Abstract: Increases in pathogenesis-related (PR) transcripts are commonly interpreted as evidence of plants' resistance responses to pathogens; however, few studies have examined whether increases in PR proteins protect plants growing under natural conditions. Pseudomonas syringae pv. tomato DC3,000, which is virulent and causes disease in Arabidopsis, is also pathogenic to the native tobacco Nicotiana attenuata. N. attenuata responds to P. syringae pv. tomato DC3,000's challenges with increases in salicylic acid and transcripts of at least two PR genes, PR-1 and PR13/Thionin. To determine if either of these PR proteins functions in bacterial resistance, we independently silenced both genes by RNAi and found that only PR-13/Thionin mediates resistance to P. syringae pv. tomato DC3,000 in glasshouse experiments. When NaPR-1- and NaThionin-silenced plants were transplanted into the plant's native habitat in the Great Basin Desert of Utah, opportunistic Pseudomonas spp. performed better on NaThionin-silenced plants compared with NaPR-1-silenced and wild-type (WT) plants, and accounted for increased plant mortality. The native herbivore community of N. attenuata attacked both NaPR-1- and PR-13/NaThionin-silenced plants to the same degree as it did in WT plants, indicating that neither PR protein provides resistance to herbivores. Although PR-1 is generally considered a marker gene of disease resistance, we found no evidence that it has an antimicrobial function. In contrast, PR-13/NaThionin is clearly an ecologically relevant defense protein involved in resisting pathogens in N. attenuata.

Abstract: The phytohormone jasmonic acid (JA) is known to mediate herbivore resistance, while salicylic acid (SA) and non-expressor of PR-1 (NPR1) mediate pathogen resistance in many plants. Herbivore attack on Nicotiana attenuata elicits increases in JA and JA-mediated defenses, but also increases SA levels and Na-NPR1 transcripts from the plant's single genomic copy. SA treatment of wild-type plants increases Na-NPR1 and Na-PR1 transcripts. Plants silenced in NPR1 accumulation by RNAi (ir-npr1) are highly susceptible to herbivore and pathogen attack when planted in their native habitat in Utah. They are also impaired in their ability to attract Geocorus pallens predators, due to their decreased ability to release cis-alpha-bergamotene, a JA-elicited volatile 'alarm call'. In the glasshouse, Spodoptera exigua larvae grew better on ir-npr1 plants, which had low levels of JA, JA-isoleucine/leucine, lipoxygenase-3 (LOX3) transcripts and JA-elicited direct defense metabolites (nicotine, caffeoyl putrescine and rutin), but high levels of SA and isochorismate synthase (ICS) transcripts, suggesting de novo biosynthesis of SA. A microarray analysis revealed downregulation of many JA-elicited genes and upregulation of SA biosynthetic genes. JA treatment restored nicotine levels and resistance to S. exigua in ir-npr1 plants. We conclude that, during herbivore attack, NPR1 negatively regulates SA production, allowing the unfettered elicitation of JA-mediated defenses; when NPR1 is silenced, the elicited increases in SA production antagonize JA and JA-related defenses, making the plants susceptible to herbivores.

Abstract: Induced resistance to biotic attackers is thought to be mediated by responses elicited by jasmonic acid (JA), a subset of which are lipoxygenase 3 (LOX3) dependent. To understand the importance of LOX3-mediated insect resistance, we analysed the performance of Manduca sexta larvae on wild-type (WT) and on isogenic Nicotiana attenuata plants silenced in NaLOX3 expression and JA signalling, and we used Waldbauer nutritional indices to measure the pre- and post-ingestive effects. LOX3-mediated defenses reduced larval growth, consumption and frass production. These defenses reduced how efficiently late-instar larvae converted digested food to body mass (ECD). In contrast, LOX3-mediated defenses decreased approximate digestibility (AD) in early instar larvae without affecting the ECD and total food consumption. Larvae of all instars feeding on defended WT plants behaviourally compensate for their reduced body mass by consuming more food per unit of body mass gain. We suggest that larvae feeding on plants silenced in NaLOX3 expression (as-lox) initially increase their AD, which in turn enables them to consume more food in the later stages and consequently, to increase their ECD and efficiency of conversion of ingested food (ECI). We conclude that N. attenuata's oxylipin-mediated defenses are important for resisting attack from M. sexta larvae, and that Waldbauer nutritional assays reveal behavioural and physiological counter responses of insects to these plant defenses.