNAs actually

interfere with viral replication. A reliable approach to identify these immunologically effective

siRNAs (esiRNAs) and to define the characteristics underlying their activity has not been available

so far. We developed a novel screening approach that enables a rapid functional identification of

antiviral esiRNAs. Tests on the efficacy of such identified esiRNAs of a model virus achieved a

virtual full protection of plants against a massive subsequent infection in transient applications. We

find that the functionality of esiRNAs depends crucially on two properties, the binding affinity to

Argonaute proteins and the ability to access the target RNA. The ability to rapidly identify functional

esiRNAs is expected to be of great benefit for all RNA silencing-based plant protection measures,

against viruses as well as against fungi, nematodes or other pests.

https://doi.org/10.1093/nar/gkz678 https://www.sciencemag.org/news/2019/08/new-medicine-could-vaccinate-plants-

against-devastating-viruses)

PLANT mRNA ADENOSINE METHYLASE (MTA) DEPOSITS m6AON pri-miRNAs TO

MODULATE MICROPROCESSOR ASSEMBLY AND miRNA BIOGENESIS

SS BHAT1, D BIELEWICZ1, T GULANICZ1,2, Z BODI3, X Yu4, L SZEWC1, M BAJCZYK1, J DOLATA1,

N GRZELAK1, DJ SMOLINSKI2, BD GREGORY4, RG FRAY3, A JARMOLOWSKI1, Z

SZWEYKOWSKA-KULINSKA1

1 Department of Gene Expression, Faculty of Biology, Adam Mickiewicz University, Poznan 61-614, Poland;2 Centre

For Modern Interdisciplinary Technologies, Nicolaus Copernicus University, Torun 87-100, Poland.3 School of

Biosciences, Plant Science Division, University of Nottingham, Sutton Bonington, Loughborough LE12 5RD, UK;4

Department of Biology, University of Pennsylvania, 433 South University Avenue, Philadelphia, PA 19104, USA;

microRNAs, biogenesis, microprocessor, plant development

N6-Methyladenosine (m6A), one of the most abundant mRNA modifications in eukaryotes, has been

associated with various metabolic processes in plants. We provide evidence showing that m6A plays

a role in miRNA biogenesis in plants. We show that plants with globally reduced m6A level are not

able to produce mature miRNAs up to the levels of WT plants, while pri-miRNAs are accumulated

in such hypomorphic mta mutant plants. Direct interactions between MTA and pri-miRNAs, along

with the presence of m6A mark in pri-miRNAs provide proof that pri-miRNAs are novel substrates

for m6A methylation catalyzed by MTA. PIP-Seq analysis revealed a significant decrease in

prevalence of secondary structures within stem-loop region of miRNA precursors in mta plants,

which negatively impacts miRNA biogenesis. The in vivo interaction between MTA and RNA Pol II

indicates that m6A methylation in plants is a co-transcriptional process. In addition, the Tough protein

(TGH), a known miRNA biogenesis related protein, directly interacts with MTA. Interestingly, both

MTA and TGH are necessary for efficient co-localization of DCL1 and HYL1 with RNA Pol II.

Hence, we propose that changes in secondary structure of miRNA precursors and the resulting

inefficient recruitment of Microprocessor components to pri-miRNAs, result in the inefficient

processing of pri-miRNAs and decreased miRNA production in mta plants. Lastly, the low m6A

methylation level causes strong inhibition of auxin signaling in hypomorphic mta mutant plants. We

show that the reduced level of miRNA393b, caused by the absence of MTA/m6A, is one of the factors

responsible for this inhibition.

EFFICIENT IN PLANTA AND SPRAYABLE DELIVERY OF dsRNAS AGAINST

COLORADO POTATO BEETLE, LEPTINOTARSA DECEMLINEATA SAY

(COLEOPTERA: CHRYSOMELIDAE)

ALLAH BAKHSH AND EMRE AKSOY

Department of Agricultural Genetic Engineering, Faculty of Agricultural Genetic Engineering, Nigde Omer Halisdemir

University, Nigde, Turkey , Email: abthebest@gmail.com

Potato, In planta RNAi, Ecdysone receptor, sprayable, insect pest management

Colorado potato beetle (CPB) is devastating insect pest of potato all over the world and has developed

resistant against more than 50 active compounds belonging to various chemical groups. The increased

pest pressure and resistantance to chemical insecticides provide impetus for the development of

alternate management strategies. In addition to its applications in other biological fields, RNAi has

also proved its usefulness in functional genomic research on insects and proved itself a potential

strategy in crop improvement for the control of insect pests. We have carried out two independent

research projects where using in planta and sprayable dsRNA delivery, we were able to induce

mortality in Colorado potato beetle. In plant based RNAi approach, we knocked down insect molting

associated Ecdysone receptor gene successfully. Transgenic potato lines showed enhanced resistant

against CPB with reduced EcR transcipts levels in insect feeding on transgenic lines. Whearase in

other project, the transcript levels of imidacloprid resistant genes (Cuticular protein, Cytochrome

P450 monoxygenases and Glutathione synthetase) were downregulated in CPB under laboratory

conditions. The bio-assays conducted on various stages of imidacloprid resistant CPB lab population

by applying HT115 expressing dsRNA on potato leaflets exhibited upto 50% mortality rates. More

interesting, synergism of RNAi with imidacloprid conducted on the 2nd instar larvae, exhibited 100%

mortality of larvae reduced doses of dsRNAs along with imidacloprid were used.

HIGS AND SIGS APPROACHES TO INDUCE RESISTANCE AGAINST BOTRYTIS

CINEREA IN STRAWBERRY (FRAGRARIA X ANANASSA) AND GRAPEVINE

Capriotti L.1, Sabbadini S.1, Jin H.2, Mezzetti B.1

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

2Department of Microbiology and Plant Pathology, Center for Plant Cell Biology, Institute for Integrative Genome

Biology, University of California, Riverside, California, United States of America

RNAi, HIGS, SIGS, Botrytis cinerea, genetic transformation

Grey mold disease, caused by the ascomycete Botrytis cinerea, is one of the most economically

impactful pathogen for several fruit tree crops worldwide, causing severe losses on both cultivated

plants and on harvested fruits. Recently a new biotechnological approach called Spray-induced gene

silencing (SIGS), based on RNAi mechanism, resulted to be effective in containing this disease on

leaves and fruits of different plant species (Wang et al., 2016). The resistant effect observed in these

studies has been obtained by the exogenous application of dsRNA molecules targeting dicer like 1

(DCL 1) and 2 (DCL2) genes of B. cinerea. We are validating these RNAi molecules on strawberry

(Fragraria x ananassa) and grapevine cultivars (Vitis vinifera) through laboratory trials (on different

detached organs), and by in Planta tests on potted plants in the greenhouse. In parallel, genetic

transformation trials have been carried out on both strawberry (Fragraria x ananassa) cv. Sveva, and

Vitis vinifera cv. Thompson seedless explants, to attempt the stable expression of the hairpin-based

gene construct targeting B.cinerea DCL1/2 genes (Host-induced gene silencing (HIGS)). The

obtained transgenic lines will be molecularly characterized and evaluated for their resistance against

B.cinerea infection in comparison with the optimized SIGS method.

ENHANCED ANTIVIRAL RESISTANCE IN PLANTS THROUGH THE MULTI-

TARGETING OF VIRAL RNAs WITH HIGHLY EFFECTIVE ARTIFICIAL sRNAs

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

Artificial small RNAs (art-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 highly 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 art-sRNA construct. Therefore, there is a need

to develop strategies for the identification of highly effective antiviral art-sRNAs 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 art-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 art-sRNAs, together with a resistance

transient assay in Nicotiana benthamiana. Following this methodology, we have identified highly

effective art-sRNAs against Potato spindle tuber viroid (PSTVd) and Tomato spotted wilt virus

(TSWV). Moreover, most effective anti-TSWV sRNAs induced high levels of antiviral resistance

when expressed in tomato plants from a single syn-tasiRNA construct. Our results highlight the

usefulness of this high-throughput methodology for the fast-forward identification of art-sRNAs with

high antiviral activity prior to plant transformation. They also suggest that syn-tasiRNAs induce

enhanced antiviral resistance because of the combined silencing effect of each individual art-sRNA,

which minimizes the possibility that the virus simultaneously mutates all different target sites to fully

escape each art-sRNA species expressed from the syn-tasiRNA precursor.

