Pgapza expression system

SnTox3 protein (SNOG_08981; GenBank acc; XP_001799284) was produced in Pichia pastoris using the pGAPzA expression system (Thermo Fisher Scientific, MA, USA)^{23 (link),25 (link)}. The P. pastoris culture filtrate (CF) containing the Tox3 expressed protein was harvested and desalted with 10 mm sodium phosphate buffer pH 7.0 as previously described^{25 (link)}. Crude CFs containing necrosis‐inducing factors produced by the five wild-type P. nodorum isolates (Table 1) were generated in Fries 3 broth as previously described^{21 (link),69 (link)}. The CFs were filter sterilised prior to plant infiltration.
A simple leaf infiltration technique as described in Oliver et al.^{70 (link)} was used for SnTox3 and the crude CFs. A needleless 1 ml plastic syringe was used to infiltrate the expressed proteins into the first leaf of 2-week-old wheat seedlings. Infiltrated plants were kept in a Conviron growth chamber for 4 days for SnTox3-induced necrosis and 7 days for crude CFs from wild-type isolates under a 12 h photoperiod prior to scoring. Sensitivity was visually evaluated using a scale of 0–4, where a score of 0 indicates no observable reactions; 1, mild chlorosis; 2, chlorosis; 3, chlorosis with mild necrosis; 4, necrosis^{71 (link)}. All infiltrations were carried out in biological triplicates.

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 ).