1 Molecular Plant Pathology Laboratory, USDA/ARS,Beltsville, MD 20705, USA
2 Biometrical Consulting Service, USDA/ARS,Beltsville, MD 20705, USA
Correspondence
Robert Owens
owensr{at}ba.ars.usda.gov
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ABSTRACT |
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MAIN TEXT |
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Previous studies of viroid sequence variation have addressed several aspects of hostpathogen interaction. Differences in overall sequence homology have been used to divide isolates of Hop stunt viroid (HSVd) into host-specific groups (Shikata, 1990; Kofalvi et al., 1997
; Sano et al., 2001
; Amari et al., 2001
). Co-variation in nucleotide sequence provides evidence for the role of alternative structures such as secondary hairpins I (Polivka et al., 1996
) and II (Loss et al., 1991
; Qu et al., 1993
) in viroid replication. A recent study by Matousek et al. (2001)
provides insight into the role of environmental stresses in generating viroid sequence diversity. These authors showed that heat treatment of hop plants infected with Hop latent viroid (HLVd) results in a rapid decrease in viroid titre and a dramatic increase in sequence variability. The prevalence of HLVd variants containing three or four changes suggests that mutations accumulate during successive replication cycles. For genomes like that of influenza virus A where certain codons are subject to positive selection, phylogenetic methods can be used to predict the future course of evolution (Bush et al., 1999
; Farci et al., 2000
).
To better understand the selective pressures guiding viroid evolution in vivo, we have examined the fitness landscape surrounding two well-characterized, naturally occurring strains of PSTVd. PSTVd is the type member of the genus Pospiviroid whose members possess a highly base-paired, rod-like secondary structure. RG1 is a highly pathogenic variant that appeared spontaneously during repeated passage of PSTVd-Int(ermediate) in tomato (Gruner et al., 1995). PSTVd-Int and RG1 differ at only three positions within a portion of the molecule known as the pathogenicity domain. Our results indicate that these variants are surrounded by a network of neutral or near-neutral mutants that allows the population to move from peak to peak in the fitness landscape.
Fig. 1 compares the pattern of sequence variation among more than 40 natural isolates of PSTVd (panel A) with that introduced by oligonucleotide-directed randomization of the pathogenicity domain of PSTVd-Int (panel B). Natural variability is concentrated in the lower portion of the pathogenicity domain as well as positions 120 and 123 of the variable domain. To investigate the feasibility of screening large libraries of viroid sequences for variants with altered biological properties, we randomized five positions in PSTVd-Int. PSTVd-Int and RG1 differ at positions 46, 47 and 317; positions 315 and 316 were also mutagenized to produce a more complex mixture of variants. Wild-type viroid was eliminated from the potential progeny by replacing the G residue at position 46 with an equimolar ratio of A, C and T, resulting in an equimolar mixture of 3x4x4x4x4=768 sequences. Three bioassays were carried out using RNA transcripts synthesized from independent cDNA template preparations to inoculate either Rutgers or Microtom (Meissner et al., 1997
) tomato seedlings. Infected plants were identified by dot blot hybridization (Podleckis et al., 1993
) and the progeny from 50 individual plants was characterized by RT-PCR (Hu et al., 1996
). By examining uncloned PCR products rather than individual cloned cDNAs, we would expect to detect only the predominant PSTVd variant(s) in each infected plant.
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The 24 infected plants analysed in the third bioassay contained 16 different PSTVd variants. Variant M24/M7 was recovered from three different plants and seven other variants were recovered twice. Taken together, our three bioassays yielded 23 viable variants of which 20 were previously unknown. Because relatively few of the 768 possible variants were recovered (and several more than once), the total number of neutral mutants seemed likely to be small. Because all but two of the 50 plants analysed contained only a single variant, their competitive abilities appeared to be ordered. Viroid symptom expression is almost completely suppressed in dwarf tomato varieties like Microtom; thus, the relative severity of each of these variants could not be directly assessed.