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

DERIVED SMALL INTERFERING RNAs TO INDUCE SHARKA RESISTANCE IN NON-

TRANSGENIC FRUIT TREES

NURIA ALBURQUERQUE, LYDIA FAIZE, LORENZO BURGOS

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

30.100 Murcia (Spain)

Grafting, siRNA, Resistance, Plum Pox Virus

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. Unfortunately the high viral inoculum compromised

viability of graftings and only a low number of plants could be evaluated. As a resume 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. We decided to try a more realistic approach and

healthy buds were used for grafting transgenic plums. After artificial winter in cold chamber and

sprouting of buds in the greenhouse, plants were infected by chip budding and transferred again to

the cold chamber. Again in the greenhouse after the artificial winter they were evaluated for virus

presence by symptoms, ELISA and confirmed by RT-PCR. Virus was transmitted to all scions grafted

onto the susceptible St5’-7 rootstocks with only one exception out of 18 graftings. ELISA averaged

2.11 in a scale of 0 to 3. In the intermediate St5’-6 line the virus was not found in 12 out of 38 infected

scions and the average value for ELISA was 0.87. Both resistant lines St5’-1 and St5’-9 had 9/36 and

9/23 plants without virus and average ELISA values of 1.17 and 0.78, respectively. Plants are again

in the cold chamber and in spring will be evaluated again for the virus presence. Since it is a silencing

mechanism it may work on the long term, eliminating viral particles from the apricot grafted onto

resistant plums rootstocks. Therefore, at least an additional evaluation is necessary.

NOVEL miRNAs REGULATES FLORAL ORGAN DEVELOPMENT BY SUPPRESSING

EXPRESSION OF A MADS-BOX GENE IN PISTACHIO

S. KAFKAS, H. TOPCU, H. KARCI, A. PAIZILA, E.KAFKAS, L. GUNDUZ AND O.F. BILGIN

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

Pistachio, sex expression, dioecious, miRNA

The pistachio (Pistacia vera L.) belongs to Anacardiacdeae family and it is only cultivated and

commercially important species for its nuts. 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 scanning electron microscopy study revealed two stages of organ development in

staminate and pistillate inflorescence buds: the first one is from early spring to dormant stage and the

second one is from late winter till inflorescence bud burst time in the next spring. The buds were

sampled weekly or 15 days intervals with two replicates from three female and three male cultivars

and RNA extractions were performed. Equal amount of RNAs from each sampling date from three

cultivars were bulked for each sex and for two different stages with two replicates. RNA and small

RNA sequencing was performed in a total of 8 bulked samples. Differentially expressed genes and

miRNAs between female and male were determined. There were 3,123 up-regulated and 2,411 down-

regulated DEGs, and 88 differentially expressed miRNAs between female and male samples. Of

them, 24 were known miRNAs and 64 were novel miRNAs, and 42 were down-regulated and 46

were up-regulated miRNAs. Seventy miRNAs targeted a total of 758 genes in cv. Siirt genome. Two

novel miRNAs targeted a same MADS-box gene by suppressing its expression which was confirmed

by RNAseq and qPCR analysis.

STUDYING TOMATO WRKYS IMPLICATION IN REGULATING ROOT KNOT

NEMATODE DISEASE USING RNAI INTERFERENCE

BRAUN MIYARA SIGAL, CHINNAPANDI BHARATHIRAJA, PATRICIA BUCKI AND NATALIA

FOITOUSSI

Department of Entomology, Volcani Center, Rishon LeZion, Israel

Root knot nematodes, WRKY transcription factors, plant immunity

Disease caused by plant parasitic nematodes in vegetables leading to extensive yield decline, among

them Meloidogyne spp., commonly known as a root-knot nematode (RKN), are widely distributed

and are developing into a major problem for agriculture. Thus molecular understanding the

mechanisms underlying plant innate resistance is a choice method for developing safe alternative to

harmful chemical nematicides to control RKN. Prospecting for genes involved in transcriptional

regulation of plant defences against RKN disease exposed a tight implication of WRKYs transcription

factor in RKN parasitism on tomato roots. Herein we investigate the function role of tomato WRKYs

transcription factors SlWRK3 and SlWRKY35 in regulating nematode disease development. By using

promoter-GUS reporter gene fusions, we show that both genes WRKY3 and WRKY35 are induced

within 5 days upon infection and continued through feeding-site development and gall maturation

with much stronger response of WRKY3 compared to WRKY35 to nematode infection. Histological

analysis of nematode feeding sites indicated that WRKY3 was highly expressed within developing

and mature feeding cells and associated vasculature cells while WRKY35 expression was observed

only within mature feeding sites. Responses of SlWRKY3 and SlWRKY35 promoters to several

phytohormones showed that both induced by the defence-signaling molecule salicylic acid salicylic

acid (SA) and Indole-3-butyric acid (IBA) while application of MeJA lead to WRKY3 and WRKY35

repression. Overexpressing SlWRKY3 resulted in lower infection of the RKN M. javanica while

knocking down SlWRKY3 resulted in increased infection. Hormone and oxylipins profile conducted

by LC-MS analysis have shown that enhanced resistance observed in tomato roots overexpressing

SlWRKY3 is coupled with increased accumulation of defence molecules parts of the shikimate and

oxylipins pathways. In conclusion our results pinpoint, SlWRKY3 as a positively regulator operating

the induced systemic resistance against nematodes with a major impact during the early stages of

nematode infection.

THE INFLUENCE OF LEAD AND ACYRTHOSIPHON PISUM (HARRIS) ON

EXPRESSION OF GENES ENCODING ENZYMES INVOLVED IN THE BIOSYNTHESIS

OF PHYTOHORMONES

A. WOŹNIAK1, W. NOWAK2, D. NAROŻNA3, JACEK KĘSY4, I. MORKUNAS1

1 Department of Plant Physiology, Poznań University of Life Sciences, Wołyńska 35, 60-637 Poznań, Poland;

2 Laboratory of Molecular Biology Techniques, Faculty of Biology, Adam Mickiewicz University in Poznań,

Umultowska 89, Poznań 60-614, Poland,

3 Department of Biochemistry and Biotechnology, Poznań University of Life Sciences, Dojazd 11, 60-632 Poznań,

Poland;

4 Chair of Plant Physiology and Biotechnology, Nicolaus Copernicus University, Gagarina 9, 87-100 Toruń

Acyrthosiphon pisum, lead, enzymes of the biosynthesis of phytohormones, Pisum sativum

Under natural conditions we may frequently observe the impact of multiple stressors occurring

simultaneously or sequentially. The main aim of this study was to understand the regulation of

biosynthesis of phytohormones - signalling molecules in the defence mechanism of a plant during the

interaction of an abiotic and biotic factor. It was important to recognize this regulation at the

molecular level in Pisum sativum L. under the influence of various concentrations of lead, low

inducing metabolic status of the plant, causing hormetic effect and high causing toxic effect, and

during feeding of the phytophagus insect with piercing-sucking mouthpart, i.e. pea aphid

[Acyrthosiphon pisum (Harris)]. The aim of the study was to determine expression of genes encoding

enzymes of the biosynthesis of signalling molecules such as phytohormones, i.e. jasmonates

(JA/MeJA), abscisic acid (ABA) and ethylene (ET). Real-Time qPCR was applied to analyse the

expression of genes coding enzymes involved in the regulation of biosynthesis of jasmonates

[lipoxygenase (LOX2); reductase of 12-oxo-phyto-dienoic acid (OPR3); allene oxide synthase (AOS)

and synthase of the jasmonic acid - isoleucin conjugate (JAR), ET [ACC synthase - synthase of 1-

aminocyclopropane-1-carboxylic acid (ACS); ACC oxidase (ACO); ABA (9-cis-epoxycarotenoid

dioxygenase (NCED); aldehyde oxidase 3 (AO3)]. In response to the above stress factors, i.e. abiotic

and biotic stressors acting independently or simultaneously, the level of expression genes encoding

enzymes of the biosynthesis of phytohormones increased. In parallel, an increase in abscisic acid

(ABA) and jasmonates (JA and MeJA) have been found. The results of this study indicate that the

response of pea seedlings to the heavy metal-lead and A. pisum aphid differed greatly at low and

sublethal Pb concentration. The intensity of these stress responses depended on the organ, the metal

dose and direct contact of the stress factor with the organ.

Acknowledgements:

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

ANALYSIS OF dsRNAs TARGETING DICER-LIKE AND CELLULOSE SYNTHASE

GENES OF GRAPEVINE-DOWNY MILDEW (Plasmopara viticola)

MEHARI Z.1, GEBREMICHAEL D.1, CAPRIOTTI L.2, SABBADINI S.2, MA L.1, MEZZETTI B.2,

BARALDI E.1

1Laboratory of Biotechnology and Plant Pathology, University of Bologna, Bologna, Italy

2Dipartimento di Scienze Agrarie, Alimentari ed Ambientali, Università Politecnica delle Marche, Ancona, Italy

dsRNA, Dicer-like, Cellulose synthase, Plasmopara viticola, grapevine

Downy mildew caused by Plasmopara viticola, an Oomycota, is one of the major challenges in grape

production. The pathogen affects all green parts of the grapevine. Severe infection around flowering

can lead to high crop loss; heavily infected leaves can fall prematurely, reducing yields and exposing

bunches to sunburn. Both protective and systemic/penetrant fungicides are widely used for the control

of downy mildew, which raises consumer health and environmental pollution issue. In search of

alternatives to P. viticola management options, the effect of small RNAs targeting Dicer-like proteins

(DCL) and cellulose synthesis pathway on disease causing ability of the pathogen are being studied.

