Legumes can meet their nitrogen requirements through root nodule symbiosis, which could also trigger plant systemic resistance against pests. The pea aphid Acyrthosiphon pisum, a legume pest, can harbour different facultative symbionts (FS) influencing various traits of their hosts. It is, therefore, worth determining if and how the symbionts of the plant and the aphid modulate their interaction. We used different pea aphid lines without FS or with a single one (Hamiltonella defensa, Regiella insecticola, Serratia symbiotica) to infest Medicago truncatula plants inoculated with Sinorhizobium meliloti (symbiotic nitrogen fixation (SNF)) or supplemented with nitrate (non-inoculated (NI)). The growth of SNF and NI plants was reduced by aphid infestation, while aphid weight (but not survival) was lowered on SNF compared to NI plants. Aphids strongly affected the plant nitrogen fixation depending on their symbiotic status, suggesting indirect relationships between aphid- and plant-associated microbes. Finally, all aphid lines triggered expression of Pathogenesis-Related Protein 1 (PR1) and Proteinase Inhibitor (PI), respective marker for salicylic and jasmonic pathways, in SNF plants, compared to only PR1 in NI plants. We demonstrate that the plant symbiotic status influences plant–aphid interactions while that of the aphid can modulate the amplitude of the plant’s defence response.

Data for Figure 1A:  Data correspond to the number of aphid survival after 12 days of infestation (dpi) with the different aphid lines on Symbiotic Nitrogen-Fixing (SNF) and nitrogen fed (NI) plants. Numbers for the 18 separated experiments.  (At t=0, 10 L1 aphids were introduced for each condition).

Data for Figure 1B: Data correspond to the mean weight of aphid surviving after 12 days from the different aphid lines on Symbiotic Nitrogen-Fixing (SNF) and nitrogen fed (NI) plants. Data for the 18 separated experiments. 

Data for Figure 2:  Plant dry weight of aphid infested and non-infested SNF and NI. For dry weight measurement plant shoots from SNF and NI were placed in a drying oven at 80°C for 3 days and weighed on a precision balance (OHAUS Corp, PA214; accuracy ±0.1mg). The dry weight of the shoots of the six individual plants from the three pots for each condition was measured on three separated experiments (18 plants weighed in total).

Figure 3: SNF plant root nodules and nitrogen fixing ability. The root nodules from SNF plants were collected we counted and weighed as they are macroscopic markers for the establishment of SNF (Figure 3A and B). The nitrogen fixing ability of the nodules was estimated indirectly by the reduction of acetylene to ethylene by the nitrogenase (acetylene reduction assay: ARA) (Figure 3C and D). For MtLb1 and Cp6, the root nodules were directly harvested after the aphid removal, pooled and frozen in liquid nitrogen before extraction of mRNA (Figure 3E and F). The data generated from the qPCR experiments analyzed through R-studio (RqPCRBase package). Cycle threshold values (Ct) were normalized using two housekeeping genes MtC27 and a38. The mean qPCR value for the control conditions was set up to one.

Figure 4:  Pathogenesis Related 1 protein (PR1) gene Protease inhibitor (PI) expression analysis by qPCR. qPCR data obtained from aphid infested and non-infested SNF and NI plant shoots. The data generated from the qPCR experiments analyzed through R-studio (RqPCRBase package). Cycle threshold values (Ct) were normalized using two housekeeping genes MtC27 and a38. The mean qPCR value for the control conditions was set up to one.

Figure S2: The Fresh and dry weight of 2-week-old SNF and NI plants were measured as in Figure 2 before aphid infestation (used as control for other conditions).

Figure S3: Relative gene expression analysis for PR1 and PI on non-infested SNF and NI plants measured as in Figure 3.

Figure S4: Relative gene expression of PI in the roots of SNF plants measured as in Figure 3.   Control: non infested plant.