Electronic supplementary materials: Archives of Virology Non-targeted effects of VIGS vectors on host endogenous gene expression Enikő Oláh, Réka Pesti, Dénes Taller, Zoltán Havelda and Éva Várallyay*
Corresponding author: *Éva Várallyay National Agricultural Research and Innovation Centre – Agricultural Biotechnology Research Institute, Szent-Györgyi A. utca 4., Gödöllő H-2100, Hungary e-mail:
[email protected]
Supplementary Table1 Representative collection of VIGS vectors used in different hosts: reference genes are identified and the method(s) used for gene expression studies indicated. Northern blot (N), semiquantiative RT-PCR (S), real-time RT-PCR (Q) N.benthamiana Virus vector PVX TMV
Reference gene Rubisco Actin Ubiquitin Actin
TRV Ubiquitin Elongation factor1 S.lycopersicum Virus vector PVX TMV
Reference gene rRNA Actin
TRV
Ubiquitin
Elongation factor
Tubulin
C.annuum Virus vector
Reference gene
Actin Elongation factor
Reference gene 18S rRNA
Reference S-(Yuan et al., 2011)
Actin
Q-(Ma et al., 2012)
Gapdh
Q-(Bennypaul et al., 2012) Q-(Manmathan et al., 2013) S,Q-(Lacomme et al., 2003)
18S rRNA TRV
BSMV
Ubiquitin
References:
Reference N-(Lin et al., 2008) none S-(Rivas et al., 2004) Q-(Chiasson et al., 2005) Q-(Rotenberg et al., 2006) S-(Kandoth et al., 2007) S-(Eybishtz et al., 2009) S-(Lim et al., 2010) Q-(Wangdi et al., 2010) Q-(Li et al., 2011) Q-(Uppalapati et al., 2011) S-(Fu et al., 2005) Q-(Rotenberg et al., 2006) Q-(Mantelin et al., 2011) S-(Liu et al., 2002) S-(Mayrose et al., 2006) Q-(Rotenberg et al., 2006) S-(Li et al., 2011) Q-(Eybishtz et al., 2009) Q-(Wangdi et al., 2010)
Reference none N-(Chung et al., 2004) Q-(Lee et al., 2010) N-(Choi and Hwang, 2012) Q-(Lim and Lee, 2014) Q-(Lim and Lee, 2014)
TMV
T.aestivum Virus vector
Reference S-(Saitoh and Terauchi, 2002) S-(Hirano et al., 2007) Q-(Hiriart et al., 2003) S,Q-(Lacomme et al., 2003) Q-(Ku et al., 2011) Q-(Rotenberg et al., 2006) S,Q-(Slocombe et al., 2008) S-(Malinowski et al., 2009) Q-(Rotenberg et al., 2006) Q-(Rotenberg et al., 2006)
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Supplementary Table 2 List of primers used for cloning parts of endogenous genes or viruses. The cloned DNA was used in a PCR reaction with the same primers to generate a template for the synthesis of radioactively labelled probes, which were used in Northern hybridizations.
