**2. Results**

#### *2.1. The M. truncatula Sickle Mutant Modulates Aphid Resistance*

To examine if the ethylene-insensitive, *M. truncatula sickle* mutant affects the moderate aphid resistance observed in A17, the performance of three aphid species, *A. kondoi*, *T. trifolii* and *A. pisum* was measured. The highly resistant accession, Jester, which carries single dominant resistance genes to all three aphid species and is near isogenic to A17, was included and aphid performance was measured with single trifoliate leaves and subsequently with whole plant assays. Although the durations of aphid infestation were different, seven days in single leaf experiments and 14 days in the whole plant assays, for each aphid species and each *M. truncatula* accession, the results were consistent between these two experiments. As shown in Figures 1 and 2, with all three aphid species, the aphid population weight were significantly higher on the moderately resistant A17 than the highly resistant Jester, which was consistent with our previous reports [17,21,31]. Interestingly, for both *A. kondoi* and *T. trifolii*, the aphid weight was significantly lower (*p* < 0.05) on *sickle* than its wild-type parent, A17, but significantly higher (*p* < 0.05) than on Jester plants. The reduction of aphid population on the *sickle* plants was most pronounced for *T. trifolii* (Figures 1 and 3). At seven days following aphid infestation on single trifoliate leaves, the average aphid population weight per trifoliate leaf on *sickle* was reduced to one third that on A17. In the whole plant experiments, on A17, aphids were able to feed but performed poorly with low aphid weight per plant dry weight (2.36 mg/g) (Figure 2). In contrast, no aphids were observed on *sickle* or on Jester plants after 14 days of aphid infestation. However, for *A. pisum*, the aphid weight did not differ significantly between *sickle* and A17 but was significantly higher than on Jester. Consistent results were obtained in repeat experiments.

**Figure 1.** Aphid performance on single intact trifoliate leaves of *Medicago truncatula* genotypes A17, *sickle* and Jester. The aphid performance was shown as aphid fresh weight per trifoliate leaf at seven days following infestation with *Acyrthosiphon kondoi* (five aphids), *Therioaphis trifolii* (seven aphids) and *A. pisum* (four aphids). The values depict the mean and standard error of six biological replicates. The means were only compared among *M. truncatula* genotypes within each aphid species and means with different letters indicate the differences are significant as determined by ANOVA, GenStat (*p* < 0.05).

**Figure 2.** Aphid performance on *Medicago truncatula* genotypes A17, *sickle* and Jester with whole plant assays. The aphid performance was shown as aphid fresh weight per plant dry weight at 14 days following infestation with *Acyrthosiphon kondoi* (five aphids), *Therioaphis trifolii* (seven aphids) and *A. pisum* (four aphids). The values depict the mean and standard error of six biological replicates. The means were only compared among *M. truncatula* genotypes within each aphid species and means with different letters indicate the differences are significant as determined by ANOVA, GenStat (*p* < 0.05).

**Figure 3.** Aphid performance on *Medicago truncatula* accession A17, *sickle*, Jester and three independent F3 lines of Jester x *sickle* crosses, JxS 44, JxS 5 and JxS 70, containing the homozygous *sickle* mutation and all three aphid resistance genes, *AKR* (*Acyrthosiphon kondoi* resistance), *APR* (*Acyrthosiphon pisum* resistance) and *TTR* (*Therioaphis trifolii* resistance), seven days after the infestation by *A. kondoi* (five aphids) (**A**), *T. trifolii* (seven aphids) (**B**) and *A. pisum* (four aphids) (**C**). The values depict the mean and standard error of six biological replicates. For each aphid species, different letters indicate significant differences in the aphid weight between *M. truncatula* genotypes as determined by ANOVA, GenStat (*p* < 0.05).

#### *2.2. E*ff*ect of M. truncatula Sickle Mutant on Plant Tolerance to Aphid Feeding*

Prior to aphid infestation, the leaves of A17, *sickle* and Jester plants (three weeks after planting) showed no noticeable phenotypical difference. Experiments with single trifoliate leaves demonstrated that following aphid feeding for seven days all three aphid species caused significant damage to the leaves of A17, resulting in reduced leaf size and leaf senescence (Figure 4). The infestation of *A. kondoi* and *A. pisum* also caused leaf senescence on the resistant Jester plants despite much lower aphid population weight on Jester than on A17. Interestingly, with all three aphid species, the leaf senescence was not noticeable on the leaves of *sickle*, even though higher aphid populations were observed on *sickle* than on the resistant Jester plants. This was most apparent with *A. pisum* where on *sickle* leaves the aphid population levels were comparable to A17, and significantly higher than on Jester (Figures 1 and 4). Consistent outcomes were obtained under both growth cabinet and glasshouse conditions in three repeat experiments.

**Figure 4.** The damage symptoms on single intact trifoliate leaves of *Medicago truncatula* genotypes A17, *sickle* and Jester (left panels). The necrotic flecks (indicated by arrows) on each *M. truncatula* genotype are depicted on the right. The photos were taken at seven days following infestation with *Acyrthosiphon kondoi* (five aphids) (**A**), *Therioaphis trifolii* (seven aphids) (**B**) and *A. pisum* (four aphids) (**C**).

The tolerance responses of *sickle*, A17 and Jester plants to aphids when measured with whole plants were consistent with the results on single trifoliate leaves. As illustrated in Figure 5, for each aphid species, the degree of plant biomass reduction caused by aphid infestation correlated with the aphid population levels shown in Figure 2. A17 showed the highest plant biomass reduction relative to the un-infested control plants. In response to the infestation by both *A. kondoi* and *T. trifolii*, *sickle* demonstrated significantly (*p* < 0.05) lower plant biomass reduction than A17. With *A. kondoi*, the biomass reduction in *sickle* was significantly (*p* < 0.05) higher than Jester; however, with *T. trifolii*, the plant biomass of *sickle* or Jester was not significantly different between aphid-infested and control plants. In contrast, the plant biomass reduction caused by *A. pisum* infestation did not differ between *sickle* and A17, which was significantly (*p* < 0.05) higher than that of Jester (Figure 5).

