Plants are attacked in nature by a diverse suite of biotic challenges from pathogens and herbivores which can be devastating. But when plants are attacked by pathogens and herbivores, they mount defense responses which can slow an herbivore's feeding and also the rate of disease spread. For example, in response to herbivore attack, plants produce a broad spectrum of defense compounds that are elicited by a jasmonic acid-dependent signaling pathway. Tomato plants produce potent anti-herbivore defense metabolites such as proteinase inhibitors and polyphenoloxidase when attacked by Spodoptera exigua 1. In response to damage by the solanaceous specialist herbivore Manduca sexta, Nicotiana attenuata produces anti-herbivore defense metabolites such as nicotine 23, caffeoyl putrescine, rutin, and diterpene glycoside 4, as well as anti-digestive trypsin protease inhibitors (TPIs) 56. On the other hand, plants infected with fungi, bacteria or viruses produce several types of pathogenesis-related proteins (PRs) belonging to at least 17 families 7. Most PR proteins are known to possess antimicrobial characteristics. For instance, PR-2 (glucanases) of tobacco, barley, alfalfa and soybean have been shown to suppress diseases caused by Phytophthora megasperma f. sp. medicaginins, Rhizoctonia solani and Alternaria alternata 7. PR-3 (chitinases) isolated from bean can suppress Rhizoctonia solani in tobacco and canola when overexpressed 8. PR-13/Na-defensins in N. attenuata have been shown to suppress Pseudomonas syrinage pvtomato-DC3000 (PST DC3000) 9.

These examples clearly suggest that depending on the type of the attacker, plants can produce different blends of defense metabolites. But in nature, plants often have to deal with not one but several natural enemies, and these can occur either simultaneously or one after the other, with one enemy facilitating or eliciting resistance to the attack of subsequent attackers. Insects are also well-known vectors for pathogens 10; moreover, attack by rust fungi can influence subsequent herbivory in the same plant species 11. The co-evolution of plants and their natural enemies makes it likely that plant responses related to one attacker have far-reaching consequences for subsequent attackers. Plants often produce secondary metabolites with generalized detrimental effects on herbivores as well as pathogens. The phenolic compound rutin is such an example 12. Constitutive levels of iridoid glycosides among natural variants of Plantago lanceolata have been shown to confer resistance to the herbivore Spodoptera exigua as well as to the biotropic fungal pathogen Diaporthe adunca 11. Recently it was shown that caterpillar feeding significantly reduced the extent of disease caused by the bacterial pathogens PST DC3000 and Xanthomonas campestris pvarmoraciae in Arabidopsis 13.

Although many studies have shown that pathogen- and herbivore-elicited plant responses can have detrimental effects on subsequent attacker(s), few have established causal associations with the induction of a specific metabolite(s) by the first attacker with a detrimental effect (direct or indirect) on subsequent attacker(s). Some studies have causally linked the signaling pathways that mediate effects against herbivores and pathogens 13, others have found associations among the amounts of constitutively expressed metabolites (iridoid glycosides) known to affect both herbivores and pathogens; but all these studies have been conducted with genotypes selected over generations for high- and low-leaf iridoid glycoside concentrations 11 and are not iso-genic. Therefore, they may differ in resistance traits other than the measured metabolites. As with constitutively produced metabolites, little is known about the cross effects of inducible defense metabolites.

Here we 1) identify a defense metabolite (Na-defensin) in WT N. attenuata that is elicited in response to attack from both pathogens and herbivores and 2) examine the consequences of this metabolite for both pathogen and herbivore resistance by comparing the resistance of WT and iso-genic plants transformed to silence the expression of the metabolite. N. attenuata, a solanaceous annual originating from the Great Basin Desert or North America, dramatically increases Nadefensin (also known as PR-13) levels when attacked by herbivores such as Manduca sexta larvae 1415, Tupiocorus notatus, Myzus nicotianae, Spodoptera littoralis and Trichoplusia ni 16 as well as the pathogen PST DC3000 9. Previous work from our lab has shown that WT N. attenuata plants silenced for Nadefensin made WT plants increasingly susceptible to PST DC3000 9. Our main objectives were to study 1) the role of defensin in N. attenuata's induced resistance to M. sexta and 2) the influence of defensin on M. sexta growth when elicited by PST DC3000 and vice versa. We began by observing the patterns of Nadefensin expression in N. attenuata in response to M. sexta and PST DC3000 and then studied the effect of Nadefensin on M. sexta's resistance to plants that had been previously infected with PST DC3000 as well as the effects of Nadefensin on PST DC3000 resistance when plants had been previously damaged by M. sexta.

Two observations motivated us to study the direct and indirect roles of Nadefensin in resistance to pathogens and herbivores in N. attenuata: 1) Nadefensin levels are increased in N. attenuata in response to attack from these two natural enemies and 2) Nadefensin is effective in resisting PST DC3000 in N. attenuata but not herbivores 9. Therefore we asked: Does Nadefensin (an anti-bacterial defense gene) have a function during herbivore attack? This question led us to ask if Nadefensin indirectly affects interactions between M. sexta and PST DC3000.

