Infection-responsive pre-mRNA splicing and developmentally dynamic intron-derived RNA species of the grapevine CYP736B defense gene

Abstract Cytochrome P450 defense genes are transcriptionally reprogrammed during pathogen attack, but the fate of their intron-derived RNA as the plant grows is rarely examined. The single-intron grapevine gene CYP736B is up-regulated in Pierce’s-disease–resistant vines and down-regulated in susceptible vines after infection by Xylella fastidiosa (Xf). Re-analyzing a 2008–2009 dataset, we used reverse-transcription PCR with two divergently oriented intron-internal primers across the 71-nt CYP736B intron and detected multiple discrete cDNA products whose abundance varied reproducibly with genotype (resistant 9621-67 vs susceptible 9621-94) and, across a 1-, 6- and 10-week post-inoculation series spanning the vine’s vegetative growth, with developmental time. Products formed only from intron-containing RNA templates, not from genomic DNA, RNase-treated genomic DNA, or oligo(dT)-primed cDNA. Cloning and Sanger sequencing resolved two recurrent junction classes: short intron-only loops and longer chimeras additionally containing upstream exon-1 sequence, joined near a candidate branch-point adenosine within a CUGAG motif ~ 18 nt upstream of the 3′ AG acceptor. The resistant and susceptible lines yielded overlapping but non-identical junction repertoires with reproducible genotype-associated sequence differences. We interpret the exon-containing junctions with an exon-docking (hybrid-lariat) hypothesis—an exonic site 5′ of the intron receiving 2′–5′ attack from the intronic branch point—and weigh it equally against the leading alternative, reverse-transcriptase template switching and PCR chimerism, which the present data cannot exclude. We define the experiments (RNase-R resistance, lariat/branch-point sequencing, primer-independent detection, debranching) that would discriminate them. The work documents developmentally dynamic, infection-responsive intron-derived RNA from a plant defense gene and offers a testable model for branch-point selection.