Silencing amplicons of 258 bp nucleotides from DCL-1 (named DCL441) and 257 bp nucleotides

from DCL-2 (named DCL331) were selected. The silencing constructs of DCL441 and chimera

(DCL441+DCL331) were produced via the Gateway™ system, using PK7GWIWG2-7F2,1 and

PK7GWIWG2_II-RedRoot vectors in DH5α Escherichia coli strain. Mirvana miRNA isolation kit

was used to extract dsRNA produced via Agroinfiltration-based transient expression in Nicotiana

benthamiana. Young fully expanded grapevine leaves were inoculated with 5µl droplets of P. viticola

sporangia (1*105 sporangia/ ml), along with a topical application of 5µl droplets of DCL441,

Chimera and ccdb (negative control) dsRNA, at different concentrations (2000 ng/ µl, 200 ng/ µl, 20

ng/ µl, and 5 ng/ µl). Disease score taken at 1 week post inoculation (wpi) and 2 wpi highlighted that

the dsRNA of DCL441 and Chimera could impair the development of P. viticola, as compared to

ccdb. No much difference among the different concentrations of DCL441 and Chimera dsRNAs was

observed. To further validate the preliminary result obtained, dsRNA production using Litmus 28i

plasmid in HT115 strain of E. coli is being optimized. Activity targeting the four cellulose synthase

genes of P. viticola, for RNAi, is ongoing.

smallRNA PROFILING AND DIFFERENTIAL EXPRESSION ANALYSIS IN SOLANUM

BETACEUM EMBRYOGENIC, NON-EMBRYOGENIC AND LONG-TERM

ESTABLISHED CALLUS CULTURES

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

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

2Institut Jean-Pierre Bourgin, INRA Centre de Versailles-Grignon, 78026 Versailles Cedex France

post-transcriptional regulation, small RNAs, somatic embryogenesis

Somatic embryogenesis (SE) is a process widely used in biotechnology as a powerful way of

regeneration, germplasm conservation, breeding for genetic engineering and artificial seed production

and 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. In this study, three callus lines, including embryogenic (EC),

non-embryogenic (NEC) and long-term established (LTC) callus, induced from leaf explants, were

prepared for high-throughput sequencing (HTS) of small RNAs libraries and functional analysis of

differentially expressed miRNAs families. The results showed higher similarities between EC and

LCT libraries, although specific miRNAs/siRNAs were differentially expressed between them. NEC

samples abundantly expressed miRNAs/siRNAs that target genes involved in plant growth,

development and maturation of the plant, such as miR393 and miR396. These results provide new

clues for future investigation of the mechanisms that control SE.

DAY 2 – THURSDAY FEBRUARY 27TH, 2020

BIOSAFETY OF GM PLANTS EXPRESSING RNAi: DATA REQUIREMENTS AND EU

REGULATORY CONSIDERATIONS

S. ARPAIA1, O. CHRISTIAENS2, K. GIDDINGS3, H. JONES4, B. MEZZETTI5, F. MORONTA-BARRIOS6,

J.N. PERRY7, J.B. SWEET8, C.N. TIZI TANING2, G. SMAGGHE2, A. DIETZ-PFEILSTETTER9

1ENEA, Italy,

2Ghent University, Belgium,

3Bayer U.S. Crop Science,

4Aberystwyth University, UK,

5Università Politecnica delle Marche, Italy,

6ICGEB, Trieste, Italy,

7Former chair EFSA GMO Panel,

8JT Environmental Consultants Ltd, Cambridge, UK, 9Julius Kuhn Institute, Braunschweig, Germany

The use of RNA interference (RNAi) mechanisms enables the silencing of target genes in plants or

plant-dwelling organisms, through the production of double stranded RNA (dsRNA) to silence genes

encoding specific genomic traits. Genetically modified (GM) crop plants displaying resistance to

viruses or insects via expression of dsRNA have recently received authorisation for cultivation

outside Europe. Some products derived from RNAi plants have also received a favourable opinion

from EFSA for import and processing in the EU. The authorization process requires applicants to

produce a risk assessment considering food/feed and environmental safety aspects. Some GM plants

expressing dsRNA have improved quality characteristics while others exploit new mechanisms with

activity in plant pests and pathogens. The presentation will alight the main aspects of the safety

assessment for RNAi-based GM plants, according to the guidelines of EFSA. The analysis of these

main topics in risk assessment indicates that the science-based regulatory process in Europe can be

applicable to RNAi plants, therefore the evaluation of their risk assessment can be effectively

conducted without changes in the authorization process. Our examination of these newly developed

GM plants has highlighted the important role that bioinformatics can play in the risk assessment

process. Food/feed safety assessment of products from RNAi plants is expected to be simplified, since

no novel proteins are produced from the dsRNA. Therefore some of the data requirements do not

apply to these cases and the comparative compositional analysis becomes the main source of evidence

for food/feed safety. During environmental risk assessment, the analysis of gene expression levels,

as well as data concerning the observable phenotype on non-target organisms will provide the

evidence for ensuring safety of species exposed to RNAi plants. Outcomes from the present paper

offer suggestions for consideration in the process of an update of the EFSA Guidance documents on

risk assessment of GM organisms.

ENVIRONMENTAL DISSIPATION OF DSRNA AND BARRIERS TO PLANT UPTAKE

PAMELA BACHMAN, JOSHUA FISCHER, ZIOHNG SONG, EWA URBANCZYK-WOCHNIAK,

MICHAEL BENNETT, JILL DEIKMAN, ALBERTO IANDOLINA, GREG WATSON

Bayer Crop Science

dsRNA, dissipation, residue, plant barriers

Two primary use patterns exist or are possible for deployment of dsRNA-based products in crop

improvement: in planta produced dsRNA such as in a genetically engineered (GE) crops; and

topically applied dsRNA. Completion of robust environmental risk assessments for and

demonstrating the envirnomental benefits of these dsRNA-base products relies on the successfull

measurement in relevant environmental compartments (soil, sediment, surface water, plant matrices).

Results from numerous environmental fate and degradation studies with naked or unformulated

dsRNAs demonstrate a high lability of these molecules and low potential for persistence in the

environment. Results of a pilot study of topically applied dsRNA on soybean plants demonstrate

similar rapid degradation under field conditions. The inability to produce biologically meaningful

systemic RNAi effects in plants treated with dsRNA applied to a leaf surface and experients showing

the majority of foliarly applied dsRNA resides on the leaf surface means that topically applied dsRNA

will be subject to environmental factors that drive dissipation and degradation. Because biologically

meaningful whole plant RNAi has not been observed for endogenous gene products, under growing

conditions including field production, the risk assessment of foliarly-applied dsRNA-based products

should not consider exposure scenarios that include systemic response to small RNAs in treated

plants. Formulations or other approaches that mitigate environmental degradation or the bypassing of

plant barriers may lead to more eficacious products but may change the known dissipation and

degradation kinetics of dsRNAs.

DSRNA-DELIVERY SYSTEMS: ELUCIDATION OF UPTAKE IN PLANT AND INSECT

CELLS

K. DE SCHUTTER1, Z.M. REYNA1, O. CHRISTIAENS1, C.N.T. TANING1, L. SWEVERS2, P.

DUBRUEL3, E.J.M. VAN DAMME4, G. SMAGGHE1

1Department of Plants and Crops, Ghent University, Ghent, Belgium

2Institute of Biosciences & Applications, National Center of Scientific Research ‘Demokritos’, Athens, Greece

3Department of Organic and Macromolecular Chemistry, Ghent University, Ghent, Belgium

4Department of Biotechnology, Ghent University, Ghent, Belgium

RNAi efficacy, cellular uptake, plant and insect cells, lectin, cell-penetrating peptide

The changing environment, with its obvious effects on abiotic stress (drought, flooding, etc) and the

introduction of novel or increased biological stresses (pests, diseases, etc), poses enormous challenges

to the agricultural sector. To cope with these challenges and the growing emergence of pesticide

resistance, new methods for pest control and improvement of plant performance are needed. In the

recent years, RNAi-based pest management has become the new hope for a sustainable agriculture,

with several examples of silencing essential genes in pests, causing mortality of the pest, or in the

plant itself, to provide desired phenotypes or resistance to (a)biotic stresses. While most examples

use GMO-plants expressing the dsRNAs, a non-GMO approach such as spray-induced gene silencing

(SIGS) could offer an easy design with high specificity and flexibility of application. However, in

this approach, a high variability in RNAi response has been observed. One main factor affecting

RNAi efficiency is the uptake into the cells. Inspired by synthetic chemistry and natural compounds,

dsRNA carrier systems have been designed to boost the RNAi efficacy. These systems are designed

to efficiently deliver their dsRNA cargo into the cells by avoiding RNAi barriers such as an inefficient

cellular uptake, a low endosomal release and extracellular degradation of the dsRNA. Here we

explore the potential of lectin- and cell-penetrating peptide-based carriers for the delivery of dsRNA

in insect and plant cells and aim to elucidate the mechanisms involved in the uptake of dsRNA and

its carriers in plant and insect cells.

