2 MATERIALS AND METHODS
2.1 Study system
The Palouse region of eastern Washington and northern Idaho, USA, is
home to many legumes including P. sativum (Black et al., 1998).
In P. sativum fields, S. lineatus , a chewing herbivore,
co-occurs with A. pisum , a phloem-feeding herbivore that can
transmit pathogens such as PEMV (Chisholm et al., 2019). PEMV is one of
several viruses that infects P. sativum , and this pathogen is
obligately transmitted by aphids in a persistent manner (Chisholm et
al., 2019).
Sitona lineatus adults overwinter outside of P. sativumfields and migrate into fields in late spring before A. pisumarrive (Cárcamo et al., 2018). After S. lineatus eggs hatch,
larvae burrow into the soil to feed and pupate before emerging as adults
in the summer (Cárcamo et al., 2018); these second-generation adults
often occur on plants under attack from A. pisum and PEMV
(Chisholm et al., 2019). Thus, S. lineatus attacks plants in the
field both before and after A. pisum and PEMV. However, it is
unknown if responses of P. sativum differ based on the number of
stressors, and their order of attack. Moreover, molecular mechanisms
that mediate interactions among these stressors are largely unknown
(Chisholm et al., 2019; Bera et al., 2020).
To address these questions, we conducted greenhouse assays to assess
interactions between S. lineatus , A. pisum, and PEMV onP. sativum plants, and molecular mechanisms affecting these
interactions. First-generation adult S. lineatus for experiments
were collected from commercial P. sativum fields, or wild patches
of Vicia villosa , immediately prior to experiments. Colonies of
infectious A. pisum with PEMV, and uninfectious A. pisum ,
were started from Palouse field-collected individuals (Chisholm et al.,
2019) and were maintained on P. sativum plants in a greenhouse
(21-24°C during day cycle, 16-18°C during dark cycle, 16:8 h
light:dark).
2.2 Experimental design
We conducted a 3 × 2 greenhouse (21-24°C day cycle, 16-18°C dark cycle,
16:8 h light:dark) experiment that varied S. lineatus , A.
pisum , and PEMV (Fig. 1). There were three S. lineatustreatments: (i) control: no adults prior to A. pisum treatments
(none), (ii) two adults that fed for 48 h prior to A. pisumtreatments (first), and (iii) two adults that fed for 48 h afterA. pisum treatments (second). The two A. pisum treatments
were: (i) sham: 10 5-d old uninfectious adults that fed for 48 h and
(ii) PEMV: 10 5-d old PEMV-infectious adults that fed for 48 h. For
treatments with S. lineatus first, they were removed by hand
prior to A. pisum treatments; for treatments with A. pisumfirst, they were removed by aspirator prior to S. lineatustreatments. Treatments were conducted on individual P. sativumplants in mesh ‘bug dorms’ (0.6 × 0.6 × 0.6 m), with six replicates
randomly assigned to each treatment in a factorial design (3 S.
lineatus treatments × 2 A. pisum treatments). After insects were
removed, plants were allowed to develop for 7 d before we harvested
tissue to assess viral titer, gene
expression, and nutrients. Tissue samples from the whole aboveground
portion plants were collected and flash frozen in liquid nitrogen and
stored in a -80°C freezer until processing. Viral titer samples
confirmed that 100% of plants in the PEMV treatments became infected
over the course of the experiment.
2.3 Analysis of plant defense and biosynthetic genes
Plant tissue was processed using liquid nitrogen in sterilized mortars
and pestles. Powdered tissue samples (50 to 100 mg) were used for RNA
extraction with Promega SV total RNA isolation kits (Promega, Madison,
WI). The quantity and quality of RNA was estimated on a NanoDrop1000 and
agarose gel electrophoresis, respectively and 1 µg of total RNA from
each sample was used for cDNA synthesis (Bio-Rad iScript cDNA Synthesis
kits). Gene specific primers (Table S3) for qRT-PCR were designed using
the IDT Primer Quest Tool. Each qRT-PCR reaction (10 µl) was set up
containing 3 µl of ddH2O, 5 µl of iTaq Univer SYBR Green Supermix
(Bio-Rad), 1 µl of specific primer mix (forward and reverse
[concentration 10µM]), and 1 µl of diluted (1: 25) cDNA template.
