For full method, please see the document titled: 'PTV A Tobravirus Vector from the Baulcombe Archive'.

Origins
PTV00 is a tobravirus vector based on RNA 2 of TRV strain PPK20. This vector was derived from pCa7, a 35S driven full length infectious clone of PPK20 RNA 2 kindly provided by Prof J Bol (Hernandez et al., 1995) To construct pTV00 the modified pCa7 insert was transferred to the pGreen 0000 RLSK Agrobacterium binary vector (Hellens et al., 2000). PTV00 requires proteins encoded by RNA1 for replication and movement. PBINTRA6 is a Bin19 based construct containing 35S RNA 1 and an intron. The intron prevents expression of the replicase in E. coli and Agrobacterium and thereby promotes stability of the DNA. PBINTRA6 must be introduced to plants as well as pTV00 to obtain infection (Ratcliff et al., 2001).

Purpose
Tobravirus derived vectors have several potential advantages over other currently available virus vectors.

• The presence of a large polylinker of unique sites, and the small overall size of the vector should -facilitate cloning of inserts.
• Virus infections that lack Orfs 2b and 2c are symptomless in N. benthamiana and very mild in Arabidopsis; the effects of protein expression or VIGS are therefore clearer in the absence of virus symptoms.
• Tobraviruses are unusual in infecting meristems, floral organs and even pollen. pTV should therefore be able to express or silence genes in these areas.
• TRV does not block the systemic signal for silencing. Even uninfected cells may therefore become silenced.

Use
The vectors are provided as DNA samples. To propagate in E. coli use 50µg/ml kanamycin for both constructs. We recommend E. coli strain DH5a, especially for pBINTRA6, since it tends to be unstable in E. coli. For propagation in A. tumefaciens use C58c1 for pBINTRA6 and GV3101 with pSa-rep (Hellens et al., 2000) for pTV00 and derivatives and select with 50µg/ml Kan and 5 µg/ml Tet and 50µg/ml Rifampicin