Alternative splicing and mRNA levels of the disease resistance gene RPS4 are induced during defense responses.

The Arabidopsis (Arabidopsis thaliana) disease resistance protein RESISTANCE TO PSEUDOMONAS SYRINGAE4 (RPS4) activates defenses in response to bacterial pathogens expressing avrRps4 in a gene-for-gene specific manner. The RPS4 gene produces multiple transcripts via alternative splicing of two regular introns flanking exon 3 and a cryptic intron within exon 3. We showed previously that RPS4-mediated resistance requires the combined presence of transcripts encoding both full-length and truncated open reading frames. Here, we demonstrate that alternative splicing of RPS4 undergoes dynamic changes specifically during the resistance response. Furthermore, RPS4 expression was induced by the presence of AvrRps4 in an EDS1-dependent manner. Interestingly, inducible alternative splicing was not limited to the avrRps4-RPS4 interaction, indicating that regulation of alternative splicing may be a general response to prime the plant stress response system. Intron-deficient transgenes lacking only one intron were previously shown to be nonfunctional. Here, we establish quantitatively that the absence of one intron had no effect on the splicing frequency of remaining introns. Given the lack of functionality of single intron-deficient transgenes, this suggests that the products of individual transcripts have distinct functions during RPS4-triggered resistance. Transient expression of truncated RPS4 proteins in Nicotiana benthamiana induced hypersensitive response-like cell death in the absence of AvrRps4. Interestingly, different truncated proteins had markedly differing stability. In summary, RPS4 function is regulated at multiple levels, including gene expression, alternative splicing, and protein stability, presumably to fine-tune activity and limit damage inflicted by activated RPS4 protein.