The CaMV-RTBV RNA leader optimal secondary structure at 20 o C (625 nt leader + 22 nt part of ORF VII) Wild type CaMV 35S RNA leader optimal secondary.

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The CaMV-RTBV RNA leader optimal secondary structure at 20 o C (625 nt leader + 22 nt part of ORF VII) Wild type CaMV 35S RNA leader optimal secondary structure at 20 o C (611 nt leader + 22 nt part of ORF VII) Predicted by MFold Program (SeqWeb Version 1.2, Wisconsin GCG Package Version 10.1) at 20 o C Secondary structure in the wild type and chimeric leaders

Wild type CaMV leader sequence (72 nt, upstream of the stem) kcal kcal-10.0 kcal kcal-19.0 kcal-18.8 kcal-19.0 kcal kcal-18.7 kcal-18.5 kcal-18.4 kcal CaMV-RTBV chimeric leader sequence (88 nt, upstream of the stem) junction Local secondary structures in the wile type and chimeric leader Optimal structure Suboptimal structures Optimal structure Suboptimal structures

Inoculations, passages and symptom observation Mechanical inoculation with the cloned viral DNA genome ~20 days Visible systemic symptoms (chlorotic mosaic and vein clearing) ~30 days The CaMV-RTBV chimera Two of four inoculated plants showed systemic symptoms with a delay of 10 days The wild type CaMV virus The CaMV-RTBV shunt chimeric virus in turnip plants

The delay in development of symptoms of the chimeric virus vanished gradually on passage, which correlated with the appearance of reversions Infected plant N1 PCR Infected plant N2 PCR 8-12 days days 8-12 days 2-6 days 8 days No delay 2-4 days 0-2 days No delay 0-2 days No delay Passaging the CaMV-RTBV chimera progeny in turnip plants ( Oct 2000 – Jan 2002) PCR PROGENY 1 1’ 2 2’ Late harvest

ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AGU GAG UAG UCGUCUU Progeny-1 (Plant N1, inoculation , symptoms – ; early harvest – ) RTBV Short ORFCaMV junction Original mutantStemLanding site I-Rev1-1 I-Rev1-6 (+ G-5A, G241A) I-Rev1-2 I-Rev1-3 I-Rev1-4 I-Rev1-5 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU GAG GUC AGU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAA GUC AGU GAG UAG UCGUCUU G 595 A c 597 A G 595 A I-Rev1-7 (+ U317A) ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU GAG GUC AGU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAA GUC AGG GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAC GUC AGU GAG UAG UCGUCUU M A Q V S E R H E E E ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUC UCC AUA AUA AUG UGU GAG UAG UUCCCA- Wild type CaMV strain 4184 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUA AUA AUG UGU GAG UAG UUCCCA- strain Strasbourg M C E

ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AGU GAG UAG UCGUCUU Progeny-1 after one passage (inoculation , symptoms – ; harvest ) RTBV Short ORFCaMV junction Original mutant II-Rev1-1 II-Rev1-2 II-Rev1-3 II-Rev1-4 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG ACU CAG GUC AGU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAA GUC AGU GAG UAG UCGUCUU G 595 A StemLanding site G 595 A ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG ACU CAG GUC AGA GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AUU GAG UAG UCGUCUU M A Q V S E R I T T ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUC UCC AUA AUA AUG UGU GAG UAG UUCCCA- Wild type CaMV strain 4184 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUA AUA AUG UGU GAG UAG UUCCCA- strain Strasbourg M C E

ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AGU GAG UAG UCGUCUU Progeny-1 after two passages (inoculation , symptoms – ; harvest – ) RTBV Short ORFCaMV junction Original mutant III-Rev1-1 III-Rev1-6 III-Rev1-2 (+ G 168 U) III-Rev1-3 (+ U 612 AU) III-Rev1-4 (+ U 612 AAU) III-Rev1-5 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAA GUC AGU GAG UAG UCGUCUU G 595 A StemLanding site G 595 A ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUUgaGAUCA AUG GCU CAA GUC AGU GAG UAG UCGUCUU G 595 A ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUG GCU CAA GUC AGU GAG UAG UCGUCUU M A Q V S E ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUC UCC AUA AUA AUG UGU GAG UAG UUCCCA- Wild type CaMV strain 4184 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUA AUA AUG UGU GAG UAG UUCCCA- strain Strasbourg M C E

ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AGU GAG UAG UCGUCUU Progeny-1 after five passages RTBV Short ORFCaMV junction Original mutant VI-Rev1-1 VI-Rev1-9 VI-Rev1-2 VI-Rev1-3 VI-Rev1-4 VI-Rev1-5 VI-Rev1-6 VI-Rev1-7 (+A 606 U) VI-Rev1-8 ACACGCUGAAAUCACCAGUCUCUCUCUACAAACCUAUCUCUCUCU AUG GCU CAA GUC AUU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUG GCU CAA GUC AGU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUG ACU CAA GUC AGU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUUgaGAUCA AUG GCU CAA GUC AGA GAG UAG UCGUCUU StemLanding site C 107 U ins(3nt) C 107 U delCG 594 C 107 U delAcg 596 delAcg 596 C 107 U delAcg 596 C 107 U delACG 588 G 595 A C 107 U delCGUU 589 G 595 A T T T T T I I ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUUgaGAUCA AUG ACU CAA GAC AGU GAG UAG UCGUCUU D M A Q V S E R R ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUC UCC AUA AUA AUG UGU GAG UAG UUCCCA- Wild type CaMV strain 4184 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUA AUA AUG UGU GAG UAG UUCCCA- strain Strasbourg Short ORF A M C E

