The green peach aphid, <em>Myzus persicae</em>, acquires a LIPOXYGENASE5-derived oxylipin from <em>Arabidopsis thaliana</em>, which promotes colonization of the host plant

A LOX5-Derived Oxylipin(s) Facilitates GPA Infestation on Arabidopsis

The Arabidopsis thaliana genome contains six LOX genes of which LOX1 and LOX5 encode 9-LOXs.⁸ Recently, we demonstrated a role for LOX5-derived oxylipins in facilitating infestation by the phloem sap-consuming insect Myzus persicae, commonly known as the green peach aphid (GPA).⁹ GPA population size was smaller on Arabidopsis lox5 mutants compared with the wild-type (WT) plant. This lox5 defect was complemented by the application of 9-hydoxy-10E, 12Z-octadecadienoic acid (9-HOD), a LOX5-derived oxylipin. LOX5 expression was rapidly induced in roots of GPA-infested WT plants.⁹ This increase in LOX5 expression was paralleled by a corresponding increase in LOX5-derived oxylipins in the roots and vascular sap-enriched petiole exudates of GPA-infested plants. Micrografting experiments confirmed that LOX5 function in roots is critical for promoting foliar infestation by GPA, thus highlighting an important role for roots in plant interaction with insects that colonize aboveground tissues.⁹

The vascular sap of Arabidopsis contains an antibiosis activity that adversely impacts GPA fecundity on an artificial diet.⁹ GPAs manipulate host physiology to counteract this antibiosis activity and successfully colonize Arabidopsis. However, insects reared on the lox5 mutant were unable to suppress this antibiotic activity. These observations suggest that a LOX5-derived factor is required for GPA to suppress this antibiosis activity.⁹ Compared with the WT plant, on the lox5 mutant the GPA also spent less time feeding from the sieve elements and was unable to tap into the xylem. As a result, the water content in lox5-reared GPA was lower than in insects reared on WT plants. The active ingestion of xylem sap is important for rehydration and osmoregulation of the insect hemolymph, thus enabling the insect to counter problems posed by the consumption of a sugar-rich diet.¹⁰ Since GPA population size on an artificial diet was enhanced when the diet was supplemented with 9-HOD,⁹ we suggested that LOX5-synthesized oxylipins, or products thereof, facilitate feeding and promote fecundity by directly impacting aphid behavior and physiology. Interestingly, although the GPA lacks a 9-HOD synthesizing activity, as shown in Figure 1, 9-HOD accumulated in GPA reared on WT plants. By contrast, 9-HOD level in insects reared on the lox5 mutant was undetectable (Fig. 1). However, in GPA reared on lox5 plants that were irrigated with 9-HOD, the level of 9-HOD was comparable to that in GPA reared on WT plants (Fig. 1), thus implying that 9-HOD synthesized by Arabidopsis is consumed by GPA. We therefore conclude that LOX5-derived oxylipins ingested by GPA, or their products in the insect, facilitate insect feeding from the phloem and xylem, and simultaneously promote insect fecundity.

Figure 1. GPA acquires LOX5-derived 9-HOD from Arabidopsis. Concentrations of 9-HOD in GPA reared on wild-type (WT) Arabidopsis accession Columbia and the lox5–1 mutant, and the lox5–1 mutant irrigated with 9-HOD (0.1 μM). Between 300–400 GPA were collected from the foliage 48 h post-infestation (hpi). Each bar represents the mean (± SE) (n = 3) presented as ng/g fresh weight (FW). GC-MS was used to determine the concentrations of 9-HOD. Chiral phase HPLC analysis was used to determine the S and R isomer composition of the oxylipins. A strong abundance of S-isomer over R-isomer was observed, suggesting that vast majority of the oxylipin detected were synthesized enzymatically. nd, none detected. 9-HOD levels in insects reared on the WT and the lox5-1 mutant irrigated with 9-HOD were not significantly different.

