N-acetyltransferase AAC(3)-I confers gentamicin resistance to Phytophthora palmivora and Phytophthora infestans

Background Oomycetes are pathogens of mammals, fish, insects and plants, and the potato late blight agent Phytophthora infestans and the oil palm and cocoa infecting pathogen Phytophthora palmivora cause economically impacting diseases on a wide range of crop plants. Increasing genomic and transcriptomic resources and recent advances in oomycete biology demand new strategies for genetic modification of oomycetes. Most oomycete transformation procedures rely on geneticin-based selection of transgenic strains. Results We established N-acetyltransferase AAC(3)-I as a gentamicin-based selectable marker for oomycete transformation without interference with existing geneticin resistance. Strains carrying gentamicin resistance are fully infectious in plants. We further demonstrate the usefulness of this new antibiotic selection to super-transform well-characterized, already fluorescently-labelled P. palmivora strains and provide a comprehensive protocol for maintenance and zoospore electro-transformation of Phytophthora strains to aid in plant-pathogen research. Conclusions N-acetyltransferase AAC(3)-I is functional in Phytophthora oomycetes. In addition, the substrate specificity of the AAC(3)-I enzyme allows for re-transformation of geneticin-resistant strains. Our findings and resources widen the possibilities to study oomycete cell biology and plant-oomycete interactions.


Table S1
Table S1. Gentamicin-based pTOR-Gateway vectors. Gentamicin resistance conferred by the aacC1 gene is indicated by the letter G, in addition to the previously described naming conventions (Evangelisti et al, 2019).

Media
Standard recipes for one liter of raw/clarified V8 agar medium are given below. For reference, see Miller PM (1955). Phytopathology 45: 461-462. 10% (raw) V8 agar medium. In a beaker, add 100 ml of V8 juice (commercially available) and 1 g of calcium carbonate (CaCO 3 , Sigma C6763). Mix thoroughly until the calcium carbonate is completely dissolved. Add 0.05 g of β-sitosterol (Sigma S1270) and bring to 1 L with distilled water. Add 15 g of agar (Sigma A1296). Sterilize by autoclaving at 15 psi for 20 min.
10% clarified V8 agar medium. Centrifuge 150 ml of V8 juice containing 1 g of calcium carbonate at 8000 g for 5 min. Transfer 100 ml of supernatant to a beaker, add β-sitosterol and agar (see above).
Sterilize by autoclaving at 15 psi for 20 min. Note: for 10% clarified liquid V8 medium, proceed exactly the same, omitting the agar.

Plasmids
Below is a list of plasmids that were successfully transformed with this protocol.

Backbone
Helper cassette Gateway cassette

P. palmivora axenic culture
Electro-transformation of P. palmivora zoospores requires production of bacteria-free, high concentration (10 6 zoospores/ml) suspensions of motile zoospores. This is achieved by (1) cleaning-up mycelium using an antibiotic cocktail to reduce bacterial cocultivation and (2) optimizing growth conditions and mycelium propagation.

Cleanup of P. palmivora mycelium
1. Inoculate a plate of V8 medium 1 containing rifampicin, cefotaxime and spectinomycin at 100 mg/L with a mycelium plug or a droplet of zoospores. Incubate at 25°C 2 for a week.
2. Excise an agar plug from the growing edge of the mycelium and transfer to a fresh plate of V8 medium containing the same antibiotics.

Axenic culture maintenance
1. Inoculate a plate of V8 medium with 4 equally spread mycelium plugs, or 1 ml of zoospore suspension. In our hands, the latter gives more sustained zoospore yield over time.

Plasmid preparation
We achieved successful P. palmivora transformation using at least 20 µg of plasmid DNA. Plasmid can be prepared using maxiprep kits from various manufacturers. Below is an alternative protocol using a plasmid miniprep kit (Qiagen, Germany) that we routinely use in the lab. Critical steps are indicated in bold.

Inoculate 100 ml of LB medium with a single
Escherichia coli colony. 12. Centrifuge at full speed for 1 min.
14. Centrifuge at full speed for 1 min.
Filter-sterilize and store at 4°C. 6 Precise counting with a haemocytometer is not required.   (Evangelisti et al, 2019).

Screening by PCR
When fluorescence is not available, presence of the transgene should be assessed by PCR. P. palmivora genomic DNA can be extracted from mycelium or zoospores using standard STES (Möller et al, 1992) or CTAB (Kim et al, 1990) protocols.
Note: Transgene expression level can vary within a single colony mycelium and also between independent transformants. We therefore do not recommend the establishment of monozoosporic lines from these transformants.

Cryo-preservation
Long-term storage of P. palmivora mycelium can be achieved through cryo-preservation in liquid nitrogen. For comparison of various cryo-preservation protocols, see Houseknecht et al, 2012.