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Nucleotide-binding leucine-rich repeat (NLR) proteins serve as immune receptors in both plants and animals. To identify components required for NLR-mediated immunity, we designed and carried out a chemical genetics screen to search for small molecules that can alter immune responses in Arabidopsis thaliana . From 13 600 compounds, we identified Ro 8-4304 that was able to specifically suppress the severe autoimmune phenotypes of chs3-2D ( chilling sensitive 3, 2D ), including the arrested growth morphology and heightened PR ( Pathogenesis Related ) gene expression. Further, six Ro 8-4304 insensitive mutants were uncovered from the Ro 8-4304-insensitive mutant ( rim ) screen using a mutagenized chs3-2D population. Positional cloning revealed that rim1 encodes an allele of AtICln (I, currents; Cl, chloride; n, nucleotide). Genetic and biochemical analysis demonstrated that AtICln is in the same protein complex with the methylosome components small nuclear ribonucleoprotein D3b (SmD3b) and protein arginine methyltransferase 5 (PRMT5), which are required for the biogenesis of small nuclear ribonucleoproteins (snRNPs) involved in mRNA splicing. Double mutant analysis revealed that SmD3b is also involved in the sensitivity to Ro 8-4304, and the prmt5-1 chs3-2D double mutant is lethal. Loss of AtICln , SmD3b , or PRMT5 function results in enhanced disease resistance against the virulent oomycete pathogen Hyaloperonospora arabidopsidis Noco2, suggesting that mRNA splicing plays a previously unknown negative role in plant immunity. The successful implementation of a high-throughput chemical genetic screen and the identification of a small-molecule compound affecting plant immunity indicate that chemical genetics is a powerful tool to study whole-organism plant defense pathways. [ABSTRACT FROM AUTHOR]