In plants, ARGONAUTEs (AGOs) are the effector proteins functioning in small RNA (sRNA)-directed gene silencing pathways controlling key biological processes such as development, chromatin remodeling, stress responses and antiviral defense. AGOs interact with other proteins, load sRNAs, and target and silence specific RNAs through sequence-specific interactions conferred by their guide sRNA. AGOs can be programmed with highly-specific, computationally-designed artificial sRNAs to silence the desired gene(s) in gene function studies or for crop improvement.
We seek to develop both new basic knowledge and biotechnological tools for crop improvement while addressing two major gaps in the plant AGO field.
1. GENOME-WIDE IDENTIFICATION OF AGO INTERACTOME
The complete repertoire of target RNAs and protein interactors of a plant AGO is currently unknown. We use a multidisciplinary approach to identify, at a genome-wide level, the complete target RNA and protein interactomes of Arabidopsis AGO1, and to assess to what extent these interactomes are dynamic during a well-characterized stress response such as high-salinity. For target RNA identification, we apply our recently developed RNA immunoprecipitation followed by high-throughput silencing (RIP-seq) methodology using AGO1 catalytic mutants to capture and sequence AGO1 target RNAs. For protein interactor identification, we affinity purify AGO1 complexes previously tagged with the highly-efficient Twin-Step tag (TST) system, and use high-performance mass spectrometry platforms to identify bona fide interactors.
2. ARTIFICIAL sRNA-BASED AGO1 PROGRAMMING FOR ANTIVIRAL RESISTANCE AND FINE-TUNE REGULATION IN PLANTS
sRNA-based gene silencing tools have been optimized for time and cost effectiveness, high-throughput applicability and gene silencing efficacy, but do not allow the fine-tune control of the activity of the artificial sRNA to induce a desired degree of gene silencing. We use our recently developed sRNA-based tools to explore if AGO1 can be selectively programmed through specific artificial sRNAs to fine-tune regulate gene expression in Arabidopsis, and generate effective and durable antiviral resistance in crops such as tomato.
In conclusion, our research will help to understanding how silencing information is processed and expressed at the AGO complex level, particularly during the response to high-salinity stress, and will provide a framework with which other biotic and abiotic stress responses can be examined. We also expect developing new artificial sRNA-based tools for fine-tune controlling gene expression and inducing strong antiviral resistance in plants. Indeed, the knowledge and tools generated here could serve to develop more effective biotechnological approaches for protecting crops against critical stresses and ensure food availability for the next years.