Reactions were set up in triplicates for each sample and ran on a CFX96
qRT-PCR machine (Biorad). The qRT-PCR program included an initial
denaturation for 3 min at 95°C, followed by 40 denaturation cycles for
15 s at 95°C, annealing for 30 s at 60°C, and extension for 30 s at
72°C. For melting curve analysis, a dissociation step cycle (55°C for 10
s and then 0.5°C for 10 s until 95°C) was added. The comparative
2−ΔΔCt method (Livak & Schmittgen, 2001; Kozera &
Rapacz, 2013) was used to calculate the relative expression level of
each gene, with β-tubulin as an endogenous control.
We assessed expression of seven genes associated with defense in peas.
Gene sequences were obtained using accession numbers (available genes)
or using Pea Marker Database (Kulaeva et al., 2017) and blast searching
through the reference genome (Kreplak et al., 2019). Four genes were
associated with plant hormone biosynthesis: (i) Isochorismate
synthase1 (ICS1 ) (salicylic acid), (ii) Lipoxygenase 2(LOX2 ) (jasmonic acid), (iii) Aldehyde oxidase 3(AO3 ) (abscisic acid), and (iv) Gibberellin 2-oxidase(GA2ox ) (gibberellic acid). ICS1 converts chorismate to
isochorismate, a precursor of salicylic acid biosynthesis (Seguel et
al., 2018), while LOX2 is a precursor to jasmonic acid
biosynthesis (Wasternack & Hause, 2013). AO3 catalyzes abscisic
acid biosynthesis by oxidizing abscisic aldehyde, and GA2OXcatalyzes bioactive giberrelic acids or their immediate precursors to
inactive forms (Zdunek-Zastocka & Sobczak, 2013; Serova, Tsyganova,
Tikhonovich, & Tsyganov, 2019; He et al., 2019). All of these gene
transcripts can affect plant defense and plant-microbe interactions (Lee
et al., 2012; Yergaliyev et al., 2016).
The three additional genes examined were associated with defense
response transcripts that occur downstream from hormone induction. One
of these genes, Pathogenesis-related protein 1 (PR1 )
affects systemic acquired resistance-mediated defense signaling and
occurs downstream in the salicylic acid pathway (Fondevilla, Küster,
Krajinski, Cubero, & Rubiales, 2011; Miranda et al., 2017). The
second defense response transcript was an antimicrobial defensin peptide
called Disease resistance response gene (DRR230), which
has been reported to provide resistance in peas against various
pathogens (Lacerda et al., 2014; Selim, Sanssené, Rossard, & Courtois,
2017). The third defense response transcript assessed was Lectin
(PsLectin) . Plant lectins are a group of carbohydrate binding proteins,
and Lectin genes can be induced by salicylic acid, jasmonic acid,
and herbivores to stimulate phytoalexin and pistatin production in peas
(Fondevilla et al., 2011; Armijo et al., 2013; Macedo, Oliveira, &
Oliveira, 2015).
2.4 Measurement of plant phytohormones
Plant tissue samples were assessed for three phytohormones: jasmonic
acid, salicylic acid, and abscisic acid following procedures of Patton,
Bak, Sayre, Heck, & Casteel (2019). Briefly, tissue samples were first
flash frozen in liquid nitrogen before being lyophilized and weighed.
