Maize CP1A and CP2 Overexpression in N. benthamiana

Maize cp1a (GRMZM2G166281) without the sequence encoding for the granulin domain and cp2 (GRMZM2G038636) genes were amplified by PCR from maize (EGB) cDNA. PCR products were directly cloned with BsaI overhangs into the level 1 binary vector (pICH47732) (Addgene) containing 2x35S promotor and a C-terminal Streptwin tag using the golden gate procedure^{43 (link)}. Binary plasmids pL1M-F1-CP2-Streptwin::2x35S and pL1M-F1-CP1A_nogran-Streptwin::2x35S were generated and transformed into Agrobacterium tumefaciens GV3101 competent cells for overexpression in N. benthamiana. GV3101 strains containing the desired plasmids were grown in liquid media overnight and diluted to OD = 2 in 10 mM magnesium chloride with 200 µM acetosyringone final concentration (Sigma-Aldrich, Taufkirchen, Germany). After 1 h incubation cultures were mixed with GV3101 strains containing the p19 construct⁴⁴ to a final OD = 1 and cultures were syringe infiltrated in leaves of 5−6-week-old N. benthamiana plants. Three days post infiltration leaves were harvested and apoplastic fluid was isolated.