Avoiding Effective Defenses: Strategies Employed by Phloem-Feeding Insects

Phytophages breach the integrity of plant tissues to recover nutrients from foliage, seeds, pollen, nectar, roots, or shoots. While many herbivores cause extensive damage, phloem-feeding insects, such as aphids and whiteflies, cause modest to barely perceptible damage, respectively. Phloem-feeding insects provide additional challenges to plants as they deplete photosynthates, vector viruses, and introduce chemical and/or protein effectors that alter plant defense signaling, infestation symptoms, and plant development (Kaloshian and Walling, 2005). When these attributes are combined with broad host ranges, breeding strategies that promote invasiveness, highly evolved feeding strategies, the ability to adapt to a wide range of plant habitats, and the emergence of insecticide-resistant strains, it is not surprising that phloem-feeding insects cause heavy losses in agriculture and horticulture (Goggin, 2007). With the tools of cell and molecular biology, genetics, genomics, electrophysiology, and biochemistry, investigators are providing novel insights into the complexity and dynamics of plant-herbivore interactions. Many of the reviews in this issue describe the initial events in perception, as well as the defense signals and biochemical reprogramming that influence direct (antibiotic and antixenotic) and indirect (interactions with natural enemies) defenses to tissue-damaging herbivores. This review will highlight intricacies of plant-/phloem-feeding insect interactions, with a primary focus on whiteflies and comparisons to aphids. Hemipteran development and SLWF-plant interactions. A, SLWF adult with arcs of eggs deposited on the abaxial side of an Arabidopsis ecotype Columbia leaf. Scale bar = 1 mm. B, Silverleaf symptoms on squash caused by a severe infestation in Riverside, CA. C, SLWF eggs and first, second, third, and early fourth instar nymphs on Arabidopsis leaves after 23 d of infestation. Scale bar = 0.7 mm. D, SLWF red-eye nymphs (late fourth instar) on Arabidopsis leaves after 28 d of SLWF infestation. Scale bar = 0.5 mm. E, Bluegreen aphid female giving birth to a nymph (John Klinger, University of Arizona). Scale bar = 1 mm. Aphids and whiteflies take advantage of their adept feeding strategies and avoid or deter many plant defenses. These insects disguise themselves and deceive their hosts and natural enemies by using their stylets to deliver salivary chemicals and/or proteins into the plant to influence wound healing, defense-signaling pathways, and volatile emissions. Similar deceptive strategies are routinely employed by phytopathogenic microbes to avoid recognition and combat plant defenses (da Cunha et al., 2007). Pathogens introduce effectors into plant cells manipulating many biochemical and cellular processes to enhance phytopathogen success on host plants. In plant-biotroph interactions, effectors influence three stages of interaction (pre-entry, entry, and colonization). These interaction stages will form the framework for discussing adaptations and evasive strategies employed by phloem-feeding insects. The plant selection mechanisms used by phloem-feeding insects vary. Whiteflies use color, while aphids use both visual and olfactory cues to direct flight responses to host plants (Gerling, 1990; Powell et al., 2006). Upon landing, adults evaluate the tactile and chemical cues of the plant surface to determine the suitability of a plant as shelter or as a feeding and/or oviposition host. For insects that have sessile instars, like whiteflies, the plant chosen for egg deposition is a crucial maternal decision. On a good host, the next generation will thrive; on a poor host, insect populations will decline. While on the leaf surface, insects are exposed to chemicals that are imbedded in the hydrophobic cuticular waxes, including nonvolatile secondary metabolites, as well as volatile and semivolatile compounds (i.e. monoterpenes and glucosinolate-derived volatiles), which serve to attract or repel insects (Müller and Riederer, 2005). Leaf trichomes contribute to this complex environment by exuding secondary metabolites and proteins that have antibiotic or antixenotic effects (Wagner et al., 2004). Glandular trichome exudates deter whitefly settling and entrap whiteflies, providing one of the most effective whitefly-resistance mechanisms known to date. Surprisingly, whiteflies prefer plants with nonglandular trichomes over glabrous plants and preferentially oviposit near trichome bases (Neal and Bentz, 1999). These trichomes provide shelter for the sessile nymphs, and trichome exudates deter natural enemies. Whiteflies are not perturbed by these exudates because whiteflies shed particles of wax from their coat to form a physical barrier to trichome exudates. Interestingly, trichomes also induce a beneficial polyphenism in whiteflies (Guershon and Gerling, 2006). On glabrous leaves, all whitefly nymph cases are flat. On leaves with trichomes, most of the fourth-instar cases are decorated with dorsal waxy projections (setae). Setea are induced by the tactile experiences (collisions with trichomes, exuvia, and eggs) of the mobile whitefly crawler. Setose nymphs are smaller, develop more rapidly, and provide less time for enemies to identify their prey. Furthermore, predators prefer non-setate nymphs. Whiteflies and aphids use tactile and gustatory cues to determine the value of a plant as a feeding and oviposition host (Gerling, 1990; Powell et al., 2006). During initial encounters with a plant, these insects often use their stylets to tap on and make shallow probes of the leaf surface. Combined with secretion of small amounts of watery saliva to dissolve surface chemicals and imbibition of the liquids at the surface (Miles, 1999), whiteflies and aphids can determine physical features and “taste” the chemical defenses of the phylloplane. These behaviors detect differences in the carbohydrate content of cell walls, epicuticular waxes, and presence or absence of secondary metabolites to determine nonhost or host status (Müller and Riederer, 2005). If the plant is unacceptable, the winged adults depart in search of a more suitable site or host. Therefore, like tissue-damaging caterpillars, the ability to move within and between plants is important for avoiding defenses in initial hemipteran-plant interactions (Paschold et al., 2007). The damage caused by cell punctures and the nature of salivary effectors will determine the defense-signaling pathways that are activated and metabolites and proteins that accumulate in the infested plant. In addition, the stylet path determines the constitutive and induced defenses an herbivore will encounter. To limit damage to epidermal cells and contact with extracellular defenses, hemiptera deposit beads of rapidly gelling saliva to form a flange at the leaf surface (to limit stylet slippage) and a sheath that insulates the stylets from apoplastic defenses, respectively (Miles, 1999). In addition, the sheath's polyphenol oxidases may polymerize apoplastic phenolics (an induced defense) to prevent damage to plant cells. Finally, the sheath provides a track along which stylets move. This limits cellular damage as evidenced by the tracks of whitefly nymph stylets after larval molts and Astegopteyx minuta's opportunistic use of dislodged aphid sheaths to guide its own stylet to an SE (Foster, 1996; Freeman et al., 2001). Aphid and whitefly stylet sheath paths are multi-branched, showing that stylets take tortuous routes to the phloem (Freeman et al., 2001; Tjallingii, 2006). During this journey, aphids puncture and “taste” virtually all mesophyll cells on their path to a major vein of the phloem. This appears to orient the stylet's progression toward an SE. Plant cell damage can be moderate to extensive depending on the mechanics and vigor of aphid stylet probing and the effectors introduced by the salivas. Therefore, it is not surprising that wound-signaling pathways are transiently activated by aphids (Martinez de Ilarduya et al., 2003). In contrast, whiteflies rarely puncture mesophyll cells and, thereby, avoid activation of wound responses and contact with the potent defenses that are stored within vacuoles and apoplasts of these cells (Walling, 2000; Kempema et al., 2007). Like aphids, whiteflies secrete saliva to allow appraisal of the chemical composition of the apoplast (Lei et al., 1998); these gustatory cues may provide directionality to stylet movement and provide up-to-date information about host suitability for feeding and oviposition. When a stylet pierces a phloem SE, the plasma membrane lesion must be rapidly sealed to prevent leakage of phloem sap into the apoplast (Will and van Bel, 2006). Plants repair SE wounds by depositing callose and proteins, and hemiptera modify these responses to enhance their own success. Gelling saliva cements the stylet sheath to the SE, rapidly sealing the puncture site. Apoplastic callose deposits also reinforce this repair (Kempema et al., 2007; Saheed et al., 2007). In the cytoplasm, proteins aggregate at an SE lesion to occlude the wound. In the Fabaceae, a unique protein complex (forisome), which changes conformation in response to wound-induced alterations in free Ca2+ or redox state, is used to seal wounds (Will and van Bel, 2006). Infestations and symptoms on aphid-resistant and -susceptible melons, tomatoes, and Medicago. Cotton aphid (Aphis gossypii)-melon interactions: A and B, Cotton aphid infestation of the resistant melon ‘AR5’ line (Vat gene; A) and susceptible melon ‘PMR5’ line (B; G. Thompson, Oklahoma State University). Notice lack of overt symptoms in the incompatible and compatible interactions. Potato aphid-tomato interactions: C and D, Potato aphid infestation of the resistant tomato variety ‘Motelle’ (Mi-1.2 gene; C) and susceptible ‘Moneymaker’ (mi-1.2; D; Isgouhi Kaloshian, UC Riverside). Aphid-M. truncatula interactions: E, Bluegreen aphid infestation of the susceptible ‘A17’ line causes necrosis. F, Spotted alfalfa aphid-infested ‘Borung’ (spotted alfalfa aphid-susceptible line; ttk AKR) displaying local chlorosis. G, Spotted alfalfa aphid infestation of the resistant ‘Mogul’ line (TTR AKR) displaying local purple haze. H, Spotted alfalfa aphid infestation of the susceptible ‘A20’ line displaying systemic vein chlorosis. All Medicago-aphid interaction photos were provided by John Klinger (University of Arizona). Most insects are deterred by the chemical complexity of a plant's phylloplane. However, some insects tolerate these constitutive defenses and use a plant as a host (a compatible interaction). During compatible interactions, plants perceive the amount of tissue damage, the quality and quantity of salivary signals (effectors), and the magnitude of electrical and/or hydraulic signals caused by hemipteran attack (Walling, 2000). After integration of this suite of signals, plants deploy signal transduction pathways to regulate large cohorts of genes to provide the “best” defense response to its intruder. Many induced genes appear to address the changes in physiological status imposed by hemipteran feeding, and defense-response genes are activated or suppressed (Thompson and Goggin, 2006). Almost without exception, pathogenesis-response (PR) gene RNAs, proteins, and/or activities are elevated after phloem-feeding insect attack (Walling, 2000). Salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) control many of the cellular and biochemical responses to pathogens and pests. These signaling pathways cross talk and may act antagonistically or synergistically (Koornneef and Pieterse, 2008). The SA, JA, and ET networks also liaise with other known (i.e. auxin, abscisic acid, and brassinosteroids) and novel defense-signaling networks to provide the innate immunity to pests/pathogens (Robert-Seilaniantz et al., 2007). The integration of defense networks may minimize expression of costly and ineffective defenses that divert carbon and nitrogen resources from plant growth and reproduction. However, similar to pathogens, insects have leveraged the molecular communication between the signaling networks to enhance their success on host plants. The Bemisia tabaci biotype B (silverleaf whitefly [SLWF])-Arabidopsis (Arabidopsis thaliana) interaction is a clear example of an insect manipulating plant signaling to suppress effective defenses, increase plant susceptibility, and enhance insect performance (Kempema et al., 2007; Zarate et al., 2007). In response to SLWF nymph feeding, SA-regulated RNAs increase locally and systemically, while JA- and ET-regulated RNAs are unchanged or decline. The correlation of defense gene RNA levels and SLWF performance on JA and SA defense mutants show that JA-regulated defenses are important in deterring SLWF nymph development. Furthermore, when the npr1 mutant, which impairs SA-regulated defenses and uncouples SA-JA cross talk, is treated with methyl jasmonate (MeJA), SLWF nymph development is severely delayed, demonstrating that JA controls defenses that actively thwart whitefly development. SLWFs deceive Arabidopsis plants and prevent the activation of the JA-regulated defenses that actively deter nymph development. SLWFs may merely evade JA-regulated defenses due to the absence of tissue damage once a feeding site is established. Alternatively, nymph saliva may contain effectors that directly antagonize JA-regulated defenses. There is precedent for an insect effector (Glc oxidase) to suppress effective direct (nicotine production) and indirect (volatile biosynthesis) defenses (Musser et al., 2002; Bede et al., 2006). It is also possible that a nymph effector could act indirectly by increasing SA levels and leveraging SA-JA cross talk mechanisms to inhibit the expression of JA-regulated defenses. This is supported by the fact that SLWFs increase SA levels locally and systemically during infestation of Arabidopsis (S.I. Zarate, D.A. Navarre, and L.L. Walling, unpublished results). Similar evasive strategies appear to be active during the aphid interactions with Arabidopsis, sorghum (Sorghum bicolor), and Medicago truncatula. Behavioral studies on aphid-preinfested plants indicate that aphid feeding reduces the defenses that deter aphids (Prado and Tjallingii, 2007). In addition, SA-regulated RNAs increase and JA-regulated RNAs are reduced or increase modestly in aphid-infested leaves (Moran and Thompson, 2001; Ellis et al., 2002; Zhu-Salzman et al., 2004; De Vos et al., 2005; Gao et al., 2007). Unlike whiteflies, only modest systemic responses occur, and changes in SA or JA levels are not detected after Myzus persicae infestation of Arabidopsis (De Vos et al., 2005). Aphid performance on a large number of Arabidopsis mutants has been reported (Thompson and Goggin, 2006; de Vos et al., 2007). While the impact of SA and JA defense mutants on aphid population growth has varied, JA-regulated defenses appear to be important in deterring aphid population expansion in Arabidopsis. In addition, MeJA treatment of Arabidopsis, sorghum, and Medicago plants retards aphid population expansion (Ellis et al., 2002; Zhu-Salzman et al., 2004; Gao et al., 2007). Collectively, these data indicate that aphids, like whiteflies, express “decoy” defenses and suppress or avoid the JA-regulated defenses that antagonize insect performance (Thompson and Goggin, 2006). The identities of most of the JA-regulated suppressed during whitefly and aphid are However, one of these appears to be of which is by SA, JA, and ET et al., 2006). Upon tissue damage, and their to highly Vos et al., 2007). avoid cellular damage and, not not and levels et al., 2006; Kempema et al., 2007; and 2007). A in levels a more environment for which are by However, reduced may be a for aphids that are to and these compounds for their own defense et al., While aphids appear to most one is at elevated levels after aphid infestation and is a potent aphid and that some effective constitutive defenses can be in response to hemipteran It is not clear all suppress a of plant defenses to enhance their success. These can be only when defense mutants are used in performance and RNAs for genes for defense-signaling are This is a in most Arabidopsis, networks are complex and novel defense-signaling networks are (Robert-Seilaniantz et al., 2007). In the of the and networks are not and appear to and 2007). Therefore, it is not surprising to to the For both SA and JA appear important in innate immunity to the aphid and antibiotic and antixenotic respectively et al., 2006; et al., Whiteflies and aphids increase and RNA levels et al., 2005; Kempema et al., 2007). The in RNAs, and SA controls a to aphids that is of Surprisingly, not influence SLWF nymph development. SLWFs may lack effectors to or effectors to deter the Alternatively, SLWFs may tolerate the using chemical and/or which are effective strategies to plant defenses. While defenses effective aphids and whiteflies it is to the of and in the molecular responses to phloem (De Vos et al., 2005; Kempema et al., 2007). This is due to the wide variety of that influence these including plants for infestation of of feeding, and in and However, in plant and physiological responses to insects is in volatile and interactions et al., and is also supported by interactions. plants can between whitefly and tabaci biotype A) as evidenced by the of of genes and de et al., 2000; During incompatible interactions, a plant with a gene rapidly an insect and the infestation is (Kaloshian and Walling, 2005). While is known about the genes effectors in insects, genes effective insects have been and and strategies are employed to identify these To only one gene that to insects is at the molecular plants are resistant to the whitefly and biotype a and three and the mechanisms of appear (Kaloshian and Walling, 2005; et al., and For to a response which not in the response to aphids Potato aphid is antibiotic and phloem while to phloem-feeding is Finally, to whiteflies