ENVIRONMENTAL RISK ASSESSMENT OF RNA SPRAYS – APPLICABILITY OF

DATA REQUIREMENTS FOR CHEMICAL PLANT PROTECTION PRODUCTS

C.P.E. VAN RIJN1, J. W. A. SCHEEPMAKER2, D.C.M. GLANDORF1

1Dept. of Gene Technology and Biological Safety, National Institute of Public Health and the Environment, Bilthoven,

NL.

2Dept. of Environmental Risks of Substances and Products, National Institute of Public Health and the Environment,

Bilthoven,, NL.

Environmental risk assessment, RNA sprays, data requirements

Application of RNA sprays for plant protection is expected in the near future and questions have been

raised on their safe environmental use. There is little experience with the environmental risk

assessment (ERA) and use of data requirements for risk assessment of RNA-based products.

Therefore data requirements for chemical plant protection products were evaluated for their use for

RNA sprays. These sprays are targeted against specific insect pests on plants. Bottlenecks in

complying with data requirements were identified and alternative tests are proposed.

VIRUS-INDUCED GENE SILENCING OF THE VESICULAR TRAFFICKING GENE

TaMIN7 IN WHEAT COMPROMISES HOST IMMUNITY RESULTING IN HIGHER

DISEASE SUSCEPTABILITY TO FUSARIUM GRAMINEARUM

VINAY PANWAR, A.K.M. WOOD, K.E. HAMMOND-KOSACK, AND KOSTYA KANYUKA

Biointeractions and Crop Protection, Rothamsted Research, Harpenden AL5 2JQ, United Kingdom

Vesicular Trafficking, VIGS, RNAi, Wheat-FHB interaction

Plants have developed intricate defence mechanisms, referred to as innate immunity, to identify and

defend themselves against a wide range of pathogens. In the event of pathogen attack, plants can

prime appropriate defences including the synthesis and secretion of antimicrobial compounds at the

primary infection site. This channelling of defence-related proteins in plants relies on the formation

and transport of membrane-bound vesicles and proteins involved in vesicle formation. By adjusting

cellular trafficking plants can quickly respond to microbe confrontation whereas, inhibition of host

vesicle-mediated trafficking is one counteractive strategy of microbes to suppress host defence.

Recent findings indicate that vesicular trafficking is also involved in the transport of RNA

interference (RNAi) signals in cross-kingdom RNAi. Much of the evidence regarding the importance

of vesicular trafficking in plant comes from studies involving model plants and our understanding of

the mechanism of this complex network and its role during plant-pathogen interactions remains

largely uncharacterized in crop plants. Earlier an AtMIN7 gene involved in vesicle trafficking was

identified in Arabidopsis and found to interact with the HopA1 effector from Psedomonas syringae

triggering the effector triggered immunity responses. To test whether the components of vesicular

trafficking are involved in plant-microbe interactions in higher plants we identified the AtMIN7

ortholog in wheat, TaMIN7, and studied its role during interaction with the fungus Fusarium

graminearum the causative agent of the wheat Fusarium head blight (FHB) disease. Our results show

that knock-down of the TaMIN7 expression in wheat using virus induced gene silencing (VIGS)

significantly supported more FHB growth. This suggests that disruption of TaMIN7 function in wheat

compromised the trafficking of innate immunity signals resulting in higher disease susceptibility.

Overall, understanding the role of genes involved in vesicular trafficking may help in developing

effective biotechnological methods for controlling agronomically important diseases in various crops.

CONTRIBUTION OF DSRNA-BASED PRODUCTS TO AGRICULTURAL

SUSTAINABILITY IS DEPENDENT ON REGULATORY PROCESS

GREG WATSON

Bayer Crop Science

dsRNA, biologicals, biopesticides

Regulatory approval of biologically-based active ingredients for use in agriculture is normally

required before products containing these active ingredients can be made available for use; therefore,

the ability for biologically-based products to contribute to the sustainability of agriculture is

dependent on the regulatory processes and procedures that are implemented to enable regulatory

approval. This is particularly true for biologically-based active ingredients that are aimed at the

management or control of plant pests. Because regulatory authorities have acknowledged the

potential benefits of biologically-based active ingredient to agriculture, there have been multiple

international efforts undertaken to develop relevant regulatory processes for biologically-based active

ingredients, including the work of the OECD Expert Group BioPesticides (EGBP; working under the

leadership of the OECD Working Group Pesticide (WGP)). Among these efforts have been

comparisons of the regulatory processes of different countries and regions, such as the Regulation of

Biological Control Agents project (REBECA; initiated by an EU Commission action in 2006);

normally one of the goals of these comparisons is to identify potential opportunities for improving

exisiting regulatory processes. For example, at the time of the final REBECA report, the average

time needed for review and approval of a biologically-based plant protection active ingredient was

23 months for US EPA and 86 months for EU Annex I inclusion. Using the REBECA report and

other publicly available information, an overview of the recent regulatory history of biologically-

based active ingredients meant for plant protection use can inform the potential regulatory processes

and procedures that will impact the ability of dsRNA-based active ingredients to contribute to

sustainable agriculture.

A NOVEL GENOME EDITING TECHNOLOGY: GENE EDITING INDUCED GENE

SILENCING (GEIGS)

DARIO BREITEL, KANU PATEL, FEDERICO ALFANO, JACK PEART, OFIR MEIR & EYAL MAORI

Tropic Biosciences Ltd. | Innovation Centre, Norwich Research Park, NR4 7GJ, United Kingdom

GEiGS, Gene Editing, RNAi, Crop Protection, Crop Improvement, Disease Resistance

Transgenic crops carry foreign DNA within their genomes to express specific improvements such as

yield increases or disease resistance. However, the commercialization of this methodology requires

significant investment of resources and time due to extensive regulatory approval processes and

public stigmatization.

RNA interference (RNAi) has been applied to agriculture in recent years, providing a practical

approach to control various pest and viral diseases and could increase food security. However, these

attempts have been limited mainly to host-mediated transgenic dsRNA expression. We have

developed GEiGSTM, a unique and versatile technology that enables non-transgenic heritable RNAi

applications through minimalistic editing of non-coding RNA genes. In this presentation I will give

an overview of the current barriers in using RNAi or gene editing for crop protection and

improvement and discuss how the GEiGSTM platform combines the benefits of RNAi & gene

editing, while overcoming their challenges. The presentation will also indicate how the GEiGSTM

platform enables numerous applications for crop protection and improvement in plants and animals

and how GEiGSTM could also be used to study basic non-coding RNA biology in diverse biological

contexts.

STRATEGIES FOR STINK BUG (PIEZODORUS GUILDINII) CONTROL IN SOYBEAN

CROPS BY RNAi.

SCHVARTZMAN C.1, FEIJÓO M.2, FRESIA P.1, MURCHIO S.1, PESSIO M.1, MUJICA V.1, DALLA-

RIZZA M.1

1Instituto Nacional de Investigación Agropecuaria, INIA, Uruguay. 2CURE Treinta y Tres – UdelaR.

Pest control, hemiptera, soybean, gene silencing,

Soybean became the largest cultivated crop in Uruguay occupying more than 60% of the total

agricultural land. Piezodorus guildinii (Westwood) is a Neotropical stink bug species ranging from

Argentina to southern USA. It is an important pest of soybean in the Southern Cone of South America

and the most abundant in Uruguay. It causes damages to the surface and depth of grains, reducing

their viability, vigor and affecting grain production and quality. Chemical control used based on

piretroids and neonicotinoids is inefficient, environmentally unfriendly and susceptible to resistance

emergence. In this work we seek to use the potential of RNAi as a non-genetically modified pesticide.

To achieve this, we generated a P. guildinii transcriptome database, from total mRNA extracted from

all life stages, cDNA synthesis and Illumina sequencing. De novo transcriptome assembly and gene

annotation were performed, candidate genes selected and dsRNA designed. Effect of oral

administration of dsRNAs will be evaluated by qPCR and viability of stink bugs will be determined

over time and concentration. The successful implementation of this strategy will open a completely

new approach to combat soybean stink bugs and boost sustainable production of soy bean crops.