Host plant Nicotiana benthamiana
Endogenous gene
Primer
Sequence
Rubisco
F: 5' CTTTCCTCTGCAGCAGTTGCC 3' R: 5' GCACTTGACGCACGTTGTCG 3' F: 5' TCTCAAATATGACTCCACCC 3' R: 5' ACCCCATTCATTGTCATACC 3' F: 5' GGCATTCAGCCTGATGGC 3' R: 5' AATGTCCAATGAGCGCCTGC 3' F: 5' GCTGAGATGAACAAGAGGTC 3' R: 5' GAGCATATCCATTTCCAATC 3' F: 5' CATAAACTGGTAGAAGTAGAACC 3' R: 5' GGAGCTCTATGCCGACACCG 3' F: 5' ATGCCTATGTTGGTGATGAAGC 3' R: 5' CCACATGACACTTCATGATTG 3' F: 5' GCCCTTCATCTAAGATGAGGC 3' R: 5' TCGCTAAGCAATTGCTCATCG 3' F: 5' TCTCAAATATGACTCCACCC 3' R: 5' ACCCCATTCATTGTCATACC 3' F: 5' GGCATTCAGCCTGATGGC 3' R: 5' AATGTCCAATGAGCGCCTGC 3' F: 5' GCTGAGATGAACAAGAGGTC 3' R: 5' GAGCATATCCATTTCCAATC 3' F: 5' GCTGGTCGTGTGGTGATGGAG 3' R: 5' CGGTTAACCCTAGAACTCGACCCG 3' F: 5' ATGCCTATGTTGGTGATGAAGC 3' R: 5' CCACATGACACTTCATGATTG 3' F: 5' GCCCTTCATCTAAGATGAGGC 3' R: 5' TCGCTAAGCAATTGCTCATCG 3' F: 5' TCTCAAATATGACTCCACCC 3' R: 5' ACCCCATTCATTGTCATACC 3' F: 5' CGCTAGCCTCGGCAGCGTCAG 3' R: 5' AATCCGATGATGCGGACATAC 3' F: 5' ACTTCCTCCGGTGCTGGCACG 3' R: 5' CGTTGTCGTACCAGGCGACGA 3' F: 5' CTGAGGAGCACCCAGTACTGC 3' R: 5' ATAGTTGAGCCACCACTGAGC 3'
region, size in bp X02353.1 1063-1943, 880bp AB937979.1 330-972, 642bp AJ421413.1 139-711, 572bp D63396.1 166-1066, 900bp AY368274.1 96-706, 610bp U60493.1 184-971, 787bp X05982.1 158-690, 532bp XM_004236801.2 443-1265, 822bp XM_004244485.1 241-813, 572bp X53043.1 2592-3572, 980bp NM001247559 60-661, 601bp XM_010321768.1 269-979, 710bp X05982.1 158-690, 532bp AJ246008.1 123-942, 819bp AB042066.1 99-660, 561bp AK334943.1 293-1207, 911bp GQ339780.1 41-662, 621bp
Gapdh Tubulin Ef Cph Actin Solanum lycopersicum
Rubisco Gapdh Tubulin Ef Cph Actin
Capsicum annuum
Rubisco Gapdh
Triticum aestivum
Rubisco Gapdh Actin
List of primers used for cloning parts of the virus vectors. Virus
Primer
Sequence
PVX
F: 5' GGACATGAAGGTGCCCAC 3' R: 5' GAAACTGGGGTAGGCGTC 3' F: 5' GCTATAACCACCCAGGACGCGA 3' R: 5' TGGGCCCCTACCGGGGGTAACG 3' F: 5' GCTGCTAGTTCATCTGCAC 3' R: 5' GCACGGATCTACTTAAAGAAC 3' F: 5' CAATACGTAAGTCCGTAGCTTC 3' R: 5' CATATGGTTGATGGGCACCATC 3'
region, size in bp KJ534604.1 5850-6397, 547bp X68110.1 5278-6395, 1117bp AF406991.1 1116-1752, 636bp M16576.1 2832-2972, 140bp
TMV TRV BSMV
Supplementary Figure 1 Virus specific symptoms on N.benthamiana
Virus specific symptoms on N.benthamiana infected with empty or PDS-containing PVX, TMV and TRV-VIGS vectors. Plants were infected with empty (E) or PDS-containing (PDS) PVX, TMV or TRV-VIGS vector. Photos were taken at 7dpi from PVX and TMV-infected plants and at 10dpi from TRV-infected plants.
Supplementary Figure 2 Virus specific symptoms on S.lycopersicum Virus specific symptoms on S.lycopersicum infected with empty or PDS-containing PVX, TMV and TRV-VIGS vectors. Plants were infected with empty (E) or PDS-containing (PDS) PVX, TMV or TRV-VIGS vector. Photos were taken at 14dpi from PVX and TMV-infected plants and at 24dpi from TRV-infected plants.
Supplementary Figure 3 Virus specific symptoms on C.annuum and on T.aestivum
a/Virus specific symptoms on C.annuum infected with empty or PDS-containing PVX, TMV and TRV-VIGS vectors. Plants were infected with empty (E) or PDS-containing (PDS) PVX, TMV or TRV-VIGS vector. Photos were taken at 20dpi from PVX and TMV-infected plants and at 44dpi from TRV-infected plants. b/ Virus specific symptoms on T.aestivum infected with empty or PDS-containing BSMV VIGS vector. Photos were taken at 20dpi.
Supplementary Figure 4 Virus specific symptoms on N.benthamiana infected with TRV vector carrying different target sequences.