I-Rev1-2 (G 69 A), I-Rev1-1 I-Rev1-4 (G 69 C) I-Rev1-3 (G 69 A + U 75 G) I-Rev1-5,6,7 (C 67 G) kcal kcal kcal kcal Progeny 1 II-Rev1-1 (G 69 A) II-Rev1-2 (G 64 A ) kcal kcal II-Rev1-3 (G 64 A + U 75 A) kcal II-Rev1-4 (G 74 U) kcal III-Rev1-1,2,3,4 (G 69 A) kcal kcal III-Rev1-5 (C 53 A + G 69 A) kcal III-Rev1-6 (del[A A 6o ] + G 69 A) New junction kcal kcal -17 kcal New junction VI-Rev1-1 (C 53 A + G 69 A + U 75 A) VI-Rev1-2 (C 53 A + G 64 A + G 69 A + U 71 A) VI-Rev1-3, 4, 5, 6 (del[A A 6o ] + G 64 A + G 69 A) VI-Rev1-8, 9 (U 33 C + del[A A 6o ] + G 69 A + U 75 A) kcal VI-Rev1-7 (del[A A 6o ] + G 69 A) New junction Early infection 1st passage 2nd passage5th passage

-19.8 / junction / –24.8 junction / junction /-24.2 junction kcal Wild type -17 kcal New junction Dominant after 5 passages Progeny 1 Early infection 1st passage 2nd passage5th passage Only the deletion in late passages could lead to restoration of more relaxed secondary structure, whereas dominant point mutations in the sORF would rather further stabilize the structure VI-Rev1-3, 4, 5, 6 (del[A A 6o ] + G 64 A + G 69 A)

ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AGU GAG UAG UCGUCUU Progeny-1’ (Plant N1, inoculation , symptoms – , late harvest – ) RTBV Short ORFCaMV junction Original mutant I-Rev1’-1 (+ G 260 A) I-Rev1’-2 I-Rev1’-5 (+ delU 605 ) I-Rev1’-4 I-Rev1’-6 (+ U 317 A) ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AUU GAG UAG UCGUCUU StemLanding site G 595 A delAcg 596 ACA--CUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AGA GAG UAG UCGUCUU I-Rev1’-3 (+ U 317 A) ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU--UUUCUCgaGAUCA AUG ACU CAG GUC AGG GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU GAG GUC AGU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG ACU CAA GUC AGU GAG UAG UCGUCUU delCGAcg 594 ACACACUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUU----aGAUCA AUG GCU CAG GUC AGA GAG UAG UCGUCUU I R R R M A Q V S E T T E ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUC UCC AUA AUA AUG UGU GAG UAG UUCCCA- Wild type CaMV strain 4184 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUA AUA AUG UGU GAG UAG UUCCCA- strain Strasbourg M C E

ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AGU GAG UAG UCGUCUU Progeny-1’ after one passages (inoculation , symptoms – ; harvest – ) RTBV Short ORFCaMV junction Original mutant II-Rev1’-1 (+ A 483 C) II-Rev1’-2 II-Rev1’-5 (+ U 612 AAAU) II-Rev1’-4 II-Rev1’-6 (+ U 612 AU) ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG ACU CAG GUC AGA GAG UAG UCGUCUU StemLanding site ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG UCU CAG GUC AGA GAG UAG UCGUCUU delAcg 596 II-Rev1’-3 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG UCU CAG GUC AGA GAG UAG UCGUCUU delCGAcg 596 delAcg 596 ACACACUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUAU-UUUUCUCgaGAUCA AUG ACU CAG GUC AGA GAG UAG UCGUCUU delAcg 596 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GAG UAG UCGUCUUU 98 C G 595 A + delAcg 596 M A Q V S E RT RT RS RS RS RS II-Rev1’-7 (+ G -2 A, ins 21 nt b/w A 608 and A 609 ) - - ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUC UCC AUA AUA AUG UGU GAG UAG UUCCCA- Wild type CaMV strain 4184 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUA AUA AUG UGU GAG UAG UUCCCA- strain Strasbourg M C E

ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AGU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUC UCC AUA AUA AUG UGU GAG UAG UUCCCA- Progeny-1’ after four passages RTBV Short ORFCaMV junction Original mutant Wild type CaMV strain 4184 V-Rev1’-1 V-Rev1’-9 V-Rev1’-2 (+ G -2 A, G 380 A) V-Rev1’-3 (+ A 175 C, T 612 AT) V-Rev1’-4 V-Rev1’-5 V-Rev1’-6 V-Rev1’-7 V-Rev1’-8 A 557 G delAcg 596 Stem ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUA AUA AUG UGU GAG UAG UUCCCA- strain Strasbourg Landing site delAcg 596 A 557 G delAcg 596 delAcg 596 ACGAcgtc 593 to CCGCUA ACGAcgtc 593 to CCGCUA G 590 U + delCGAcg 594 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUG --U CA- GUG AGU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUG --U CA- GUG AGU GAG UAG UAGUCUU ACACGCUAAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUG --U CA- GUG AGU GAG UAG UAGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUG --U CA- GUG AGU GAG UAG UCGUCUU M A Q V S E S S S S S S S S S S S M C E

