11RNA-based silencing functions as an important antiviral immunity mechanism in plants. 12 Plant viruses evolved to encode viral suppressors of RNA silencing (VSRs) that 13interfere with the function of key components in the silencing pathway. As effectors in 14 the RNA silencing pathway, ARGONAUTE (AGO) proteins are targeted of by some 15VSRs, such as that encoded by Turnip crinkle virus (TCV). A VSR-deficient TCV mutant 16 was used to identify AGO proteins with antiviral activities during infection. A quantitative 17phenotyping protocol using an image-based color trait analysis pipeline on the PlantCV 18 platform, with temporal red, green and blue (RGB) imaging and a computational 19 segmentation algorithm, was used to measure plant disease after TCV inoculation. This 20 process captured and analyzed growth and leaf color of Arabidopsis plants in response 21to virus infection over time. By combining this quantitative phenotypic data with 22 molecular assays to detect local and systemic virus accumulation, AGO2, AGO3, and 23AGO7 were shown to play antiviral roles during TCV infection. In leaves, AGO2 and 24 AGO7 functioned as prominent non-additive, anti-TCV effectors, while AGO3 played a 25 minor role. Other AGOs were required to protect inflorescence tissues against TCV. 26Overall, these results indicate that distinct AGO proteins have specialized, modular 27 roles in antiviral defense across different tissues, and demonstrate the effectiveness of 28image-based phenotyping to quantify disease progression. 29 30
Author Summary
31Plant viruses caused substantial losses in crop production and quality worldwide. 32Precisely measuring plant health is critical for better understanding the mechanisms 33underlying plant virus and host interactions. Advances in high-resolution imaging 34technologies and deep-learning tools have made acquiring and analyzing "big data" of 35 disease traits possible. In this study, we have developed a high-throughput, image-36 based trait phenotyping pipeline to quantify disease severity in Arabidopsis thaliana 37 infected by Turnip Crinkle Virus (TCV). Our aim is to understand how the antiviral RNA 38 silencing machinery is tuned to protect the host from invading virus infection. We 39 focused on ARGONAUTE proteins, which are the effectors in the RNA silencing 40 pathway. A mutant line of TCV with a dysfunctional silencing suppressor (P38) was 41 used to investigate which ago mutation could compensate for the dysfunctional 42 silencing suppressor and facilitate the development of disease symptoms. We 43 demonstrated that specific AGO proteins contribute to protecting leaves from TCV 44 infection in a non-additive manner. Our results also implied that distinct AGOs are 45 required to function collectively to silence TCV in inflorescence tissues. More evidence 46is still needed to further understand how these antiviral AGOs interact with suppressor 47 proteins molecularly during TCV infection. 48