1.13.12
TRANSMISSION BY NEMATODES OF TRV ISOLATE PAY4 CARRYING MUTATIONS IN THE NON-STRUCTURAL GENES OF RNA2

N VASSILAKOS, S MACFARLANE and DJF BROWN

Scottish Crop Research Institute, lnvergowrie, Dundee DD2 5DA, Scotland, UK

Background and objectives
Tobraviruses are transmitted between plants by root-feeding nematodes belonging to the genera Trichodorus and Paratrichodorus. The association between virus and nematode is highly specific, with particular virus isolates being transmitted by only one, or occasionally a few, species of nematode. Studies of mutant viruses, derived from infectious cDNA clones, have shown that for pea early-browning tobravirus (PEBV) the virus coat protein and at least two other viral proteins are involved in the transmission process. However, for the PpK2O isolate of tobacco rattle tobravirus (TRV), probably only the coat protein and one other protein are necessary for transmission. To further investigate tobravirus transmission we have cloned, sequenced and analysed mutants of a new isolate (PaY4) of TRV. This particular virus isolate has two known nematode vector species, P. pachydermus (which it shares with TRV PpK20) and P. anemones (which it shares with PEBV). Sequence analysis and mutagenesis of the non-structural genes carried on PaY4 RNA2 will allow us to comment on the specificity of the interaction between virus and vector nematode.

Results and conclusions
TRV PaY4 RNA2 was found to be 3926 nt in length, the largest tobravirus RNA analysed to date. It contains three open reading frames encoding (from 5' to 3') the coat protein (209 amino acids, 22.5K) and two other proteins, 27K (237aa) and 32K (286aa). The 27K protein has extensive sequence homology (39% identity) with the PEBV 29K nematode transmission protein, and more limited homology (19% identity) with the TRV PpK2O 37K nematode transmission protein. The 32K protein has no sequence homology with other tobravirus proteins. Mutations were introduced in the full-length cDNA clone of PaY4 RNA2, at existing restriction sites in the 27K and 32K genes. The 27K gene was deleted between Mscl and PflMI restriction sites (nt 1503 and 1675), and a frameshift was created by digesting an XbaI site (nt 1608) followed by Klenow treatment and religation. The 32K gene was deleted between the AatII and NsiI sites (nt 2580 and 2956), and a frameshift was also created at the AatII site. Transcripts were synthesized from the mutant clones, mixed with wild-type TRV RNA1 and inoculated to Nicotiana benthamiana plants. Stable, mutant virus was recovered from the 27K frameshift, 32K frameshift and 32K deletion transcripts; however, virus derived from the 27K deletion mutant RNA was unstable and was not examined further. The three stable mutants were inoculated to N. clevelandii plants and tested for transmission by P. anemones nematodes. The 27K frameshift mutant was transmitted from none of 14 plants, whereas the 32K deletion mutant was transmitted from eight of 15 plants, and the 32K frameshift mutant was transmitted from four of 13 plants. In the same series of tests, wild-type TRV PaY4 was transmitted from nine of 15 plants. Thus the TRV PaY4 27K gene is essential for transmission by P. anemones, whereas the 32K gene is not required for nematode transmission. TRV PaY4, therefore, resembles TRV PpK20 in that only one of the RNA2-encoded non-structural proteins is involved in nematode transmission. Similar results were obtained when the PaY4 mutants were tested for transmission by P. pachydermus. Current experiments aim to identify how the two TRV isolates (PaY4 and PpK20) maintain a specific interaction with the shared vector nematode (P. pachydermus), while only the PaY4 isolate can interact with P. anemones.