SUPPORTING INFORMATION TO MOLECULAR ECOLOGY ARTICLE
Potato Virus Y infection hinders potato defence response and renders plants more
vulnerable to Colorado potato beetle attack
Marko Petek1*, Ana Rotter1, Polona Kogovšek1, Špela Baebler1, Axel Mithöfer2, Kristina
Gruden1
Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana,
Slovenia
2
Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, 07745 Jena,
Germany
1
Supporting information is available at Molecular Ecology online.
List of supporting Figures and Tables:
 Figure S1: Experimental set-up for the collection and analysis of the potato leaves and
VOCs, and the CPB midgut tissue.





Table S1: qPCR assays used in this study (available as separate .xlsx file)
Table S2: Potato RNAseq mapping statistics
Table S3: Statistical evaluation of larval weight gain in feeding assays
Table S4: Potato qPCR gene expression matrix (available as separate .xlsx file)
Table S5: Potato RNAseq defence pathway expression matrix (available as separate .xlsx
file)
 Table S6: CPB gene expression matrix (available as separate .xlsx file)
 Table S7: VOC release data matrix (available as separate .xlsx file)
1
Supplementary materials and methods
Plant growth, larval feeding assays, and tissue sampling
Potato plants of cv. ‘Igor’ (healthy and secondary PVYNTN-infected) and cv. ‘Désirée’ (nontransgenic and coi1 plants; Halim et al. 2009) were grown under conditions described in Petek et
al. (2012). Secondary PVYNTN-infected plants were grown from infected potato tubers and
propagated in tissue culture (Pompe-Novak et al. 2006). For the feeding assays, 22 potted plants
were placed into each of four glass containers (volume, 72 L) that were open at the top. The CPB
eggs were obtained from the Department of Agriculture, New Jersey, USA, and were hatched at
room temperature. The larvae were fed fresh healthy potato cv. ‘Igor’ leaves for first 24 h after
hatching.
As shown in Figure S1, the sampling of the potato leaves for qPCR was performed at three time
points: 0 hours post infestation (hpi; before larval infestation, 11 a.m.), 3 hpi (2 p.m.) and 24 hpi
(11 a.m. the next day). As well as the infested leaves, leaves from neighbouring non-infested
plants (less than 15 cm away) and leaves from plants grown outside the glass containers (control
leaves) were sampled. The sampling was performed in triplicate for all of the potato plant groups.
The leaf samples were immediately frozen in liquid nitrogen and stored at –80 °C.
2
Figure S1. Experimental set-up for the collection and analysis of the potato leaves and
VOCs, and the CPB midgut tissue
For VOC sampling, the intact plants and plants infested with two 4th instar CPB larvae were
headspace sampled for 24 h or 48 h. For the CPB larval differential feeding assays, the depicted
sampling timeline indicates the days on which the CPB larvae were weighed and the potato
leaves and larval midguts were sampled. hpi, hours post CPB larvae infestation; dpi, days post
CPB larvae infestation.
collective weighting
of CPB larval groups
0 3
hpi
24
hpi
2
dpi
weighting of individual CPB
larvae
4
dpi
5
dpi
6
dpi
7
dpi
8
dpi
9
dpi
10
dpi
qPCR gene expression analysis
For potato genes, the following were used: previously designed SybrGreen qPCR assays for
BASIC PATHOGENESIS-RELATED PROTEIN 1 (PR1b; Baebler et al. 2011), β-1,3GLUCANASE I (Glu I; Oufir et al. 2008), β-1,3-GLUCANASE II (Glu II), CHLOROPHYLL A/BBINDING PROTEIN (CAB4), RUBISCO ACIVASE (RA; Kogovšek et al. 2010), and a TaqMan
3
MGB qPCR assay for AUXIN RESPONSE FACTOR 2 (ARF2; Baebler et al. 2014) (Table S1).
The following were designed by Applied Biosystems: TaqMan MGB qPCR assays for potato
genes 13-LIPOXYGENASE H3 (13-LOX), ALLENE OXIDE SYNTHASE 2 (AOS2), WOUNDINDUCED PROTEIN KINASE (WIPK), JASMONATE RESISTANT 1 (JAR1), CORONATINE
INSENSITIVE 1 (COI1), JASMONATE-ZIM-DOMAIN PROTEIN 1 (JAZ1), TRANSCRIPTION
FACTOR MYC2 (MYC2), ETHYLENE RECEPTOR 1 (ETR1), ETHYLENE RECEPTOR 4
(ETR4), CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1), ETHYLENE-RESPONSIVE
TRANSCRIPTION FACTOR 1 (ERF1), and AUXIN-REPRESSED PROTEIN (ARP). The
following were additionally designed in-house by PrimerExpress 2.0 software (Applied
Biosystems, USA) to study gene expression of defence pathways (Table S1): TaqMan qPCR
assays for CELL WALL INVERTASE (CW_INV), PHENYLALANINE AMMONIA-LYASE (PAL),
CINNAMIC ACID 4-HYDROXYLASE (C4H), HYDROXYCINNAMOYL TRANSFERASE (HCT),
NON-EXPRESSOR OF PR1 (NPR1), LEUCINE ZIPPER TRANSCRIPTION FACTOR TGA2
(TGA2) and RNA-DEPENDENT RNA-POLYMERASE 1 (RdR1), and a SybrGreen assay for the
POTATO CYSTEINE PROTEINASE INHIBITORS (PCPI) gene family. The assays were
designed based on potato sequences from the NCBI (Nucleotide or dbEST), the DFCI Potato
Gene Index, the Potato Oligo Chip Initiative (POCI), and the Potato Genome Sequencing
Consortium (PGSC) databases (Table S1). Sequences from different databases with nucleotide
identity above 95% were aligned and assays were designed on regions showing low
polymorphism.
The following were used for CPB genes SybrGreen or TaqMan qPCR previously designed assays
for intestains C (IntC), intestains D (IntD), intestains C (IntE), serine proteases (Ld_ser_prot),
glycoside hydrolase family 28 pectinase 11 (Ld_GH28Pect-11), juvenile hormone binding
protein-like genes (Ld_jhbp-like), glycoside hydrolase family 45 cellulase 6 (Ld_GH45-6; Petek
et al. 2012), intestains A (IntA), intestains B (IntB), glycoside hydrolase family 48 cellulase 1
(Ld_GH48-1), glycoside hydrolase family 48 cellulase 2 (Ld_GH48-2; Šmid et al. 2013). A
SybrGreen assay for the reference gene ubiquitin-like smt3 (Ld_smt3) was designed using
PrimerExpress 2.0 (forward primer: TACCGATACCCCAACCACATTAG, reverse primer:
CCAGTTTGCTGTTGGTATACTTCAA). PVYNTN infection of secondary infected cv. ‘Igor’
plants was confirmed by qPCR, as described previously (Kogovšek et al. 2008).
Potato leaf and CPB gut samples were analysed on a LightCycler 480 real-time PCR system
(Roche Applied Science, USA) in 5-µl reaction volume using the universal PCR conditions
described in Petek et al. (2010). Ld_smt3 and 18S rRNA (Eukaryotic 18S rRNA TaqMan
endogenous control; Applied Biosystems, USA) were used as reference genes for CPB samples,
and cytochrome oxidase (COX; Weller et al. 2000), 18S rRNA (Applied Biosystems, USA) and
elongation factor 1 (EF-1; Baebler et al. 2009) were used as reference genes for potato samples.
For every gene, the limit of quantification (LOQ) was determined from the standard curve. If the
determined Cq value of a sample was below the LOQ, the sample copy number was assigned the
4
LOQ copy number. Expression values normalized to reference genes for potato and CPB
experiments are available in Table S4 and Table S6, respectively.
