The bacterium Photorhabdus luminescens is mutualistically associated with entomopathogenetic nematodes. These nematodes invade insect larvae and release the bacteria from their intestine, which kills the insects through the action of toxin complexes. We elucidated the mode of action of two of these insecticidal toxins from P. luminescens. We identified the biologically active components TccC3 and TccC5 as adenosine diphosphate (ADP)-ribosyltransferases, which modify unusual amino acids. TccC3 ADP-ribosylated threonine-148 of actin, resulting in actin polymerization. TccC5 ADP-ribosylated Rho guanosine triphosphatase proteins at glutamine-61 and glutamine-63, inducing their activation. The concerted action of both toxins inhibited phagocytosis of target insect cells and induced extensive intracellular polymerization and clustering of actin. Several human pathogenic bacteria produce related toxins.

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We have cloned and sequenced the maize chloroplast genome fragment Eco RI e which contains the 2.2 kb transcript previously reported (Link, G. and Bogorad, L. (1980) Proc. Nat. Acad. Sci. 77 6821-6825) to lie next to the maize gene for the large subunit of ribulose bisphosphate carboxylase (LS) and to be transcribed divergently. Immunochemical and sequencing data show that the gene codes for the beta subunit of the maize chloroplast coupling factor complex (CF1). The derived amino acid sequence is highly homologous to that of the corresponding E. coli protein (Saraste et al. (1981) Nucleic Acids Res. 9 5287-5296). The last base of the codon for the terminal lysine residue of the beta subunit of CF1 is the first base of the codon for the initiating methionine of an open reading frame whose derived amino acid composition and size closely match that reported for the epsilon subunit (Binder et al. (1978) J. Biol. Chem. 253 3094-3100). The close coupling of the two genes may serve to in sure their stoichiometric production.

The effect of the herbicide isoxaben on the incorporation of radiolabeled glucose, leucine, uracil, and acetate into acid insoluble cell wall material, protein, nucleic acids, and fatty acids, respectively, was measured. Dichlobenil, cycloheximide, actinomycin D, and cerulenin, inhibitors of the incorporation of these precursors into these macromolecular components, functioned as expected, providing positive controls. The incorporation of radiolabeled glucose into an acid insoluble cell wall fraction was severely inhibited by isoxaben at nanomolar concentrations. Amitrole, fluridone, ethalfluralin, and chlorsulfuron, as well as cycloheximide, actinomycin D, and cerulenin did not inhibit incorporation of glucose into this fraction, ruling out a general nonspecific effect of herbicides on glucose incorporation. The evidence thus suggests that isoxaben is an extremely powerful and specific inhibitor of cell wall biosynthesis.

Expressing double-stranded RNA (dsRNA) in transgenic plants to silence essential genes within herbivorous pests is referred to as trans-kingdom RNA interference (TK-RNAi) and has emerged as a promising strategy for crop protection. However, the dicing of dsRNA into siRNAs by the plant’s intrinsic RNAi machinery may reduce this pesticidal activity. Therefore, genetic constructs, encoding ∼200 nt duplex-stemmed-hairpin (hp) RNAs, targeting the acetylcholinesterase gene of the cotton bollworm, Helicoverpa armigera, were integrated into either the nuclear or the chloroplast genome of Nicotiana benthamiana. Undiced, full-length hpRNAs accumulated in transplastomic lines of N. benthamiana and conferred strong protection against H. armigera herbivory while the hpRNAs of nuclear-transformed plants were processed into siRNAs and gave more modest anti-feeding activity. This suggests that there is little or no RNAi machinery or activity in the chloroplast, that hpRNAs produced within this organelle do not enter the cytoplasm, and that oral delivery of chloroplast-packaged intact hpRNA is a more effective means of delivering TK-RNAi than using nuclear encoded hpRNAs. This contrasts with a recently reported correlation between siRNA expression and effectiveness of TK-RNAi targeting the chitinase gene of H. armigera, but is consistent with reports of efficient TK-RNAi by dsRNA generated in chloroplasts by converging promoters flanking a pest gene sequence and from very small (21 nt-stem) hpRNAs resembling artificial miRNAs. Here we demonstrate that hpRNAs, constructed along the conventional design principles of plant RNAi constructs but integrated into the chloroplast genome, are stable and effective over multiple generations, and hold the promise of providing durable pest resistance in crops.