Out of 83 clones and a total of 135
207 nucleotides sequenced, only four mutations were observed, yielding a mutation rate (±s.e.m.) of μmax
m/b/r. Two mutations were found in the NIb
gene, one synonymous (C8029U) and the other (C8248U) with a probable major effect on the protein folding, since it replaces P2750 by S, an amino acid with a small polar radical. Two other non-synonymous mutations were found in the CP gene, C8980A (Q2994K) and C9008A (A3003D). The first mutation replaced a positively charged side chain by another one with similar characteristics. The second one may have a stronger effect on fitness since it replaced a small apolar radical by a larger side chain with net negative charge. Assuming that only P2750S and A3003D may be lethal for the virus, then the estimated mutation rate would be μ
m/b/r. This value is in good agreement with the indirect estimate reported by Carrasco et al. (2007)
for the deleterious genomic mutation rate.
The use of a high-fidelity DNA polymerase minimizes the probability that the observed mutations were due to PCR errors, but reverse transcription artefacts are more likely since the error rate of MMLV RT is probably higher than 3.3×10−5
m/b/r (Arezi & Hogrefe 2007
). Therefore, we can only safely conclude that the obtained μmax
, at least for the sequenced region, in the particular host employed and experimental conditions, is as low as that for a common RT enzyme.
How do our data compare with the levels of genetic variability reported for other plant RNA viruses? compiles μmax
for four different viruses on different hosts. These values were in all cases estimated after characterizing the mutant spectrum from individual plants. The median μmax
across viruses and experiments is 4.74×10−4
m/b/r, which is in the range of estimated rates for animal RNA viruses and some RNA bacteriophages (Drake & Holland 1999
). It is worth noting that estimates are homogeneous for the five different viruses (two-way non-parametric ANOVA: H
=1.140, d.f.=2, p
=0.566), although they significantly vary across host plants (H
=22.424, d.f.=9, p
=0.008), suggesting that virus mutation rates depend on the environment in which they replicate, as suggested by Pita et al. (2007)
Upper-limit estimate for the mutation rate (μmax) for several RNA plant viruses.
The approximately 16-fold lower μmax
obtained for TEV compared with the above median value can be attributed to several factors. It is possible that TEV shows an unusually low rate of spontaneous mutation or that tobacco plants are hosts in which mutation rate is particularly low. It must also be noted that the region we have sequenced contains cis
-acting secondary structure motifs necessary for genome replication (Haldeman-Cahill et al. 1998
), and mutations affecting these motifs render replication-incompetent viruses. Therefore, our μmax
value might be closer to the actual mutation rate than μmax
values obtained in studies where a less-constrained region was sequenced. Hence, most of the genetic variation sampled in the studies are listed in , and in previous work with animal RNA viruses and bacteriophages might correspond to neutral or nearly neutral mutations that had been segregating in the population for long periods of time, thus reaching relatively high frequencies, or that some of these mutations were beneficial. In our experiments, by contrast, long-term variation could not be present because infections were initiated from a single lesion-forming unit. Furthermore, the virus used in our experiments had been replicating in tobacco plants for 30 passages prior to the assays without showing significant changes in several fitness traits (data not shown), suggesting that the mutations sampled were probably not beneficial.
Our data allow us to conclude that the mutation rate of TEV is in the lower range of those reported for other RNA viruses, but, obviously, we cannot conclude that plant RNA viruses show low mutation rates in general. However, the fact that our estimate is very close to the only previous estimate available for a plant RNA virus (Malpica et al. 2002
) at least suggests so. Finally, slow rates of molecular evolution in plant viruses could be partly explained by low mutation rates, but the complex relationships between mutation and evolution need to be considered in detail to explain why some plant viruses show unusually high levels of genetic stability while others evolve fast.