Ommochrome pathway genes kynurenine 3-hydroxylase and cardinal participate in eye pigmentation in Plutella xylostella

Abstract

Background Eye pigmentation genes have been utilized as visible markers for constructing genetic control prototypes in several insect vectors of human disease. Here, orthologs of two ommochrome pathway genes, kynurenine 3-hydroxylase (kmo) and cardinal, were investigated in Plutella xylostella, a globally distributed, economically important pest of Brassica crops. Results Both somatic mosaic and germline mutations were efficiently created using the CRISPR/Cas9 system, and null mutant strains of Pxkmo and Pxcardinal were obtained. A frame-shift mutation in Pxkmo caused yellow compound eyes at adult stage while an in-frame mutation lacking two amino acids resulted in a hypomorphic red eye phenotypes. In contrast, Pxcardinal-deficient moths with a frame-shift mutation exhibited yellow eye pigmentation in newly emerged adults which turned to red as the adults aged. Additionally, differences were observed in the coloration of larval ocelli, brains and testes in Pxkmo and Pxcardinal yellow-eye mutant lines. Conclusions Our work identifies the important roles of Pxkmo and Pxcardinal in P. xylostella eye pigmentation and provides tools for future genetic manipulation of this important crop pest.

Towards CRISPR/Cas9-based gene drive in the diamondback moth Plutella xylostella

“…A random selection of WT F2 knockout mutants were individually sequenced at the relevant target site. Multiple indel alleles were observed separate from those previously established as occurring in each knockout line 26 (Figure S6).…”

“…DBM transgenic lines were generated from the Vero Beach wildtype strain. Rearing conditions were as described previously 25,26 .…”

“…For constructing homing elements, sgRNAs were in vitro transcribed (150 ng/ul), complexed with 300 ng/ul Cas9 protein 26 and injected alongside the relevant sgRNA plasmid (800 ng/ul). Injection, transformant and insertion-site identification were as previous 26,32 .…”

“…For constructing homing elements, sgRNAs were in vitro transcribed (150 ng/ul), complexed with 300 ng/ul Cas9 protein 26 and injected alongside the relevant sgRNA plasmid (800 ng/ul). Injection, transformant and insertion-site identification were as previous 26,32 . For resolving the 5' junction between the integrated homology arm and the genomic flanking sequence in 1619P15, the Cas9 Sequencing Kit (Oxford Nanopore Technologies, UK) was used, as PCR could not confirm the junction.…”

“…Homozygous null-mutations in Pxyellow or Pxkmo cause visible phenotypes in the body or eye pigmentation of DBM, respectively, making them scorable markers for genome editing events 26,35 . As such we chose these two genes as targets for building sgRNA expression lines.…”