Conserved exitrons of FLAGELLIN-SENSING 2 (FLS2) across dicot plants and their functions

The alternative splicing of pattern recognition receptor genes regulates immune signalling in mammals, but in plants its role is still unknown. Here, we detected alternatively spliced introns (exitrons) in the first annotated exons of FLAGELLIN-SENSING 2 (FLS2) genes in all the examined dicot plants across nine families. The 5' splice site (SS) regions were conserved and with rare synonymous substitutions. Point mutations and gene swaps indicated that the position and efficiency of exitron splicing primarily depended on the nucleotide sequences of FLS2 genes. Single-nucleotide mutations in the invariable codon carrying 5' SS dramatically altered the accumulation of poplar and tomato FLS2 transcripts, indicating the 5'-proximal exitrons of FLS2 function as stimulatory introns on gene expression. The 3' SSs of exitrons are diverse and can be changed by 1-2 nucleotide mutations in Salicaceae FLS2. The alternative transcripts (ATs) of poplar and tobacco FLS2, which encode small secreted proteins, were specifically induced by flg22, and one such AT from tobacco FLS2 suppressed flg22-induced response. Our results indicated that the exitrons of FLS2 genes regulate the accumulation of transcripts by an intron mediated enhancement (IME) mechanism and some ATs have the potential to encode suppressors for FLS2 pathway.