Resistance management in a native plant: nicotine prevents herbivores from compensating for plant protease inhibitors

Abstract

Plants deploy chemical defenses in complex mixtures, which are thought to be adaptive, but experimental tests have used artificial diets rather than plants. Herbivore attack on Nicotiana attenuata rapidly increases the production and accumulation of trypsin proteinase inhibitors (TPI) and the toxic alkaloid nicotine. By transgenically silencing their respective biosynthetic genes, we were able to abolish TPI activity and reduce inducible nicotine by 85%. Nicotine production was not affected by silencing pi or vice versa, and transformation did not alter levels of other metabolites examined. Spodoptera exigua, a native generalist herbivore that can compensate for heterologous TPI expression, performed better on TPI- or nicotine-deficient plants compared with the wild-type. Because of a compensatory feeding response to TPI when nicotine is absent, larvae performed better on nicotine-deficient plants than they did on plants silenced in both defenses. The antifeedant toxin, nicotine, prevents this compensatory response. We conclude that N. attenuata counters an insect adaptation with a defensive synergism.

Herbivory-induced volatiles function as defenses increasing fitness of the native plant Nicotiana attenuata in nature

“…We chose a line of ir PI plants with no detectable TPI activity (Steppuhn and Baldwin, 2007), and a line of ir LOX2 plants with GLV emissions <20% of WT (Allmann et al, 2010); non-target defense metabolites are not affected in either line (Steppuhn and Baldwin, 2007; Allmann and Baldwin, 2010), including emission of ( E )-α-bergamotene measured in a glasshouse characterization of all lines prior to field release (see 'Non-target metabolites are not affected in ir LOX2 , hemi-ir LOX2 or ir PI plants'). Because of the importance of GLVs for the plant-herbivore interaction, we used both homozygous (Allmann et al, 2010) and hemizygous ir LOX2 plants to provide different levels of GLV silencing.…”

“…The ir PI plants (Steppuhn and Baldwin, 2007) had no detectable TPI activity in the glasshouse or throughout the field experiment in 2011, and PI transcripts accumulated to only 0.3% of WT levels in ir PI (transcripts, N=5, p<0.001 in Scheffe post hoc tests following two-way ANOVAs on log 2 -transformed data with factors W+OS treatment and genotype: treatment F 1,29 =75.909, p<0.001; genotype F 3,29 =174.077, p<0.001); in contrast, TPI activity and PI transcripts were similar to WT plants in ir LOX2 and hemi-ir LOX2 (transcripts, N=5, p>0.2 in Scheffe post hoc tests following two-way ANOVAs on log 2 -transformed data with factors W+OS treatment and genotype; activity, N=10–17, p>0.05 in one-way ANOVAs with factor genotype) (Figure 1). …”

“…The ir PI line A-04-186-1 (Steppuhn and Baldwin, 2007) and ir LOX2 line A-04-52-2 (Allmann et al, 2010) have been characterized previously, and neither is affected in jasmonate signaling. Particularly, the emission of ( E )-α-bergamotene, the best-characterized HIPV in N. attenuata apart from GLVs (Halitschke et al, 2000; Kessler et al, 2004; Halitschke et al, 2008; Skibbe et al, 2008), does not differ significantly among the lines used (N=4 measured 24–32 hr after W+OS treatment as according to Halitschke et al (2000) and normalized as a percentage of the internal standard peak: WT, 67.9±17.1%; ir PI , 30.2±13.2%; ir LOX2 , 26.6±5.8%; hemi-ir LOX2 , 42.7±23.3%; ANOVA: F 3,12 =1.338, p=0.308).…”

“…We used previously characterized, homozygous, inverted-repeat (ir) RNAi transformed lines of the second transformed generation (T 2 ) to silence GLV biosynthesis: ir LOX2 line number A-04-52-2 (Allmann et al, 2010), and TPI activity: ir PI line number A-04-186-1 (Steppuhn and Baldwin, 2007). Vector construction and the pSOL3 plasmid have been described previously (Bubner et al, 2006).…”

“…Non-toxic protease inhibitor (PI) proteins, which inhibit protein digestion and thus decrease the availability of organic nitrogen in the form of amino acids (Zavala et al, 2008), are widespread in flowering plants (Hartl et al, 2011), and trypsin protease inhibitors (TPIs) slow the growth of M. sexta on N. attenuata (Zavala et al, 2008). However, herbivores can overcome PIs by producing insensitive or desensitized proteases, inactivating or degrading PIs, eating more plant tissue, and eating more nutritious young tissue (Winterer and Bergelson, 2001; Steppuhn and Baldwin, 2007; Zavala et al, 2008). In the latter two cases, PIs could reduce plant fitness.…”