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. 2019 Mar 5;93(6):e01675-18. doi: 10.1128/JVI.01675-18

TABLE 1.

Primers and probes used in this study

Name Sequence (5′ to 3′) Purpose
HindIII-virus construction fragment 1F′ AAGCTTAGATTTGCATTTAAATTATGA CLCuMuV infectious clone
XbaI-virus construction fragment 1R′ TCTAGACAGGATCTTTCAGGAGC
XbaI-virus construction fragment 2F′ TCTAGATTTGCATTTAAATTATGAAATTG
KpnI-virus construction fragment 2R′ GGTACCAATCAAAGTACAGCACA
V1 ATG-ACG mutant F′ CGTAATTACGTCGAAGCGAGCT CLCuMuV ΔV1
V1 ATG-ACG mutant R′ AGCTCGCTTCGACGTAATTAC
V2L76S mutation F′ GGCGTCTGAATCTCGGC CLCuMuV V2L76S
V2L76S mutation R′ GCTGCCGAGATTCAGACG
Lic1 + AGO4-2 F′ CGACGACAAGACCGTCACCATGGCTGAAGAAGATAAGAATGG pcLUC-NbAGO4-2, pMYC-NbAGO4-2, pAD-NbAGO4-2, pBD-NbAGO4-2, pMYC-NbAGO4-1, cLUC-NbAGO4-1
Lic2 + AGO4-2 R′ GAGGAGAAGAGCCGTCGTTAACAAAAGAACATGGAACTGG
Lic1 + AGO4-1 F′ CGACGACAAGACCGTCACCATGGCTGAAGAAGACAATGGT
Lic2 + AGO4-1 R′ GAGGAGAAGAGCCGTCGCTAACAGAAGAACATGGAACTAGAAA
Lic1 + AGO4 PAZ F′ CGACGACAAGACCGTCACCATGGGGCCTGTTGTGG pMYC-NbAGO4 PAZ domain, pAD-NbAGO4 PAZ domain, pAD-NbAGO4 PIWI domain
Lic2 + AGO4 PAZ R′ GAGGAGAAGAGCCGTCGCTATGACACCAAATTGCATAGCTC
Lic1 + AGO4 PIWI F′ CGACGACAAGACCGTCACCATGCTGCTTCGTGCCTG
Lic2 + AGO4 PIWI R′ GAGGAGAAGAGCCGTCGCTTCATCCACTGTCCCATCT
Lic1 + V2 F′ CGACGACAAGACCGTCACCATGTGGGATCCATTGTTAAACGAAT pGFP-V2, pnLUC-V2, pGST-V2, PVX-V2, pGFP-V2L76S, pnLUC-V2L76S, pGST-V2L76S, PVX-V2L76S
Lic2 + V2 with stop codon R′ GAGGAGAAGAGCCGTCGTCACAACCCTTTGGAACATCCGGACT
Lic1 + V1 F′ CGACGACAAGACCGTCACCATGTCGAAGCGAGCTGC PVX-V1, PVX-V1F23L
Lic2 + V1 with stop codon R′ GAGGAGAAGAGCCGTCGTCAATTCGTTACAGAGTCATAAAAATATAT
V1F23L mutation F′ GGCGTCTGAATCTCGGC PVX-V1F23L
V1F23L mutation R′ GCTGCCGAGATTCAGACG
Lic1 + AGO4 VIGS F′ CGACGACAAGACCGTCACCTCGTTGGAGCTATGCATTG pTRV-NbAGO4
Lic2 + AGO4 VIGS R′ GAGGAGAAGAGCCGTCGTGTTTGAAGAAGTCCAGTCAAA
35S BIS F′ AAGGCAAGTAATAGAGATTGGAGT 35S promoter bisulfate sequence
35S BIS R′ CACCTTCCTTTTCCACTATCTTCACAAT
CLCuMuV IR BIS F′ TGATTTTGGTTAATTGGAGATAA CLCuMuV IR bisulfate sequence
CLCuMuV IR BIS R′ TAAATTTAAAACTTAATACACAA
Tnt1 BIS F′ GTGATGGGGTTTAGTTTTTTTAAAT Tnt1 bisulfate sequence
Tnt1 BIS R′ TTACTCTTTATTTTCTCTCTTTTATTAATA
TE1 BIS F′ GAGGTTGTTTATTTTGGAGATTTGA TE1 bisulfate sequence
TE1 BIS R′ TATTTAACAACCTACCCACCCTAAA
qeIF4a F′ CCCAGAGAGGAAATACAGTG RT-qPCR and qPCR
qeIF4a R′ CAATAGACGGACCAGATTCG
qCLCuMuV C3 F′ GGCGCATATATCTGGGAGGT qPCR
qCLCuMuV C3 R′ GTCTGAGGGTGTAAGGTCGT
qGFP F′ ATGGGCACAAATTTTCTGTCA RT-qPCR
qGFP R′ TCCTCTCCTGCACGTATCC
qNbAGO1 F′ TGCGGAAGAAGAGAACTGATGT
qNbAGO1 R′ CACGGCCACCATGTCCTC
qNbAGO2 F′ AATCGTGGTGGTGGTGGTG
qNbAGO2 R′ GGCCTTGCTCCTGTTCGTAT
qNbAGO4-2 F′ GCTGTCTACTTGTTCGTCAGTC
qNbAGO4-2 R′ GCTATCAGAAAGTCCACAACAGG
qNbAGO4-1 F′ GCTTTGCGAGGTCAAGAGTC
qNbAGO4-1 R′ CACCTCCAACTTCCGCAAAA
32P-probe F′ ATGTGGGATCCATTGTTAAAC Viral Southern blot label
32P-probe R′ ACATGGGCCTTCACAACC