Supporting information
Method S1. Details for construction of satBaMV, BaMV, and TRV vectors.
A 319-bp fragment of Sul gene was amplified from N. benthamiana cDNA with the primer
pairs NbSU-1 and NbSU-2. The amplified DNAs were cloned into the T&A cloning vector
(Yestern Biotech Co., Taiwan). To construct staBaMV vectors containing Sul gene inserted into
different sites (pCF4:Sul-C2, pCF4:Sul-N4 and pCF4:Sul-N5), DNA fragments were amplified
with cloned DNA as a template and the primer pairs NbSU-7 and NbSU-2 for pCF4:Sul-C2,
NbSU-1 and NbSU-2 for pCF4:Sul-N4 and NbSU-5 and NbSU-8 for pCF4:Sul-N5. The DNA
products were double-digested with BstXI and EcoNI or NotI and NcoI or single-digested with
EcoNI and ligated to pCF4 restricted with the same enzyme (Fig. 1A) to construct a number of
satBaMV deletion-containing Sul genes (pCF4:Sul-C1, pCF4:Sul-N1, pCF4:Sul-N2, and
pCF4:Sul-N3). P20 fragments were amplified with pCF4 as a template and the primer pairs
satBaMV-2 and satBaMV-22 for pCF4:Sul-N1, satBaMV-2 and satBaMV-10 for pCF4:Sul-N2,
and satBaMV-2 and satBaMV-23 for pCF4:Sul-N3. The DNA products were double-digested
with BstXI and NcoI and ligated to pCF4:Sul-C2 restricted with the same enzyme. To construct
pCF4:Sul-C1, DNA fragments were amplified with pCF4 as a template and the primer pairs
satBaMV-24 and satBaMV-25. The resulting PCR product was cloned into EcoNI-digested
pCF4:Sul-C2 plasmid.
The sequence of tobacco PDS gene was amplified from N. benthamiana cDNA with the
primer pairs PDS-1 and PDS-2. The amplified DNAs were cloned into the T&A cloning vector
for sequencing. To construct a plasmid for pCF4:NbPDS, a DNA fragment was amplified with
cloned DNA as a template and the primer pairs PDS-41 and PDS-44. The DNA product was
single-digested with EcoNI and ligated to pCF4 restricted with the same enzyme.
To construct a plasmid for pCF4:GFP that contains a 319-bp fragment of the mGFP gene, a
DNA fragment was amplified with pTRV2/GFP (kindly provided by Dr. C. H. Tsai, National
Chung Hsing University, Taiwan) as a template and the primer pairs mGFP-1 and mGFP-2.
The DNA product was single-digested with EcoNI and ligated to pCF4 restricted with the same
enzyme.
A 319-bp DNA fragment of tobacco Hsp70 gene was amplified from N. benthamiana cDNA
with the primer pair Hsp70-A and Hsp70-2. The resulting PCR product was cloned into the
EcoNI-digested pCF4 plasmid to generate the Hsp70 gene-silencing construct pCF4:Hsp70.
A 319-bp DNA fragment of tobacco Hsp90 was amplified from N. benthamiana cDNA with
the primer pair Hsp90-A and Hsp90-2. The resulting PCR product was cloned into the
EcoNI-digested pCF4 plasmid to generate the Hsp90 gene-silencing construct pCF4:Hsp90.
The sequences of Sul gene were amplified from B. distachyon cDNA with the primer pair
BdSU-F3 and BdSU-R1. The amplified DNAs were cloned and sequenced as described above.
To construct the satBaMV vector containing BdSul (pCF4:BdSul), a DNA fragment was
amplified with cloned DNA as a template and the primer pair BdSU-5 and BdSU-6. The DNA
product was single-digested with EcoNI and ligated to pCF4 restricted with the same enzyme.
The partial sequence of PDS was amplified from B. distachyon cDNA with the primer pair
PDS-B1 and PDS-B2. The amplified DNA was cloned and sequenced as described above. To
construct the satBaMV vector containing BdPDS (pCF4:BdPDS), a DNA fragment was
amplified with cloned DNA as a template and the primer pair PDS-B16 and PDS-B2. The DNA
product was single-digested with EcoNI and ligated to pCF4 restricted with the same enzyme.
For agro-infiltration, plasmids pCF4:NbSul, pCF4:NbPDS, pCF4:GFP, pCF4:Hsp70, and
pCF4:Hsp90 were double-digested with XhoI and BglII and ligated to pKn restricted with the
same enzymes to generate pKF4:NbSul, pKF4:NbPDS, pKF4:GFP, pKF4:Hsp70, and
pKF4:Hsp90.
To construct BaMV vectors containing Sul from N. benthamiana, DNA fragments were
amplified from pCF4:NbSul as a template with the primer pair NbSU-5 and NbSU-6. The DNA
product was digested with NotI and ligated to pCBV restricted with the same enzyme to
generate pCBV:NbSul. Next, to construct a plasmid for pCBV:NbPDS that contains a 319-bp
fragment of PDS, a DNA fragment was amplified with pCF4:NbPDS as a template and the
primer pair NbPDS-1 and NbPDS-2. The DNA product was double-digested with PspOMI and
NheI and ligated to pCBV restricted with NotI and NheI. For agro-infiltration, plasmids
pCBV:NbSul and pCBV:NbPDS were double-digested with NsiI and SacI and ligated to
pKYLX7 restricted with the same enzymes to generate pKBV:NbSul and pKBV:NbPDS.
To construct BaMV vectors containing PDS from B. distachyon, a DNA fragment was
amplified from pCF4:BdPDS as a template and the primer pair PDS-B4 and PDS-B14. The
DNA product was digested with NotI and ligated to pCBV restricted with the same enzyme to
generate pCBV:BdPDS.
Plasmids pTRV1, pTRV2, and pTRV2/PDS were kindly provided by Dr. D. C. Baulcombe
(University of Cambridge, UK). To construct the pTRV2 vector containing Hsp70 and Hsp90
(TRV:Hsp70 and TRV:Hsp90), a DNA fragment was amplified with cloned DNA as a template
and the primer pairs Hsp70-F and Hsp70-R for Hsp70 or Hsp90-F and Hsp90-R for Hsp90.
The DNA products were double-digested with XbaI and BamHI and ligated to a pTRV2
restricted with the same enzyme.