Appendix S1: Experimental Procedures
Plant and insect materials
N. attenuata plants were from the 17th or 22nd inbred generation of seeds
originally collected from a native population from Utah, USA. All plants were grown
according to the conditions described earlier (Kruegel et al., 2002). M. sexta eggs
were obtained from North Carolina State University (Raleigh, NC, USA).
Isolation of the N. attenuata RdR2 gene
A previously described PCR-based strategy was used to isolate the RdR2 from
N. attenuata (Pandey and Baldwin, 2007). Briefly, identical or complementary
primers were designed from the corresponding RdR2 sequences of the close relatives
of N. attenuata: N. benthamiana and N. tabacum. PCR was performed on the genomic
DNA; single bands were gel-purified and sequenced. Fragments were aligned with the
cDNA sequences and Fourier transformed (Tiwari et al., 1997) to determine if they
were coding or non-coding.
Generation and characterization of RdR2-silenced transgenic plants
Plants stably silenced for RdR2 gene expression were produced by cloning an
RdR2 gene fragment in an inverted-repeat orientation in pRESC5 transformation
vector (Bubner et al., 2006). Agrobacterium tumifaciens–mediated transformation
was performed as described earlier (Kruegel et al., 2002); T1 plants were screened for
hygromycin resistance, and homozygosity was determined by segregation analysis of
T2 plants. Two independently transformed, homozygous lines (irRdR2; 271-7 and
303-1), harboring single transgene inserts (determined by Southern blot analysis) and
having reduced accumulated transcripts (determined by qPCR assay) were further
characterized in T2 generation.
DNA gel blots and expression analysis by quantitative real-time PCR
Southern blot analysis to determine the copy number of the endogenous RdR2
gene or transgene was done as described earlier (Bubner et al., 2004). 10 μg genomic
DNA after digestion was separated on 0.8% agarose gel, blotted onto a nylon
membrane (GeneScreen plus, NEN, Boston, MA, USA), hybridized overnight with
radio-labeled gene-specific fragments (350bp RdR2 specific fragment for endogenous
NaRdR2 copy number determination or a hygromycin gene-specific fragment for
transgene copy number determination), and washed and analyzed on a Phosphoimager
FLA 3000 (Fujifilm Europe, Düsseldorf, Germany).
NaRdR1 transcript levels are rapidly elevated in response to herbivore-specific
elicitors (Pandey and Baldwin, 2007), but it is also known that plant responses to
herbivory and UV overlap (Izaguirre et al., 2003; Stratmann, 2003). To determine if
these signals of herbivory also elicit NaRdR2 transcript accumulation, we performed a
time course analyses of RdR2 transcripts using quantitative real-time PCR after
treating the plants with different elicitors. To simulate herbivory, the second fully
expanded (+2) leaves (van Dam et al., 2001) were either mechanically wounded with
a fabric pattern wheel followed by the immediate application of 20 μL M. sexta-OS,
diluted 1:1 (v/v) with water to the puncture wounds (Halitschke et al., 2001), or the
application of 20 μL of pure water. JA and SA are phytohormones that are known to
regulate plant defense responses. Therefore we asked if RdR2 transcript levels
respond when plants are sprayed with 1mM JA and 2mM SA solutions (Pandey and
Baldwin, 2007). Treated leaf tissue was harvested from three to four biological
replicates 0, 1, 4, 8, 12, 24, and 48 h after each treatment.
Total RNA was isolated using the Trizol method and reversed-transcribed to
prepare first strand cDNA (SuperScript, Invitrogen) using oligo(dT) as primers.
SYBR green assays (qPCR core kit for SYBR Green I, Eurogentec, Belgium) were
developed to test the efficiency of amplicons. All the qPCR assays for RdR2
transcripts used 100 ng RNA before transcription as a template. For the remaining
assays, 20 ng RNA before transcription was used as template and amplified with a
qPCRTM core reagent kit (Eurogentec, Belgium) and gene-specific primers and
probes. Each biological replicate was used twice. The N. attenuata sulfite reductase
(ECI) gene was used as an endogenous reference. ECI is a house-keeping gene
involved in sulfur metabolism. Both the endogenous control (ECI) and target genes
were shown to be similarly efficient, as suggested in the manufacturer’s manual. The
2-ΔΔCT method was used for data analysis, as recommended by the manufacturer and
described previously (Bubner et al., 2004; Pandey and Baldwin, 2007). For the MYB
transcription factor, qPCR analysis was conducted with a SYBR green assay.
