Virus-Induced Gene Silencing for Identifying Autoimmune Regulators in Arabidopsis.

Virus-induced gene silencing (VIGS) has been applied as a functional genomics tool across diverse plant species. Integrated with the Arabidopsis sequence-tagged T-DNA homozygous mutant library, VIGS enables an efficient screening approach that combines features of both forward and reverse genetics, facilitating the identification of novel regulators in plant immunity. Plant defense against pathogens relies on a two-layered immune system, classified as pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Dysregulation of key PTI or ETI components can lead to excessive or uncontrolled cell death. The cell death phenotype offers a unique avenue for genetic screens aimed at identifying suppressors of immune-related cell death. However, conventional genetic approaches face limitations due to seedling lethality and the consequent lack of viable seeds, restricting their efficiency. Here, we describe an Agrobacterium-mediated transient VIGS assay optimized for systematic gene silencing at seedling stages, leading to cell death phenotypes. This method enables high-throughput screening for cell death suppressors using T-DNA homozygous mutant collections. The platform provides a rapid, cost-efficient strategy for uncovering key regulators of plant immune signaling, offering new insights into mechanisms governing immune homeostasis and cell death suppression.