Table 1.
Isolate details for the six Phytophthora species used in this study.
Table 2.
Primer pairs used for diagnostic PCR to detect plasmid DNA in Phytophthora transformants.
Fig 1.
Minimum inhibitory concentrations (MICs) of geneticin (G418) for Phytophthora cultures.
The G418 MIC was determined for each of the five forest Phytophthora species tested in this study and the agricultural pathogen P. sojae (used as a transformation control). Two replicates were tested for each species/isolate. *Two isolates of P. ramorum were tested, NA2 16_0386_0016 and NA2 17_0134_0030, and growth stopped for both at 5 μg/mL of G418. Figure created with BioRender.com (Agreement #MS26FYBSTT).
Fig 2.
Diagnostic PCR results for Phytophthora sojae wildtype and pYF2-PsCG transformants.
Gel electrophoresis of products from a PCR performed on DNA from Phytophthora sojae P6497 wildtype (PsWT; well 4) and 12 pYF2-PsCG transformant (pYF2-PsCG PsT; wells 5–10 and 12–17) cultures using the pYF_Cas9_Diag_F2/R2 primer pair (expected product size is 731 bp). The negative PCR control was a reaction using molecular-grade water instead of DNA (well 2). The positive PCR control was a pYF2-PsCG plasmid DNA extraction (wells 3). Note that the DNA used for the plasmid control was the same preparation that was used for both P. sojae transformations. The gel was run at 100 volts for 75 minutes on 1.75% agarose in 0.5X TBE buffer with a 100 bp DNA Ladder (Thermo Fisher Scientific, Waltham, MA, USA). All samples were loaded with Safe-Green™ stain (Applied Biological Materials Inc., Richmond, BC, Canada) at a ratio of 1 μL Safe-Green: 5 μL diluted PCR product.
Table 3.
Summary of Phytophthora transformation results.
Fig 3.
Growth of Phytophthora cactorum wildtype and pYF2-PsCG transformant cultures.
Transformations of Phytophthora cactorum FF42 with the pYF2-PsCG plasmid yielded stable transformants. (A) No growth of wildtype (untransformed) cultures on V8 agar + 40 μg/mL G418 relative to (B) growth of transformant cultures on the same medium. Photos were taken five days post plating. (C) Transformants growing stably on V8 agar + 40 μg/mL G418 after multiple rounds of sub-culturing. Photos were taken ten days post plating.
Fig 4.
Diagnostic PCR results for Phytophthora cactorum wildtype and pYF2-PsCG transformants.
Gel electrophoresis of products from a PCR performed on DNA from Phytophthora cactorum FF42 wildtype (PcWT; well 4) and pYF2-PsCG transformant (pYF2-PsCG PcT; wells 5–12 and 14–17) cultures using the eGFP_Diag_F1/R1 primer pair (expected product size is 274 bp). The negative PCR control was a reaction using molecular-grade water instead of DNA (well 2), and the positive PCR control was purified pYF2-PsCG plasmid DNA (well 3). The gel was run at 100 volts for 60 minutes on 1% agarose in 0.5X TBE buffer with a 100 bp DNA Ladder (Thermo Fisher Scientific, Waltham, MA, USA). All samples were loaded with Safe-Green™ stain (Applied Biological Materials Inc., Richmond, BC, Canada) at a ratio of 1 μL Safe-Green: 5 μL diluted PCR product. The pYF2-PsCG PcT4-S (well 15) transformant is highlighted in red because it struggled to grow through the second round of G418 selection (40 μg/mL), with roughly only 25% mycelial growth relative to the other three transformants in the same experiment (T1-S, T5-S, T8-S), however, a strong band amplified in the diagnostic PCR.
Fig 5.
Comparison of Phytophthora sojae and P. ramorum putative transformant mycelial growth.
Putative transformants (PTs) of the control species Phytophthora sojae P6497 (A) and P. ramorum NA2_17 (B) growing on V8 agar supplemented with 50 μg/mL G418 and 30 μg/mL G418, respectively, four days post transformation. While putative P. ramorum transformants did grow on three of the plates, the mycelial growth was weak and irregular (see arrows) relative to the P. sojae control. The white arrows indicate PTs that would have been considered “strong” for P. ramorum, whereas the grey arrows indicate very weak PTs. All P. ramorum PTs were taken through a second round of selection, but most did not grow at the increased concentration of G418; none of the “weak” PTs (grey arrows) grew, and only two of the “strong” PTs (white arrows) grew. The same pattern emerged for every P. ramorum transformation attempted.
Fig 6.
Diagnostic PCR results for Phytophthora ramorum wildtype and plasmid (pYF2-PsCG, pYF515) transformants.
Gel electrophoresis of products from a PCR performed on DNA from P. ramorum NA2_17 wildtype (PrWT; well 4), pYF515 transformant (pYF515 PrT; wells 5–7), and pYF2-PsCG transformant (pYF2-PsCG PrT; wells 10–14) cultures using the pYF_Cas9_Diag_F3/R3 primer pair (expected product size is 824 bp). The negative PCR control was a reaction using molecular-grade water instead of DNA (well 2). The positive PCR controls are pYF515 plasmid DNA (well 2) and pYF2-PsCG plasmid DNA (well 9). The gel was run at 50 volts for 90 minutes on 1.25% agarose in 0.5X TBE buffer with a 100 bp DNA Ladder (Thermo Fisher Scientific, Waltham, MA, USA). All samples were loaded with Safe-Green™ stain (Applied Biological Materials Inc., Richmond, BC, Canada) at a ratio of 1 μL Safe-Green: 5 μL diluted PCR product.