EVALUATION OF PRODUCTION OF VIRUS-LIKE PARTICLES’ COMPONENTS BY

THE BACULOVIRUS EXPRESSION VECTOR SYSTEM FOR EFFICIENT DSRNA

DELIVERY IN INSECTS

DIMITRIOS KONTOGIANNATOS2, ANNA KOLLIOPOULOU2, YONGCHAO ZHAO1, 2, FEIFEI REN1, 2,

QIUYUAN LU1, JINGCHEN SUN1 & LUC SWEVERS2

1College of Animal Science, South China Agricultural University, Guangzhou 510642 Guangdong,

People’s Republic of China 2Institute of Biosciences & Applications, National Centre for Scientific Research “Demokritos”, Aghia Paraskevi, Greece

RNA interference; double stranded RNA; Virus-like particles; Baculovirus Expression Vector

System; Cypovirus

For the past 22 years, RNA interference (RNAi) became a potent molecular tool for the selective

down-regulation of gene expression in almost all eukaryotes. This technique has found applications

that cover the entire spectrum of biological sciences, from functional genomics and crop engineering

to its recent use in pest control science. RNAi is triggered by double stranded RNA (dsRNA)

molecules that enter the insect’s cells and activate the endogenous RNAi core machinery;

consequently these molecules “silence” the targeted gene expression resulting in developmental arrest

and finally in mortality. Although RNAi is a promising tool to be used in Integrated Pest Management

(IPM) improvements are needed in order for this technique to be potent and reliable. There are many

limitations that decrease RNAi efficiency in insects, such as the biochemical nature of the insect gut,

the chemical instability of dsRNA molecules and their decreased capability to penetrate eukaryotic

cells. Therefore there is an urgent need for new methodologies that ascertain that the dsRNA will be

transferred intact, stable and in high amounts to the targeted insect cells.

In this work we investigate the possibility of using virus-like particles (VLPs) as nano-delivery

vectors of dsRNA molecules in insect cells. For that reason we have expressed via the Baculovirus

Expression Vector System (BEVS) the most important molecular components of Bombyx mori

Cypovirus and are addressing the potential of this biotechnological platform in vitro. Moreover, we

intend to develop analytic methodologies and bioassays to measure dsRNA activities in vitro and in

vivo and discuss further experimentation needed in order to engineer the efficient production of VLPs

and their encapsidation of dsRNA.

BIOINFORMATICS FOR SUSTAINABLE AGRICULTURE: EXPLORING A NOVEL

NETWORK-BASED APPROACH OF MIRNA TARGETS IN THE MODEL LEGUME

MEDICAGO TRUNCATULA

A. MACOVEI1, E. SAUTA2, C. GUALTIERI1, D. DE MARCHI2, M. BELLATO2, P. MAGNI2, A.

BALESTRAZZI1, L. PASOTTI2

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

Bioinformatics, microRNA, co-expression networks, gene expression, Medicago truncatula

The current scenario of global climatic changes demands the development of more sustainable

agricultural practices. One solution to sustainably increase plant yield is to optimize plants based on

the molecular understanding of gene function and regulatory networks involved in stress tolerance,

development and growth. Through the application of informatics technology at a molecular level,

bioinformatics can provide predictive tools to this purpose. MicroRNAs, acting as post-

transcriptional regulators of many biological functions, regulate gene expression through interactions

with their target mRNAs. By repressing the activity of their gene targets, miRNAs function similarly

to RNAi. In this study, a Medicago truncatula co-expression network was constructed to investigate

the regulatory potential of miRNAs in multiple biological processes. To build this network, the

ARACNE tool was employed to shape publicly available microarray datasets (Affymetrix GeneChip

Medicago Genome array). The miRNA targets, predicted using psRNATarget, were mapped into the

network to extract their co-expression interactome which was then analyzed via the gLay clustering

procedure and subjected to ClueGO enrichment. Biological processes like cell growth, exocytosis,

thylakoid membrane organization, hormone-mediated signaling pathways, mRNA metabolism, DNA

replication, amino acid activation, cellulose metabolism, fruit and seed development, were associated

with miRNA targeted genes. The expression of the targeted genes, retrieved from publicly available

database (Medicago eFP Browser), was used to generate heatmaps and illustrate changes in different

developmental stages and stress treatments. This investigation provides novel data relative to miRNA

coordinated networks to be further experimentally validated and used to boost crop productivity and

adaptation to stress.

FURTHER EVIDENCE OF DURABLE RESISTANCE IN HONEYSWEET TRANSGENIC

PLUM UNDER NATURAL INFECTION WITH D AND REC STRAINS OF PLUM POX

VIRUS

L. A. ZAGRAI, I. ZAGRAI

Fruit Research & Development Station Bistrita, Romania

Genetic engineering, RNA-i, plum pox virus, -D and -Rec strains, resistance

'HoneySweet' is a transgenic plum protected against Plum pox virus (PPV) based on RNA

interference. Its behaviour to natural PPV infection was the subject of different field trials undertaken

in several endemic European countries. The first experiment in Romania was performed between

1996-2006 and no 'HoneySweet' tree was found infected under high natural PPV infection pressure.

To further asses the durability of resistance to PPV of 'HoneySweet' a new field trial was established

in 2013, and a new cycle of investigation was undertaken until 2019. Tolerant plum cultivars were

used as controls. The field trial was statistically designed in 12 blocks of 4 trees (two trees of 'Honey

Sweet' and two trees of conventional plums cultivars: 'Stanley' and 'Reine Claude d’Althan'). As

inoculum sources of PPV plants previously artificially inoculated with -D or -Rec strains of PPV were

grown in pots which were then placed inside of the blocks, one infector per block, alternately each

one of the two strains. Limited treatments with insecticides were made within the plot in order to

stimulate the virus spread by aphids. The monitoring of PPV spreading was made by DAS-ELISA

combined with IC-RT-PCR. PPV strain discrimination was made by IC/RT-PCR using P1/PD,

P1/PM, CIP-M/CIP-MR and CIP-D/CIP-DR primer sets. The temporal spread of PPV revealed a

continual evolution of infection in both conventional plums. Thus, 13 out of 24 conventional plums,

seven trees of 'Stanley' and six of 'Reine Claude d’Althan', became infected during the six years of

field testing. Seven out of thirteen trees confirmed the presence of -Rec strain, and the other six, -D

strain. Three trees from each cultivar proved to be infected with -Rec strain. No trees of transgenic

plum 'HoneySweet' expressed PPVsymptoms or were found to be infected by serological and

molecular assays, confirming its durable resistance to natural PPV infection with -D or -Rec strains

A NEW APPROACH OF USING RNAi AGAINST PLUM POX VIRUS: FROM

HONEYSWEET TRANSGENIC PLUM TO MALE STERILE HYBRIDS

I. ZAGRAI1, L. ZAGRAI1, M. RAVELONANDRO2, R. SCORZA3, A CALLAHAN3, C. DARDICK3

1Fruit Research & Development Station Bistrita, Romania

2Institut National de la Recherches Agronomique Bordeaux, France

3 Ralph Scorza LLC , Shepherdstown, WV USA (formerly USDA-ARS)

Plum pox virus, genetic engineering, resistance, male sterile hybrids, coexistance

Pathogen derived resistance through RNAi technology has been shown to be an efficient solution to

controlling Plum pox virus (PPV), the most detrimental viral pathogten of stone fruits. 'HoneySweet’

transgenic plum has for over 20 years demonstrated a high, stable, and durable resistance to Plum pox

virus in different PPV endemic areas. Regulatory authorities in the U.S. have found no safety

concerns and approved ‘HoneySweet’ for cultivation. Research on ‘HoneySweet’ has continued since

the U.S. approval and additional data demonstrating its good agronomic performance and the

potential for the cultivation of ‘HoneySweet’ to reduce environmental and health impacts of pesticide

use. The real challenge to the practical application of the RNAi mechanism of ‘HoneySweet’ remains

its approvals in European countries highly affected by PPV. To address this challenge, a new

approach to use the silencing mechanism is in progress. We propose to use 'HoneySweet' as the donor

of PPV resistance in conventional breeding to develop male sterile PPV resistant hybrids. This is

possible by using as parents in crossing with ‘HoneySweet’ cytoplasmic male sterile Romanian plum

varieties many of which are already being cultivated in Romania. This approach may be useful

because, farmers may more readily accept easier these GMO plum varieties since they will not

produce pollen and isolation to minimize the pollen dispersal would not be an issue. Consequently,

monitoring plans associated with cultivation could be significantly simpler. The use of male sterile

transgenic hybrids will contribute to the avoidance of controversy on coexistence issues, which is an

important concern in the EU. To perform this study a new permission was granted by the Ministry of