Virus specific symptoms on TRV transgenic N.benthamiana infected with TRV-VIGS vector, carrying 100bp and 300bp of GFP and a 582bp part of UPF1 in its cloning site. Photos were taken at 12dpi under normal and UV light to demonstrate not only symptoms, but efficient silencing of GFP in three different plants.
Supplementary Figure 5 Biological replicates of Northern blot analysis of gene expression changes in N.benthamiana infected with different VIGS vectors.
Investigation of typical endogenous reference gene expressions in N.benthamiana infected with empty or PDS-containing PVX, TMV and TRV-VIGS vectors in two different, independent experiments. Accumulation of Rubisco, Gapdh, Tubulin, Elongation factor (Ef), Cyclophillin (Cph) and Actin mRNA was investigated in systemically infected leaves of empty (E) or PDS-containing (PDS) PVX, TMV or TRV-VIGS vector infected N.benthamiana by Northern blot analyses and compared to mock-inoculated plants (M) in two biological replicate experiments. Samples were taken at 7dpi from PVX and TMV-infected plants and at 10dpi from TRV-infected plants. Virus infection was monitored by Northern blot analyses of PVX, TMV and TRV specific viral RNAs. Ethidium-bromide stained agarose gel served as a loading control. In the extra blocks Northern blot analyses of RNA samples from individual infected plants were made and hybridized for Gapdh and Tubulin.
Supplementary Figure 6 Biological replicates of Northern blot analysis of gene expression changes in S.lycopersicum infected with different VIGS vectors.
Investigation of typical endogenous reference gene expressions in S.lycopersicum infected with empty or PDS-containing PVX, TMV and TRV-VIGS vectors in two different independent experiments. Accumulation of Rubisco, Gapdh, Tubulin, Elongation factor (Ef), Cyclophillin (Cph) and Actin mRNA was investigated in systemically infected leaves of empty (E) or PDS-containing (PDS) PVX, TMV or TRV-VIGS vector infected S.lycopersicum by Northern blot analyses and compared to mock-inoculated plants (M) in two biological replicate experiments. Samples were taken at 14dpi (PVX, TMV) and 24dpi (TRV). Virus infection was monitored by Northern blot analyses of PVX, TMV and TRV specific viral RNAs. Ethidium-bromide stained agarose gel served as a loading control. In the extra blocks Northern blot analyses of RNA samples from individual infected plants were made and hybridized for Rubisco and Cph.
Supplementary Figure 7 Biological replicates of Northern blot analysis of gene expression changes in C. annuum and T. aestivum infected with different VIGS vectors.
Investigation of typical endogenous reference gene expression in C.annuum and in T.aestivum infected by appropriate VIGS vectors two different, independent experiments. a/ Accumulation of Rubisco and Gapdh mRNA was investigated in systemic leaves of empty (E) or PDS containing (PDS) TMV or TRV vector infected C.annuum by Northern blot analyses and compared to mock-inoculated plants (M) at 20dpi (TMV) and 44dpi (TRV) in two biological replicate experiments. Virus infection was monitored by Northern blot analyses against TMV and TRV. Ethidium-bromide stained agarose gel served as a loading control. b/ Accumulation of Rubisco, Gapdh and Actin mRNA was investigated in systemic leaves of empty (E) or PDS containing (PDS) BSMV vector infected T.aestivum by Northern blot analyses and compared to mock-inoculated plants (M) at 20dpi. in two biological replicate experiments Virus infection was monitored by Northern blot analyses against BSMV. Ethidium-bromide stained agarose gel served as a loading control. Numbers show relative intensity of the bands in VIGS-vector and mock experiments compared with the loading control. In the extra blocks Northern blot analyses of RNA samples from individual infected plants were made and hybridized for Rubisco and Gapdh.
Supplementary Figure 8 Biological replicates of Northern blot analysis of gene expression changes in N.benthamiana infected with TRV vector carrying different target sequences.
GFP transgenic N.benthamiana was infected with TRV-VIGS vector carrying 100bp and 300bp of GFP and a 582bp part of UPF1. Accumulation of Rubisco and Gapdh mRNA was investigated in systemic leaves at 12dpi by Northern blot in two different independent experiments. Virus infection was monitored by Northern blot analyses against TRV. Ethidium-bromide stained agarose gel served as a loading control.
