**1. Introduction**

Aphids (Hemiptera: Aphidoidea) constitute a large group of sap-sucking insect pests that cause substantial losses to agriculture worldwide by draining plant nutrients and transmitting pathogenic viruses [1–3]. The plant-aphid interaction is distinctive from plant interactions with microbial pathogens and chewing insects mainly because aphid infestation instigates very little physical damage to the plant. With their stylets, aphids penetrate plant tissues by piercing intercellularly through epidermal and mesophyll cell layers and ultimately feed specifically from the phloem sieve element [1–5]. A number of plant genes or loci, including Resistance (*R*) genes, that modulate plant defenses against aphids have been identified in a range of plant species against various aphid species ([6–8]. Molecular studies have revealed that plant phytohormones are also involved in the regulation of plant interactions with

aphids. However, in many cases, the specific roles that phytohormone pathways play in basal and *R* gene-mediated aphid resistance remains largely unknown.

Ethylene (ET) is a gaseous plant hormone which is involved in the regulation of various developmental as well as abiotic and biotic stress responses. Studies using mutants impaired in ET biosynthesis and signalling demonstrated a direct role for ET in plant defence against microbial pathogens and insect pests and in the control of plant association with beneficial microbes, such as rhizobia and mycorrhizas. ET signalling has also been shown to regulate plant interactions with insect herbivores [4].

A number of studies have been conducted to investigate the role that ET plays in both compatible and incompatible plant−aphid interactions. For compatible interactions, most studies were carried out with the generalist aphid *Myzus persicae* (Sulzer) for which there is no natural genetic resistance. However, the results from these studies were inconsistent. Divol et al. [5] and Moran et al. [6]showed that genes involved in ET biosynthesis and signalling were induced in both celery and *Arabidopsis* following infestation by *M. persicae*. Other studies showed that ET accumulation remained unchanged in both *M. persicae*-infested *Arabidopsis* and *Nicotiana attenuate* compared to the un-infested control plants [7,8] When the performance of *M. persicae* was compared between the *Arabidopsis* wild-type and ET-insensitive *etr* or *ein2* mutant plants, Kettles et al. [9]. found that the aphid fecundity did not di ffer between the *Arabidopsis* wild-type and *etr1* mutant, whilst the *ein2* mutant did show higher *M. persicae* fecundity than wild-type plants. In contrast, Mewis et al. [10] found that the fecundity of both *M. persicae* and *Brevicoryne brassicae* was reduced on the *etr1* mutant compared to wild-type *Arabidopsis* plants. These contradictory results in the *Arabidopsis*−*M. persicae* interaction highlight the need to further study the role of ET in plant-aphid interactions.

Studies also indicated that ET may modulate *R* gene-mediated plant defence against aphids. In aphid-resistant barley plants, ET production was significantly induced following the infestation by *Schizaphis graminum*, *Rhopalosiphum padi*, and *Diuraphis noxia* [11,12]. Upon feeding by *D. noxia*, transcript levels of ET-related genes increased in aphid resistant wheat plants [13]. Furthermore, the induction of genes involved in ET signalling and downstream responses was also found in both susceptible and resistant interactions of tomato with *Macrosiphum euphorbiae* and in melon with *Aphis gossypii* [14]. However, in melon with *A. gossypii* stronger induction of ET pathway genes was shown in the resistant variety than the susceptible plants, but this was not the case in tomato with *M. euphorbiae*.

*Medicago truncatula* is a model legume species for studying plant interaction with aphids [15,16]. *M. truncatula* is a host to several important aphid species including *Acyrthosiphon kondoi* (bluegreen aphid), *Therioaphis trifolii* (spotted alfalfa aphid) and *A. pisum* (pea aphid). In *M. truncatula*, various types of resistance against these aphid species have been identified and the resistance is controlled either through major dominant resistance genes and/or quantitative loci [17–24]. In *M. truncatula* cv. Jester, three single dominant resistance genes, named *AKR* (*A. kondoi* resistance), *APR* (*A. pisum* resistance) and *TTR* (*T. trifolii* resistance), provide strong resistance to *A. kondoi*, *A. pisum* and *T. trifolii*, respectively [17,21,22]. In addition to the major dominant resistance genes for *A. kondoi* and *A. pisum*, a second semi-dominant resistance gene termed *AIN* (*Acyrthosiphon induced necrosis*) has been identified [23]. *AIN* confers a moderate level of resistance to both aphid species and forms hypersensitive response (HR-like) necrotic lesions at the site of infestation by both *A. kondoi* and *A. pisum* [23,25]. This locus is present in both Jester and the reference *M. truncatula* accession, Jemalong (A17) which lacks the three major resistance genes, *AKR*, *APR* and *TTR*. The *M. truncatula* cv. Jester is closely related to A17 [21]. When compared with the highly susceptible *M. truncatula* accession A20, A17 shows moderate resistance to all three aphid species [15]. In A17, in addition to the antibiosis resistance conferred by the *AIN* locus, two distinct quantitative trait loci (QTLs) have been identified for tolerance to *A. kondoi* and *A. pisum*, respectively [18]. Furthermore, three QTL involved in the moderate antibiosis and tolerance to *T. trifolii* have also been identified in A17 [20].

The molecular mechanisms underlying the various types of aphid resistance in *M. truncatula* are largely unknown. Expression analysis of genes involved in defence signalling pathways indicated

that salicylic acid (SA)-related genes were induced in both A17 and Jester following the infestation by *A. kondoi* and *A. pisum* [17,26,27]. However, jasmonic acid (JA)-related genes were highly induced only in Jester when infested by *A. kondoi* suggesting that JA might be involved in the *AKR*-mediated resistance to *A. kondoi*. In the interactions between *M. truncatula* and European biotypes of *A. pisum*, the induction of phytohormones in *Medicago truncatula* was dependent upon the genotypes of both plant and insect as well the time post-infestation and aphid density [28]. There was some induction of hormones in the compatible interaction but higher concentration of JA, SA and medicarpin exhibited during the incompatible interaction. Although Gao et al. [26] showed an induction of ET associated genes following *A. kondoi* infestation of both Jester and A17, little is known about the role of the ET signalling pathway in moderate or *R-*gene mediated responses in *M. truncatula* following aphid predation.

The primary aim of this study was to determine the role of ET signalling in the different modes of resistance in *M. truncatula* against the three aphid species, *A. kondoi*, *A. pisum* and *T. trifolii*. The ET insensitive mutant *sickle* in the A17 background, provides a useful genetic tool to decipher the function of ET signalling in the control of different plant-aphid interactions [16,29,30]. Therefore, the role of ET in the moderate resistance to aphids found in A17[18,20,23], was tested by comparing aphid performance and plant tolerance in *sickle* to A17 wild-type plants. To examine the role of *R* gene mediated resistance, crosses were made between Jester, which harbours the major resistance genes and *sickle.* Offspring that carry both the homozygous *sickle* mutation and homozygous resistance genes for the respective aphid species were then tested for aphid performance and plant damage caused by aphid feeding. We found that ET is a negative regulator of moderate resistance to *A. kondoi* and *T. trifolii* but not to the Australian, *A. pisum* biotype. Our results also showed that ET is not essential for *R* gene mediated resistance against the three aphid species or for the *AIN* mediated HR-like response to both *A. kondoi* and *A. pisum*.
