Methods
Aphids and Plants. Pea aphids (A. pisum ) in red color used in experiments were collected from Medicago sativa fields in Yinchuan (106.27°N, 38.47°E), Ningxia, China in 2015.Serratia -infected aphids were parthenogenetic descendants from a single isolated female. We established Serratia -free aphids by injecting ampicillin into Serratia -infected aphids. To minimize the effect of the antibiotic on experiments, we reestablishedSerratia -carrying aphids, namely Serratia -rebuilt aphids, by injecting hemolymph of Serratia -infected aphids intoSerratia -free (see Supplementary Information for details).M. truncatula plants (cv. A17) was kindly provided by Professor Wenhao Zhang, Institute of Botany, Chinese Academy of Sciences, China. Transgenic Nicotiana benthamiana overexpressing GCaMP3used to detect Ca2+ sparks, was kindly provided by Department of Cell & Systems Biology, University of Toronto, Toronto, Canada (Defalco et al., 2017). Details of plant growth conditions were described in Supplementary Information .
Aphid Mean Relative Growth Rate (MRGR), Offspring Number and Development Duration. To measure pea aphid MRGR, 10 2nd instar nymphs fromSerratia -free or Serratia -infected aphids were weighed with an automatic electrobalance before and after feeding on M. truncatula plants for 5 days. The MRGR was calculated as previously described (Leather & Dixon, 1984): MRGR = (ln W2 − ln W1)/t, where W1 was the initial weight, W2 was the final weight, and t represented days between weighing, i.e. t=5. Data were collected from 50 aphids.
For reproduction and developmental duration, one adult fromSerratia -free or Serratia -infected aphids was placed on a fifth trifoliate leaf (counting from the base) of 4-week-old M. truncatula to reproduce. Newborns were counted and moved to anotherM. truncatula plant . Fifty newborn nymphs were individually recorded every 7 to 9 hours for their developmental status. Data were collected from 50 aphids for both aphid populations.
Quantitative PCR. For gene expression analyses of plants and aphids, total RNA of each sample was isolated from 100 mg leaves of plant fed by aphid for 24 h using the RNA Easy Mini Kit (Qiagen, Valencia, CA, USA), 5 pea aphids using TRNzol A+ (Tiangen, Beijing, China) or 20 salivary glands using the Absolutely RNA Nanoprep Kit (Agilent Technologies Inc., Santa Clara, United States), respectively. RNA (1 μg) was used to synthesize cDNAs (20μl) with FastQuant RT Kit (Tiangen, Beijing, China) for each sample. To detect gene expression, qPCR was performed in a 20 μL reaction volume with 2×SYBR Premix EX TaqTM (Qiagen, USA) master mix using gene-specific primers (Table S4) for phenylalanine ammonia lyase (PAL ), nonexpresser of PR genes 1 (NPR1 ), pathogenesis-related protein 1 (PR1 ), lipoxygenase 2 (LOX2 ), allene oxide synthase 2 (AOS2 ) in plants andApHRC in aphids and the salivary glands. Reactions were carried out on the Mx 3500P detection system (Stratagene, La Jolla, CA, USA): 3 min at 95 °C; followed by 40 cycles of 30 s at 95 °C, 30 s at 56 °C and 30 s at 72 °C, and finally stay at 72 °C for 3 min. Six biologically replicates were conducted for each treatment and each biological replicate contained three technical repeats.
Measurement of ROS Production. ROS production in M. truncatula leaves was monitored using 2’7’-dichlorofluorescin diacetate (DCFH-DA) (Beyotime Institute of Biotechnology, China). Its oxidation by ROS generates fluorescent 2′,7′-dichlorofluorescein (DCF). Briefly, adaxial leaves were shaved to remove trichome. They were then soaked in either 1 ml aphid saliva or 1 ml 15% source solution for 6 h in dark, followed by infiltration with 10 μM of DCFH-DA in phosphate buffer (PBS, pH 7.4) for 90 s in 5ml syringes, and 3 washes with phosphate buffer. These leaves then placed in dark for 20 min before fluorescence detection with a Zeiss LSM710 laser confocal microscope (Zeiss, Germany). Leaf cuttings were sandwiched between two microscope coverslips with the adaxial side facing the 488-nm argon laser. Once excited, the fluorescence emissions were detected through wavelength bands 510-550 nm for dichlorofluorescein and 650-750 nm for the red fluorescence for chloroplasts (Lei et al., 2016). Images were analyzed using imaging system software (Zen, Zeiss, Germany).
Expression of ApHRC in M. truncatula. The full-length cDNA of ApHRC was amplified and ligated into a binary vector pBWA(V)HS. The construct was sequence-validated and transformed into Agrobacterium tumefaciens strain GV3101. When recombinantAgrobateria were grown in liquid LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 50µg/ml kanamycin) at 28 °C to an OD600 of 1, cells were collected by centrifugation and resuspended in infiltration buffer (10 mM MES, 10 mM MgCl2, 200 µM acetosyringone, pH5.7). Leaves of 4 week-old M. truncatula were infiltrated with A. tumefaciens . The uninfiltrated leaf area was harvested after 12 h for RNA isolation and cDNA synthesis. The cDNA samples were subjected to qPCR to detect ApHRC expression. Aphid feeding behavior, expression of JA- and SA-pathway genes and H2O2 production were recorded 12 h after infiltration.
Fluorescent Signal Analysis. TIFF files were imported into Fiji (Image J) v1.52n (National Institutes of Health, USA). Fluorescence values were analyzed by GFP fluorescence of every photo as a whole. ΔF/F0 was calculated according to the equation ΔF/F0 = (Ft - F0)/F0, where F0 was the first fluorescence value of the series photos and Ft was the current fluorescence value.