RNAseq library preparation and data analysis
Following a standard Illumina RNAseq protocol, pooled mRNA-enriched samples were
fragmented and cDNA was synthesised using random hexamer primers. cDNA fragments were
purified, the ends were repaired, and a single adenine nucleotide was added before adapters were
ligated. Fragments were size-selected and PCR amplified.
Raw Illumina reads were imported to CLC Genomics Workbench 6. Reads were quality (limit =
0.01) and ambiguity (no ambiguous nucleotides allowed) trimmed, and overlapping pairs were
merged using default parameters. The mapping was performed using the following parameters:
minimum length fraction, 0.8; minimum similarity fraction, 0.8; unspecific match limit, 10; use
strand specific assembly, No; count paired reads as two, No; and organism type, PROKARYOTE
(because transcript sequences were used as reference). Using these parameters, approximately
70% of reads were mapped to the StNIB transcriptome reference (Table S2). The read counts for
all of the transcripts were exported to Excel files.
Analysis of volatile compounds
Identification of compounds was performed in AMDIS version 2.69 (NIST, USA). For both
potato cultivars, sample chromatograms with the highest peaks in relation to internal standards
(n-bromodecane, 100 ng/μl) were used for compound identification. The AMDIS deconvolution
algorithm was used to separate overlapping peaks. Compounds were identified by comparing
mass spectrometry spectral data and the calculated retention indices for those in the ADAMS
essential oils library 4th edition. Compounds with best hit scores <700 were marked as tentative,
and hits with best hit scores <500 were designated as unknown. Identification of limonene, βcaryophyllene, germacrene D, and α-copaene was confirmed using authentic standards (all from
Sigma, Germany).
Peak alignment and relative quantification was performed in LCquan version 2.6.0.1128 (Thermo
Scientific, USA). For each identified compound, an expected retention time and specific fragment
mass (m/z) was determined. The Interactive Chemical Integration System peak detection
algorithm was used to integrate the peaks using: baseline window, 40 scans; area noise factor, 5;
and peak noise factor, 10. The peak edges were constrained to 5% of peak height using a tailing
factor value of 1.2. For most compounds, the retention time window was set to 3 s and the
minimal signal-to-noise ratio to 10. All of the compound ‘area under the curve’ (AUC) values
were expressed relative to the AUC of the internal standard n-Br-decane, to obtain response ratios
that were comparable between samples. The scaled VOC response ratios for cv. ‘Igor’ and cv.
‘Désirée’ are given in Table S7.
Statistical analysis of the VOC release levels was performed in the R statistical software
environment (R Core Team 2012). Eight VOCs from potato cv. ‘Igor’ and ten VOCs from potato
5
cv. ‘Désirée’ were omitted from the statistical analysis because they were quantified in <10% of
the samples, and 30 VOCs were omitted due to uncertain compound identification (Table S7).
The data were preprocessed by first imputing values under LOD by replacing them with half of
the local minimum; i.e., the column-wise (metabolite-wise) minimum of quantified compounds.
Secondly, the data were log10-transformed. A “golden standard” subset (gs) was chosen; i.e.,
healthy/ non-transformed plants before CPB infestation. The mean and standard deviation for
each volatile were calculated for the gs samples. Ten volatiles with zero standard deviation in gs
samples and over 70% of values imputed were omitted from further analyses. Finally, the data
were standardised using the equation 𝑥𝑠𝑡𝑎𝑛𝑑 =
xi −x̅gs
sdgs
, where x̅gs is the mean of the gs samples,
and sdgs is the standard deviation of the gs samples.
Mixed effects models (Pinheiro & Bates 2000) were used to determine statistically significant
differences in the VOC release. The factors used in the statistical models were plant genotype
(coi1 or non-transgenic), infestation (non-infested or CPB infested) and virus (healthy or
PVYNTN-infected plants).
Supplementary results
Table S2: Potato RNAseq mapping statistics
Mapping was performed in CLC Genomics Workbench, using: StNIB transcriptome reference;
minimum length fraction, 0.8; and minimum similarity fraction, 0.8.
