Spider venom neurotoxin based bioinsecticides: A novel bioactive for the control of the Asian citrus psyllid Diaphorina citri (Hemiptera).

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a key vector of the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas) associated with huanglongbing (HLB), the most serious and currently incurable disease of citrus worldwide. Here we report the first investigation into the potential use of a spider venom-derived recombinant neurotoxin, ω/κ-HxTx-Hv1h (hereafter HxTx-Hv1h) when delivered alone or when fused to snowdrop lectin (Galanthus nivalis agglutinin; GNA) to control D. citri. Proteins, including GNA alone, were purified from fermented transformed yeast Pichia pastoris cultures. Recombinant HxTx-Hv1h, HxTx-Hv1h/GNA and GNA were all orally toxic to D. citri, with Day 5 median lethal concentrations (LC50) derived from dose-response artificial diet assays of 27, 20 and 52 µM, respectively. Western analysis of whole insect protein extracts confirmed that psyllid mortality was attributable to protein ingestion and that the fusion protein was stable to cleavage by D. citri proteases. When applied topically (either via droplet or spray) HxTx-Hv1h/GNA was the most effective of the proteins causing >70 % mortality 5 days post treatment, some 2 to 3-fold higher levels of mortality as compared to the toxin alone. By contrast, no significant mortality or phenotypic effects were observed for bumble bees (Bombus terrestris L.) fed on the recombinant proteins in acute toxicity assays. This suggests that HxTx-Hv1h/GNA has potential as a novel bioinsecticide for the management of D. citri offering both enhanced target specificity as compared to chemical pesticides and compatibility with integrated pest management (IPM) strategies.

Enhancing the oral and topical insecticidal efficacy of a commercialized spider venom peptide biopesticide via fusion to the carrier snowdrop lectin (Galanthus nivalis agglutinin).

BACKGROUND: Spear®-T sold as a contact foliar spray for the control of glasshouse pests such as aphids, thrips, spider mites and whiteflies, contains the recombinant spider venom peptide GS-ω/κ-HxTx-Hv1h (named as GS-ω/κ-HxTx-Hv1a by Vestaron) as the active ingredient. Here we investigate whether fusion of the peptide to snowdrop lectin, (Galanthus nivalis agglutinin; GNA) enhances the efficacy of this venom peptide towards aphid pests. RESULTS: Recombinant GS-ω/κ-HxTx-Hv1h (HxTx-Hv1h) and an HxTx-Hv1h/GNA fusion protein were produced using the yeast Pichia pastoris. Purified proteins showed comparable toxicity when injected into lepidopteran (Mamestra brassicae) larvae, but significant differences in oral and contact activity towards aphids. HxTx-Hv1h had comparable acute oral toxicity to pea (Acyrthosiphon pisum) and peach potato (Myzus persicae) aphids with respective Day (2) median lethal concentration (LC50 ) values of 111 and 108 µm derived from diet assays. The fusion protein also showed comparable oral toxicity to both species but D2 LC50 values were >3-fold lower (35 and 33 µm for pea and peach potato aphids, respectively) as compared to HxTx-Hv1h. Topically applied toxin and fusion protein, but not GNA, caused significant reductions in pea aphid survival. Contact effects on mortality were significantly greater for aphids exposed to fusion protein as compared to toxin alone. Whole aphid fluorescence microscopy and immunoblotting suggest that improved efficacy is due to enhanced persistence of HxTx-Hv1h when fused to GNA following internalisation of ingested or topically applied proteins. CONCLUSIONS: This is the first study to report on the insecticidal activity of HxTx-Hv1h towards aphids and results suggest that a fusion protein-based approach offers opportunities to significantly enhance oral and contact efficacy of naturally derived toxins, such as HxTx-Hv1h, towards crop pests. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Plant growth promotion by the interaction of a novel synthetic small molecule with GA-DELLA function.

Synthesized small molecules are useful as tools to investigate hormonal signaling involved in plant growth and development. They are also important as agrochemicals to promote beneficial properties of crops in the field. We describe here the synthesis and mode of action of a novel growth-promoting chemical, A1. A1 stimulates enhanced growth in both shoot and root tissues of plants, acting by increasing both dry and fresh weight. This suggests that A1 not only promotes uptake of water but also increases production of cellular material. A1 treatment of Arabidopsisleads to the degradation of DELLA growth-inhibitory proteins suggesting that A1-mediated growth promotion is dependent upon this mechanism. We performed genetic analysis to confirm this and further dissect the mechanism of A1 action upon growth in Arabidopsis. A quintuple dellamutant was insensitive to A1, confirming that the mode of action was indeed via a DELLA-dependent mechanism. The ga1-5gibberellin synthesis mutant was similarly insensitive, suggesting that to promote growth in ArabidopsisA1 requires the presence of endogenous gibberellins. This was further suggested by the observation that double mutants of GID1 gibberellin receptor genes were insensitive to A1. Taken together, our data suggest that A1 acts to enhance sensitivity to endogenous gibberellins thus leading to observed enhanced growth via DELLA degradation. A1 and related compounds will be useful to identify novel signaling components involved in plant growth and development, and as agrochemicals suitable for a wide range of crop species.

