No abstract

Binding studies were performed with two '251-labeled Bacillus thuringiensis -endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One S-endotoxin, Bt2-protoxin, is a 130-kDa recombinant crystalline protein from B. thuringiensis subsp. berliner. It kills larvae of both insect species. The active Bt2-toxin is a 60-kDa proteolytic fragment of the Bt2-protoxin. It binds saturably and with high affinity to brush border membrane vesicles from the midgut of both species. The other 6-endotoxin, Bt4412-protoxin, is a 136-kDa crystalline protein from B. thuringiensis subsp. thuringiensis, which is highly toxic for P. brassicae, but not for M. sexta larvae. Bt4412-toxin, obtained after proteolytic activation of Bt4412-protoxin, shows high-affinity saturable binding to P. brassicae vesicles but not to M. sexta vesicles. The correlation between toxicity and specific binding is further strengthened by competition studies. Other B. thuringiensis 6-endotoxins active against M. sexta compete for binding of 125I-labeled Bt2-toxin to M. sexta vesicles, whereas toxins active against dipteran or coleopteran larvae do not compete. Bt2-toxin and Bt4412-toxin bind to different sites on P. brassicae vesicles.Bacillus thuringiensis produces crystalline parasporal inclusions containing insecticidal proteins called 8-endotoxins. Most S-endotoxins are protoxins, which are proteolytically activated in the insect midgut to smaller active toxins (1). 8-Endotoxins produced by different B. thuringiensis strains may exhibit different insecticidal spectra. Toxins active toward lepidopteran, dipteran, or coleopteran larvae have been described (2-4). Among &endotoxins specific for Lepidoptera, marked differences exist in the relative levels of toxicity toward different species of this order (5-7).Several factors such as the solubilization and proteolytic activation of the crystals in the insect midgut (5, 8) and the presence of specific cell membrane receptors for different 8-endotoxins (9,10) MATERIALS AND METHODSInsect Toxicity Assays. Insect toxicity assays on M. sexta and P. brassicae have been described (12). M. sexta was reared on an artificial diet (13); P. brassicae was reared on fresh cabbage leaves (Brassica oleracea var. gemnifera D.C.).B. thuringiensis 6-Endotoxins. Cloning of bt2, the gene encoding the 130-kDa 8-endotoxin (Bt2-protoxin) from B. thuringiensis subsp. berliner strain 1715, purification of recombinant Bt2-protoxin from Escherichia coli, and generation of the toxic 60-kDa tryptic fragment (Bt2-toxin) has been described by Hofte et al. (12). For further purification Bt2-toxin was precipitated in (NH4)2SO4 (70%) and was then dissolved in Tris buffer (20 mM Tris HCl/200 mM NaCl, pH 8.65) with 5% (vol/vol) glycerol. Streptomycin sulfate was added to a concentration of 0.2% to remove contaminating nucleic acids. The solution was allowed to stand for 60 min at 4°C and the precipitate was spun down in a Sorvall SS34 rot...

Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement.