DOI: 10.5061/dryad.q2bvq83gt
title: Aphid infestation differently affects the defences of nitrate-fed and nitrogenfixing Medicago truncatula and alters symbiotic nitrogen fixation

description: 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.
description: 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
 <i>MtLb1</i> and <i>Cp6</i>, 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
 <i>MtC27</i> and <i>a38</i>. The mean qPCR value
 for the control conditions was set up to one.   Figure
 4:  <i>Pathogenesis Related 1 protein</i>
 (<i>PR1</i>) gene <i>Protease inhibitor (PI)</i>
 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
 <i>MtC27</i> and <i>a38</i>. 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 <i>PR1</i> and
 <i>PI</i> on non-infested SNF and NI plants measured as in
 Figure 3.   Figure
 S4: Relative gene expression of <i>PI</i> in the roots of SNF
 plants measured as in Figure 3.   Control: non infested plant.