Alternative splicing of mini-exons in the Arabidopsis leaf rust receptor-like kinase LRK10 genes affects subcellular localisation

Abstract

AtLRK10L1.2 produces a variety of alternatively spliced variants in the region a mini-exon and skipping of the mini-exon alters the subcellular localization of the protein. We have examined expression and alternative splicing in the gene encoding Arabidopsis LRK10-like 1 (AtLRK10L1) which is most closely related to wheat leaf rust 10 disease-resistance locus receptor-like protein kinase (LRK10). AtLRK10L1 produces two different transcripts, LRK10L1.1 and 1.2 through the use of two different promoters. We found no evidence of alternative splicing for the AtLRK10L1.1 transcript but identified numerous alternative splicing variants of AtLRK10L1.2 by sequencing of cloned cDNAs prepared from RNA isolated from whole cell, nucleolar and nucleoplasmic fractions. Many of these transcripts contained unspliced introns and accumulated differentially in the nucleolus and the nucleoplasm consistent with intron retention transcripts being retained in the nucleus (Gohring et al., Plant Cell 26:754-764, 2014). We examined the fate of different alternatively spliced transcripts by fusing variants to YFP and expressing them by agroinfiltration in Nicotiana benthamiana. AtLRK10L1 contains a 45 nt mini-exon which encodes part of a putative transmembrane domain. Full-length cDNA of LRK10L1.2 fused to YFP targeted the fusion protein to the plasma membrane while expression of transcripts where the mini-exon had been deleted, altered the localization of the fusion protein to the endoplasmic reticulum. Similarly, expression of full-length and mini-exon deleted versions of three other members of the LRK10 receptor-like kinase (RLK) gene family also showed the switch in localization. Thus, the mini-exons in Arabidopsis LRK10 genes are required for localization to the plasma membrane.