**Figure 5.** Plant tolerance of *Medicago truncatula* genotypes A17, *sickle* and Jester with whole plant assays. The plant tolerance was measured as the percentage of plant biomass reduction caused by the infestation of *Acyrthosiphon kondoi* (five aphids), *Therioaphis trifolii* (seven aphids) and *A. pisum* (four aphids) for 14 days. The values depict the mean and standard error of six biological replicates. The means were only compared among *M. truncatula* genotypes within each aphid species and means with different letters indicate the differences are significant as determined by ANOVA, GenStat (*p* < 0.05).

#### *2.3. E*ff*ect of M. truncatula Sickle Mutant on R Gene Mediated Resistance to Aphids*

To examine if the *sickle* mutation affects *R* gene mediated aphid resistance, we crossed the *sickle* locus (located on chromosome 7) to the Jester background. Jester contains all three single dominant aphid resistance genes *AKR*, *APR* and *TTR*, which are closely linked and located on chromosome 3 [17,22,32,33]. The F2 plants from these crosses were first screened for the *sickle* locus using 1-aminocyclopropane-1-carboxylic acid (ACC) (see Materials and Methods). Out of 512 F2 seedlings screened, 163 showed normal embryonic root growth similar to ACC treated *sickle* mutants and untreated controls, which is consistent with a 1:3 segregation ratio (Chi-square = 2.34, *p* = 0.125) for the 163 lines containing the homozygous *sickle* allele. A subset (90) of 163 pre-selected *sickle* mutant lines were further assessed using high throughput, Multiplex-Ready marker technology and molecular markers linked to these resistance gene loci. Nine homozygous *sickle* plants also contained homozyogous alleles of all three aphid resistance genes (Supplementary Table S2).

To investigate if the *sickle* mutation affects *R* gene mediated resistance to the three aphid species, aphid performance and leaf tolerance were first measured on single trifoliate leaves of individual F2 plants with the *sickle* mutation and the three aphid resistance loci and the results compared to *sickle*, A17 and Jester. A follow-up experiment with three randomly selected F3 lines, each containing homozygous *sickle*, *AKR*, *APR* and *TTR* alleles was conducted. The results were consistent in both studies using F2 or F3 plants, but only the results using the F3 homozygous lines are presented in Figure 3. With all three aphid species, aphid weights on the control plants of *sickle*, A17 and Jester were consistent to the results shown in Figure 1. For each aphid species, the aphid weights on the three independent F3 lines were not significantly different (*p* > 0.05) from each other and did not differ significantly from the aphid weight on the Jester plants (Figure 3). The results demonstrate that the ET insensitive *sickle* mutation has no impact on the antibiosis effect conferred by the three aphid *R* genes; *AKR*, *TTR* and *APR*, against *A. kondoi*, *T. trifolii* and *A. pisum*, respectively.

#### *2.4. The Role of Ethylene Insensitivity in the AIN-Mediated Hypersensitive Response to A. kondoi and A. pisum Infestation*

Both *M. truncatula* A17 and Jester carry the semi-dominant *AIN* gene (*Acyrthosiphon*-induced necrosis) which causes HR-like necrotic flecks upon feeding by both *A. kondoi* and *A. pisum* [23]. To evaluate if the *sickle* mutant interacts with AIN-mediated necrosis, the number of necrotic flecks per trifoliate leaf were recorded. *A. kondoi* and *A. pisum* both induced necrotic like spots on A17 and *sickle* (Figures 4 and 6). As shown in Figure 4, when infested with *A. kondoi*, the average number of necrotic flecks per leaf varied significantly (*p* < 0.05) between A17 and *sickle* with 19.5 and 12 per leaf, respectively (Figure 6). In contrast, upon feeding by *A. kondoi* there were no macroscopic lesions observed on Jester nor on leaves of F2 and F3 lines containing both homozygous *sickle* and *AKR* loci (Figures 4 and 6). When infested with *A. pisum*, necrotic spots were observed on the leaves of all *M. truncatula* accessions examined. The number of necrotic spots per leaf did not differ significantly (*p* > 0.05) between A17 and *sickle* or between Jester and lines carrying both the *sickle* and *APR* loci (Figure 6).

**Figure 6.** The numbers of necrotic flecks on *Medicago truncatula* accessions A17, *sickle*, Jester and three independent F3 lines of Jester x *sickle* crosses, JxS 44, JxS 5 and JxS 70, containing the homozygous *sickle* mutation and all three aphid resistance genes, *AKR* (*Acyrthosiphon kondoi* resistance) and *APR* (*A. pisum* resistance), seven days after the infestation by *A. kondoi* (five aphids) and *A. pisum* (four aphids). The values depict the mean and standard error of six biological replicates. The means were only compared among *M. truncatula* accessions within each aphid species. The di fferences in the number of necrotic flecks caused by *A. pisum* are not significant as determined by ANOVA, GenStat (*p* > 0.05). With *A. kondoi*, the necrotic flecks were not observed on *M. truncatula* accession, Jester, JxS 44, JxS 5 and JxS 70, containing homozygous *sickle* and *AKR* alleles. \* indicates that with *A. kondoi* the numbers of necrotic flecks on A17 and *sickle* are significantly di fferent as determined by ANOVA, GenStat (*p* < 0.05).
