Supporting Information
Supporting Methods
Construction of plant expression vectors and rice transformation
The mpi sequence comprising the promoter region and mpi cDNA was generated by
PCR (primers 2PMPI5 and MPI3, supplementary Table S1) from a genomic clone
containing the mpi gene (Cordero et al. 1994), and inserted into the pBSK vector to
obtain the pBSK::mpi plasmid. This DNA fragment contained 1,872 bp of the mpi
promoter, the 197 bp of the 5’ UT sequence of the mpi gene, including the intron of 108
bp that is present in the 5’ UT region, and the 219 bp of the mpi coding sequence. On
the other hand, the 2A linker sequence was obtained by PCR (primers 2A5 and 2A3,
supplementary Table S1) from the plasmid pGUS2AGFP (kindly provided by Dr. Claire
Halpin, Dundee University; Halpin et al. 1999), and inserted into the pBSK::mpi
plasmid to obtain the pBSK::mpi::2A construct.
The pci cDNA (117 pb) was obtained by PCR (primers PCI5 and PCI3,
supplementary Table S1) from the pCubi::pci::nos vector (Quilis et al. 2007) and
inserted into the pBSK vector to get the pBSK::pci construct. Next, the DNA fragment
covering 450 bp of the mpi terminator region was PCR amplified (primers TMPI5 and
TMPI3, supplementary Table S1) from the pCC1mpi vector (Vila et al. 2005) and
cloned downstream of the pci sequence in the pBSK::pci vector (pBSK::pci::ter
construct). Finally, the mpi-2A and pci-ter DNA fragments were obtained from the
pBSK::mpi::2A using primers 2PMIP5 and 2A3, and pBSK::pci::ter constructs using
primers PCI5-2 and TMPI3-2, and inserted into the pCAMBIA vector to obtain the
pC1300::mpi::2A::pci::ter construct (plasmid for expression of the mpi-2A-pci fusion
gene in rice plants).
A similar strategy was followed to prepare the mpi-C-pci fusion gene. In this case,
the nucleotide sequence coding for the processing site of the Cry1B precursor was
obtained from the plasmid pBSK::ubi::Cry1B-Cry1Aa::nos using primers PS5 and PS3
(Bohorova et al. 2001; M. Royer, unpublished results). The C linker sequence was fused
to the mpi sequence, and then to the pci-ter sequence as described above obtaining the
pC1300::mpi-C-pci::ter construct (plasmid for expression of the mpi-C-pci fusion gene
in rice plants).
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Transformation was carried out using the Mediterranean elite japonica rice (Oryza
sativa L.) cultivar Ariete. Transgenic plants were generated through Agrobacteriummediated transformation of embryonic callus derived from mature embryos, as
described previously (Sallaud et al. 2003). Each transformation vector (pC1300::mpi-Cpci::ter, and pC1300::mpi::2A::pci::ter) was transferred to Agrobacterium tumefaciens
EAH105 strain. The parent pCAMBIA1300 vector contains the hptII (hygromycin
phosphotransferase) gene encoding hygromycin resistance in the T-DNA region. Rice
plants expressing either the mpi gene under the control of its own regulatory sequences
(Cordero et al. 1994), or the pci gene under the control of the maize ubiquitin promoter
(Quilis et al. 2007) were also used in this study.
Wounding of plant leaves
To induce the mpi-pci gene expression, the third leaf of 3 week-old plants was
wounded by making small perpendicular cuts along both edges with a scalpel blade as
described (Breitler et al. 2001). Blade tissue was collected 8h or 18h after wounding and
used for Northern or Western blot analysis, respectively.
Immunoblot analysis
Protein extracts were subjected to SDS-PAGE and electroblotted to nitrocellulose
membrane. Protein determination was performed by the dye-binding assay (Bio-Rad,
Munchen, Germany). Blots were incubated for 2h at room temperature with the antiMPI antiserum at a dilution of 1:500 (Tamayo et al. 2000). Following that they were
washed four times in PBS-T [PBS (1.6mM NaH2PO4, 8.4mM Na2HPO4, 0.15mM NaCl)
pH 7.4 + 0.1% Tween 20] and then incubated with horseradish peroxidise-conjugated
goat anti-rabbit antibody (Pierce, Rockford, IL, USA) at a dilution of 1:10.000 for 1 h at
room temperature. Peroxidase activity was made visible by incubating the blot with
Enhanced Chemiluminescence Western Blotting Substrate (Pierce, Rockford, IL, USA)
for 5 min on an LAS-4000 Image analyzer (Fujifilm).
Insect bioassays
Larvae of C. suppressalis were field collected, C. suppressalis larvae at the secondinstar stage (L2) were placed on transgenic (five independent T2 homozygous lines) and
wild type rice plants at the tillering stage. For each transgenic line and for control
plants, mesh cages containing at least 27 plants and four larvae per plant were used
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(Vila et al. 2005). Two weeks after infestation, larvae found inside the stems were
collected and recorded. Mean values for the data on weight gain were analysed by
Student´s t-test. Leaves from rice plants used in insect bioassays were also collected and
assayed for transgene expression.
The percentage of white panicles in rice plants used in feeding bioassays was also
determined. For this, a subset of non infested and C. suppressalis-infested transgenic
and wild-type plants were allowed to continue growth and further analyzed for the
production of white panicles.
Blast resistance assays
For blast resistance assays, the second leaves of 2-week-old soil-grown rice plants
were placed into plate dishes with 1% (w/v) water agar containing kinetine at 2 mg/l.
Whatman filter paper discs saturated with a M. oryzae spore suspension at the
appropriate concentration (5x105, 106, and 3x106 spores/ml) were placed onto the upper
face of the leaf for 36 hours and then removed. The inoculated leaves were maintained
under high-humidity conditions at 28ºC under a 16h light and 8h dark cycle for the
required time. Blast resistance assays were carried out with mpi-2A-pci, mpi-C-pci and
pci lines (three independent T2 homozygous lines for each transgene, and at least 12
plants for each line). Experiments were repeated three times. Disease severity was
inferred from the lesion size developed at the inoculated spots on the leaves after 6 days
of infection. Lesion size was determined on three leaves and three inoculation sites
each, and three plants per each transgenic line and wild-type plant. Pictures of the
inoculated leaves were taken with a Nikon camera D7000. Microscopic examinations of
the leaf lesions were carried out by light and fluorescent visualization using an Olympus
Stereoscope microscope SZX16. Lesion areas were quantified by Image Analysis
Software, Assess 2.0, for plant disease quantification (Campos-Soriano and San
Segundo et al. 2009).
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