Pooled sample name
Bases
(Gbp)
Total
reads
GC
(%)
Reads
mapped
Unmapped
reads
4,38
Reads
above
Q20 (%)
98.6
24,332,852
43.5
18,076,297
6,256,555
Reads
mapped
(%)
74.3
Desiree_control_NT
Desiree_control_coi1
4,42
98.9
24,575,804
42.8
17,499,213
7,076,591
71.2
Desiree_cpb_NT
4,43
98.9
23,965,275
42.8
17,639,922
6,950,478
71.7
Desiree_cpb_coi1
4,32
98.7
24,590,400
43.2
17,322,949
6,642,326
72.3
Igor_control_HEALTHY
4,46
98.6
24,779,711
43.5
18,620,057
6,159,654
75.1
Igor_control_PVY
4,31
98.6
23,916,983
43.0
16,678,519
7,238,464
69.7
Igor_CPB_HEALTHY
4,50
98.6
24,963,450
42.5
17,819,262
7,144,188
71.4
Igor_CPB_PVY
4,46
98.6
24,780,048
42.9
17,971,418
6,808,630
72.5
6
Table S3: Statistical evaluation of larval weight gain in feeding assays
Comparison of larval weight for CPB larvae reared under the different conditions. Testing for
significant differences in larval weight was performed using Student's t-test statistics assuming
equal variances (** p <0.01; *** p <0.001).
Days post
infection
5
6
7
8
9
p value for larval weight comparisons according to CPB larvae rearing
PVY-infected/
healthy cv. ‘Igor’
potato plants
0.07
0.67
0.75
0.0002 ***
2.80 E-07 ***
coi1/ non-transgenic
cv. ‘Désirée’ potato
plants
0.007 **
0.00001 ***
0.0003 ***
0.0009 ***
2.08 E-06 ***
Healthy, fresh leaves
exchanged every 24 h/ healthy
cv. ‘Igor’ potato plants
6.60 E-09 ***
3.18 E-07 ***
2.54 E-08 ***
3.02 E-08 ***
5.13 E-06 ***
Potato genetic background dependent responses to CPB infestation
The response of potato to CPB attack differs between cultivars as was noticed when comparing
the transcriptional changes and VOC release from NT cv. 'Désirée' plants and healthy cv. 'Igor'
plants. Here we present and discuss the most prominent differences observed at the gene
expression level.
Expression profiles of majority of JA biosynthesis and signalling genes at 24 hpi showed little
differences between both cultivars. Evidently, the expression of JA methyltransferase, which
transforms JA to a volatile MeJA, was induced at a higher extent in cv. 'Désirée' but had a much
higher constitutive expression in cv. 'Igor' (Table S5). On the other hand, the constitutive
expression of many JA-responsive antinutritional protein and terpene synthase genes was higher
in cv. 'Désirée'. Additionally, the qPCR data show that the 13-LOX gene was induced at 3 hpi in
cv. 'Désirée' but remained at the same level in cv. 'Igor'. At 24 hpi, WRKY70 and NIMIN2, both
involved in SA signalling, were downregulated in cv. 'Désirée' but in cv. 'Igor' their expression
did not change. In cv. 'Désirée' the PR genes were induced at a higher extent than in cv. 'Igor'
which however was again mostly due to a higher constitutive expression in cv. 'Igor' (Table S5).
Cultivar or ecotype genetic diversity is known to influence herbivore defence response also in
other plants (Kusnierczyk et al. 2007; Wu et al. 2008). Transcriptional profiles obtained in our
study suggest that the SA pathway was constitutively induced at higher extent in cv. 'Igor' than in
cv. 'Désirée'. This, in turn, may contribute to a faster and more efficient induction of JA response
pathway in cv. 'Désirée' upon CPB infestation and possibly a higher resistance of this cultivar to
CPB as suggested by weight gains in the differential feeding assays.
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