I-Rev1’-6 (C 67 G) kcal kcal I-Rev1’-1 (G 74 U) kcal I-Rev1’-2 (U 75 A + del[CG 5,6 ]) kcal I-Rev1’-3 (U 75 A + del[C g 54 ] +G 5 A) kcal I-Rev1’-4 (G 64 A + U 75 G + del[AU 46,47 ]) kcal I-Rev1’-5 (G 64 A + G 69 A) II-Rev1’-1 (G 64 A + U 75 A) kcal kcal II-Rev1’-2 (G 64 A + U 75 A + G 5 A + C 44 A + delA 46 ) kcal II-Rev1’-3, 4, 5, 6 (G 64 U + U 75 A) kcal II-Rev1’-7 (del[ G U 75 ]) kcal kcal kcal V-Rev1’-1,2,3,4,7,8,9 (del[A A 60 ] + del 3 nt +C 72 G) V-Rev1’-5 (del[A A 60 ] + del 3 nt +C 72 G + C 83 A) V-Rev1’-6 (del[A A 60 ] + del 3 nt +C 72 G + C 83 A + G 5 A) New junction Progeny 1’ Late infection 1st passage 4th passage

-19.9 / junction kcal Wild type / junction / junction Progeny 1’ Late infection1st passage4th passage kcal V-Rev1’-1,2,3,4,7,8,9 (del[A A 60 ] + del 3 nt +C 72 G) New junction Dominant after 4 passages

ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AGU GAG UAG UCGUCUU Progeny-2 (Plant N2, inoculation , symptoms – ; early harvest – ) RTBV Short ORFCaMV junction Original mutant = original mutant + (G 162 A) I-Rev2-2 I-Rev2-3 + (UU 604,605 A) I-Rev2-4 I-Rev2-5 + (G -4 U) I-Rev2-6 I-Rev2-7 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AGG GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG ACU CAG GUC AGU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU GAG GUC AGU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AGU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGCUCA AUG GCU CAG GUC AGU GAG UAG UCGUCUU StemLanding site delAcg 596 G 595 A C 574 U + del [C c 597 ] I-Rev2-1 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AUU GAG UAG UCGUCUU M A Q V S E E T T R I ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUC UCC AUA AUA AUG UGU GAG UAG UUCCCA- Wild type CaMV strain 4184 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUA AUA AUG UGU GAG UAG UUCCCA- strain Strasbourg M C E

ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AGU GAG UAG UCGUCUU Progeny-2, first passage (inoculation , symptoms – ; harvest ) RTBV Short ORFCaMV junction Original mutant II-Rev2-1 + (ins[A 611 UAAAAAA]) II-Rev2-3 + (ins[ U 612 AU]) ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAC GUC AGU GAG UAG UCGUCUU StemLanding site delAcg 596 II-Rev2-2 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AUU GAG UAG UCGUCUU M A Q V S E H I I I ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUC UCC AUA AUA AUG UGU GAG UAG UUCCCA- Wild type CaMV strain 4184 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUA AUA AUG UGU GAG UAG UUCCCA- strain Strasbourg M C E

ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AGU GAG UAG UCGUCUU Progeny-2 after two passages (inoculation , symptoms – ; harvest – ) RTBV Short ORFCaMV junction Original mutant III-Rev2-1 III-Rev2-3 + (C 292 A) III-Rev2-4 + (U 612 AU) III-Rev2-5 + (U 627 G) III-Rev2-6 + (UU 604,605 AA) ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAU CA AUG GCU CAG GUC AUU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AUU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAA GUC AGU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AUU GAG UAG UCGUCUU StemLanding site delAcg 596 U 586 A + delAcg 596 III-Rev2-2 M A Q V S E I I I I I I I I ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUC UCC AUA AUA AUG UGU GAG UAG UUCCCA- Wild type CaMV strain 4184 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUA AUA AUG UGU GAG UAG UUCCCA- strain Strasbourg M C E

ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AGU GAG UAG UCGUCUU Progeny-2 after five passages RTBV Short ORFCaMV junction Original mutant VI-Rev2-1 VI-Rev2-9 VI-Rev2-2 (U 612 AU) VI-Rev2-3 (del[U U 160 ]) VI-Rev2-4 VI-Rev2-5 (G -2 U) VI-Rev2-6 VI-Rev2-7 (U 604 A) VI-Rev2-8 (U 612 AU) ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCU--AUUUUCUCgaGAUCA AUG GCU CAG GUC AUU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAU CA AUG GCU CAG GUC AUU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUA GAUCA AUG GCU CAG GUC AUU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCU A AUG GCU CAC GUC AGU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAC GUC AGU GAG UAG UUGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCU A AUG GCU CAC GUC AGU GAG UAG UCGUCUU StemLanding site C 109 U delAcg 596 C 107 U delAcg 596 C 107 U C 594 U delAcg 596 C 594 U delAcg 596 U 581 C delAcg 596 C 107 U delAcg 596 G 585 A delAcg 596 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUU--CUCgaGAUCA AUG ACU CAG GUC AGA GAU UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUC UCC AUA AUA AUG UGU GAG UAG UUCCCA- Wild type CaMV strain 4184 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUA AUA AUG UGU GAG UAG UUCCCA- strain Strasbourg M A Q V S E M C E I I I RDT H H H H H H

