A Ralstonia solanacearum effector targets splicing factor SR34a to reprograms alternative splicing and regulates host immunity

Alternative splicing (AS) is a critical post-transcriptional regulatory mechanism in eukaryotes. While infection with Ralstonia solanacearum GMI1000 significantly alters plant AS patterns, the underlying molecular mechanisms remain unclear. Herein, we investigate the effects of GMI1000 Type III secretion system (T3SS) effector proteins on AS in the tomato cultivar Heinz 1706. RNA sequencing (RNA-seq) confirmed genome-wide AS changes induced by the T3SS in tomato, in-cluding 1386 differential alternative splicing events in 1023 differentially alternatively spliced genes, many of which are associated with plant defense. Seven nucleus-localized Type III effec-tors(T3Es) were transiently expressed in an RLPK splicing reporter system transgenic tobacco, identifying RipP2 as an effector that modulates AS levels. Sequence analysis, protein-protein in-teraction assays, and AlphaFold2 structural predictions revealed that RipP2 interacts with the tomato splicing factor SR34a.Furthermore, RipP2 acetylates a conserved lysine at position 132 within the SWQDLKD motif of SR34a, regulating its splicing pattern in defense-related genes and modulating plant immunity. This study elucidates how the "Rip-SR34a module" influences plant immune responses by regulating the alternative splicing of immune-related genes, providing new insights into the molecular mechanisms of pathogen-plant interactions, particularly in splicing regulation. The study includes RNA samples extracted from Solanum lycopersicum (tomato plants) to investigate the regulatory effects of Type III secretion system effectors from Ralstonia solanacearum strain GMI1000 on alternative splicing (AS). The experimental design comprises two main groups: a control group and an experimental group. Each group includes three biological replicates to ensure reproducibility and accuracy. Tomato plants treated with a non-virulent strain (hrcV mutant) of Ralstonia solanacearum (Samples: hrcV-1, hrcV-2, hrcV-3). Tomato plants treated with the wild-type strain GMI1000 of Ralstonia solanacearum (Samples: GMI1000-1, GMI1000-2, GMI1000-3). The variables under investigation include the presence or absence of the effector proteins from R. solanacearum and their impact on alternative splicing patterns in the host plant. Control samples help to establish a baseline for comparison with the experimental samples, which are expected to exhibit changes in splicing patterns due to the presence of the effectors.