Abstract
RNA viruses and viroids replicate with high mutation rates, forming quasispecies, population of variants centered around dominant sequences. The mechanisms governing quasispecies remain unclear. Plasmodesmata regulate viroid movement and were hypothesized to impact viroid quasispecies. Here, we sequenced the progeny of potato spindle tuber viroid intermediate (PSTVd-I) strain from mature guard cells lacking plasmodesmal connections and from in vitro-cultivated mesophyll cell protoplasts from systemic leaves of early-infected tomato (Solanum lycopersicum) plants. Remarkably, more variants accumulated in guard cells compared to whole leaves. Similarly, after extended cell culture, we observed more variants in cultivated mesophyll protoplasts. Coinfection and single-cell sequencing experiments demonstrated that the same plant cell can be infected multiple times by the same or different PSTVd sequences. To study the impact of initial population composition on PSTVd-I quasispecies, we conducted coinfections with PSTVd-I and variants. Two inoculum ratios (10:1 or 1:10) established quasispecies with or without PSTVd-I as the master sequence. In the absence of the master sequence, the percentage of novel variants initially increased. Moreover, a 1:1 PSTVd-I/variant RNA ratio resulted in PSTVd-I dominating (>50%), while the variants reached 20%. After PSTVd-I-only infection, the variants reached around 10%, while after variant-only infection, the variants were significantly more than 10%. These results emphasize the role of cell-to-cell communication and initial population composition in shaping PSTVd quasispecies.
Cell-to-cell communication mediated by plasmodesmata and the initial sequence composition of the PSTVd quasispecies are significant constraints on the PSTVd sequence population.
IN A NUTSHELL.
Background: RNA viroids, minute infectious agents, have high mutation rates, resulting in diverse populations known as quasispecies. Despite this mutability, viroid quasispecies exhibit remarkable stability in sequence structure. Typically, a few master sequences dominate, while numerous variants also exist. Plasmodesmata, which enable communication between cells and regulate RNA movement, may play an important role in constraining viroid sequence diversity.
Question: This study employed potato spindle tuber viroid (PSTVd) to explore constraints on viroid quasispecies. In particular, we examined the role of plasmodesmata and initial viroid population sequence structure.
Findings: Unexpectedly, a higher accumulation of PSTVd variants was observed in isolated guard cells and in vitro-cultivated mesophyll protoplasts compared to whole leaves. Remarkably, plant cells were found to be susceptible to multiple infections by the same or different variants, enabling the coexistence of multiple variants within a single cell. Coinfection experiments revealed a higher emergence of novel variants in populations initially lacking a master sequence. This underscores the significance of plasmodesmata-mediated cell-to-cell communication and the initial sequence composition as 2 key constraints on PSTVd quasispecies.
Next steps: Subsequent research should delve into the molecular intricacies involved, unraveling the contributions of initial viroid sequences and cell-to-cell communication to quasispecies evolution. This deeper understanding holds potential implications for devising strategies to manage viroid infections in plants.
Introduction
RNA viruses and viroids replicate with extremely high mutation rates, leading to enhanced evolvability and virulence. As a result, they exist and replicate as dynamic and complex variant clouds known as quasispecies, which can be defined as a distribution of variants shaped by error-prone replication and selection pressure (Domingo et al. 2008, 2012). Viral quasispecies typically consist of 1 or a few master sequences that appear most frequently and a large number of variants derived from the master sequence(s) (Domingo and Perales 2019). Master sequences and variants act in concert as a single entity. Although variants only occupy a small sequence space in an established population, some may demonstrate higher fitness in new quasispecies that might arise when the host environment undergoes shifts in the fitness landscape, allowing them to dominate the population (Lauring and Andino 2010). Such shifts could occur as a result of immune responses or human intervention (e.g. vaccines). Additionally, variants within quasispecies have been observed to facilitate transmission to new hosts (Domingo et al. 2021). In relatively constant environments, however, the master sequence remains dominant within the population over time even though variants are continuously generated (Lauring and Andino 2010).