I-Rev2-3 (C 67 G) kcal I-Rev2-4, 5 (G 64 A ) kcal kcal I-Rev2-7 (G 74 U) kcal I-Rev2-6 (U 75 G) kcal I-Rev2-2 (A 57 C) Progeny kcal II-Rev2-2, 3 (G 74 U) II-Rev2-1 (G 69 C) kcal kcal III-Rev2-2, 3, 4, 5 (G 74 U) kcal III-Rev2-1 (G 69 A) kcal III-Rev2-6 (G 74 U + del[U U 58 ]) New junction VI-Rev2-1 (G 69 C + C 83 U) kcal kcal VI-Rev2-2, 3, 4, 5 (G 69 C + del[C C 59 ]) kcal VI-Rev2-6 (G 74 U + del[U a 55 ]) kcal VI-Rev2-7 (G 74 U + del[U U 58 ]) kcal VI-Rev2-8 (G 74 U + del[CU 44,45 ]) kcal VI-Rev2-9 (G 64 A + U 75 A + G 78 U + del[UU 49,50 ]) New junction New junction New junction Early infection 1st passage 2nd passage5th passage

-21.1 / junction kcal Wild type / junction / junction / junction kcal VI-Rev2-2, 3, 4, 5 (G 69 C + del[C C 59 ]) New junction Dominant after five passages Progeny 2 Early infection 1st passage 2nd passage5ht passage

ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUC UCC AUA AUA AUG UGU GAG UAG UUCCCA- Progeny-2’ (Plant N2, inoculation , symptoms – ; late harvest – ) Wild type CaMV strain 4184 I-Rev2’-5 I-Rev2’-6 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUA AUA AUG UGU GAG UAG UUCCCA- strain Strasbourg delAcg 596 I-Rev2’-1 delAcg 596 I-Rev2’-2 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG ACU CAG GUC AGU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG ACU CAG AUC AGU GAG UAG UCGUCUU I-Rev2’-3 + (UU 604,605 A) G 583 U ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG ACU CAA GUC AGU GAG UAG UCGUCUC ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG ACU CGG GUC AGU GAG UAG UCGUCUU I-Rev2’-4 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG ACU CAG GUC AGA GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUA GAUCA AUG GCU CAG GUC AUU GAG UAG UCGUCUU T T T T T R I R I ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AGU GAG UAG UCGUCUU RTBV Short ORFCaMV junction Original mutantStemLanding site M A Q V S E M C E

ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AGU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUC UCC AUA AUA AUG UGU GAG UAG UUCCCA- Progeny-2’ after one passage inoculation , symptoms – ; harvest (one plant)– RTBV Short ORFCaMV junction Original mutant Wild type CaMV strain 4184 II-Rev2’-3 + (C -58 U + UU 604,605 AA) II-Rev2’-4 Stem ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUA AUA AUG UGU GAG UAG UUCCCA- strain Strasbourg Landing site delAcg 596 II-Rev2’-1 delAcg 596 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG ACU CAG GUC AGA GAG UAG UCGUCUU ACACACUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG ACU CAG GUC AGA GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG ACU CAG GUC AGA GAG UAG UCGUCUU 5 nt del CACGUUACGAcg 587 AAAUUGU II-Rev2’-2 delAcg 596 ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCCAUUUUCUCgaGAUCA AUG ACU CAG GUC AGA GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG ACU CAG GCC AGA GAG UAG UCGUCUU II-Rev2’-5 AGACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG ACU CAG GUC AGA GAG UAG UCGUCUU C 99 U M A Q V S E T T T T T T T T T T R R R R R R R R R R A M C E II-Rev2’-6 + (U 605 A)

ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUCUCgaGAUCA AUG GCU CAG GUC AGU GAG UAG UCGUCUU ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAUUUUC UCC AUA AUA AUG UGU GAG UAG UUCCCA- Progeny-2’ after four passages RTBV Short ORFCaMV junction Original mutant Wild type CaMV strain 4184 V-Rev2’-1 V-Rev2’-5 + (U 612 AAU) V-Rev2’-6 + (UA 605,606 AC) V-Rev2’-7 + (U 605 AA) V-Rev2’-8 + (U 605 A) Stem ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCU AUA AUA AUG UGU GAG UAG UUCCCA- strain Strasbourg Landing site delAcg nt del [CGAcg 594 GU] ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUAU CA AUG ACU CAG GUC AGA GAG UAG UCGCCUU V-Rev2’-2 delAcg 596 V-Rev2’-3 + (U 174 G, U 605 AA) ACACGCUGAAAUCACCAGUCUCUCUCUACAAAUCUAUCUCUCUCUA AUG ACU CAG GUC AGA GAG UAG UCGUCUU V-Rev2’-4 3 nt del [CGAcg 594 GU] 3 nt del [CGAcg 594 GU] 3 nt del [CGAcg 594 GU] 3 nt del [CGAcg 594 GU] 3 nt del [CGAcg 594 GU] 3 nt del [CGAcg 594 GU] 3 nt del [CGAcg 594 GU] 3 nt del [CGAcg 594 GU] M A Q V S E T T T T T T T T T T R R R R R R R R R R T T R R M C E

