A robust high-throughput functional screening assay for plant pathogen effectors using the TMV-GFP vector.

Abstract

Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant-pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)-GFP vector and Agrobacterium-mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV-GFP system, we initially tested it with well-described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV-GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV-GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV-GFP system in identifying apoplastic effectors. The easy operation, time-saving nature, broad effectiveness, and low technical requirements of the TMV-GFP system make it a promising approach for high-throughput screening of effectors with immune interference activity from various pathogens.

Plant phenotyping relevance

植物免疫干渉をGFP蛍光で評価する高スループットスクリーニング系を開発・検証しており、植物状態の取得法が研究の中心である。

Code and data availability

The article describes a TMV-GFP effector screening assay with GFP fluorescence quantification via ImageJ/Fiji, but no paper-specific public dataset, image repository, analysis code, or trained model is deposited. The data availability statement only points to the paper and its supplementary figures/tables (primers, CDS

No evidence-backed public reproduction asset is currently recorded.