Environmental-Romania, and European Commission was notified (B/RO/19/01). Thus, a new field

trial of the first generation of 'HoneySweet' male sterile hybrids is planned to be developed and

evaluated during the next decade.

dsRNA STABILITY DURING EXTERNAL APPLICATIONS – AN OVERVIEW

IVELIN PANTCHEV, GORITSA RAKLEOVA 2, ATANAS ATANASSOV 2

1 Department of Biochemistry, Sofia University, 8 Dragan Tzankov, 1164 Sofia, Bulgaria

2 Joint Genomic Center Ltd., 8 Dragan Tzankov, 1164 Sofia, Bulgaria

dsRNA, RNA interference, RNA stability,

Research community is deeply convinced that RNA is unstable in the environment. Its roots rise from

numerous failed attempts to isolate functional cellular RNA molecules. Further support had originated

from the fast turnover of RNA in the cells. The situation changed recently with the discovery that

externally applied dsRNA can produce targeted gene silencing in plant-feeding insects. First results

have demonstrated that external dsRNA can successfully pass the insect gastrointestinal tract and

reach its final destination within the body cells. This was somewhat unexpected and sparkled new

interest on RNA stability in the environmental and its fate in the insect organism.

dsRNA stability in the environment became a core issue for RNAi based pesticides. Also, relatively

little is known about the factors determining the pesticide effect. In our report we will try to

summarize the available knowledge about the fate of dsRNA in the environment during external

applications as well as in the insect body upon feeding.

MUTATIONAL ANALYSIS OF ARABIDOPSIS THALIANA ABCE2 IDENTIFIES

IMPORTANT MOTIFS FOR ITS RNA SILENCING SUPPRESSOR FUNCTION

JELENA MÕTTUS, SIIM MAISTE, PRIIT EEK, ERKKI TRUVE, CECILIA SARMIENTO

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

Estonia

ABCE1; ABCE2; suppression of RNA silencing; Arabidopsis thaliana

ATP-binding cassette sub-family E member 1 (ABCE1) is recognized as a strongly conserved

ribosome recycling factor indispensable for translation in archaea and eukaryotes, however, its role

in plants remains largely unidentified. Arabidopsis thaliana encodes two paralogous ABCE proteins

(AtABCE1 and AtABCE2), sharing 81% identity. AtABCE2 has ubiquitous expression pattern and

functions as a suppressor of RNA silencing. Here we describe the structural requirements of

AtABCE2 for its suppressor function. Using agroinfiltration assay, we transiently overexpressed

mutated versions of AtABCE2 together with the inducer of GFP silencing in GFP transgenic

Nicotiana benthamiana leaves. Mutants of AtABCE2 with impaired ATP binding in either active site

I or II failed to suppress GFP RNA silencing. The disruption of ATP hydrolysis had a less pronounced

effect. Moreover, the functional asymmetry of the active sites as demonstrated earlier in the case of

archaeal ABCE1 was confirmed for AtABCE2. We also found that the N-terminal iron-sulfur cluster

domain of AtABCE2 is crucial for its suppressor function. Altogether, structural requirements of

AtABCE2 for RNA silencing suppression were similar to those of archaeal ABCE1 needed for

ribosome recycling. This suggests that AtABCE2 might suppress RNA silencing via supporting the

competing RNA degradation mechanisms associated with ribosome recycling.

RELEVANCY OF THE BIOTECHNOLOGICAL APPROACH TO TACKLE THE PLUM

POX VIRUS GENOME WITH RNAi SILENCING

M. RAVELONANDRO1, R. SCORZA2, A CALLAHAN2, P. BRIARD1, C. DARDICK2

1UMR-1332, Biologie du Fruit et Pathologie, INRA-Bordeaux, France

2Appalachian Fruit Research, USDA-ARS, Kearneysville, WV USA

RNAi, sharka virus, sustainability, resistance, methylation

Plum pox potyvirus (PPV) is one of the most damaging pests of Prunus species. Since the discovery

of sharka disease, one hundred years ago, PPV continues its spread across the world demonstrating

the challenges of controlling PPV. While many researchers are seeking potential resistance genes by

exploring plant genomes, there is a real need to develop practical control strategies as quickly as

possible as stone fruit production world-wide is threatened. Expertise about Prunus transformation

coupled with the molecular knowledge of plant gene silencing led to the development of highly

resistant Prunus domestica clones more than 10 years ago. In order to investigate the robustness of

the RNAi technology, a few resistant plum clones engineered with different RNAi constructs were

molecularly studied. During this last decade, the behaviour of different clones harboring variable gene

constructs developed to resist PPV infection were analysed in a high containment greenhouse. In

these studies the PPV-Prunus interactions developed as expected in terms of the systemic spread of

PPV in trees and the physical contrast between a susceptible versus a resistant clone. Similarly to the

outstanding behaviour of the HoneySweet resistant clone to PPV infection, constructs based on the

latest advances in molecular RNAi expression in virus resistant plants (amisiCPRNA-3 , -4,- and -7;

P1CP plums) have confirmed the effectiveness of the RNAi mediated resistance. Beyond the

variability and size of the virus transgene insert (from one to 2.5 Kbp) that was engineered, the levels

of RNAi expression, the role of tasiRNA mediated DNA transgene methylation were highlighted.

Overall, we conclude that classical breeding involving natural genes is time-consuming because it

requires an extensive characterization of plants including the field assays in different ecosystem

(including PPV strains, different aphid species, variable climates…) prior to their release to end-

users. Conversely, two significant benefits can be earned with the bioengineering of RNAi plants;

first to rapidly produce RNAi plants and second to efficiently prevent infection via the specific target

and degradation of the PPV genome.

DROSOPHILA SUZUKII: AN EMERGENCY IN BERRY CULTIVATION, STATE OF

CONTROL SYSTEMS

ANNA ALDRIGHETTI

Sant’Orsola S.C.A., Trento, Italy

Drosophila suzukii, control systems, berry cultivation

The fruit fly D. suzukii is considered a threatening fruit crop pest, causing in Europe serious economic

losses in berry fruit production. D. suzukii is a damaging pest due to its high reproduction rate and its

ability to infest thin-skinned fruits before harvest. Moreover, being D. suzukii an invasive and alien

species from Asia, a natural enemy is not available, allowing this fruit fly to easily reproduce itself.

Unfortunately, nowadays control systems are still ineffective and not protecting crops efficiently.

Chemicals are continuously used, even if they are not able to maintain Drosophila under the

damaging threshold. Nets could be considered the only effective controlling system, but they are

expensive and not always applicable. For these reasons, could RNAi technologies be helpful in berry

crop protection management?

FROM DISCOVERY TO DEVELOPMENT OF RNA-BASED FOLIAR FUNGICIDES

KESTREL MCCORKLE1, MARY MILNER1, ADRIANA ARANGO-VELEZ1, CHRISTOPHER

LAWRENCE1

1GreenLight Biosciences, 5 Laboratory Drive Suite 1250, Research Triangle Park, NC 27709

dsRNA, exogenous, fungicide, Botrytis

RNAi is a biological solution for pest control that is target specific, biodegradable, and has the

capability for rapid discovery of candidate sequences for gene knockdown. GreenLight Biosciences

has developed a proprietary, scalable process for producing cost-effective RNA products for

agricultural management of insect pests and plant pathogenic fungi. GreenLight’s first exogenously

applied dsRNA product for Colorado potato beetle (CPB) control has shown comparable defoliation

and yield protection to leading synthetic chemistry market products. Success with insect control has

prompted research of dsRNA application for fungal pathogen management by targeting essential and

pathogenicity genes. A high-throughput method of producing hundreds of dsRNAs and screening

them in an in vitro assay for control of the gray mold pathogen, Botrytis cinerea, was developed and

implemented. Actives from the in vitro screen were screened on detached tomato leaflets and fruit for

Botrytis control with the most efficacious leads selected for field testing in 2020. The use of

exogenously applied dsRNA can be expanded to additional target fungal pathogens and other

agricultural pests. The future use of dsRNAs could give growers an additional disease management

tool that is safe, sustainable, and effective.

