Supplemental data
Methods
Plant material and growth condition
Nicotiana benthamiana plants were grown in soil at 26°C with a 16 h light / 8 h dark
photocycle.
Overexpression of SlbZIP1 and SlbZIP2 in Nicotiana benthamiana
ClaI-NotI fragments containing the coding regions of SlbZIP1 and SlbZIP2 were
individually subcloned into the pSfinx vector (provided by Prof. David Baulcombe,
University of Cambridge, Cambridge, UK) (Takken et al. 2000). A ClaI-NotI GFP
fragment was separately cloned into the pSfinx vector and was used as a negative
control. Recombinant plasmids were introduced into Agrobacterium tumefaciens strain
GV3101 (provided by Prof. David Baulcombe). Each transformant was grown
overnight in YPE medium containing 50 μl.ml-1 kanamycin. Cells were harvested by
centrifugation (4,000 × g), resuspended at OD600 = 1.0 in a buffer containing 10 mM
MES-KOH, pH 5.6, 10 mM MgCl2 and 100 μM acetosyringone, and infiltrated into
fully expanded N. benthamiana leaves with a needleless syringe as described in Zhu et
al. (2012). After 24 h incubation, leaf samples were collected, immediately frozen in
liquid nitrogen, and stored at −80°C until required.
RT-PCR analysis
Total RNA samples were prepared from N. benthamiana plants using Sepasol-RNA I
Super (Nacalai Tesque, Kyoto, Japan). RNA was reverse-transcribed as described
previously (Zhu et al. 2012) and the resultant cDNA was used for PCR analysis with the
primers listed in Table S1.
Table S1. Primers used in this study
(continued)
(continued).
Fig. S1.
SlbZIP1 and SlbZIP2 transcriptionally activate the expression of ProDH and ASN. The
coding regions of SlbZIP1 and SlbZIP2 were separately cloned into the pGR106 vector
(provided by Dr. David Baulcombe). The GFP fragment was also cloned into the
pGR106 vector as a control. Plasmids were introduced into Agrobacterium tumefaciens
GV3101 and the bacterial cultures used to infiltrate Nicotiana benthamiana leaves. Leaf
discs (12 mm diameter) were removed using a cork-borer 24 h after infiltration. Total
RNA was extracted from leaf discs and was subjected to RT-PCR analysis. ProDH,
proline dehydrogenase gene; ASN, asparagine synthase gene. EF1-α was used as a
loading control. Cycle numbers of PCR were displayed in right margin.
References
Takken FLW, Luderer R, Gabriels SJEJ, Westerink N, Lu R, de Wit PJGM, Joosten
MHAJ (2000) A functional cloning strategy, based on a binary PVX-expression
vector, to isolate HR-inducing cDNAs of plant pathogens. Plant J 24:275–283.
Zhu XJ, Thalor SK, Takahashi Y, Berberich T, Kusano T (2012) An inhibitory effect of
the sequence-conserved upstream open reading frame on the translation of the main
open-reading frame of HsfB1 transcripts in Arabidopsis. Plant Cell Environm
35:2014-2030.