Supplemental detailed methods
Plant culture and inoculation
Prior to inoculations, squash seeds were germinated then planted in 10 cm3 pots of
Pro-Mix potting soil with 3 g of Osmocote slow-release fertilizer (NPK:14-14-14),
supplemented with micronutrients. The plants were grown at 25ºC in a climatecontrolled chamber under fluorescent and incandescent lights with a 16h:8h
(light:dark) photoperiod for 7 days until inoculation. Leaf tissue from symptomatic C.
pepo leaves of the 2nd generation were maintained at -80 ºC and used as the viral
source for all inoculations. The inoculum solution was prepared by grinding
approximately 2 g of frozen symptomatic plant tissue (previously stored at -80⁰C) in
10 mL of 0.1M of potassium-phosphate buffer (pH 7). This solution was then applied
to the two cotyledons of 7-day-old seedlings by dusting the cotyledon surface with
carborundum powder and wiping the inoculum over the surface with a cotton-tipped
applicator. Mock-inoculated controls were obtained by the same procedure, using
clean buffer for the leaf application. Since infection success was slightly lower than
100% (92.3% over all the experiments), only plants showing mosaic symptoms two
weeks after inoculation were used in all the following experiments.
Quantitative PCR of WMV and ZYMV RNA
RNA extractions and cDNA reverse-transcription of plant and aphid tissues: All RNA
extractions of plant tissues were performed using the RNeasy Plant Mini kit (Qiagen)
following the manufacturer’s instructions, on frozen ground samples. The recovered
RNA was eluted in 60 µL of H20. Following RNA quantification by Nanodrop, a
standardized amount of 1500 ng of total RNA was used in 20 µL reactions of cDNA
first-strand reverse-transcription by MMRLV reverse-transcriptase (Clontech) with
random hexamer primers.
Genotyping of the coat proteins: Coat protein genes of ZYMV and WMV isolates
were amplified using primers specific to each species (Table S1), in order to confirm
that our isolates were not carrying other WMV or ZYMV strains and to design qPCR
primers capable of amplifying the two isolates of each species used in the viral
replication experiment with similar efficiencies. WMV primers were designed with
the software Primer3 (v. 0.4.0) in conserved regions of the available Genbank
complete genome sequences for WMV: DQ399708, NC006262, AY437609,
AB218280 and EU660578-90. Primers sequences from Simmons et al. [1] were used
for ZYMV coat protein. After RNA extraction and cDNA synthesis, the target cDNA
was amplified by RT-PCR using the high fidelity Phusion® DNA polymerase (New
England Biolabs), purified and directly sequenced in both senses using the same
reverse and forward primers.
Quantitative RT-PCR on WMV and ZYMV coat protein sequences: To determine the
absolute amount of RNA of each virus species, two quantitative PCR tests with
florescent probes were designed with primers and hybridization probes annealing
specifically to the WMV and ZYMV coat protein sequences, respectively, with
similar annealing temperature and product size to approach similar efficiencies (see
table 1). Primers and probes specific to each virus species but indiscriminant between
the isolates of each species used in our experiment were designed using the Beacon
Designer 7.51 software, (Premier Biosoft International). A qPCR assay for the NADH
housekeeping gene of C. pepo was also designed in order to control the success of
RNA extraction and cDNA synthesis. Fluorescent probes with BQ1 quencher and
either FAM (for the WCP4 probe) or CAL Fluor-Gold (for the ZYCP4 probe)
fluorescence where obtained from Biosearch Technologies. Standard curves of known
concentrations of recombinant DNA of the WMV and ZYMV target templates were
used for absolute quantification. Briefly, the target product generated from PCR
amplification was ligated into pGEM®-T Easy vector plasmids (Promega) and
transformed in DH5α Escherichia coli competent cells. Plasmids extracted from a
few selected cell lines were sequenced, linearized by digestion with the SacI
restriction endonucleases (New England Biolabs), purified, and the amount of
recDNA was quantified three times by Nanodrop and converted to molar
concentration. Aliquots of 2.10-3 pmol/µL DNA stock dilution were used for standard
curves of tenfold 1.10-4 to 1.10-9 pmol DNA dilutions (1.10-4 to 1.10-10 pmol for aphid
tests) that were run in triplicates in each plate of qPCR assays. All assays were run in
singleplex 20 µL reactions on duplicate samples with 75 ng of template cDNA
(except aphid assays that had lower amounts), 300nM of each forward and reverse
primer, 200nM of fluorescent probes, and SsoFast probes supermix (Bio-Rad
Laboratories,) on a Bio-Rad iQ5 cycler with the following program: 3 min @ 95C, 40
cycles of 10 sec. @ 95C, 30 sec @ 59C. Validation trials showed similar efficiencies
(between 96 and 103%, with r2>0.99) for the standard curves of recDNA plasmids,
and for serial dilutions of cDNA templates obtained from C. pepo plants singly
infected with WMV, ZYMV, or mixtures of these templates. The number of viral
RNAs was estimated, from its equivalent in picomoles of double stranded recDNA
standards interpolated from the standard curve, using the Avogadro constant
(6.023.1011 molecules/pmol). To homogenize the calculation of absolute RNA copy
numbers with the standard curve, all the standard curve data from all plates of each
assay was pooled to calculate the virus RNA copy numbers. Overall, the efficiency
was 100.5%, with an r2 of 0.985, for the WC4 assay; and 101.2%, with an r2 of 0.998
for ZYCP4. The assays were sensitive to a starting number of 10 and 140 RNA
molecules per ng of cDNA for the WMV and ZYM assays respectively.
Organic volatile collection and quantification
The daytime emission of organic volatiles was collected in a greenhouse, under
natural light and fluorescent/incandescent supplements, by a closed push-pull system
using filtered air. Whole plants between three and four weeks old—inoculated at one
week old—were placed in individual 9-L glass chambers at the centre of a Teflon
base. Filtered air was pushed into the chamber through a port at the rate of 4.0 L.min1
. We collected the emitted volatiles for six consecutive daytime hours (between 9 am
and 3 pm) by vacuuming the air of the chamber through a filter, containing 150µg of
HaysepQ, at a rate of 1.0 L.min-1. The high airflow arriving in the chamber prevented
contamination from outside air and build-up of excess humidity and heat that could
affect the plant’s metabolism, and hence the volatiles produced. After the collection,
the aboveground part of each plant was cut and dried in a 50ºC oven, then weighed.
The filters were eluted with 150 µL of dichloromethane to recover the trapped
compounds and 5 µL of internal standard (80 ng. µL-1 of nonyl-acetate and 40 ng. µL1
of n-octane) was added to this elution. These samples were injected into a gas
chromatograph fitted with a flame ionization detector an (Agilent model 6890,
column: Agilent 19091Z-331, 0.25 mm internal diameter, 0.1-μm film thickness) by 1
µL aliquots. The column was heated with the following program: 35 ºC held for 0.5
min, increase of 4 ºC per min up to 160 ºC, then 20 ºC per min until 220 ºC was
reached. The retention times and compound quantities were analyzed using MSD
Chemstation (Agilent Technologies 2003) by measuring volatile output in nanograms
relative to the internal standard and dividing this value by the dry weight of the
particular sample. The compounds were identified by comparing their retention times
to known standards common to this plant system, or their mass spectra to a library of
known compounds. We compared the volatile production among mock-inoculated
control plants, plants singly inoculated with either the WMV isolate HQ11 or the
ZYMV isolate HBCF, and plants doubly inoculated with a 1:1 mixture of these two
isolates. Seven replicates of each treatment were performed, distributed in five
consecutive blocks/days. The total volatile production of each different treatment was
log-transformed and compared by ANOVA with treatment and block as explanatory
variables. The 16 main individual compounds were analyzed by a MANOVA, with,
again, treatment and block as explanatory variables. The contribution of all these
compounds to the total variance of the experiment was investigated by a principal
component analysis.
Reference
1.
Simmons H.E., Holmes E.C., Stephenson A.G. 2011 Rapid turnover of intra-
host genetic diversity in Zucchini yellow mosaic virus. Virus Res 155(2), 389-396.
(doi:10.1016/j.virusres.2010.11.007).