is in the or mesophyll and whitefly If a whitefly a feeding it can develop on plants. The biochemical for the to and the effectors in these incompatible interactions are not The defense-signaling mechanisms that control aphid have to incompatible responses in interactions. For aphids cause more in SA levels and/or gene RNAs in resistant in susceptible plants et al., 2000; and van 2002; de Ilarduya et al., 2003). In addition, to aphids is on and SA et al., However, this with the truncatula gene that to the aphid by leveraging JA-regulated defenses et al., 2007). Bluegreen aphid and alfalfa aphid interactions with Medicago are are in both compatible and incompatible interactions It is that insect saliva the signals that the incompatible interaction using mechanisms in the (Kaloshian and Walling, 2005). on the of effectors in microbes and the known salivary effectors from herbivores that influence volatile insect effectors may be a or et al., 2005; Cunha et al., 2007; et al., 2007). While an insect effector has not been in genes from and effectors important in compatible phloem-feeding insect interactions are For a salivary protein from aphids can the symptoms of infested susceptible plants et al., 2007). after herbivore attack or egg plants stored and of for from the infested and leaves et al., 2005). plant are used by tissue-damaging and phloem-feeding herbivores and their natural enemies to between and infested host providing a potent indirect also act directly to enhance or deter feeding, and provide information about herbivore can also direct defenses by defense signal transduction In addition, similar to the induced systemic with can plants for defense responses and 2006; et al., 2008). Like caterpillars, hemipteran saliva can volatile et al., and the volatile by phloem-feeding insects have chemical similar to of tissue-damaging herbivores. contain of including methyl and While some phloem volatile compounds et al., merely the of the chemicals in the volatile et al., to Unlike tissue-damaging the of in response to phloem are and at et al., et al., et al., 2003). This is due to the damage or of salivary effectors that deter volatile the of could be volatile could in direct and indirect defenses. While the and physiological changes that the of interactions are to be it is clear in a To it is not clear all herbivores one or host responses for their success or evasive are employed only by a of Phloem-feeding whiteflies and aphids a wide variety of to avoid or suppress effective defenses. The of hemipteran salivary proteins in or plant wound and defense responses is clear and with one (i.e. proteins of aphid watery the biochemical nature of effectors from phloem-feeding insects are Therefore, a on biochemical of insect is and will from the of and and is to novel to effector These biochemical strategies will be by the resources and for and their et al., 2006). The development of RNA for aphids and whiteflies that allow of insect genes will the of the effectors that enhance hemipteran success et al., 2007). These are also to allow the of the of hemipteran saliva that infestation symptoms, and incompatible interactions 1 and the of the that to these effectors in will be studies to the complexity of plant defense-signaling networks are have of gene expression after hemipteran in Arabidopsis, resources are and expression are for interactions, have an of these defense While of JA- and SA-regulated pathways are the and to other signaling pathways during interactions have to be to of and signaling will be to the and with and insects. In addition, the of novel defense pathways will to be and with defense signal transduction The impact of chemical with gene important These can be used to identify the that induce or novel signaling pathways and provide about the chemical nature of the novel plant signal and/or the insect effectors or that biochemical Finally, resources for plants will be tools for the from studies in plants to and are for a variety of and the dynamics of defense signaling in hemipteran interactions with both and of these resources with (i.e. use of gene RNA strategies, and expression of defense gene will the signaling networks and the that innate immunity and defense to phloem-feeding insects. These when with to insect performance and interactions at the and fourth allow for the development of strategies to enhance to van de and for of whiteflies and and Isgouhi State and John (University of for interaction and of and of the for