TITLE: Protocol 5: Agrobacterium-mediated transformation of Spizellomyces punctatus (Sp)
AUTHORS: Sarah M Prostak, Edgar M Medina, Erik Kalinka, Lillian Fritz-Laylin

[DESCRIPTION]
Once you have completed protocols 1-4, you are ready for transformation. This protocol is lengthy, requires accurate timing, and takes a minimum of 4 days to see results. Zoospores will need to be harvested from the plates prepared in Protocol 2; the process of harvesting is described below. Once Agro is at the appropriate OD, transformation is carried out by co-culturing Agro and Sp on induction media (“IM”) to induce the expression of Agro’s virulence genes and transformation of Sp cells. Co-culturing must be done on IM plates with premade depressions in the agar (see Protocol 4: Creating depressions in induction media plates).

[BEFORE_START]
On transformation day, Agro must be diluted to an OD660 of 0.15 and then grown to an OD660 of 0.6. Under our laboratory conditions, this takes about 4-6 hours, but this time should be empirically tested for each lab before starting to ensure proper and repeatable results. Be sure to take this growth time into account when planning all steps on and prior to transformation day.

[GUIDELINES]
This section outlines the transformation process from growing Agro to the end of co-culturing. For harvesting the co-culture and selecting transformants, see Protocol 6: Selecting for Spizellomyces punctatus transformants and Protocol 7: Picking colonies of transformed Spizellomyces punctatus (Sp). All steps of this protocol, except necessary centrifugation, must be carried out in a sterile environment, either in a laminar flow hood or in the sterile area around an open flame.

[STEPS]
SECTION: Growing Agro to the appropriate OD660
1. Dilute each overnight Agro culture to an OD660 = 0.15 using IM. 
 

SECTION: Growing Agro to the appropriate OD660
2. Grow the Agro at 28 °C and shaking at   until OD660 = 0.6. 
 



SECTION: Harvesting Sp zoospores for transformation
3. Flood all wild type Sp plates with 1 mL of DS or IM for 60 min.
 



SECTION: Harvesting Sp zoospores for transformation
4. Harvest Sp zoospores by running 1 mL of fresh DS or IM along the agar of one plate, holding the plate at a 45 degree angle. 
 


SECTION: Harvesting Sp zoospores for transformation
5. Take the liquid from the first plate and use it to harvest the zoospores on a second plate in the same manner.
SECTION: Harvesting Sp zoospores for transformation
6. Do this for all plates of Sp that were flooded in step 3.
 

SECTION: Harvesting Sp zoospores for transformation
7. Pool all zoospores into a 50 mL conical (or other appropriately sized tube).
SECTION: Harvesting Sp zoospores for transformation
8. Pass the zoospore suspension through a 40 μm mesh filter into a new 50 mL conical tube. 
 


SECTION: Harvesting Sp zoospores for transformation
9. Use a 18 gauge needle and an appropriately sized syringe to take up all of the 40 μm-filtered zoospore suspension. 
 

SECTION: Harvesting Sp zoospores for transformation
10. Use a sterile 25 mm syringe filter preloaded with grade 1 Whatman paper to further filter the spores into a new 50 mL conical tube (or other appropriately sized tube). 
 

SECTION: Co-culturing Agro and Sp in varying ratios
11. Per each control and plasmid to be transformed, prepare and label four microcentrifuge tubes according to Figure 3.
SECTION: Co-culturing Agro and Sp in varying ratios
12. Pipette the appropriate amounts of IM and the final solutions from protocols section "Growing Agro to the appropriate OD660" and "Harvesting Sp zoospores for transformation" into the appropriate microcentrifuge tube in the order they appear in Figure 3. 
 

SECTION: Co-culturing Agro and Sp in varying ratios
13. Gently pipette to mix the contents of the microfuge tube.
SECTION: Co-culturing Agro and Sp in varying ratios
14. Transfer all 200 µL of an Agro-Sp co-culture to one of the premade depressions on a room temperature IM plate. 
 


SECTION: Co-culturing Agro and Sp in varying ratios
15. GENTLY slide the IM plates to a place where they will not be disturbed. 
 

SECTION: Co-culturing Agro and Sp in varying ratios
16. Leave the plates to dry at Room temperature for 12-24 hours.

SECTION: Co-culturing Agro and Sp in varying ratios
17. Seal plates with parafilm, invert, and grow in a closed chamber (such as a plastic storage container) at Room temperature for 4 days.