Hormones were extracted in
iso-propanol:H2O:HCL1MOL (2:1:0.005)
with 100 μl of internal standard solution (1000 pg of each). Samples
were evaporated to dryness, resuspended in 100 μl of MeOH, filtered, and
10 µl of each sample was injected into an Agilent Technologies 6420
triple quad liquid chromatography-tandem mass spectrometry instrument
(Agilent, Santa Clara, CA). A Zorbax Extend-C18 column 3.0 × 150mm
(Agilent, Santa Clara, CA) was used with 0.1% formic acid in water (A)
and 0.1% (v/v) formic acid in acetonitrile (B) at a flow rate of 600 mL
min–1. The gradient used was 0–1 min, 20% B;
1–10 min, linear gradient to 100% B; 10-13 min, 100% A. Retention
times were: jasmonic acid (D5) standard (5.740 min), jasmonic acid
(5.744 min), salicylic acid D4 standard (4.677 min), salicylic acid
(4.720 min).
2.5 Analysis of plant nutritional components
For amino acid analysis, leaf tissue was lyophilized, weighed, and
extracted with 20mM of HCL (Patton et al., 2019). Derivation was done
using AccQTag reagents following the manufacturer’s instructions
(Waters, Shinagawa-ku, Tokyo), and derivatised samples (10 μl) were then
injected. Ground tissue was extracted with 100 μl of 20 mM HCl,
centrifuged, and the supernatant was saved. Amino acids were derivatized
using AccQ-Fluor reagent kits (Waters, Milford, MA), with L-Norleucine
as an internal standard. 10 μl from each sample were injected with an
Agilent 1260 Infinity pump with a Nova-Pak C18 column and fluorescence
detector, and Agilent Chemstation software for data recording. Amino
acid derivatives were detected with an excitation wavelength of 250 nm
and an emission wavelength of 395 nm. Peak areas were compared to a
standard curve made from a serial dilution of amino acid standards
(Sigma-Aldrich, St. Louis, MO). injected into a Agilent 1260 Infinity
HPLC (Agilent, Santa Clara, CA) with a Nova-Pak C18 column (Casteel et
al., 2014). Solvent A, AccQ‐Tag Eluent A, was premixed from Waters;
Solvent B was acetonitrile:water (60:40). The gradient used was
0–0.01 min, 100% A; 0.01–0.5 min, linear gradient to 3% B;
0.5–12 min, linear gradient to 5% B; 12–15 min, linear gradient to
8% B; 15–45 min, 35% B; 45–49 min, linear gradient to 35% B;
50–60 min, 100% B. The flow rate was 1.0 ml min−1.
Amino acid derivatives were measured with an Agilent fluorescence
detector with an excitation wavelength of 250 nm and an emission
wavelength of 395 nm. For concentration calculations, standard curves
were generated for each amino acid using dilutions of the standard.
2.6 Data analysis
To evaluate effects of our treatments on host-plant defenses and
host-plant quality, we ran a series of multivariate models using R ver.
3.5.2 (R Working Group, 2018). First, gene expression was evaluated withICS1 , LOX2 , GA2ox , AO3 , PR1 ,DRR230 , and PsLectin as the responses, with MANOVA to
assess treatment effects on relative gene expression
(2–∆∆Ct ) based on cycle threshold values for
each observed gene transcript. Estimated marginal mean of Ct values, and
standard error of the mean, were generated using the emmeans package in
R (Lenth, 2016). The methodology for 2–∆∆Ct followed modified recommendations from Rao, Huang, Zhou, & Lin (2013)
and Kozera & Rapacz (2013), using housekeeping gene β-tubulin to
normalize expression and a sham aphid (non-infective Pea aphid and no
weevil addition) treatment as a control.
Hormone levels in plants were evaluated using MANOVA, with salicylic
acid, jasmonic acid, and abscisic acid as responses (3 variables). Total
amino acid content was evaluated using a generalized linear model (GLM)
with total concentration among all amino acids as the response. All
models assessed treatment effects, using S. lineatus addition,A. pisum infection status, and their interaction as predictors.
Finally, changes in the amino acid profile was evaluated using
non-metric multidimensional scaling (NMDS) with the vegan package
(Oksanen et al., 2019) following Ceulemans, Hulsmans, Ende, & Honnay
(2017).