INTEGRATED SRNA, DEGRADOME PROFILING AND RNA-SEQ ANALYSIS

IDENTIFIES PHOSPHATE STARVATION-RESPONSIVE SMALL RNAS AND GENES IN

BARLEY

PAWEŁ SEGA1, KATARZYNA KRUSZKA1, DAWID BIELEWICZ1, WOJCIECH KARŁOWSKI2,

PRZEMYSŁAW NUC1, ZOFIA SZWEYKOWSKA-KULIŃSKA1, ANDRZEJ PACAK1

1Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam

Mickiewicz University, Poznań, Uniwersytetu Poznańskiego 6, Poznań, 61-614 Poland

2Department of Computational Biology, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam

Mickiewicz University, Poznań, Uniwersytetu Poznańskiego 6, Poznań, 61-614 Poland

NGS, small RNAs, phosphate homeostasis, barley

Phosphate (Pi) homeostasis is controlled by group of genes encoding PHR transcription factors

(Phosphate Starvation Response), phosphate transporters, non-coding RNAs, microRNAs (miR399

and miR827), protein modifiers and PHO2 (PHOSPHATE 2) enzyme involved in Pi-related proteins

degradation. We used tripartite approach (sRNA-seq, degradome profiling and RNA-seq) to

characterize the set of small RNAs and transcripts differentially expressed in barley shoots under low-

Pi stress. We found that differentially expressed microRNAs were represented only by over a dozen

in number, mainly different miR399 and miR827 isomiRs. Analysis of other classes of 18-25 nt small

RNAs revealed tissue-specific adaptations to low-Pi through either activation or repression of genes

expression. In roots, more abundant and diverse set of sRNAs (1796 unique sequences) contributes

to the compensation of low-Pi stress than in shoots (199 unique sequences). More than 80% of

differentially expressed sRNAs were upregulated in both tissues. Additionally in shoots upregulation

of small RNAs level was accompanied by strong induction of nucleases (S1 endonuclease and 3’-5’

exonuclease), what reflects the way of realizing internal Pi pools through RNA degradation. We

provide comprehensive data to demonstrate complex responses on RNA level in barley shoots to

maintain Pi homeostasis.

This work was funded by the National Science Centre, Poland, on the basis of DEC-2013/11/B/NZ9/01761, UMO-

2016/23/B/NZ9/00857, and UMO-2015/19/N/NZ9/00218 and by POWR.03.02.00-00-I006/17.

BIOLOGICAL CONTROL, FUNGAL VIRUSES AND RNA SILENCING: A NOVEL

INTEGRATED PEST MANAGEMENT APPROACH?

IOLY KOTTA-LOIZOU1 & ROBERT H. A. COUTTS2

1 Imperial College London, UK;

2 University of Hertfordshire, UK

entomopathogenic fungi; polymycoviruses; insect control; virus-host interactions

Currently agricultural interest in biological control is resurging as part of integrated pest management

approaches, urgently required as existing agrochemicals are being withdrawn from the market due to

environmental and health concerns. Entomopathogenic fungi, such as the popular biocontrol agent

Beauveria bassiana, can be used as an alternative to chemical insecticides for controlling insect pests.

Here we show that members of a newly proposed virus family Polymycoviridae confer mild

hypervirulence to their fungal hosts and can be employed to improve commercially available

mycopesticides. Two B. bassiana isolates were cured from infection with respectively Beauveria

bassiana polymycovirus (BbPmV)-1 and -3 and virus-free and virus-infected isogenic lines were

generated. Comparison of the two pairs of isogenic lines revealed that in polymycovirus infection

leads to increased growth rates on liquid and solid media, increased sporulation, and increased

virulence for the greater wax moth Galleria mellonella and the army mealworm Tenebrio molitor.

Molecular mechanisms underpinning virus-host interactions in general and virus-mediated

hypervirulence in particular include protein-protein interactions and RNA silencing. The latter is a

well-established fungal defence against viruses with double-stranded (ds) RNA genomes or

replication intermediates, since the viral dsRNA serves as both trigger and target of the silencing

machinery. Polymycoviruses, which contain dsRNA, are non-conventionally encapsidated and are

silenced significantly more than encapsidated mycoviruses, potentially leading to ‘off-target’

silencing of fungal transcripts. In the future polymycoviruses will be introduced into commercially

available, virus-free B. bassiana strains in anticipation of increasing their virulence against arthropod

pests.

EXOGENOUSLY APPLIED DSRNAS ARE ABLE TO INDUCE RESISTANCE AGAINST

THE NEGATIVE/AMBISENSE RNA TOMATO SPOTTED WILT VIRUS BUT NOT

AGAINST THE CIRCULAR DNA TOMATO YELLOW LEAF CURL SARDINIA VIRUS

TABEIN S. 1, ACCOTTO GP.2, NORIS E.2, BEHJATNIA SAA.1, VAIRA AM.2, MIOZZI L.2

1 Plant Virology Research Center, College of Agriculture, Shiraz University, Shiraz, Iran

2 Institute for Sustainable Plant Protection, National Research Council of Italy, Torino, Italy

tomato spotted wilt virus, tomato leaf curl Sardinia virus, RNAi-based vaccination

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

namely RNAi-based vaccination, represents an attractive and promising alternative, already shown

to be effective against different positive-sense RNA viruses and viroids. However, information on its

efficacy against ambisense/negative-sense RNA viruses or DNA viruses is lacking. In the present

study, we evaluated the potential use of RNAi-based vaccination against two devastating plant

pathogens, the ambi/negative-sense RNA virus tomato spotted wilt virus (TSWV, Bunyaviridae

family) and the phloem-limited single-stranded circular DNA virus tomato yellow leaf curl Sardinia

virus (TYLCSV, Geminiviridae family). For TSWV, a significant reduction in TSWV-induced local

lesions at 7 days post inoculation (dpi) was observed in Nicotiana benthamiana plants treated with

N-targeting dsRNAs but not in those treated with NSm-targeting dsRNAs. Furthermore, most of the

plants treated with TSWV N-dsRNAs, but not with NSm-dsRNAs, remained asymptomatic and virus-

free until 40 dpi or showed a significant delay in the appearance of systemic symptoms. In the case

of TYLCSV, none of the tested dsRNAs showed a protective capacity, as indicated by simultaneous

systemic symptoms appearance in all plants. Moreover, Southern blot and real-time PCR showed the

TYLCSV titer was similar between all dsRNAs-treated and untreated plants. The obtained results

indicate that the efficacy of the RNAi-based-vaccination depends on the choice of the target viral

pathogen: indeed, this technique is effective against ambi/negative-sense RNA viruses, such as

TSWV, but seems to fail in the case of the phloem-limited single-stranded circular DNA viruses such

as TYLCSV. Furthermore, our results emphasize the importance of the target sequence selection in

designing dsRNA-based vaccines against RNA viruses. The obtained results are discussed in the light

of the different lifestyle of the viruses here considered.

TRANSCRIPTOME AND METABOLOME OF SALIX PURPUREA UNVEIL TISSUE-

SPECIFIC PATTERNS OF LNCRNA-MEDIATED REGULATION

ANDREA PAGANO1, 2, CAROLINA GOMES1, DARIUSZ KRUSZKA1, JORGE A. P. PAIVA1

1 Institute of Plant Genetics, Polish Academy of Sciences, ul. Strzeszyńska 34, 60-479 Poznań, Poland.

2 Present address: University of Pavia, Department of Biology and Biotechnology ‘L. Spallanzani’, Via Adolfo Ferrata,

9, 27100 Pavia (PV), Italy.

lncRNA, Salix purpurea, transcriptomics, metabolomics

The non-protein coding portion of plant transcriptomes constitutes an evolving field of study within

our understanding of plant biology. Long non-coding RNAs (lncRNAs) are broadly defined as non-

coding RNA molecules longer than 200 nucleotides. They regulate gene expression by recruiting

specific factors or promoting chromatin relaxation at the promoters of their target genes, acting as

scaffolds for the assembly of protein complexes or interacting with miRNAs. They can act ‘in cis’,

on the expression of genes located in proximate chromosomal locations, or ‘in trans’, far from the

locus from which they are transcribed. There is also strong evidence of their interaction with miRNA

and RNAi pathways. lncRNAs appear to be involved in the regulation of a variety of processes,

including plant development and reproduction, nutrient uptake and stress response. A further and

particularly promising field of study is represented by their roles in secondary growth and wood

formation, especially because of the economic relevance of these processes in woody species used as

biomass feedstock for bioenergy and biofuel production. Moreover, the bark of many arboreal species

finds application as a source of economically relevant phytochemicals. Despite the evidence of the

involvement of lncRNAs in these dynamics, a comprehensive understanding of their role in

transcriptomic and metabolic regulation is currently lacking for most model and crop species.

In the present work, we produced 48 transcriptomic libraries in different Salix purpurea tissues,

namely root, bark, xylem and whole stem. Within these libraries, we listed 5157 lncRNAs and

identified the differentially expressed ones, highlighting putative correlations with differential gene

expression and metabolite accumulation. Given the relevance of Salix spp. as biomass sources,

phytoremediation tools and producers of compounds of pharmaceutical interest, a deeper

comprehension of the regulative mechanisms operating on its transcriptome and metabolome will

provide molecular bases for future studies and applications.

