A viral chitinase enhances oral activity of TMOF

Abstract

In this study we investigate the combined effect on Heliothis virescens (Lepidoptera, Noctuidae) larvae of Aedes aegypti-Trypsin Modulating Oostatic Factor (Aea-TMOF), a peptide that inhibits trypsin synthesis by the gut, impairing insect digestive function, and Autographa californica nucleopolyhedrovirus Chitinase A (AcMNPV ChiA), an enzyme that is able to alter the permeability of the peritrophic membrane (PM). Aea-TMOF and AcMNPV ChiA were provided to the larvae by administering transgenic tobacco plants, co-expressing both molecules. Experimental larvae feeding on these plants, compared to those alimented on plants expressing only one of the two molecules considered, showed significantly stronger negative effects on growth rate, developmental time and mortality. The impact of AcMNPV ChiA on the PM of H. virescens larvae, measured as increased permeability to molecules, was evident after five days of feeding on transgenic plants expressing ChiA. This result was confirmed by in vitro treatment of PM with recombinant ChiA, extracted from the transgenic plants used for the feeding experiments. Collectively, these data indicate the occurrence of a positive interaction between the two transgenes concurrently expressed in the same plant. The hydrolytic activity of ChiA on the PM of tobacco budworm larvae enhances the permeation of TMOF molecules to the ectoperitrophic space, and its subsequent absorption. The permeation through the paracellular route of Aea-TMOF resulted in a spotted accumulation on the basolateral domain of enterocytes, which suggests the occurrence of a receptor on the gut side facing the haemocoel. The binding of the peptide, permeating at increased rates due to the ChiA activity, is considered responsible for the enhanced insecticide activity of the transgenic plants expressing both molecules. These data corroborate the idea that ChiA can be effectively used as gut permeation enhancer in oral delivery strategies of bioinsecticides targeting haemocoelic receptors. Dear Rene,We have revised the manuscript as requested. Below are the reviewers comments and for each issue raised our remedial action or an explanation to address their concerns is reported in bold.I think that this version is certainly improved, and we will be glad to consider any further suggestion you may have. All the best, FrancoReviewer #1: Fiandra et al.In this manuscript Fiandra et al present some interesting data on apparent enhancement of TMOF action by chitinase. Overall, this is a sound study that will be improved with the following modifications:1. What is the mechanism of action of TMOF? This should be introduced upfront. The action of TMOF in inhibiting trypsin synthesis and impeding larval digestive processes is key to the logic behind these experiments. Not until page 16 is this information provided for the reader! This information should actually be included in the abstract. Clearly introduce TMOF.The information requested has been added in the manuscript (Abstract and Introduction). Page 4What is the size o...

Effects of Trichoderma viride chitinases on the peritrophic matrix of Lepidoptera

“…In particular, the most detailed study of chitinase effects on PMs was performed using chitinase A from Autographa californica multicapsid nucleopolyhedrovirus ( Ac MNPV) . Its hydrolytic activity was confirmed in vitro by treatment of the PMs of B. mori and Heliothis virescens . Additionally, its in vivo insecticidal activity was proved by feeding B. mori larvae with an artificial diet supplemented with the purified chitinase, or by feeding H. virescens directly with transgenic tobacco leaves .…”

“…proteins and glycans), and acting as a physical support for digestive and detoxifying enzymes. Therefore, its disruption alters the properties of this peculiar viscous layer . Chitin, also occurring in the exoskeleton of insects, as well as in the digestive systems of nematodes and in the cell wall of fungi, is an insoluble linear homopolymer of N ‐acetylglucosamine (GlcNAc) linked by β ‐(1–4)‐glycosidic bonds, which are hydrolysed by chitinases .…”

“…Chitin, also occurring in the exoskeleton of insects, as well as in the digestive systems of nematodes and in the cell wall of fungi, is an insoluble linear homopolymer of N ‐acetylglucosamine (GlcNAc) linked by β ‐(1–4)‐glycosidic bonds, which are hydrolysed by chitinases . Considerable interest in the chitinolytic enzymes has been stimulated by their possible use in integrated pest control strategies against insects, nematodes and fungi . In fact, resistance to these pests can be achieved by degradation of their vital structures, such as the PM and cuticle in insects, and the cell wall in fungal phytopathogens…”

Honey bee larval peritrophic matrix degradation during infection with Paenibacillus larvae, the aetiological agent of American foulbrood of honey bees, is a key step in pathogenesis

“…We recently found genetic evidence for the presence of toxin genes in the genome of P. larvae (Fünfhaus et al, 2009). It is therefore conceivable that efficiency of these not yet characterized P. larvae toxins depend on the permeability of the PM that can be increased by degradation of the chitinous network (Edwards and Jacobs-Lorena, 2000;Rao et al, 2004;Fiandra et al, 2010). Further work is needed to evaluate this aspect of AFB pathogenesis.…”

Dietary and phylogenetic correlates of digestive trypsin activity in insect pests

“…Several methods have been proposed to improve TMOF delivery to hemolymph and insecticidal activity (Shen et al., ; Jeffers et al., ; Kamareddine et al., ). In addition, higher larval mortality was achieved by applying TMOF with other insecticidal proteins, such as toxins of B. thuringiensis (Borovsky et al., ; Misni et al., ; Saiful et al., ) and nucleopolyhedrovirus chitinase A (Fiandra et al., ). Transgenic plants expressing TMOF have negative effects on lepidopteran (Tortiglione et al., ) and mosquito larvae (Zaritsky et al., ).…”