An intragenic mutagenesis strategy in Physcomitrella patens to preserve intron splicing

Abstract

Gene targeting is a powerful reverse genetics technique for site-specific genome modification. Intrinsic homologous recombination in the moss Physcomitrella patens permits highly effective gene targeting, a characteristic that makes this organism a valuable model for functional genetics. Functional characterization of domains located within a multi-domain protein depends on the ability to generate mutants harboring genetic modifications at internal gene positions while maintaining the reading-frames of the flanking exons. In this study, we designed and evaluated different gene targeting constructs for targeted gene manipulation of sequences corresponding to internal domains of the DEFECTIVE KERNEL1 protein in Physcomitrella patens. Our results show that gene targeting-associated mutagenesis of introns can have adverse effects on splicing, corrupting the normal reading frame of the transcript. We show that successful genetic modification of internal sequences of multi-exon genes depends on gene-targeting strategies which insert the selection marker cassette into the 5′ end of the intron and preserve the nucleotide sequence of the targeted intron.

DEK1 displays a strong subcellular polarity during Physcomitrella patens 3D growth

“…Upon successive genotyping for the loss of the WT locus using the primer pair P‐1/P‐2, the presence of 5′ vector insertion with the primer pair P‐3/P‐5 and the presence of the vector 3′ insertion with the primer pair P‐4/P‐6 and finally the detection of a single copy of the insert using the primer pair P‐3/P‐4, three transformants were found to contain a single insertion at the locus. The observed Δdek1 phenotype indicated that the construct insertion generated a null phenotype potentially caused by the mis‐splicing of the modified intron that contained the resistance cassette, a phenomenon observed previously in Physcomitrella (Demko et al , ; Johansen et al , ; Ako et al , ). To remove the resistance cassette, we subsequently transiently expressed the Cre recombinase (Trouiller et al , ) in protoplasts of the primary transformant dek1‐pArrowf1‐1 containing a single insert at the locus.…”

“…Insertion of a fluorescent tag within a native locus can generate unwanted phenotypes either due to the vector insertion or from the tag itself (Ako et al , ). In order to minimize this risk, we adopted the two steps strategy used previously in partial gene deletion in P. patens (Demko et al , ).…”

Membrane-anchored calpains – hidden regulators of growth and development beyond plants?

“…How exactly the multi-spanning transmembrane domain and Linker domain contribute to the DEK1 function, and especifically to the calpain regulation, remains largely unknown. Important data suggesting a regulatory role of the MEM and Linker domains have been provided by targeted mutagenesis of P. patens DEK1 ( Demko et al., 2014 ; Johansen et al., 2016 ; Ako et al., 2017 ) and electrophysiology studies of plants with modified DEK1 accumulation in A. thaliana ( Tran et al., 2017 ).…”

Challenges of in vivo protein localization in plants seen through the DEK1 protein lens

“…The protein model was generated using IBS. 11 these specific loci could yield improper gene splicing 21 and thus to the absence of functional DEK1. Alternatively, the presence of a tag at these specific positions could impede totally DEK1 protein synthesis or its further function and lead to the Δdek1 phenotype.…”