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.