LOX5 is Required in GPA-Infested Arabidopsis to Enhance Expression of the Defense Regulatory Gene, PHYTOALEXIN-DEFICIENT4

Why does Arabidopsis responding to GPA induce expression of LOX5 when it is the insect that seems to benefit from 9-LOX products? It is plausible that GPA-derived effectors trick the host plant to enhance LOX5 expression. Alternatively, LOX5-derived oxylipins may confer some benefit to the plant to combat GPA infestation. Indeed, in Arabidopsis, 9-LOXs are involved in the activation of plant defense against pathogens.¹¹ On similar lines, as shown in Figure 2A, we observed that LOX5 activity was required for the induction of PHYTOALEXIN-DEFICIENT4 (PAD4) gene expression in the foliage of GPA-infested plants. By comparison to the GPA-infested WT plants, in which PAD4 expression was induced to high levels, PAD4 expression was not induced in GPA-infested lox5 leaves. PAD4, which has homology to acyl hydrolases/lipases, is required for controlling GPA infestation on Arabidopsis.¹²14 9-HOD application induced PAD4 expression in Arabidopsis leaves (Fig. 2B), lending support to our suggestion that Arabidopsis engages LOX5-derived oxylipins, or products thereof, to promote PAD4 expression for limiting the severity of GPA infestation. However, compared with the WT plant, GPA population size is significantly smaller on the lox5 mutant despite the lack of elevated PAD4 expression.⁹ A possible explanation for this paradox is presented in Figure 3. According to this model, Arabidopsis engages LOX5-derived oxylipins that have been synthesized in roots and subsequently transported to the foliage to promote PAD4 expression and, thus, aid in controlling the severity of GPA infestation. However, GPA has evolved to utilize plant LOX5-synthesized oxylipins, or products thereof, as stimulatory cues, thereby enabling it to successfully colonize Arabidopsis despite the activation of PAD4-dependent defenses. When feeding on the lox5 mutant, the insect does not receive these cues, and hence is unable to effectively colonize the mutant plant, despite the inability of the lox5 mutant to efficiently activate the PAD4-dependent defense pathway. PAD4-independent mechanisms likely also contribute to defense against GPA in the absence of LOX5 function.

Figure 2.LOX5 activity is required for PAD4 expression. (A) PAD4 expression in leaves of un-infested (-GPA) and GPA-infested (+ GPA) WT (white bars) and lox5–1 (black bars) plants. Quantitative real-time RT-PCR was performed on RNA extracted from leaves harvested at the indicated hours post-infestation (hpi). The relative expression of PAD4 to that of the EF1α control is presented. Each value is the mean ± SE (n = 3). (B) PAD4 expression in leaves of WT plants infiltrated with water or 9-HOD (1 μM). RT-PCR was performed on RNA extracted from leaves harvested at the indicated hours post-treatment (hpt). The EF1α gene provided the control for RT-PCR.

Figure 3. Model for involvement of LOX5-derived oxylipin(s) in Arabidopsis interaction with GPA. GPA infestation on the leaves results in the induction of LOX5 expression in roots and a concomitant increase in the levels of LOX5-derived oxylipins (e.g., 9-HOD). These oxylipins are transported from the roots to the foliage where one or more of these oxylipins stimulate expression of the defense regulatory gene, PAD4, thus controlling severity of the GPA infestation. One or more of these oxylipins consumed by the GPA, or products thereof, impact insect behavior resulting in the stimulation of feeding from phloem and xylem and increase in insect fecundity.

According to the co-evolution theory, a continuous struggle for dominance or “arms race” between plants and their insect pests has led to the co-evolution of defenses and counter-defenses.¹⁵ Plants have evolved various preformed constitutive defenses coupled with specific induced defenses to counter threats by insect pests.¹⁶ Insect herbivores on the other hand have acquired adaptations that enable them to overcome, manipulate or exploit the plants constitutive and induced defenses.¹⁷ Although additional studies are required to characterize the role of oxylipins in modulating PAD4-mediated defenses against GPA, our findings indicate that the ability of GPA to acquire LOX5-derived oxylipins correlates with its ability to successfully colonize Arabidopsis.