**3. Discussion**

The model legume *M. truncatula* provides a grea<sup>t</sup> opportunity to decipher the molecular mechanisms underlying plant defence against sap-sucking insects, where various types of interactions have been identified to multiple aphid species [16]. The well characterised, ET insensitive, *M. truncatula sickle* mutant allowed us to determine the specific roles that ET plays in plant-aphid interactions. We have found that the *sickle* mutant enhanced the antibiosis e ffect on *A. kondoi* and *T. trifolii* but not *A. pisum* (Figures 1 and 2), and delayed leaf senescence caused by the feeding of all three aphid species. We show that the ET signalling pathway is not essential for the function of the major aphid resistance genes, *AKR*, *APR* or *TTR* against the three aphid species and is also not required for the *AIN*-mediated hypersensitive response to *A. kondoi* or *A. pisum* infestation.

Our results showed that on *sickle*, the growth of both *A. kondoi* and *T. trifolii* colonies was significantly reduced compared to its wild-type parent, A17. The results sugges<sup>t</sup> that ET is a negative regulator of the moderate resistance in *M. truncatula* against these two aphid species. The ET signalling pathway has previously been demonstrated as a negative regulator in other plant species against sap-sucking insects. For instance, in rice, the suppression of ET biosynthesis enhanced resistance against a piercing-sucking insect, the brown planthopper (*N. lugens*) but reduced plant resistance against a chewing insect, striped stem borer (*C. suppressalis*) [34]. In *Arabidopsis*, several studies suggested that ET is a negative regulator of aphid defense responses. The fecundities of the generalist *M. persicae* and the specialist *Brevicoryne brassicae* were reduced on the ET-insensitive *etr1* mutant compared to wild-type plants [10,35]. The overexpression of a transcription factor gene, *MYB102*, which promotes ET biosynthesis by upregulation of some 1-aminocyclopropane-1-carboxylate synthase (ACS) genes in the ET-synthetic pathway led to an increase in aphid performance [36]. Furthermore, in tomato, ET signalling contributes to the susceptibility of potato aphid, *M. euphorbiae*, in the absence of the *Mi-1.2* gene. In choice assays, potato aphids preferred wild-type plants to the ET-insensitive, *Neverripe* mutant [37]. Our results with *sickle* together with other results discussed, sugges<sup>t</sup> that ET can benefit the feeding for some aphid species, and the impediment of ET pathway impairs the infestation of these aphids.

In contrast to *A. kondoi* and *T. trifolii*, the *sickle* mutant did not affect the growth of the Australian biotype of *A. pisum* (Figures 1 and 2). These findings were consistent with studies with the European *A. pisum* biotype (PS01), which is distinct from the Australian *A. pisum* biotype, and where ET was also found to not be involved in the aphid susceptible or resistant interactions [24,28]. However, *sickle* was found to promote the growth of a Chinese biotype of *A. pisum* [38–40]. These differences between the *A. pisum* biotypes and with *A. kondoi* and *T. trifolii* sugges<sup>t</sup> that the role of ethylene in the *M. truncatula*-aphid interactions is both biotype- and species-dependent.

How ET signalling modulates the moderate resistance in A17 against *A. kondoi* and *T. trifolii* is unknown. A17 carries multiple QTLs conferring antibiosis factors against these two aphid species [18,20]. The direct link between the ET signalling pathway and a specific QTL(s) in A17 is ye<sup>t</sup> to be investigated. It is possible that the suppression of the ET pathway in *sickle* led to upregulation of other signalling pathways, such as for SA and JA, which might increase plant defence mechanisms against these two aphid species, as these signalling pathways are often inter-linked and work synergistically or antagonistically [41–43]. Further research on the interactions between the ET insensitivity in *M. truncatula sickle* and other defense signalling pathways in plant-aphid interactions will facilitate the understanding of the function of ET in plant resistance to aphids.

Our results also showed that the *sickle* mutant delayed the leaf senescence caused by the feeding of all three aphid species. There was no noticeable phenotypical difference among the leaves of A17, *sickle* and Jester control plants prior to aphid infestation at three weeks after planting though *sickle* could demonstrate concomitant alternation of some ethylene related phenotypes, including delayed petal and leaf senescence and decreased abscission of seed pod and leaves at the later stage of plant growth [29]. As all our experiments were carried out with young plants of three to five weeks old, it is unlikely these concomitant ethylene related phenotypes have a direct impact on aphid performance or plant symptom in response to aphid infestation. Aphid infestation causes changes in source allocation in the host plant to direct nutrients to the insect infested tissues [44]. Premature leaf senescence has been suggested to be a plant defence mechanism used to counteract aphid feeding by redirecting the nutrients to the un-infested source tissues [45]. In *Arabidopsis,* infestation by *M. persicae* induced the transcription level of *SENESCENCE ASSOCIATED GENES* (*SAG*s). Silencing of the *SAG*s delayed plant senescence which led to an increase *M. persicae* levels [45]. Here we observed the opposite with the *M. truncatula sickle* mutant. Whether the delay in leaf senescence directly relates to the increased

antibiosis resistance to *A. kondoi* and *T. trifolii* remains unknown. Further research would help elucidate the relationship between leaf senescence and aphid feeding processes.

We have determined that ET is not essential for *AKR*, *APR* or *TTR* mediated resistance against *A. kondoi*, *A. pisum* or *T. trifolii*, respectively. ET has also been found to be dispensable for the *RAP* gene mediated resistance against the European *A. pisum* biotype [28]. However, upon infestation by *A. kondoi* or *T. trifolii*, some ET related genes were found to be induced in both A17 and Jester, with higher induction in Jester than A17 [26,27]. The lack of di fference in aphid performance between Jester and Jester with the homozygous *sickle* mutation suggests that induction of the ET related genes previously found in Jester may be insu fficient in limiting aphid feeding from the plant [26]. This might also be the case in other plant-aphid systems, such as in resistant barley plants with *Schizaphis graminum*, *Rhopalosiphum padi*, and *Diuraphis noxia* [11,12], wheat with *D. noxia* [13], tomato (*Mi1.2*) with *Macrosiphum euphorbiae* and melon (*Vat*) with *Aphis gossypii* [14]. Although in these plant-aphid systems, ET production or ET related genes were shown to be highly induced in the resistant interactions, whether ET contributes to the resistance outcome is still a question.

Both A17 and Jester carry a semi-dominant locus called *AIN* which mediates necrotic lesions resembling a hypersensitive response at the site of infestation by both *A. kondoi* and *A. pisum* [23]. It is unlikely that ET signalling is negatively regulating the activity of *AIN* in A17 for the following reasons: firstly, while *AIN* is important for resistance to both *A. kondoi* and *A. pisum* in A17, the *sickle* mutant only results in an increase in resistance *to A. kondoi*. Secondly, the *sickle* mutant still displays the same HR-like symptoms conferred by *AIN* following infestation with either aphid. While the overall number of necrotic lesions were less in *sickle* than A17 following *A. kondoi* infestation, this is most likely a reflection of the lower number of aphids feeding on *sickle*, due to the increase in aphid resistance. Importantly, the size of the necrotic lesions remained similar between *sickle* and A17. Collectively, these data sugges<sup>t</sup> that ET signalling is not a negative regulator of *AIN* activity. As discussed earlier, there are other QTLs that have been identified in A17 as being important for the moderate resistance to *A. kondoi* but not *A. pisum* [18], which may be the target of ET negative regulation, or the target(s) may be an unidentified loci.

In conclusion, the *M. truncatula sickle* mutant has previously been shown to be defective in the control of root infecting micro-organisms including beneficial rhizobia, mycorrhizal fungi, as well as infection by the fungal and oomycete pathogens, *R. solani* and *P. medicaginis* [46,47]. Here we observed a positive e ffect of the *sickle* mutant on the control of the infestation by insect herbivory, by *A. kondoi* and *T. trifolii*. While ET signaling is not essential for the activity of three R genes for resistance against *A. kondoi*, *A. pisum* and *T. trifolii,* it is also not involved in the *AIN* mediated hypersensitive response to *A. kondoi* and *A. pisum*. However, the *sickle* mutant delayed leaf senescence by all three aphid species, but enhanced tolerance only to infestation by *A. kondoi* and *T. trifolii*, The results sugges<sup>t</sup> that manipulation of the ET signaling pathway could also help provide resistance to certain aphid species and enhance plant tolerance against aphid feeding.

#### **4. Materials and Methods**

#### *4.1. Plant Materials and Growth Conditions*

Included in this study were *M. truncatula* A17 (referred to as "wild-type"), which is the reference *M. truncatula* accession, an ethylene insensitive *sickle* mutant, a *tnt1* retrotransposon mutant that arose from a single *tnt1* insertion in the genetic background of A17 [29,30] and Jester which is closely related to A17 sharing 89% genome identity [21]. In addition to ethylene insensitivity, *sickle* also demonstrates delayed petal senescence and decreased abscission of seed pod and leaves. In addition F2 and F3 progenies were generated by reciprocal crossing between Jester and *sickle* accession plants according to the methods described by [48].

Prior to planting, seeds were scarified and germinated on moist filter paper in the dark at room temperature for two days. Plants were grown in a growth chamber with 16 h light (22 ◦C)/8 h dark (20 ◦C) under metal halide and incandescent lamps producing 240 to 260 μE m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> or in a glasshouse with controlled temperature around 22 ◦C and ambient light condition. In both glasshouse and growth chamber experiments, plants were grown in individual 0.9 L pots with *Arabidopsis* soil mix (Richgrow company, Perth, WA, Australia). Plants were fertilized with liquid Nitrosol fertilizer (Amgrow Australia, Perth, WA, Australia) once planted and watered two times per week throughout the experiments.

#### *4.2. Aphid Species and Rearing Conditions*

The aphid species used were *A. kondoi* (bluegreen aphid), *A. pisum* (pea aphid.) and *T. trifolii* f. *maculate* (spotted alfalfa aphid). Aphids of each species were obtained from colonies initiated from single aphid clones collected in Western Australia and were reared on Subterranean clover (*Trifolium subterraneum*) for *A. kondoi*, faba bean (*Vicia faba* L.) for *A. pisum* and alfalfa (*M. sativa*) for *T. trifolii* with 14 h light (23 ◦C)/10 h dark (20 ◦C) under high pressure sodium lamps and fluorescent light at 280 μE m–2 s–1. Aphids were transferred to experimental plants with a fine paintbrush.

#### *4.3. Screening for F2 Plants Containing the Sickle Homozygous Allele*

To obtain lines with the homozygous *sickle* locus and all three aphid resistance genes, *AKR*, *APR* and *TTR* crosses between *sickle* and Jester were performed F2 plants were first screened for the *sickle* mutation using 1-aminocyclopropane-1-carboxylic acid (ACC) [30]. To establish an effective and reliable condition for the screening, five concentrations of ACC, 20, 40, 60, 80 and 100 ppm were initially evaluated with the *sickle* mutant, A17 and Jester with water as the control. While the *sickle* mutant showed no response to all ACC concentrations, 20 ppm ACC started to show impact on the embryonic root of A17 and Jester, while 80 ppm or 100 ppm of ACC resulted in severe stunting of root radicles of both A17 and Jester (Supplementary Figure S1). The 100 ppm was used for the screening of the Jester x *sickle* F2 population (Supplementary Figure S2).

#### *4.4. Genotyping F2 Plants with AKR, APR and TTR Loci*

In order to obtain F2 lines that combined the homozygous *sickle* allele and the three aphid resistance gene loci, 90 of the 163 pre-selected F2 *sickle* mutant plants were randomly selected and analysed using high throughput Multiplex-Ready marker technology (MRT) and molecular markers linked to these loci (Supplementary Table S1). The plants were grown in the growth room conditions as described above. Two weeks after planting, a single trifoliate leaf from each plant was collected and DNA isolated using the CTAB method as described previously [49]. DNA was subsequently diluted to a concentration of 50 ng/μ<sup>L</sup> in a 96-well plate and multiplex ready PCRs were setup using the primers in Supplementary Table S1 and the protocol described by Hayden et al. [50]. The multiplexed PCR products were subjected to fragment analysis on an ABI3730 DNA analyser (Applied Biosystems, Melbourne, Victoria, Australia) according to Hayden et al. [50] and marker allele sizing determined using the Genemarker software (SoftGenetics LLC, State College, PA, USA).. After the genotyping, seeds from the F2 plants with both the homozygous *sickle* and the three homozygous aphid resistance gene alleles, *AKR*, *APR* and *TTR*, were harvested to obtain F3 seeds for the subsequent aphid infestation experiments.

