Probing the action of Clostridium toxins B and exoenzyme C3 for detection of Rho-like motifs of alfalfa proteins

Small GTP-binding Rho proteins are involved in signalling, cell polarity, membrane outgrowths and actin stabilization in eukaryotes. Known plant homologues represent essentially the Rac subfamily and an original Rop (Rho in pollen). Mammalian Rho proteins are preferential targets of clostridial toxins. In alfalfa (Medicago sativa L.) cells, Clostridium botulinum C3-exoenzyme (C3) provoked disassembly of the actin cytoskeleton, similar to its effect in mammalian cells. In alfalfa proteins, several epitopes appear to be recognized by commercial antibodies raised against peptides characteristic for human Rho. One ≈ 40-kDa band was detected immunologically by anti-RhoB: a protein of this size was ADP-ribosylated by C3 and glucosylated in vitro by Clostridium difficile toxin B, without interference between the two nor from phosphatidyl inositide. C3 was also active upon a 34-kDa band which contained protein(s) immunoreactive with anti-Rac2 and which bound [γ35S]-GTP, but was glucosylated by neither toxin B nor Clostridium sordellii Lethal Toxin. An 18-kDa band detected by [γ35S]-GTP overlay was immunologically recognized by anti-Rac1. Anti-Cdc42Hs recognized a 54-kDa band. Substrates to toxin B and C3 were purified from alfalfa cell culture and partially sequenced: they included two proteins, P40 and P41, of ≈ 40 kDa (by SDS-electrophoresis). P40 appears to constitute a tetrameric aldolase (160 kDa by gel filtration; EC 4.1.2.13) whose activity is partially inhibited by toxin B and the anti-RhoB.