Gene editing with an oxathiapiprolin resistance selection marker reveals that PuLLP, a loricrin-like protein, is required for oospore development in Pythium ultimum

Gene editing with an oxathiapiprolin resistance selection marker reveals that PuLLP, a loricrin-like protein, is required for oospore development in Pythium ultimum

Blog Article

Abstract Oomycetes, such as Pythium species, contain numerous devastating plant pathogens that inflict substantial economic losses worldwide.Although CRISPR/Cas9-based genome editing is available, the selection markers available for genetic transformation in these species are limited.In this study, a mutated version of the Phytophthora capsici oxysterol-binding protein-related protein 1 (PcMuORP1), known to confer oxathiapiprolin resistance, was introduced into the CRISPR/Cas9 system for in situ complementation in Pythium ultimum.We targeted PuLLP, which encodes a loricrin-like protein, and showed significant downregulation when the Puf RNA-binding protein-encoding gene PuM90 was knocked out.

The PuLLP knockout mutants Girls Hats could not produce oospores, indicating a similar biological function as PuM90.The reintroduction of PuLLP into the knockout mutant using PcMuORP1 as a selection marker restored oospore production.Further comparisons with the conventional selection marker NPTII indicated that PcMuORP1 could be applied at a lower concentration and cost, resulting in a higher screening efficiency.Successive subculturing in the absence of selective pressure showed that PcMuORP1 had little long-term effect on the fitness of transformants.

Hence, it could be reused as an alternative selection marker.This study demonstrates the successful implementation of the PcMuORP1 Coaster Set gene as a selection marker in the genetic transformation of Py.ultimum and reveals the loricrin-like protein PuLLP as a sexual reproduction-related factor downstream of the Puf RNA-binding protein PuM90.Overall, these results will help accelerate the functional genomic investigation of oomycetes.