I-Rev2’-1 (G 64 A ) kcal I-Rev2’-5 (G 64 A + U 75 A) kcal I-Rev2’-6 (G 74 U + del[U a 55 ]) kcal kcal I-Rev2’-4 (G 64 A + A 68 G) kcal I-Rev2’-3 (G 64 A + G 69 A + U 88 C) kcal I-Rev2’-2 (G 64 A + G 70 A) New junction kcal II-Rev2’-1, 5 (G 64 A + U 75 A) kcal II-Rev2’-2 (G 64 A + U 75 A + G 5 A) kcal II-Rev2’-3 (G 64 A + U 75 A + U 45 A) kcal II-Rev2’-6 (G 64 A + U 75 A + C 2 G) kcal II-Rev2’-4 (G 64 A + U 75 A + U 71 C) Progeny 2’ Late infection 1st passage 4th passage kcal II-Rev2’-1 (G 64 A + U 75 A + U 85 C + del[U U 58 ]) kcal II-Rev2’- 2, 3, 4, 5, 6, 7, 8 (G 64 A + U 75 A + del[U 47 – A 60 ]) New junction

-20.5 / junction / junction / junction II-Rev2’-2, 3, 4, 5, 6, 7, 8 (G 64 A + U 75 A + del[U A 60 ]) Wild type Dominant after four passages kcal Progeny 2’ Late infection1st passage4th passage kcal New junction

Conclusions on the shunt take-off site The major tendency is to alter the primary sequence within RTBV sORF, without affecting the start and stop codons. This might have occurred to optimize a translational event at the sORF required for ribosome shunting. Various versions of amino acid sequence in different revertant progenies have confirmed previous results that the encoded peptide is not critical for shunting and viral infectivity (Pooggin et al., 1998, 2001). Rather the elongation step of sORF translation might have been improved in the revertants. However, optimization of initiation and termination rates at the sORF cannot be excluded. Notably, the translation event at the sORF is not only affecting the basal level of shunting but also the degree of shunt enhancement by the transactivator TAV. In the chimeric CaMV-RTBV leader, both basal shunt and TAV-activated shunt efficiencies are reduced. The bigger length of the RTBV sORF (7 codons vs. 4 codons for CaMV sORF) seems to be tolerated, although at least in one complete progeny it was reduced by the 3 nt deletion. Additionally (or alternatively), the purpose of most of the alterations might have been to inactivate an inefficient splice donor site present in the wild type sequence of the RTBV sORF. In fact, most mutations in all the progenies hit one or more positions of the 6 nt splice site consensus sequence. In our protoplast system, the reduced shunt efficiency in the chimeric leader could be due to an inefficient splicing event(s) using this donor site and some cryptic splice acceptors in the reporter mRNA. Another major tendency at the later stages of the evolution was to create 9 to 18 nt deletions at the chimeric junction just in front of the sORF. This would tend to restore both the wild type length of the leader and/or low index of the local secondary structure. The latter might increase the shunt efficiency.

UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU GAUUUAAAGAAAUAAUCCGCAUAAGCCC---CCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU RTBVCaMV junction Chimera Wild type CaMV strain 4184 StemsORFLanding site I-Rev1-2 I-Rev1-6 I-Rev1-1 I-Rev1-3 I-Rev1-4 I-Rev1-5 UUUCUCAAAAUAUUGAGUCACGUUACAAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU G 69 A G 69 A + U 75 G I-Rev1-7 UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUACAAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUACGAAguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU G 69 C C 67 G PBS Progeny-1 (Plant N1, inoculation , symptoms – ; early harvest – ) (+ G -5 A, G 241 A) (+ U 317 A)

UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU GAUUUAAAGAAAUAAUCCGCAUAAGCCC---CCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU RTBVCaMV junction Chimera Wild type CaMV strain 4184 II-Rev1-1 UUUCUCAAAAUAUUGAGUCACGUUACAAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU G 69 A StemsORF G 69 A UUUCUCAAAAUAUUGAGUCACGUUACAAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU Landing site II-Rev1-2 G 64 A G 64 A + U 75 A G 74 U II-Rev1-3 II-Rev1-4 PBS Progeny-1 after one passage (inoculation , symptoms – ; harvest )

UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU GAUUUAAAGAAAUAAUCCGCAUAAGCCC---CCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU RTBVCaMV junction Chimera Wild type CaMV strain 4184 III-Rev1-1 UUUCUCAAAAUAUUGAGUCACGUUACAAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU G 69 A StemsORF G 69 A UUUCUCAAAAUAUUGAGUCACGUUACAAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU G 69 A Landing site III-Rev1-2 III-Rev1-3 III-Rev1-4 III-Rev1-5 G 69 A III-Rev1-6 UUUCUCAAAAUAUUGAGUCACGUUACAAcguCCGCUUAAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUACAAcguCCGCUUAAAAAAAAUUGGUAUCAGAGCC AUG AAU PBS Progeny-1 after two passages (inoculation , symptoms – ; harvest – ) (+ C 53 U) (+ G 168 U) (+ del[A 46 –- A 60 ])

UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU GAUUUAAAGAAAUAAUCCGCAUAAGCCC---CCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU RTBVCaMV junction Chimera Wild type CaMV strain 4184 VI-Rev1-1 VI-Rev1-2 UUUCUCAAAAUAUUGAGUCAACAACGUCCGCUguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU StemsORF UUUCUCAAAAUAUUGAGUCACGUUA--AcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU G 69 A C 107 U Landing site G 69 A + G 74 U C 107 U G 69 A + U 75 A C 107 U UUUCUCAAAAUAUUGAGUCACGUUA--AcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU G 69 A + G 64 A C 107 U + U 71 A G 69 A + G 64 A C 107 U VI-Rev1-3 VI-Rev1-4 VI-Rev1-5 VI-Rev1-6 VI-Rev1-7 VI-Rev1-8 VI-Rev1-9 G 69 A + G 64 A C 107 U UUUCUCAAAAUAUUGAGUC---UUACAAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU G 69 A + G 64 A UUUCUCAAAAUAUUGAGUCA----ACAAcguCCGCUUUAAAAAUUGGUAUCAGAGCC AUG AAU PBS The overlap of sORF F and ORF VII is not required (dominant 2 nt deletion shifting the frame) However the newly introduced F’ is now shifted to overlap ORF VII Progeny-1 after five passages (+ C 53 U) (+ del[A 46 –- A 60 ]) (+ del[A 46 –- A 60 ] +U 33 C)

UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU GAUUUAAAGAAAUAAUCCGCAUAAGCCC---CCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU RTBVCaMV junction Chimera Wild type CaMV strain 4184 I-Rev1’-1 I-Rev1’-2 StemsORF UUUCUCAAAAUAUUGAGUCACGUUACAAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU Landing site G 74 U U 75 A U 75 G + G 64 A I-Rev1’-3 G 69 A + G 64 A UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCU-AAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUA-----uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU I-Rev1’-4 I-Rev1’-5 I-Rev1’-6 C 67 G PBS Progeny-1’ (Plant N1, inoculation , symptoms – , late harvest – ) (+G 260 A) (+U 317 A +G 5 A +del[C 51 –- g 54 ]) (+U 317 A) (+del[A 46 –- U 47 ]) (+del[C 4 –- G 5 ])

UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU GAUUUAAAGAAAUAAUCCGCAUAAGCCC---CCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU RTBVCaMV junction Chimera Wild type CaMV strain 4184 II-Rev1’-1 II-Rev1’-2 StemsORFLanding site G 64 A + U 75 A G 64 U + U 75 A II-Rev1’-3 G 64 U + U 75 A UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU II-Rev1’-4 II-Rev1’-5 II-Rev1’-6 G 64 U + U 75 A II-Rev1’-7 del[G 64 --U 75 ] U 98 C UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUA-----uCCGCUUAAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUACA---uCCGCUUAAA 21 nt insert AAAUUGGUAUCAGAGCC AUG AAU PBS Progeny-1’ after one passage (inoculation , symptoms – ; harvest – ) (+A 483 C) (+G 5 A +C 44 A +del[A 46 ])

UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU GAUUUAAAGAAAUAAUCCGCAUAAGCCC---CCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU RTBVCaMV junction Chimera Wild type CaMV strain 4184 V-Rev1’-1 del3nt StemsORF del3nt UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU del3nt C 83 A Landing site del3nt PBS UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU V-Rev1’-2 V-Rev1’-3 V-Rev1’-4 V-Rev1’-5 V-Rev1’-6 V-Rev1’-7 V-Rev1’-8 V-Rev1’-9 del3nt A 557 G del3nt C 83 A A 557 G UUUCUCAAAAUAUUGAGUCACGUUCCGCUA--CGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACUUUA-----uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU Progeny-1’ after four passages (+del[A 46 –-A 60 ], G -2 A, G 380 A) (+del[A 46 –-A 60 ], A 175 C) (+del[A 46 –-A 60 ]) (+del[A 46 –-A 60 ], G 8 A) (+del[A 46 –-A 60 ])

UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU GAUUUAAAGAAAUAAUCCGCAUAAGCCC---CCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU RTBVCaMV junction Chimera Wild type CaMV strain 4184 I-Rev2-1 StemsORF C 67 G UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU Landing site PBS UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCA-AAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU I-Rev2-3 I-Rev2-4 I-Rev2-5 I-Rev2-6 I-Rev2-7 (+G 162 A) (+A 57 C) G 64 A = original I-Rev2-2 G 64 A G 74 U U 75 G UUUCUCAAAAUAUUGAGUCACGUUACAAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUUAAAAUAUUGAGU guCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU (+G -4 U) Progeny-2 (Plant N2, inoculation , symptoms – ; early harvest – )

UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU GAUUUAAAGAAAUAAUCCGCAUAAGCCC---CCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU RTBVCaMV junction Chimera Wild type CaMV strain 4184 II-Rev2-1 StemsORF C 67 G UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAUAAAAAAUUGGUAUCAGAGCC AUG AAU Landing site PBS UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU II-Rev2-3 II-Rev2-2 G 74 U UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAAUUGGUAUCAGAGCC AUG AAU Progeny-2 after one passage (inoculation , symptoms – ; harvest )

UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU GAUUUAAAGAAAUAAUCCGCAUAAGCCC---CCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU RTBVCaMV junction Chimera Wild type CaMV strain 4184 III-Rev2-1 StemsORF G 69 A UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU Landing site PBS UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU III-Rev2-3 III-Rev2-2 G 74 U UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU III-Rev2-4 III-Rev2-5 III-Rev2-6 G 74 U UUUCUCAAAAUAUUGAGACACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AGG AAU UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCAAAAAAAAUUGGUAUCAGAGCC AUG AAU Progeny-2 after two passages (inoculation , symptoms – ; harvest – ) (+C 292 A) (+del[U 48 –-U 58 ])

UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU RTBVCaMV junction Chimera VI-Rev2-1 StemsORF UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU Landing site PBS UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU VI-Rev2-3 VI-Rev2-2 UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUAUG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU VI-Rev2-4 VI-Rev2-5 VI-Rev2-6 G 69 C GAUUUAAAGAAAUAAUCCGCAUAAGCCC---CCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU Wild type CaMV strain 4184 (+del[C 42 –-C 59 ] +del[U 155 –-U 160 ]) UUUCUCAAAAUAUUGAAUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU VI-Rev2-7 VI-Rev2-8 VI-Rev2-9 G 69 C C 83 U C 109 U G 69 C C 107 U G 74 U G 74 U C 107 U G 64 A+U 75 A +G 78 U (+del[C 42 –-C 59 ]) (+del[C 42 –-C 59 ] +G -2 A) (+del[C 42 –-C 59 ]) UUUCUCAAAAUAUUGAGUCACGUUAUG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAAUUGGUAUCAGAGCC AUG AAU (+del[U 47 –-a 55 ]) (+del[U 48 –-U 58 ]) (+del[C 44 –U 45 ]) (+del[U 49 –U 50 ]) UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUACUGAGUCACGUUACG---uCCGCAUAAAAAAUUGGUAUCAGAGCC AUG AAU Progeny-2 after five passages inoculation , symptoms – ; harvest –

UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU RTBVCaMV junction Chimera StemsORFLanding site PBS I-Rev2’-1 G 64 A UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCA-AAAAAAUUGGUAUCAGAGCC AUG AAU I-Rev2’-3 I-Rev2’-4 I-Rev2’-5 I-Rev2’-2 I-Rev2’-6 G 64 A+G 70 A G 64 A+A 68 G G 64 A+U 75 A G 74 U G 64 A+G 69 A U 88 C UUUCUCAAAAUAUUGAGUCACUUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU (+del[U 47 –-a 55 ]) GAUUUAAAGAAAUAAUCCGCAUAAGCCC---CCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU Wild type CaMV strain 4184 Progeny-2’ (Plant N2, inoculation , symptoms – ; late harvest – )

UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU RTBVCaMV junction Chimera StemsORFLanding site PBS II-Rev2’-1 G 64 A+U 75 A UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU II-Rev2’-2 G 64 A+U 75 A UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU GAUUUAAAGAAAUAAUCCGCAUAAGCCC---CCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU Wild type CaMV strain 4184 UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCAAAAAAAAUUGGUAUCAGAGCC AUG AAU II-Rev2’-3 II-Rev2’-4 II-Rev2’-5 II-Rev2’-6 G 64 A+U 75 A C 99 U G 64 A+U 75 A UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUAAAUUGU-----uCCGCUAAAAAAAUUGGUAUCAGAGCC AUG AAU G 64 A+U 75 A +U 71 C (+G 5 A) (+C -58 U, U 45 C) (+C 2 G) Progeny-2’ after one passage inoculation , symptoms – ; harvest (one plant)–

UUUCUCAAAAUAUUGAGUCACGUUACGAcguCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU RTBVCaMV junction Chimera StemsORFLanding site PBS V-Rev2’-1 G 64 A+U 75 A U 85 C UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUACG---uCCGCUAAAAAAAAUUGGUAUCAGAGCC AUG AAU V-Rev2’-2 G 64 A+U 75 A GAUUUAAAGAAAUAAUCCGCAUAAGCCC---CCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU Wild type CaMV strain 4184 V-Rev2’-3 V-Rev2’-4 V-Rev2’-5 V-Rev2’-6 G 64 A+U 75 A UUUCUCAAAAUAUUGAGUCACGUUAGU---uCCGCUAAAAAAAUUGGUAUCAGAGCC AUG AAU G 64 A+U 75 A V-Rev2’-7 V-Rev2’-8 (+del[U 47 –-A 60 ]) (+del[U 48 –-U 58 ]) (+del[U 47 –-A 60 ]) (+del[U 47 –-A 60 ], U 174 G) (+del[U 47 –-A 60 ]) UUUCUCAAAAUAUUGAGUCACGUUAGU---uCCGCUUAAAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUAGU---uCCGCUACAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUAGU---uCCGCUAAAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUAGU---uCCGCUUAAAAAAUUGGUAUCAGAGCC AUG AAU UUUCUCAAAAUAUUGAGUCACGUUAGU---uCCGCUAAAAAAAUUGGUAUCAGAGCC AUG AAU G 64 A+U 75 A G 64 A+U 75 A Progeny-2’ after four passages

-22.2 kcal kcal RTBV-CaMV Landing Site CaMV Landing site I-Rev1-2 (c 597 A) kcal kcal I-Rev1-1,3,4,5,6 (G 595 A) = II-Rev1-1,2,3,4 = III-Rev1-1,2,5,6 III-Rev1-3 (G 595 A+U 612 AU) = III-Rev1-4 (G 595 A+U 612 AAU) Progeny kcal VI-Rev1-1,2,3 (del[CG 594,595 ]) kcal VI-Rev1-4 (del[ACG 588 ], G 595 A) VI-Rev1-7 (del[CGUU 589 ], G 595 A, A 606 U) kcal VI-Rev1-9 (del[CGUU 589 ] followed by a 7 nt insertion) kcal VI-Rev1-5,6,8 (del[Acg 596 ]) kcal