THE DEVELOPMENT OF DSRNA CARRIERS TO IMPROVE RNAI EFFICACY IN

RECALCITRANT INSECT SPECIES

OLIVIER CHRISTIAENS1, ZAREL M. REYNA1, MIRJAM G. TARDAJOS2, MAMONI DASH2, KRISTOF

DE SCHUTTER1, LUC SWEVERS3, ELS VAN DAMME4, PETER DUBRUEL2, GUY SMAGGHE1

1Department of Plants and Crops, Ghent University, Ghent, Belgium

2Department of Organic Chemistry, Ghent University, Ghent, Belgium

3Department of biosciences and Applications, National Center of Scientific Research ‘Demokritos’, Athens, Greece

4Department of Biotechnology, Ghent University, Ghent, Belgium

RNAi efficacy, delivery, nanocarriers, cellular uptake, nucleolytic degradation

In recent years, RNA interference (RNAi)-based pest management has emerged as a novel promising

control strategy for insect pests in recent years, mainly due to its high species selectivity and the short

environmental persistence of the active double-stranded RNA (dsRNA) component. Unfortunately,

not all pest insects exhibit a highly efficient RNAi response, especially after dietary uptake of dsRNA.

Several cellular and physiological barriers such as dsRNA-degrading enzymes in the digestive system

or a suboptimal cellular uptake can result in recalcitrance to RNAi in the insect. Inspired by nucleic

acid delivery in the pharmaceutical field, diverse polymer-, protein- or peptide-based nanoparticles

can be used to extend the stability of the dsRNA in harsh environments and/or improve cellular uptake

and in that way also increase the efficacy of RNAi. Here, we present the development of a guanylated

polymer which is able to improve RNAi efficacy in recalcitrant lepidopteran insects. The polymer

not only protected dsRNA against nucleolytic degradation in the midgut of the beet armyworm

Spodoptera exigua, but also improved cellular uptake in lepidopteran midgut cells. Furthermore, we

also present our first in vitro and in vivo data of recombinant lectin- and cell-penetrating peptide-

based carriers developed with the aim of improving RNAi in these lepidopteran insects.

IMPROVEMENT OF ORAL dsRNA UPTAKE IN TRIBOLIUM CASTANEUM BY USING A

GUANYLATED POLYMER FORMULATION STRATEGY

E. KNORR1, C.N.T. TANING2, O. CHRISTIAENS2, K. DE SCHUTTER2, M. PETEK3, G. SMAGGHE2

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

2Ghent University, Ghent, Belgium

3National Institute of Biology, Ljubljana, Slovenia

Insect pests cause a tremendous loss to annual crop production worldwide. A promising novel pest

control strategy is the exploitation of RNA interference (RNAi), a post-transcriptional gene silencing

mechanism, mediated by double-stranded RNA (dsRNA). However, certain insects are refractory to

RNAi, often due to dsRNA-degrading nucleases in their digestive system or limited cellular uptake.

A possible strategy to overcome these obstacles is the formulation of the dsRNA with nanocarriers.

In this research, we complexed long dsRNA designed from the enhanced green fluorescent protein

gene (eGFP) with either a cell membrane penetrating peptide (CMPP) or a lectin from Galanthus

nivalis (GNA), and analyzed their ability to enhance the cellular uptake of the nanocarrier-dsRNA

complexes into insect cells. The cell lines used for the experiments originated from different insect

orders: Tca cells from Tribolium castaneum (Coleoptera), Ag3C cells from Anthonomus grandis

(Coleoptera), S2 and Sl2 cells from Drosophila melanogaster (Diptera) and BMN4 cells from

Bombyx mori (Lepidoptera). Confocal microscopy analysis demonstrated an enhanced uptake of

CMPP formulated dsRNA into Ag3C and BMN4 cells. Similar results were obtained from GNA

complexed dsRNA, showing an enhance dsRNA uptake into both Ag3C and BMN4 cells, but also

generating agglomeration in several cases. Thus, only CMPP formulated dsRNA was analyzed in

subsequent in vivo experiments. An unpublished target gene, which is resulting in high pupal

mortality upon knockdown, was used for feeding bioassays with young T. castaneum larvae. Animals

that fed on the CMPP complexed dsRNA displayed deformations similar to those observed in

injection experiments targeting the same gene. More experiments will provide a detailed picture of

the function of the nanocarriers and verify these initial results. The formulation may provide a tool

for gene silencing in insects that are refractory to RNAi and could lead to future applications for

RNAi-based pest control in the field.

PIGGYBACKING ON THE TRANSFORMING GM STORY OF IMPOSSIBLE FOOD

DEMANDS

HILDE-GUNN OPSAHL SORTEBERG

Dept of Plant Science, BIOVIT, NMBU, Ås, Norway

Science communication, Impossible foods, Innovation, Sustainability, Macro algae

The iPLANTA communication working group meeting is focusing on good stories and how to

maximize impact. The main story served, while covering different aspects, is the Impossible Burger

(IB) and dissemination options to increase outreach. The Impossible Food (IF) and Beyond Meat are

illustrating how truly new technical innovation based on science can spread impressively, and you

will be taken through the impossible route with an emphasis on communication. The development

from the birth of a Silicon Valley start-up in 2011 by Patrick O Brown, professor emeritus biochemist

at Stanford, to beyond any precedented growth phase will be illustrated. The IB hit No1 item from

release in retail stores in 2019 (FDA GRAS 2019) and 17 000 restaurants (GRAS 2018) where Burger

King has problems meeting the demand. Despite attempts to stop it arguing it uses GM heme from

yeast to appear meaty, public perception is viral like. The success of IF and alike show consumers

crave for sustainable, new food solutions, and such plant products are expected to turn food trends

fast even if using biotechnology. IF is targeting flexitarians to improve health, and all to improve

sustainable food production. This transformation is happening globally, and how Europe will deal

with it concerns our activities: science, technology, breeding, public perception, socioeconomics,

food security and safety, risk and politics. This change can increase our outreach and effect.

Additionally, alternative macroalgae options and European suggested guide to controlled industrial

upscaling will also be covered.

RISK ASSESSMENT OF GENETICALLY MODIFIED LEGHAEMOGLOBIN AS A

FLAVOURING IN MEAT ANALOGUE PRODUCTS

HUW JONES

IBERS, Aberystwyth University UK

Impossible burger; GMO risk assessment; Pichia; leghaemoglobin

The Impossible Foods Company of Redwood City, California, USA have developed a plant-based

meat analogue product called the Impossible Burger which they claim provides the flavour and aroma

of a traditional animal-derived counterpart. An important ingredient in this product, which provides

both flavour and colour, is a haemoglobin compound (leghaemoglobin) found naturally at low

concentrations in the nitrogen fixing root nodules of the soybean plant. To manufacture sufficiently

large quantities of this product, the Glycine max LGB2 gene encoding leghaemoglobin, was codon-

optimised and inserted into the yeast Pichia pastoris for transgenic expression.

The company claim that over 20 million ¼-pound servings of the Impossible Burger have been sold

in USA since June 2016 and that they have also been available in Hong Kong and Macau since the

middle of 2019. In Oct 2019 Impossible Foods filed an application to the EU under the regulation

1829/2003 for the import and food use of soy leghaemoglobin produced from GM Pichia cells.

Bearing in mind the significant interest in veganism and non-meat dietary alternatives in the EU, if

authorised, the Impossible Burger may be major turning point for GMO food in some member states.

I will discuss the risk assessment process that EFSA will follow and offer some thoughts on how this

product may be received in the EU.

iPLANT BOOK: RNAi FOR IMPROVED CROP PERFORMANCE AND PROTECTION

JEREMY SWEET, JTEC Ltd, Cambridge, UK

BRUNO MEZZETTI, UNIVPM, Ancona, Italy

LORENZO BURGOS, Centro de Edafología y Biología Aplicada del Segura, Spanish National Research Council.

In 2016 the iPlanta COST Action1 CA15223 “ Modifying plants to produce interfering RNA” was

established with the objective of bringing together experts from a wide range of fields to develop a

deeper understanding of the science of RNA, the applications of RNAi, the biosafety of these

applications and the socio-economic aspects of these potential applications. A product of this COST

action will be a book containing a series of chapters by experts from many countries, who are

participating in iPlanta, to review the current scientific knowledge on RNAi, methods for developing

RNAi systems in GM plants and a range of applications for crop improvement, crop production and

crop protection. Chapters will examine both endogenous systems in GM plants and exogenous

systems where interfering RNAs are applied to target plants, pests and pathogens. The biosafety of

these different systems is examined and methods for risk assessment for food, feed and environmental

safety are discussed. Finally aspects of the regulation of technologies exploiting RNAi, the socio-

economic impacts of RNAi technologies and communication of the outcomes of this COST Action

are discussed.

1https://iplanta.univpm.it/

IN COLLABORATION WITH: