Ignacio M. Larrinua

Photorhabdus luminescens Toxins ADP-Ribosylate Actin and RhoA to Force Actin Clustering

Tripartite Toxin Luminescent bacterial symbionts of nematode worms that attack insects have long stirred interest in their possibilities for biological control. The bacteria produce a family of toxins composed of at least three subunits that resemble a widely occurring class of bacterial toxins also produced by human pathogens. Lang et al. (p. 1139) have elucidated the mode of action and structural interactions of some of these tripartite protein toxins and found that they poison the cells actin cytoskeleton by catalyzing unusual reactions. One toxin mediated adenosine diphosphate (ADP)–ribosylation at threonine-148 to cause actin polymerization, another ADP-ribosylated Rho protein at glutamine-63, and both synergized to cause actin clustering and cell paralysis. A bacterial toxin targets and modifies the actin cytoskeleton in insect larvae. 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.

A spinosyn-sensitive Drosophila melanogaster nicotinic acetylcholine receptor identified through chemically induced target site resistance, resistance gene identification, and heterologous expression

Strains of Drosophila melanogaster with resistance to the insecticides spinosyn A, spinosad, and spinetoram were produced by chemical mutagenesis. These spinosyn-resistant strains were not cross-resistant to other insecticides. The two strains that were initially characterized were subsequently found to have mutations in the gene encoding the nicotinic acetylcholine receptor (nAChR) subunit Dalpha6. Subsequently, additional spinosyn-resistant alleles were generated by chemical mutagenesis and were also found to have mutations in the gene encoding Dalpha6, providing convincing evidence that Dalpha6 is a target site for the spinosyns in D. melanogaster. Although a spinosyn-sensitive receptor could not be generated in Xenopus laevis oocytes simply by expressing Dalpha6 alone, co-expression of Dalpha6 with an additional nAChR subunit, Dalpha5, and the chaperone protein ric-3 resulted in an acetylcholine- and spinosyn-sensitive receptor with the pharmacological properties anticipated for a native nAChR.

Differential response to isoxaben of cellulose biosynthesis by wild-type and resistant strains of Arabidopsis thaliana

Abstract Isoxaben-resistant mutants of Arabidopsis thaliana did not exhibit differences in their uptake, accumulation, or metabolism of isoxaben when compared to the sensitive parent wild-type strain. We have previously shown that isoxaben inhibits incorporation of glucose into an acid-insoluble cell wall fraction thought to be cellulose. Careful cell wall fractionation of the wild-type strain indicates that this is the only component whose biosynthesis is inhibited by isoxaben. The inhibition of this component is relieved in the resistant strains. These mutants are defined by two independent genes. Since neither is involved in the metabolic fate of isoxaben, both should directly affect the binding of isoxaben. This strongly implicates the existence of a multimeric complex as the binding site of isoxaben.

In-Plant Protection against Helicoverpa armigera by Production of Long hpRNA in Chloroplasts

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.

Preliminary safety assessment of a membrane-bound delta 9 desaturase candidate protein for transgenic oilseed crops.

A gene encoding delta 9 desaturase (D9DS), an integral membrane protein, is being considered for incorporation into oilseed crops to reduce saturated fatty acids and thus improve human nutritional value. Typically, a safety assessment for transgenic crops involves purifying heterologously produced transgenic proteins in an active form for use in safety studies. Membrane-bound proteins have been very difficult to isolate in an active form due to their inherent physicochemical properties. Described here are methods used to derive enriched preparations of the active D9DS protein for use in early stage safety studies. Results of these studies, in combination with bioinformatic results and knowledge of the mode of action of the protein, along with a history of safe consumption of related proteins, provides a weight of evidence supporting the safety of the D9DS protein in food and feed.

Functional genomics for plant trait discovery

Functional genomics, as the name implies, approaches gene discovery on a genomewide scale. Our understanding of the complex genetic interrelationships underlying plant traits such as yield or stress tolerance is being shaped by new technologies that facilitate the analysis of thousands of genes in a single experiment.