Wheat Effector Protein Infiltration Assay

Effector assays were performed using a simple leaf infiltration technique (Oliver et al. 2009 (link)). SnTox1 (SNOG_20078) and SnTox3 (SNOG_08981) effectors were produced in Pichia pastoris using the pGAPzA expression system (Thermo Fisher Scientific, MA, USA) (Liu et al. 2009 (link), 2012 (link)). SnToxA was produced in E. coli using the pET system (Novagen) by the UQ Protein Expression Facility at the University of Queensland, Australia (Tan et al. 2012 (link)). All proteins were in 20 mM sodium phosphate buffer pH 7.0 prior to infiltration. A needleless one cc plastic syringe was used to infiltrate the expressed proteins into the first leaf of 2-week-old wheat seedlings. Plants were returned to the Conviron growth chamber for 4 days for SnToxA- and SnTox3-induced necrosis and 7 days for SnTox1. Wheat plants were visually evaluated for effector sensitivity using a scale of ‘0’ to ‘4’, where a score of 0 indicates no observable reactions; 1, mild chlorosis; 2, chlorosis; 3, chlorosis with mild necrosis; 4, necrosis (Tan et al. 2012 (link)). All infiltrations were carried out in biological triplicates where possible. SnToxA infiltration data were adapted from Dinglasan et al. (2018 ).