PcMuORP1, an Oxathiapiprolin-Resistance Gene, Functions as a Novel Selection Marker for Phytophthora Transformation and CRISPR/Cas9 Mediated Genome Editing

• Published in: Frontiers in microbiology Vol. 10; p. 2402
• Main Authors: Wang, Weizhen, Xue, Zhaolin, Miao, Jianqiang, Cai, Meng, Zhang, Can, Li, Tengjiao, Zhang, Borui, Tyler, Brett M., Liu, Xili
• Format: Journal Article
• Language: English
• Published: Frontiers Media S.A 22-10-2019
• Subjects: CRISPR/Cas9; Microbiology; oomycetes; oxathiapiprolin-resistance; Phytophthora; selection marker; transformation
• ISSN: 1664-302X; 1664-302X
• DOI: 10.3389/fmicb.2019.02402

Phytophthora
, a genus of oomycetes, contains many devastating plant pathogens, which cause substantial economic losses worldwide. Recently, CRISPR/Cas9-based genome editing tool was introduced into
Phytophthora
to delineate the functionality of individual genes. The available selection markers for
Phytophthora
transformation, however, are limited, which can restrain transgenic manipulation in some cases. We hypothesized that
PcMuORP1
, an endogenous fungicide resistance gene from
P. capsici
that confers resistance to the fungicide oxathiapiprolin via an altered target site in the ORP1 protein, could be used as an alternative marker. To test this hypothesis, the gene
PcMuORP1
was introduced into the CRISPR/Cas9 system and complementation of a deleted gene in
P. capsici
was achieved using it as a selection marker. All of the oxathiapiprolin-resistant transformants were confirmed to contain the marker gene, indicating that the positive screening rate was 100%. The novel selection marker could also be used in other representative
Phytophthora
species including
P. sojae
and
P. litchii
, also with 100% positive screening rate. Furthermore, comparative studies indicated that use of
PcMuORP1
resulted in a much higher efficiency of screening compared to the conventional selection marker
NPT II
, especially in
P. capsici.
Successive subculture and asexual reproduction in the absence of selective pressure were found to result in the loss of the selection marker from the transformants, which indicates that the
PcMuORP1
gene would have little long term influence on the fitness of transformants and could be reused as the selection marker in subsequent projects. Thus, we have created an alternative selection marker for
Phytophthora
transformation by using a fungicide resistance gene, which would accelerate functional studies of genes in these species.