FIGURE 9.

RISP is not detected in poplar leaves during immune responses and its C-terminus is cleaved by a plant-encoded mechanism. (A) Western blot showing the expression of RISP in non-infected poplar leaves and at three time-points of poplar leaves during an incompatible interaction with M. larici-populina (see main text for details). (B) Western blot showing the expression of RISP over the whole time-course experiments of poplar leaves during an incompatible interaction with M. larici-populina. In A and B, proteins isolated from poplar leaves (30 μg: 15 μg of soluble proteins + 15 μg of insoluble proteins) were separated on 15% SDS-PAGE and transferred on nitrocellulose membrane. The His-RISP recombinant protein (20 ng in A and 10 ng in B) was used as a reference. Anti-RISP polyclonal antibodies were used for primary detection of RISP. Numbers presented indicate hpi. (C) Five percent (w/w) of purified RISP and His-RISP were incubated with soluble protein extracts from poplar leaves for 30 min at room temperature under gentle agitation and with 1 mM phenylmethanesulfonylfluoride (PMSF). (D) RISP time-course incubation with soluble protein extracts from poplar leaves. Five percent (w/w) of purified His-RISP was incubated with untreated or denatured soluble protein extracts from poplar leaves for up to 24 h at room temperature under gentle agitation and with 1 mM PMSF. (E) His-tagged thioredoxin h1 (His-Trxh1) time-course incubation with soluble protein extracts from poplar leaves. Five percent (w/w) of purified His-Trxh1 was incubated with soluble protein extracts from poplar leaves for up to 24 h at room temperature under gentle agitation and with 1 mM PMSF. Protein integrity was followed by western blot. ^∗Indicates the lane occupied by the molecular weight marker.