PT - JOURNAL ARTICLE AU - Xingguo Zheng AU - Noah Fahlgren AU - Arash Abbasi AU - Jeffrey C. Berry AU - James C. Carrington TI - Antiviral Functions of ARGONAUTE Proteins During <em>Turnip Crinkle Virus</em> Infection Revealed by Image-based Trait Analysis in <em>Arabidopsis</em> AID - 10.1101/487322 DP - 2018 Jan 01 TA - bioRxiv PG - 487322 4099 - http://biorxiv.org/content/early/2018/12/04/487322.short 4100 - http://biorxiv.org/content/early/2018/12/04/487322.full AB - RNA-based silencing functions as an important antiviral immunity mechanism in plants. Plant viruses evolved to encode viral suppressors of RNA silencing (VSRs) that interfere with the function of key components in the silencing pathway. As effectors in the RNA silencing pathway, ARGONAUTE (AGO) proteins are targeted of by some VSRs, such as that encoded by Turnip crinkle virus (TCV). A VSR-deficient TCV mutant was used to identify AGO proteins with antiviral activities during infection. A quantitative phenotyping protocol using an image-based color trait analysis pipeline on the PlantCV platform, with temporal red, green and blue (RGB) imaging and a computational segmentation algorithm, was used to measure plant disease after TCV inoculation. This process captured and analyzed growth and leaf color of Arabidopsis plants in response to virus infection over time. By combining this quantitative phenotypic data with molecular assays to detect local and systemic virus accumulation, AGO2, AGO3, and AGO7 were shown to play antiviral roles during TCV infection. In leaves, AGO2 and AGO7 functioned as prominent non-additive, anti-TCV effectors, while AGO3 played a minor role. Other AGOs were required to protect inflorescence tissues against TCV. Overall, these results indicate that distinct AGO proteins have specialized, modular roles in antiviral defense across different tissues, and demonstrate the effectiveness of image-based phenotyping to quantify disease progression.Author Summary Plant viruses caused substantial losses in crop production and quality worldwide. Precisely measuring plant health is critical for better understanding the mechanisms underlying plant virus and host interactions. Advances in high-resolution imaging technologies and deep-learning tools have made acquiring and analyzing “big data” of disease traits possible. In this study, we have developed a high-throughput, image-based trait phenotyping pipeline to quantify disease severity in Arabidopsis thaliana infected by Turnip Crinkle Virus (TCV). Our aim is to understand how the antiviral RNA silencing machinery is tuned to protect the host from invading virus infection. We focused on ARGONAUTE proteins, which are the effectors in the RNA silencing pathway. A mutant line of TCV with a dysfunctional silencing suppressor (P38) was used to investigate which ago mutation could compensate for the dysfunctional silencing suppressor and facilitate the development of disease symptoms. We demonstrated that specific AGO proteins contribute to protecting leaves from TCV infection in a non-additive manner. Our results also implied that distinct AGOs are required to function collectively to silence TCV in inflorescence tissues. More evidence is still needed to further understand how these antiviral AGOs interact with suppressor proteins molecularly during TCV infection.