Identification of neutral mutants surrounding two naturally occurring variants of Potato spindle tuber viroid

R. A. Owens1, S. M. Thompson1 and M. Kramer2

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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Single point mutations in the pathogenicity domain of Potato spindle tuber viroid (PSTVd) can have a dramatic effect on disease expression, and only three substitutions are required for the spontaneous conversion of the type strain PSTVd-Intermediate to the rapidly replicating, highly pathogenic variant RG1 (Gruner et al., Virology 209, 60–69, 1995). To identify available evolutionary pathways linking these two variants, we mutagenized five positions in an infectious cDNA copy of PSTVd-Intermediate and screened the resulting mixture of 768 sequences for neutral or near-neutral mutants. Numerical simulations based on the bioassay data indicate that the 23 variants recovered represent >80 % of all such sequences. RG1 was the only naturally occurring variant recovered, and the overall pattern of sequence changes observed indicates that PSTVd-Int occupies a comparatively steep peak within the fitness landscape.


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Viroids are the smallest autonomously replicating pathogens yet described – small (246–399 nt), unencapsidated, circular RNAs that lack mRNA activity yet are able to induce a full range of disease symptoms in susceptible host plants (Flores et al., 1997; Owens, 1999). Like other RNA genomes, viroids propagate in vivo as quasispecies, populations of similar but non-identical sequences resulting from the inherently high error rate of RNA replication (Eigen, 1993). Evolution of viroid and viral satellite RNA sequences appears to be constrained by the need to preserve certain secondary structural features (Fraile & Garcia-Arenal, 1991; Fontana & Schuster, 1998; Ambrós et al., 1998); nevertheless, cloned Potato spindle tuber viroid (PSTVd) variants often give rise to complex populations of variants within a single passage (Góra-Sochacka et al., 1997). Such populations contain all possible single and double mutants as well as many triple mutants.

Previous studies of viroid sequence variation have addressed several aspects of host–pathogen 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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Fig. 1. PSTVd sequence variability. (A) Variation among 48 naturally occurring isolates. Bar heights are proportional to the number of isolates differing from PSTVd-Int (Gross et al., 1978) at individual positions. Numbers indicate boundaries of the five structural/functional domains proposed by Keese & Symons (1985): terminal left (T1), pathogenicity (P), central conserved (C), variable (V) and terminal right (T2) domains. Vertical arrows indicate nucleotide positions mutagenized by oligonucleotide-directed randomization (Reidhaar-Olson & Sauer, 1988) in the present study. (B) Pathogenicity domains of PSTVd-Int and RG1. Horizontal arrows indicate locations of pre-melting loop 1 (PM1) and the virulence-modulating (VM) region as described by Schnölzer et al. (1985).

 
Table 1 summarizes the results from automated sequence analysis of uncloned PCR products. In experiment 1, eight variants were recovered from eleven infected plants; i.e. three variants were present in more than one plant. There appeared to be a general tendency for CA to replace GC at positions 46–47, and spontaneous mutations were detected at several nearby positions. These may represent compensatory mutations that offset the deleterious effects of mutations at the targeted positions (Matousek et al., 2001). The ID50 for unmutagenized PSTVd-Int was approximately 1 pg µl-1, while that of the mutagenized transcript mixture appeared to be >10-fold higher (results not shown). Variant M3/M1 was previously described by Hammond (1992).


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Table 1. PSTVd variants recovered from randomized inocula

Newly described variants deposited in GenBank (accession numbers AF458986–459004).

 
Progeny from the second bioassay included four other variants in addition to M3/M1. One plant contained a mixture of RG1 and M1, another variant first described by Hammond (1992). These two variants differ by only a single U->C change at position 317, suggesting that M1 may represent an evolutionary intermediate between PSTVd-Int and RG1. The most striking result from this experiment, however, was the presence of M1 in ten separate plants. This was a most improbable outcome, and sequence analysis of this particular cDNA template preparation subsequently revealed a marked overrepresentation of C at position 46 as well as very low levels of substitution at the other mutagenized positions (results not shown). No such bias was detected in the template preparations used in the two other bioassays.

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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Fig. 2. Sequence and structural relationships among PSTVd sequence variants. (A) Two-dimensional network connecting infectious variants generated by randomization/in vivo selection. Note that M14/M1 and M23/M1 (shaded) are each connected to two other variants by only single nucleotide changes. PSTVd-D is a nonviable variant described by Hammond (1992). (B and C) Computer-predicted secondary structures of viable variants including PSTVd-Int and RG1 (B) and 25 randomly selected noninfectious variants (C). Red squares represent base pairs present in all sequences in the data set, and blue squares denote base pairs present in PSTVd-Int. Green squares denote base pairs present in sequences other than PSTVd-Int. Smaller squares indicate less probable base pairings; i.e. suboptimal structures. The nine red squares at the lower left corner of the diagonal denote a 9 bp helix involving nucleotides 60–68 and 292–300; the six red squares in the upper right corner denote a helix at the left side of the pathogenicity domain containing nucleotides 30–35 and 326–331 (see Fig. 1).

 
Because so few mutagenesis-derived PSTVd variants were detected, it seemed possible that the competitive sequences might share some common structural feature(s). To address this possibility, we compared the secondary structures of their pathogenicity domains using CONSTRUCT, a software tool developed by Luck et al. (1999). Fig. 2(B) shows the optimal and suboptimal structures for PSTVd-Int, RG1 and the 23 nearby neutral mutants; Fig. 2(C) shows the corresponding structures for 25 randomly chosen noncompetitive sequences. Overall, the two structure distributions were very similar. The two additional base pairs visible on the diagonal in Fig. 2(B) were present in only a single neutral mutant, M31/M5b. Likewise, the eight base pairs above the main diagonal in Fig. 2(C) were present in only one noncompetitive sequence. In PSTVd-Int, nucleotides 46 and 315 are predicted to form a G : C pair in the 6 bp helix visible in the upper right hand corner of Fig. 2(B). Compensatory mutations in variants M11/M8 and M31/M5 result in the conversion of this G : C pair into either a U : A or an A : U pair, but neither change is predicted to have significant structural effects. No structural features unique to competitive variants were obvious.

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.