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. 2020 Feb 5;18(8):1810–1829. doi: 10.1111/pbi.13343

Figure 2.

Figure 2

Interaction analyses of GmSNAP18 and GmSHMT08 proteins by co‐agroinfiltration (Co‐Ag) in N. benthamiana. The Agrobacterium mixture containing p35S‐GmSNAP18 and p35S‐GmSHMT08‐HA constructs was mixed with the P19 (suppression of gene silencing) and then incubated for 4 h at 28 ºC before infiltration. (a) After co‐agroinfiltration, total protein of the tobacco (Nicotiana bentamiana) leaves was extracted and co‐immunoprecipitated with anti‐SNAP18 PA or anti‐Rubisco PA (control) and blots from the eluted fractions were probed with anti‐HA, anti‐SHMT08 or anti‐SNAP18 antibodies. Only IgG or beads were used for Co‐IP experiments as a negative control and technical control, respectively. The Co‐Ag results confirmed that GmSNAP18 interacts physically with GmSHMT08. N. benthamiana leaves after 5 days were co‐agroinfiltrated to express either the indicated GmSNAP18 and/or the GmSHMT08 from (b) Forrest or (c) Essex. (b) Cell death and necrosis symptoms caused by the GmSNAP18 were intensified when both GmSNAP18 and GmSHMT08 were co‐agroinfiltrated in N. benthamiana and co‐expressed under the control of the constitutive promoter p35S. (c) Cell death symptoms were very limited in Essex. E, Essex; F, Forrest; EV, empty pGWB vector; AB, agroinfiltration buffer; GV3101, Agrobacterium GV3101 strain, WB, Western blot.