The results demonstrate that PST DC3000-infection significantly reduces M. sexta's leaf consumption and growth; its effects are most dramatically seen in the Nadefensin-silenced plants. Since Nadefensin-silenced plants are more susceptible than WT plants to PST DC3000 9, we inferred that either the presence of PST DC3000 in the larval diet or the altered leaf chemistry resulting from PST DC3000 infection in Nadefensin-silenced plants was responsible for the poor larval performance. Larvae that fed on PST DC3000-infected WT plants, which are resistant to PST DC3000, consumed significantly more than those that fed on PST DC3000-infected Nadefensin-silenced plants, which are highly susceptible to PST DC3000 (Fig. 2A and 2B); however, we found no difference in the titers of PST DC3000 in the guts of the larvae (Fig. 4A and 4B) that had consumed either WT or Nadefensin-silenced plants. We propose that changes in plant chemistry associated with differences in PST DC3000 resistance negatively affect leaf consumption, but not the pathogen per se. This scenario is consistent with earlier studies in which tomato leaflets that had an increase in polyphenol oxidase after being infected by PST DC3000 decreased the suitability of non-inoculated leaflets of the same leaf for Helicoverpa zea 1. Interestingly, our results show that despite differences in leaf consumption between M. sexta that fed on either uninduced WT or Nadefensin-silenced plants and those that fed on PST DC3000-infected WT plants, larvae nevertheless gained the same amount of body mass (Fig. 3A). Since body mass is maintained at lower levels of consumption, PST DC3000 infection of WT plants appears to increase the efficiency by which ingested food creates body mass for M. sexta larvae.

We noticed that larvae avoided consuming the portions of the leaf exhibiting disease symptoms (Fig. 2B) and found leaf consumption to be inversely related to the size of the disease lesions. Why do larvae feeding on PST DC3000-infected WT plants gain more mass even when they consume less? Plant responses to pathogen infection such as increased salicylic acid (SA), which is produced in response to PST DC3000 infection, could be involved 9. Several studies have shown that growth in herbivores is enhanced in plants that have elevated SA levels because SA can antagonize the oxylipin signaling which mediates herbivore resistance 202122. However, WT and Nadefensin-silenced plants infected by PST DC3000 have the same level of SA 9, so other unmeasured changes in leaf chemistry are likely involved.

Nadefensin can function as an anti-bacterial protein and is capable of inhibiting PST DC3000 growth. Since Nadefensin is also induced after M. sexta damage 1415, we hypothesized that after herbivore damage Nadefensin-silenced plants should also be susceptible to PST DC3000. Indeed, Nadefensin-silenced plants that were uninduced or previously damaged by M. sexta were more susceptible to PST DC3000 than the WT plants that were uninduced or previously damaged by M. sexta, respectively. Clearly, Na-defensin functions as an anti-bacterial defense protein regardless of whether it is elicited by either pathogens or herbivores. Moreover, it appears that herbivore-mediated cellular changes do not affect Nadefensin's known anti-bacterial function. This is not surprising given the structure of the protein. Nadefensin belongs to the γ-Na-defensin class, which is 40–45 amino acids long, with 8 conserved cysteine residues that form 4 disulphide bridges; these disulphide bridges are thought to contribute to the stability of the protein. Moreover, Na-defensin is a cationic-basic protein and thus can be attracted to bacterial or fungal cell membranes, where it interacts directly 23. We do not know exactly what feature of plant responses (other than jasmonates) towards pathogens or herbivores causes the induction of Nadefensin, but plants may elicit Nadefensin to prime their defense responses to possible bacterial damage. The wounds that herbivore feeding causes are likely entry points for many pathogens. This scenario is consistent with the lower PST DC3000 growth in WT or Nadefensin-silenced plants that were previously fed on by M. sexta compared to WT or Nadefensin-silenced plants that were previously undamaged. M. sexta-damaged plants had reduced PST DC3000 CFUs, which is consistent with other studies reporting that herbivore-damaged plants may be less suitable for pathogens 2425. Herbivore damage is associated with the production of several defense metabolites that can have detrimental effects on herbivores as well as pathogens. For instance, M. sexta damage increases nicotine production in N. sylvestris26 and N. attenuata 3, and nicotine was found to inhibit the growth of five species of Pseudomonas bacterial pathogens 12. Similarly, many phenolic compounds such as rutin and chlorogenic acid are also produced in N. attenuata 4. Rutin, for instance, is a broad spectrum defense metabolite 12. Accordingly, after herbivory plants likely elicit herbivore-specific metabolites as well as anti-bacterial defense proteins such as defensin. The latter may be elicited in anticipation that the wounds created during herbivory might provide an opening for pathogenic bacteria.

We studied the three-way interaction between a plant (N. attenuata), an herbivore (M. sexta) and pathogenic bacteria (PST DC3000) with reference to a known anti-bacterial defense protein, Na-defensin. Na-defensin was found to be effective in containing PST DC3000 growth but ineffective in preventing M. sexta from feeding and gaining body mass. When the indirect effects of Nadefensin on herbivore performance were analyzed in PST DC3000-infected WT leaves, we found that the increased expression of Nadefensin increased resistance to PST DC3000 and also reduced M. sexta's leaf consumption but not its larval mass gain. Reduced Nadefensin expression in PST DC3000-infected Nadefensin-silenced plants decreased resistance to PST DC3000 but hindered M. sexta's leaf consumption and its mass gain. When we analyzed the Nadefensin-mediated responses in M. sexta-damaged leaves to PST DC3000 resistance, we found that damage by M. sexta increased the resistance of both WT and Nadefensin-silenced plants to PST DC3000. This enhanced resistance in M. sexta-damaged plants may result from the elicitation of a large set of anti-herbivory defense metabolites that can affect both herbivores as well as pathogens and to some specific anti-bacterial defense proteins such as defensin.

CR carried out the molecular studies, herbivore and pathogen bio-assays and statistical analysis. ITB designed and coordinated the experiments and also helped to draft the manuscript. CR and ITB read and approved the final manuscript