#### *4.5. Aphid Performance and Plant Damage on Single Trifoliate Leaves*

To assess the aphid performance and leaf tolerance of *sickle* in comparison with its wild-type, parent A17 and Jester against three aphid species, three experiments were conducted, one in the glasshouse and two in growth chambers. Six replicate plants of each *M. truncatula* accession were randomly arranged. For all three experiments, three weeks after sowing, a single trifoliate leaf of similar age (fourth or fifth trifoliate leaf to emerge on the primary stem) of each plant was infested with four, five or seven adults of *A. pisum*, *A. kondoi* or *T. trifolii*, respectively. The number for each aphid species was determined based on our previous experiences with regards to the aphid size, the speed of aphid reproduction and degree of leaf damage caused to create a condition that allowed the aphid

growth and leaf damage to be fully expressed to make comparison between the three *M. truncatula* accessions [17,22,23,26,31–33]. The aphids were caged on a single trifoliate leaf in a linen mesh cage (35 × 200 mm) per plant. A wooden stake supported the stem and cage [26]. Seven days after aphid infestation, the aphids on each leaf were collected and weighed. The damage on each leaf was visually assessed after the removal of the aphids.

The e ffect of the *sickle* mutant on *R* gene mediated aphid resistance was first measured using the F2 plants after the genotyping. This was followed by an experiment using three independent F3 lines which contained the *sickle*, *AKR*, *APR* and *TTR* homozygous alleles. A17, Jester and the *sickle* mutant were included for comparison. For each accession/F3 line, six replicate plants were set up for the aphid infestation as described above with single trifoliate leaves. The aphid population weight and leaf damage symptoms, such as leaf senescence and necrosis, were assessed. To examine if the *sickle* mutant a ffects the *AIN* -mediated hypersensitive response to *A. kondoi* and *A. pisum*, the numbers of macroscopic necrotic flecks per single trifoliate leaves were also recorded.

#### *4.6. Aphid Performance and Plant Tolerance Experiments on Whole Plants*

To assess the aphid performance and plant tolerance on whole plants, non-choice experiments were conducted under glasshouse conditions. Plants of *M. truncatula sickle* mutant, A17 and Jester were grown as described above. Two weeks after planting, each plant was infested with four, five or seven adults of *A. pisum*, *A. kondoi* or *T. trifolii*, respectively. Six replicate plants were set up for each *M. truncatula* accession with or without aphid infestation. Plants were randomly arranged. Fourteen days after the aphid infestation, aphids were collected from each plant and weighed immediately. After the removal of the aphids, the aerial part of all the plants including the non-infested control plants were dried in the oven at 50◦C for two days. The dried weight of each plant was recorded. For each plant, aphid fresh weight per plant dry weight was calculated to determine aphid performance on the plant. For each *M. truncatula* accession, the tolerance of individual plants to aphid infestation was measured as the percentage of plant biomass reduction (PBR) relative to mean biomass of the control plants of the same *M. truncatula* accession using the formula: PBR = [(A − B)/A] × 100, in which A: average of the non-infested plant dry weight; B: dry weight of individual aphid-infested plant.

With each experiment, the aphid weight, the number of necrotic flecks or plant biomass reduction were analyzed by one-way ANOVA and compared by the LSD test at a 5% significance level using GenStat (VSN International, Rothamsted Research, Hertfordshire, UK).

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1422-0067/21/13/4657/s1. Table S1. Overview of the PCR primers used; Table S2. Overview of the genotyping data. Supplementary Figure S1. The radicle root growth of *Medicago truncatula* A17, Jester and *sickle* mutant. Supplementary Figure S2. Screening of *Medicago truncatula* F2 plants of crosses between Jester and *sickle*

**Author Contributions:** Conceptualization, L.-L.G. and K.B.S.; investigation, L.Z., L.-L.G., L.G.K., and Y.G.; formal analysis, L.Z., L.-L.G., L.G.K..; resources, K.B.S.; writing—original draft preparation, L.-L.G.; review and editing, L.Z., L.G.K., S.J. and K.B.S.; supervision L.-L.G. and K.B.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding

**Acknowledgments:** The authors thank Elaine Smith for technical assistance and Jonathan Anderson, Louise Thatcher and Kemal Kazan CSIRO for critical review of the manuscript. This research was financially supported by CSIRO A&F. The authors also would like to acknowledge the Shanxi Agricultural University and Scholarship Council of Shanxi Province, China to support L.J. Zhang and Y.Q. Guo during their visit to CSIRO.

**Conflicts of Interest:** The authors declare no conflict of interest.
