Project

Genome-wide analysis of RNA and protein interacting profiles during a plant virus infection (InterAcTEV, H2020 Marie S. Curie Reintegration Grant 655841)

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

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Alberto Carbonell
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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. 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, virus-derived sRNAs (vsRNAs) associate with a host ARGONAUTE (AGO) protein, and sRNAs guide the AGO to interact and repress viral RNAs sharing sequence complementary with them. Once the ternary complex is formed, the viral RNA is ‘sliced’ (AGOs possess endonucleolytic activity), translationally repressed, or inactivated through other mechanisms. The second antiviral mechanism 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.
Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are two classes of artificial sRNAs engineered to silence specific transcripts in plants. They are produced in planta by expressing a functional miRNA or tasiRNA precursor with modified miRNA/miRNA* or tasiRNA sequences, respectively. AmiRNAs and syn-tasiRNAs are generated differently, but function similarly. One of the strands of the amiRNA or syn-tasiRNA duplex associates with an AGO protein to silence highly sequence complementary transcripts usually through AGO-mediated endonucleolytic cleavage. Both classes of artificial sRNAs have been used to selectively inactivate endogenous and reporter genes, as well as viral RNAs to confer antiviral resistance in transgenic plants.
This project intends to better understand the molecular interplay occurring during plant/virus interactions. Our main goal is to identify new viral targets to develop effective antiviral resistance for crop protection. In particular, we want 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. The stable expression in tomato plants of specific amiRNAs and/or syn-tasiRNAs against these target sites should induce high levels of antiviral/antiviroid resistance. 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. Our goal is to create a protein interactor map for each tagged TEV protein. This is currently being done through a proteomics approach including affinity purification and mass spectrometry analysis of host proteins associating with each TST-tagged TEV protein, and through a systems biology approach by generating the network maps through collaboration with in-house expertise.
 
Alberto Carbonell
added a project goal
This project intends to better understand the molecular interplay occurring during plant/virus interactions. Our main goal is to identify new viral targets to develop effective antiviral resistance for crop protection. In particular, we want to identify AGO target sites highly susceptible to artificial sRNA-mediated inactivation, as well as plant proteins interacting with viral proteins upon infection.