Chloroplast calcium signalling regulates thermomemory.

After an episode of heat stress plants retain a cellular "memory" of this event, a phenomenon known as thermomemory. This mechanism allows plants to better cope against a subsequent heat event. Thermomemory occurs through the persistence of heat shock proteins (HSPs) synthesized after the first "priming" event. Maintenance of this thermomemory comes at the cost to growth though, therefore it is vital that the memory is reset when no longer required. Recently, it has been reported that autophagy is important for resetting the thermomemory. It has also been shown recently that in response to heat, Arabidopsis displays an increase in chloroplast free calcium concentration which is partially dependent on calcium sensing receptor (CAS) protein. It is not known what the purpose of this heat-activated calcium signal is. Therefore, we compared downstream responses to heat in wild type (WT) and cas mutants, as the latter produce a reduced chloroplast calcium signal to heat. We found that after thermopriming the cas mutants displayed a greater biomass and a reduced level of the small heat shock protein HSP 17.6 degradation compared to WT. cas mutants did not show an increase in free amino acid levels after thermopriming, suggesting reduced autophagy. These results suggest that heat-induced chloroplast calcium elevation is a positive signal for resetting of the thermomemory.

The insecticidal activity of recombinant nemertide toxin α-1 from Lineus longissimus towards pests and beneficial species.

The nemertide toxins from the phylum Nemertea are a little researched family of neurotoxins with potential for development as biopesticides. Here we report the recombinant production of nemertide α-1 (α-1), a 65-residue inhibitor cystine knot (ICK) peptide from Lineus longissimus, known to target insect voltage-gated sodium channels. The insecticidal activity of α-1 was assessed and compared with the well characterised ICK venom peptide, ω-atracotoxin/hexatoxin-Hv1a (Hv1a). α-1 elicited potent spastic paralysis when injected into cabbage moth (Mamestra brassicae) larvae; conferring an ED50 3.90 µg/larva (10.30 nmol/g larva), followed by mortality (60% within 48 h after 10 µg injection). By comparison, injection of M. brassicae larvae with recombinant Hv1a produced short-lived flaccid paralysis with an ED50 over 6 times greater than that of α-1 at 26.20 µg/larva (64.70 nmol/g larva). Oral toxicity of α-1 was demonstrated against two aphid species (Myzus persicae and Acyrthosiphon pisum), with respective LC50 values of 0.35 and 0.14 mg/mL, some 6-fold lower than those derived for recombinant Hv1a. When delivered orally to M. brassicae larvae, α-1 caused both paralysis (ED50 11.93 µg/larva, 31.5 nmol/g larva) and mortality. This contrasts with the lack of oral activity of Hv1a, which when fed to M. brassicae larvae had no effect on feeding or survival. Hv1a has previously been shown to be non-toxic by injection to the beneficial honeybee (Apis mellifera). By contrast, rapid paralysis and 100% mortality was observed following injection of α-1 (31.6 nmol/g insect). These results demonstrate the great potential of naturally occurring non-venomous peptides, such as α-1, for development as novel effective biopesticides, but equally highlights the importance of understanding the phyletic specificity of a given toxin at an early stage in the quest to discover and develop safe and sustainable pesticides.

Basal stomatal aperture is regulated by GA-DELLAs in Arabidopsis.

Stomatal aperture is tightly regulated in order to achieve the best compromise between gas exchange and water conservation. Steady-state (basal) stomatal aperture is therefore understandably a key component in plant fitness. It has been shown previously in tomato that DELLA proteins act as positive regulators of closure of stomata, and their action is enhanced by the hormone ABA, which is itself important in mediating drought stress tolerance. DELLAs are regulated by a variety of signals which promote plant growth, most notably the hormones gibberellins, which have been shown to promote stomatal opening. We have found that DELLA proteins are also used in Arabidopsis for regulating basal stomatal aperture. We also discovered that the perception of endogenous gibberellins via the GID1 receptors is necessary for optimal basal stomatal aperture.