Viroids are a group of circular single-stranded RNA molecules that are considerably smaller than viruses (size range 250 to 400 nucleotides [nts]). Unlike viruses, they do not encode proteins and lack a protein coat. Nevertheless, viroids systemically infect a range of plant species and induce a variety of symptoms, such as stunted growth, yellowing, and other abnormalities (Ding 2009). In nature, most viroids are transmitted by mechanical abrasion. In agricultural settings, they are typically spread through plant vegetative propagation or contaminated tools and, in some cases, through seeds (Hadidi et al. 2022). Replication is accomplished by exploiting host cellular machinery. In addition, viroid RNAs traffic between cells through plasmodesmata, gated intercellular channels that allow regulated passage of small molecules as well as RNA and protein (Ding et al. 1997). They also move systemically through the vascular system, specifically the phloem (Navarro et al. 2021). In short, viroids are infectious long noncoding RNAs and the smallest known infectious agents (Adkar-Purushothama and Perreault 2020). Viroids were previously thought to only infect angiosperms. However, recent discoveries of viroid-like RNAs in a large number of metatranscriptomes and plant transcriptomes suggest that the host range of viroids may have been underestimated (Lee et al. 2023). In addition, a recent study characterized a viroid-like RNA that naturally infects a fungus (Dong et al. 2023).
We study viroid biology using potato spindle tuber viroid (PSTVd, genus Pospiviroid) as a model. PSTVd replicates in the nucleus of infected cells by coopting host DNA-dependent RNA polymerase II (Pol II), DNA ligase I, and other cellular components (Ding 2009). In a single replication cycle, PSTVd generates at least 7 new genomes, and de novo mutations occur on average at a rate of 1 per every 919 nts synthesized (Wu and Bisaro 2020). Since the PSTVd genome consists of ∼359 nts, this corresponds to a de novo mutation rate of about 1 per 2.5 genomes during Pol II-catalyzed replication. Extreme mutation rates have been observed for other viroids as well (Gago et al. 2009; Lopez-Carrasco et al. 2017). Notably, the PSTVd mutation rate is approximately 10 times higher than that of single-stranded RNA viruses, making it a promising model for evolutionary studies. Based on this extremely high rate, we calculated that the proportion of the sequence population represented by a PSTVd master sequence in the quasispecies derived from it should be less than 50% after 2 replication cycles (Fig. 1). However, our previous research, which utilized a novel library preparation technique and whole-genome deep sequencing, revealed that the proportion of master sequence to variants remains consistently around 9:1 at various time intervals following inoculation (Wu et al. 2020). Consequently, mutations occur at a high rate but exist at a low frequency within a sequence population due to the influence of selective forces.
Figure 1.
A hypothetical model of PSTVd quasispecies structure after 2 replication cycles. PSTVd replicates at an error rate of about 1 per 2.5 genomes. In each replication cycle, PSTVd produces at least 7 monomers. Theoretically, about 2.5 mutant genomes (red ovals) will be generated after 1 replication cycle. After 2 cycles, more than half of the genomes will have experienced a mutation.
The evolution of RNA viruses and viroids is a dynamic and complex process (Holland et al. 1982, 1992). Quasispecies theory has been utilized to build mathematical models that investigate viral evolution under conditions of near-infinite population sizes and error-prone replication (Eigen 1993; Biebricher and Eigen 2006; Domingo et al. 2006). In this framework, the error threshold is the maximum number of mutations that a replicating population can tolerate before losing genetic information and descending into genetic chaos (Biebricher and Eigen 2005). However, a viral quasispecies that occupies the entire or even most sequence space has never been observed. In reality, certain variants are selectively maintained, exploring only a small fraction of the sequence space, while others are lost (Holmes 2003). This discrepancy may be due in part to the insufficient depth of deep sequencing platforms. Additionally, mechanisms likely exist that select for or against certain variants in quasispecies. As we observed with PSTVd, de novo mutations occur less frequently in replication-critical genome regions, and genome-wide most de novo variants are lost due to selection (Wu and Bisaro 2020). Thus, there are constraints on the diversity of quasispecies, although mechanisms that restrict sequence space remain unclear. Several theories have been proposed, including those related to fitness landscapes, RNA secondary structures, and the error threshold, in an attempt to elucidate the factors contributing to the stable structure of viral quasispecies (Nilsson and Snoad 2000; Domingo and Schuster 2016). However, replicative capacity is typically employed as a proxy for fitness, while other crucial factors for viral infection, including spread within the host, cellular tropism, transmissibility, and immune evasion, are often overlooked (Domingo and Holland 1997). Moreover, RNA secondary structure and error threshold theory describe phenomena without providing mechanistic explanations (Nilsson and Snoad 2000; Holmes 2003; Domingo and Schuster 2016).
