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 described25 (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 described21 (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, necrosis71 (link). All infiltrations were carried out in biological triplicates.
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, necrosis71 (link). All infiltrations were carried out in biological triplicates.
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