To estimate the true number of neutral mutants, we simulated the inoculation and subsequent infection processes based on the following assumptions: (i) variants are initially present in equal numbers in the inoculum; (ii) the number of variants present in an inoculum is a sample from a Poisson distribution (i.e. variants are distributed randomly in the solution and the Poisson parameter depends on solution concentration); and (iii) the competitive ability of variants is ordered (i.e. if two variants are present in the same inoculum, only the most competitive will later be detected). Sim2, written in Perl, allows one to specify the number of simulations, number of plants used, true number of infectious variants and proportion of uninfected plants [by setting the Poisson parameter to -ln (proportion of uninfected plants)]. For each simulation, the program (i) creates an inoculum, (ii) draws from the inoculum and applies it to the plant and (iii), if the plant becomes infected, identifies the most competitive variant. For each true number of infectious variants in the range producing results similar to our data, we ran 1000 simulations. When we set the true number of infectious variants at 28, the results matched those of our largest data set (experiment 3) exactly. In this data set, inoculation of 24 tomato seedlings with 1000 pg mutagenized RNA transcripts µl-1 resulted in 14 infected plants containing 11 different variants. For these parameters and with the true number of viable sequences set to 28, Sim2 calculated the mean number of different variants to be 11·0±1·92. Sim2 also allowed us to estimate the power associated with various experimental designs. We found that high infection rates and a sample size of 100 plants is necessary for a precise estimate of the true number of neutral mutants.
Possible relationships among mutagenesis-derived and naturally occurring PSTVd sequence variants were analysed using a combination of neighbour-joining and maximum-parsimony (Swofford, 1998) and split decomposition (Huson, 1998
) methods. The results indicated the presence of networks rather than bifurcating trees. As shown in Fig. 2
(A), noteworthy features of these networks included (i) the complex relationships between certain variants and (ii) the relatively isolated position occupied by PSTVd-Int. In many cases, conversion of one variant to another requires only a single substitution. For example, only a single substitution at position 315 separates RG1 from its two nearest neighbours, and M14/M1 and M23/M1 can each be derived from at least two other variants by single nucleotide substitutions. All three pathways leading away from PSTVd-Int, in contrast, begin with a double change. A two-dimensional representation can depict only the lowest transition orders of such a complex network.
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To describe the informational complexity of RNA and DNA genomes, Eigen introduced the concept of a multi-dimensional sequence space (Eigen & Biebricher, 1988; Eigen, 1993
). Sequences differing at one to n positions (where n is the total number of nucleotides in the molecule) occupy positions that are located, respectively, one to n units from each other. Collapsing this multidimensional space to two dimensions and assigning a fitness value to each sequence results in a fitness landscape consisting of peaks connected by ridges and separated by valleys or planes. Implicit in the n-dimensional nature of sequence space is the possibility that sequence changes in distant portions of an RNA molecule may be functionally linked. PSTVd-Int and RG1 differ at only three positions, and randomization of two additional positions could open evolutionary pathways to other previously described variants. If PSTVd-Int and RG1 were located on a flat, comparatively featureless portion of the fitness landscape, one might expect the number of neutral mutants to approach the theoretical population size of 768. The much lower estimate derived from our data (i.e. 28) together with the scarcity of variants containing only a single nucleotide change provides further evidence for a rugged topology surrounding PSTVd-Int. In contrast to the comparatively isolated position of PSTVd-Int, RG1 is separated from its three closest neighbours by only a single nucleotide change.
Although most studies have focused on the diseases they cause, viroid infection can also have beneficial effects. For example, viroid infection can dwarf citrus growing on certain rootstocks, thereby increasing productivity and lowering production costs (reviewed by Hutton et al., 2000). PCR-mediated DNA shuffling has been used to balance the complex viral functions necessary for gene therapy and vaccine applications (Soong et al., 2000
), and we are investigating the use of a similar mutagenesis-selection strategy to produce improved Citrus viroid III (CVd-III) dwarfing agents (Owens et al., 2003
). In preliminary studies, limited sequence randomization resulted in dramatic changes in biological properties. Provided that the appropriate genome regions for mutagenesis can be identified, our results suggest that randomization/selection will yield improved citrus dwarfing agents and other viroid variants with specific biological properties.
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Received 8 August 2002;
accepted 25 October 2002.