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Genome-wide analysis of RNA and protein interacting profiles during a plant virus infection

Periodic Reporting for period 1 - InterAcTEV (Genome-wide analysis of RNA and protein interacting profiles during a plant virus infection)

Reporting period: 2015-05-01 to 2017-04-30

Viruses constitute a major class among plant pathogens. RNA viruses encompass the majority of plant viruses, accounting for substantial losses in crop yields worldwide. Understanding how viruses incite diseases and how plants defend themselves against them is critical to develop effective antiviral approaches for crop protection that ensure food availability in the following years.

Plants have evolved two highly specific adaptive immunity pathways to face viruses, viroids and other pathogens. First, RNA silencing is an ancient antiviral mechanism induced by viral double-stranded RNAs (dsRNAs) that are processed into 21 to 24 nt small RNAs (sRNAs). In the antiviral RNA-silencing pathway model (see Figure), virus-derived sRNAs (vsRNAs) associate with a plant ARGONAUTE (AGO) protein, and sRNAs guide the AGO to interact and repress viral RNAs sharing sequence complementary with them. Second, plants have evolved involves disease resistance (R) proteins that recognize viral-encoded proteins through direct protein-protein interactions or through an indirect effect due to the interaction of a viral protein with a host protein included in a larger complex containing an R protein.

This project intended to better understand the molecular interplay occurring during plant/virus interactions. Our main goal was to identify new viral targets to develop effective antiviral resistance for crop protection. In particular, we wanted to identify AGO target sites highly susceptible to artificial sRNA-mediated inactivation, as well as plant proteins interacting with viral proteins upon infection.

To accomplish these objectives, first we explored the possibility of using amiRNAs and syn-tasiRNAs to specifically interfere with infections by economically important viruses and viroids. The combined use of recent high-throughput methods for artificial sRNA construct generation and of the Nicotiana benthamiana/Tomato spotted wilt virus (TSWV) and the N. benthamiana/Potato spindle tuber viroid (PSTVd) pathosystems allowed for the simple and time-effective screening of multiple artificial sRNAs targeting sites distributed along TSWV and PSTVd RNAs. We have identified several AGO target sites in TSWV and PSTVd RNAs that are efficiently targeted by specific amiRNAs or syn-tasiRNAs. Second, we have further optimized our RNA immunoprecipitation followed by high-throughput sequencing (RIP-Seq) approach. Our current methodology has already identified potential new AGO1 target transcripts that are currently being investigated, and will serve to detect AGO target sites in viral RNAs which could be used as ideal targets for artificial sRNA-mediated inactivation. And third, we have used the well-characterized N. benthamiana/Tobacco etch virus (TEV) pathosystem to analyse the protein interactors of several TEV proteins. Several TEV infectious clones including TEV proteins tagged with the Twin-Strep tag (TST) system have been generated and are being analyzed.
1) We have used recent high-throughput methods to evaluate in a simple and time-effective manner the antivirus and antiviroid activity of multiple artificial sRNA constructs designed to target specifically AGO target sites located in TSWV or PSTVd genomic RNAs. Our results show that both classes of artificial sRNAs expressed transiently in N. benthamiana have been effective in interfering specifically with TSWV or PSTVd infection.

2) We have also further developed our RNA immunoprecipitation followed by high-throughput sequencing approach for identifying AGO target transcripts. The efficiency of the recovery of known AGO1 miRNA target sites was dramatically increased by using Micrococcal Nuclease.

3) We tagged several TEV proteins with the TST affinity epitope. Our goal is to create a protein interactor map for each tagged TEV through a proteomics approach including affinity purification from infected tissue and mass spectrometry analysis of host proteins associating with each TST-tagged TEV protein.

Results were disseminated in scientific meetings and workshops:
• Carbonell, A. Nuevas herramientas para el control de la expresión génica en plantas basadas en pequeños RNAs artificiales. Master en Biotecnología y Bioingeniería (06/05/2016), Instituto de Biología y Bioingeniería, Universidad Miguel Hernández, Elche, Spain.
• Cordero, T., Cerdán, L., Carbonell, A., Katsarou, K., Kalantidis, K. and Daròs, J.A. DICER-LIKE4 está implicado en la restricción del movimiento del virus del mosaico amarillo del calabacín en Nicotiana benthamiana. Oral presentation. XVIII Congreso Nacional de la Sociedad Española de Fitopatología (20/09/16-23/09/16), Palencia, Spain.
• Cordero, T., Rosado, A., Carbonell, A., Aragonés, V., Monzó, I., Jaramillo, A., Rodrigo, G. and Daròs, J.A. Detección de patógenos mediante circuitos reguladores de plantas. Poster. XVIII Congreso Nacional de la Sociedad Española de Fitopatología (20/09/16-23/09/16), Palencia, Spain.
• Carbonell, A., Fahlgren, N. Carrington, J.C. and Daròs J.A. Genome-wide identificatioon of ARGONAUTE-bound target RNAs in Arabidopsis. Oral presentation. XIII Reunión de Biología Molecular de Plantas (22/06/16-24/06/16), Oviedo, Spain.
• Carbonell, A. and Daròs JA. Producción de pequeños RNAs artificiales para el control de la expresión génica y defensa antiviral en plantas. Oral presentation. XII Reunión Ribored. (06/02/16-06/03/16), Madrid, Spain.

Results were published in peer-reviewed publications:
• Carbonell (2017). Methods in Molecular Biology 1640: 1-21.
• Carbonell (2017). Methods in Molecular Biology 1640: 93-112.
• Kénesi et al. (2017. Nucleic Acids Research (in press).
• Carbonell&Daròs (2017). Molecular Plant Pathology 18: 746-753.
• Cordero et al. Molecular Plant-Microbe Interactions 30: 63-71.
• Carbonell et al. Plant virus RNA replication. In: eLS.
• Carbonell et al. (2016). RNA and Disease 3: e1130.
• Carbonell&Carrington (2015). Current Opinion in Plant Biology 27: 111-117.
We identified a series of AGO target sites located in TSWV and PSTVd RNAs that are efficiently targeted by transiently expressed amiRNAs and syn-tasiRNAs. The stable expression in tomato plants of specific artificial sRNAs against these target sites should induce high levels of resistance.

Moreover, while the proteomics part of this project is aimed to identify multiple new AGO interactors, the transcriptomics approach based on the use of the optimized RIP-Seq methodology should identify authentic AGO target sites in viral RNAs. In both cases, the identification of these new viral RNA or protein interactors will facilitate the successful design and testing of new antiviral strategies for antiviral resistance, such as the use of amiRNAs or syn-tasiRNAs to target viral RNAs directly or to repress the transcripts of those interactors that are susceptibility factors.

Our project has also impacted positively the European society/economy in several ways. For example, several EU researchers have been trained at the host institution, and results have been communicated through periodical departmental/center seminars.By generating knowledge on how plant-virus interactions function and by identifying new viral targets from which engineer novel and effective antiviral tools to protect crops, our project is ensuring the availability and access to sufficient safe and nutritious food, which is a key priority that impacts all EU citizens and needs to be ensured today and in the future.
Schematic representation of key steps in antiviral RNA silencing in plants