Digestive Duet: Midgut Digestive Proteinases of Manduca sexta Ingesting Nicotiana attenuata with Manipulated Trypsin Proteinase Inhibitor Expression

Abstract

BackgroundThe defensive effect of endogenous trypsin proteinase inhibitors (NaTPIs) on the herbivore Manduca sexta was demonstrated by genetically altering NaTPI production in M. sexta's host plant, Nicotiana attenuata. To understand how this defense works, we studied the effects of NaTPI on M. sexta gut proteinase activity levels in different larval instars of caterpillars feeding freely on untransformed and transformed plants.Methodology/ Principal FindingsSecond and third instars larvae that fed on NaTPI-producing (WT) genotypes were lighter and had less gut proteinase activity compared to those that fed on genotypes with either little or no NaTPI activity. Unexpectedly, NaTPI activity in vitro assays not only inhibited the trypsin sensitive fraction of gut proteinase activity but also halved the NaTPI-insensitive fraction in third-instar larvae. Unable to degrade NaTPI, larvae apparently lacked the means to adapt to NaTPI in their diet. However, caterpillars recovered at least part of their gut proteinase activity when they were transferred from NaTPI-producing host plants to NaTPI-free host plants. In addition extracts of basal leaves inhibited more gut proteinase activity than did extracts of middle stem leaves with the same protein content.Conclusions/ SignificanceAlthough larvae can minimize the effects of high NaTPI levels by feeding on leaves with high protein and low NaTPI activity, the host plant's endogenous NaTPIs remain an effective defense against M. sexta, inhibiting gut proteinase and affecting larval performance.

Serine Protease Inhibitors Specifically Defend Solanum nigrum against Generalist Herbivores but Do Not Influence Plant Growth and Development

“…Both M. sexta and a Spodoptera sp were observed to occur and feed on S. nigrum in Utah. M. sexta is a solanaceous specialist and to some extent adapted to PI-II inhibitors (Zavala et al, 2008). S. exigua is considered a generalist with a wide host range that includes solanaceous crop plants (Brown and Dewhurst, 1975).…”

Pithy Protection: Nicotiana attenuata’s Jasmonic Acid-Mediated Defenses Are Required to Resist Stem-Boring Weevil Larvae

“…To investigate the effect of PI production in pith on T. mucorea behavior, the plant piths were analyzed for PI activity after wounding and M. sexta oral secretion (OS) elicitation, a treatment that is known to dramatically increase PI transcripts and activity in leaves (Zavala and Baldwin, 2004;Zavala et al, 2008). Pith of N. attenuata wild-type and ir-pi plants was wounded by making punctures into the stem and adding water only or water and M. sexta OS (as a positive control) to the holes, or left unwounded in controls.…”

“…attenuata uses a combination of toxins and digestibility reducing defenses, both of which are elicited by herbivore attack. Trypsin protease inhibitors (TPIs) are an effective component of this inducible defensive system that reduces the performance of folivores by targeting their main proteolytic digestive enzymes (van Dam et al, 2000;Glawe et al, 2003;Zavala and Baldwin, 2004;Horn et al, 2005;Zavala et al, 2008;Bezzi et al, 2010). Nicotine, a neurotoxin, generally reduces consumption by a variety of herbivores (Steppuhn et al, 2004).…”

Silencing NaTPI Expression Increases Nectar Germin, Nectarins, and Hydrogen Peroxide Levels and Inhibits Nectar Removal from Plants in Nature

“…Trypsin protease inhibitors (TPIs) are an effective component of this inducible defensive system that reduces the performance of folivores by targeting their main proteolytic digestive enzymes and is strongly induced by herbivore attack (van Dam et al, 2000;Glawe et al, 2003;Zavala et al, 2004bZavala et al, , 2008Horn et al, 2005). However, in N. attenuata, the biosynthesis of TPIs incurs substantial fitness costs (Zavala et al, 2004a); silencing the TPI gene in N. attenuata abolishes the plant's capacity to produce TPIs and allows it to grow faster, flower earlier, and produce more seed capsules compared with TPI-producing genotypes (Zavala et al, 2004a).…”

“…No significant difference in TPI activity was found between anther heads and ovaries of wild-type flower parts; however, anther heads and ovaries had significantly higher TPI activity than other wild-type flower parts. B and D, X-ray film assay (Zavala et al, 2008). Forty micrograms of total protein (determined by the Bradford assay) from each flower part was loaded on a 12% native gel for electrophoresis.…”