Insect performance and competition assays
Insect performance assays were conducted by challenging irRdR2 and WT
plants with M. sexta larvae. A freshly hatched larva was placed on the +2 leaf of each
of the genotypes. 11-15 replicate plants were used for each genotype. Starting from
the third day, caterpillar mass was recorded every 2 days for 11 days.
In order to compare the competitive ability of the irRdR2 and WT plants, we
grew initially size-matched seedlings in 2 L pots as described earlier (Zavala et al.,
2004). The seeds were germinated as previously described (Kruegel et al., 2002) and
20 days after germination, sized-matched seedling pairs were transplanted, ~7 cm
apart in 2 L pots. Plant growth was recorded 18 days after transplanting, when all the
plants had started to elongate. Two subsequent readings were taken at 7-day intervals.
Phytohormone analysis
Phytohormone analysis was performed as previously described (Schmidt and
Baldwin, 2006). Briefly, around 200mg of tissue from four to five replicate plants
from both genotypes was homogenized in 1mL ethyl acetate, and spiked with 200ng
mL-1 methanolic [13C2] JA and D4-SA as internal standards for JA and SA. The
homogenate was centrifuged (13000 rpm, 20 min); the supernatant was evaporated
and the dried extract suspended in 1mL ethyl acetate and centrifuged again; the
supernatant was re-dried, this time suspended in 500 μL of 70% methanol (v/v). 15 μL
of this clear suspension was injected in a Varian 1200L Triple-Quadrapol-MS, using a
Pursuit C8 column for high-performance liquid chromatography. Parameters for each
run were as described in Schmidt and Baldwin (2006).
Proteomics analysis
The 2-D electrophoresis of the proteins extracted from WT and irRdR2 plants
was carried out as previously described by Giri et al. (2006). First stem leaves were
harvested from three biologically replicate WT and 271-7 plants (26 days after
transplanting in a York Chamber) for one set of 2-D gels. Total leaf proteins were
phenol-extracted and quantified with a 2-DE quant kit (GE Healthcare Biosciences).
For the first-dimension separation, IPG strips, pH 3-7 (Bio-Rad) were used. SDS-page
separation for second dimension was done on 12% gels. The gels were stained with
Bio-safe Coomassie G-250 (Bio-Rad) according to the manufacturer’s instructions,
and gel images were created with GS-800 densitometer (Bio-Rad). Gels were
analyzed with PDQuest 7.3.1. Spots differentially regulated on the 2 gels were
marked. The entire analysis was repeated on biologically replicated samples.
Differentially regulated spots were picked and tripsin-digested before being subjected
to MALDI-TOF and LC-MS/MS analyses as previously described (Giri et al., 2006).
Virus-induced gene silencing (VIGS) of MYB
As described earlier (Ratcliff et al., 2001; Saedler and Baldwin, 2004), VIGS,
based on tobacco rattle virus (TRV), was used to silence the R2R3-MYB homolog of
N. tabacum (Galis et al., 2006) in N. attenuata. A MYB fragment homologous to the
R2R3-MYB in N. tabacum (Galis et al., 2006) was cloned in a pTV vector (Ratcliff et
al., 2001), the empty vector (EV; pTV00) constructs served as controls. Ten 25- to
28-day-old WT plants per construct were inoculated with both of the constructs at
200C, 65% relative humidity, and allowed stand in the dark for two days, after which
light levels were returned to normal. Samples for transcriptional and metabolite
analyses were collected 35 days after inoculating the MYB-silencing (ptvMYB) and
EV (ptv00) constructs.
Statistical analysis
Data (transformed, wherever they did not meet assumptions of normality)
were analyzed with StatView (Abacus Concepts, Inc., Berkeley, CA, USA). All the
field data or data derived from samples from field were tested with a paired t-test
because in all the field experiments, a transgenic line and WT control plant were
planted as a single pair. Insect assays and other studies in the glasshouse were
analyzed with an Analysis of Variance (ANOVA) or Repeated Measures ANOVA as
applicable.