Figure 2.

Production of AfpB in the apoplast of Nicotiana benthamiana leaves. (a) Schematic representation of the two constructs prepared and designed to accumulate AfpB in the apoplast or endoplasmic reticulum (ER) of plant cells. Both incorporate the signal peptide of tobacco AP24 protein at the N‐terminus of the mature AfpB protein, and the one designed for ER accumulation also contains the ER‐retention signal KDEL at its C‐terminus. RB and LB, right and left border T‐DNA; P35S and T35S, CaMV 35S promoter and terminator; δ, ribozyme; MP, movement protein; CP, coat protein. (b) Appearance of N. benthamiana leaves 7 days after agroinfiltration with the indicated constructs or with the agrobacterium induction media (mock). (c‐f) Analysis of AfpB accumulation in agroinfiltrated leaves from two independent plants (1 and 2). Proteins from extracellular fluids (c, d) or acid extraction (e, f) were separated by tricine‐SDS‐PAGE and Coomassie‐blue stained (c, e) or immunodetected using antibodies anti‐PAFB (d, f). Purified AfpB (3.5 or 7 μg) from Penicillium digitatum cultures (AfpB[Pd]) was run in parallel as a control. ECFs from plants accumulating a cysteine‐rich protein of unknown function (UP, see Figure 3) was also run in parallel for comparative purposes. Molecular weight markers are shown in kDa on the left.