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

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 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 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.

Automated summary

In this study, a mutated version of the Phytophthora capsici oxysterol-binding protein-related protein 1 (PcMuORP1) was used as a selection marker for genetic transformation in Pythium ultimum. The knockout of PuM90 gene resulted in significant downregulation of PuLLP expression, and reintroduction of PuLLP using PcMuORP1 restored oospore production. PcMuORP1 showed higher screening efficiency and little long-term effect on fitness, making it a potential alternative selection marker. This study contributes to the functional genomic investigation of oomycetes.