-22.6 kcal I-Rev1’-1 (G 595 A) kcal kcal I-Rev1’-5 (del U 605 ) I-Rev1’-6 (del[CGAcg 594 ]) kcal I-Rev1’-2,3,4 (del[Acg 596 ]) = II-Rev3-1,2,3,4 = V-Rev3-1,2,3,4,5,6 II-Rev1’-5 (del[Acg 596 ], U 612 AAAU) kcal II-Rev1’-6 (del[CGAcg 594 ], U 612 AU) kcal kcal V-Rev1’-7,8 (ACGAcguC to CCGUA) kcal V-Rev1’-3 (del[Acg 596 ], U 612 AU) kcal V-Rev1’-9 (del[CGAcg 594 ], G 590 U) kcal RTBV-CaMV Landing Site kcal CaMV Landing site Progeny-1’

-20.4 kcal I-Rev2-1,2 (del[Acg 596 ]) = II-Rev2-2 = III-Rev2-1,2,3 = VI-Rev2-1,5, kcal I-Rev2-6 (G 595 A) kcal I-Rev2-3 (UU 604,605 A) kcal I-Rev2-7 (C 574 U, del[C 587 – c 597 ]) kcal II-Rev2-1 (A 611 UAAAAAA) kcal II-Rev2-3 (U 612 AU) Progeny kcal III-Rev2-5 (del[Acg 596 ], U 586 A, U 627 G) kcal III-Rev2-6 (del[Acg 596 ], UU 604,605 AA) kcal III-Rev2-4 (del[Acg 596 ], U 612 AU) = VI-Rev2-2, kcal VI-Rev2-3,4 (del[Acg 596 ], C 594 U) kcal VI-Rev2-7 (del[Acg 596 ],U 581 C, U 604 A) kcal VI-Rev2-9 (del[Acg 596 ], G 585 A) kcal RTBV-CaMV Landing Site kcal CaMV Landing site

-20.4 kcal I-Rev2’-1,2 (del[Acg 596 ]) = II-Rev4-1,2,4 = V-Rev4-1, kcal I-Rev2’-3 (UU 604,605 A) kcal II-Rev2’-3 (del[Acg 596 ], UU 604,605 AA) kcal I-Rev2’-6 (G 590 U) kcal II-Rev2’-6 ( CACGUUAAGAcg to AAAUUGU], U 605 A) kcal V-Rev2’-3 (del[Acg 596 ], U 605 AA) Progeny-2’ kcal V-Rev2’-4 (GCAcg to GU]) kcal V-Rev2’-5 (GCAcg to GU], U 612 AAU) kcal V-Rev2’-6 (GCAcg to GU], U 605 AC) kcal V-Rev2’-8 (GCAcg to GU], U 605 A) kcal V-Rev2’-7 (GCAcg to GU], U 605 AA) kcal RTBV-CaMV Landing Site kcal CaMV Landing site

Summary and Conclusions on the shunt landing site G595A occurred in all the progenies; dominated in the first two passages of Progeny-1. In the last passages it was combined with 3 or 4 nt deletions in the upstream sequence - del[ ] or del[ ] or in the downstream sequence - del[ ] del[ ] – the second most frequent event of the evolution in all the progenies, always more competitive compared to G595A. In the progeny-4, it was combined with CG594,595 to GU del[ ] occurred in 40% clones of the last passage of Progeny-1 A few occasional point mutations, or short deletions and insertions, which were never dominating, would usually hit G or C or GC-rich regions Frequently, insertions of a few nts and point mutation occur in the AAAAAA stretch just upstream of the primer binding site (PBS). This mutation hot spot has been observed before during forced evolution of other CaMV mutants (Pooggin et al., 1998) and therefore it is not related to a particular case of RTBV-shunt elements in the CaMV background The original chimeric mutant sequence was present in subpopulations at earlier passages Tendencies for most reversions are: (1) to weaken the local secondary structure (also G595A, but less significantly) (2) to restore the wild type spacing in the leader between the shunt landing site and the ORFVII start codon

Late alterations in the RTBV stem section 1 U U A A U G U U G C U CC A A G G A U C U C C U G U G A U A UUCUGCU A G A G G A U A C U U A CUCUUU A G U U C C u A U U A C A U A U G U U G G G A U U A U C UU G G G A A U U A G G G A A U A G ACCCUUG A G U C C C U U A U C AUUUAG C A A C C C U U A C C C U A U A U A U C G U U Only after several passages (i.e. the latest events in the evolution of the chimeric virus) C107U is the only dominant mutation and the only one found in two independent progenies C109U is found in two independent clones of the same revertant A557G is found in two clones representing different revertants U98C and C99U are occasional mutations (each found only once in one clone; disappeared on the next passages) Occurred once Dominant in two progenies Occurred once Occurred twice in one progeny Occurred twice in one progeny Wild type CaMV stem

VI-Rev1-1,2,3,4,5,7,8,9 VI-Rev2-2,3,8 (C 107 U) kcal RTBV stem section kcal II-Rev1’-7 (U 98 C) kcal VI-Rev2-1 (C 109 U) V-Rev1’-4, 5 (A 557 G) kcal II-Rev2’-5 (C 99 U) kcal kcal-16.8 kcal kcal kcal stem base helix kcal kcal CaMV stem section 1 stem base helix kcal Late alterations in the RTBV stem