Successful infection by viruses and viroids relies on their ability to replicate at the initial site of infection, move between cells, and efficiently traffic to remote sites (Trono et al. 2010; Tilsner and Oparka 2012; Gaudin et al. 2013; Heinlein 2015). The movement of plant viruses and viroids between cells occurs via plasmodesmata (Ding 2009; Benitez-Alfonso et al. 2010; Lee and Lu 2011; Heinlein 2015; Navarro et al. 2021). We have been using PSTVd to investigate plasmodesmal gating systems involved in the regulation of RNA trafficking. In previous studies, unidirectional plasmodesmal gates have been identified between epidermal and palisade mesophyll cells (Wu et al. 2019; Wu and Bisaro 2022), palisade and spongy mesophyll cells (Takeda et al. 2011), mesophyll cells and bundle sheath (Qi et al. 2004), and bundle sheath and phloem (Zhong et al. 2007; Wu et al. 2020; Wu and Bisaro 2022). These gates interact with 3D RNA motifs, as well as G-U base pairs, formed by PSTVd to regulate its movement between different cell types. The role of plasmodesmal gates in regulating the movement of viral and viroid RNAs is not the only factor that can affect infection. Plasmodesmal transport of signaling molecules may also impact viral infection by influencing plant immunity (Brunkard and Zambryski 2017; Cheval et al. 2020).
Based on these observations, we hypothesize that the ability of variants within PSTVd quasispecies to traffic between cells may be an important element shaping the structure of the sequence population. In this scenario, competence of variants to form specific RNA 2D or 3D structures capable of interacting with host factors that permit cell-to-cell and systemic trafficking is crucial. To this end, variants present in PSTVd quasispecies derived from a PSTVd intermediate (PSTVd-I) strain master sequence in mature tomato (Solanum lycopersicum) guard cells lacking plasmodesmal connections to other cells were analyzed. Remarkably, higher variant levels in guard cell quasispecies compared to the entire leaf were observed. Similarly, extended in vitro culture of mesophyll cell protoplasts isolated from PSTVd-infected tomato plants showed increased variant accumulation compared to whole leaf samples. These results underscore the role of plasmodesmata-mediated cell-to-cell trafficking in shaping PSTVd quasispecies.
Additionally, interactions among variants in quasispecies could also have an impact on the sequence population (Arbiza et al. 2010). Hosts are infected by multiple genotypes that differ in their ability to move, replicate, or evade the immune system (Schröter et al. 2003). Coexistence of multiple variants presents opportunities for interaction between themselves and with cellular machinery, shaping the evolutionary dynamics of sequence populations. Some variants might contribute to the quasispecies population in a positive manner, while others might be detrimental or exclude other genotypes (superinfection exclusion; Laskus et al. 2001; Domingo et al. 2012; Moreno et al. 2012). Further, superinfection exclusion, a phenomenon in which a prior viral infection impedes subsequent infection by the same or a closely related virus, may restrict the migration of multiple PSTVd-I variants from neighboring cells into the same cell. The phenomenon of superinfection exclusion has been extensively documented in studies of viruses (Perdoncini Carvalho et al. 2022); however, its applicability to viroids remains unknown, despite the presence of potential evidence (Serra et al. 2023). In this study, coinfection experiments, coupled with single-cell sequencing analysis, revealed that a plant cell can be infected multiple times by the same or different PSTVd sequences. To simulate natural infections with multiple variants, PSTVd-I was coinoculated to Nicotiana benthamiana plants with a mixture containing an equal amount of 12 functional variants in different ratios (10:1, 1:1, and 1:10), forming initial quasispecies with or without PSTVd-I as the master sequence. Inoculations with only PSTVd-I or variants were also conducted. Notably, quasispecies without a master sequence accumulated a greater number of novel variants. These findings indicate that cell-to-cell communication and initial population composition shape PSTVd quasispecies structure.
Results
The proportion of variants in quasispecies does not increase with increasing accumulation of PSTVd in infected plants
In a previous study, we demonstrated that the ratio of the master sequence to variants consistently remained at approximately 9:1 at various time intervals following inoculation of PSTVd-I (Wu et al. 2020). This suggests that the proportion of variants within PSTVd-I quasispecies is not linked to infection duration or, possibly, the accumulation of viroid RNA. To test this assumption, systemic leaves were collected from tomato plants infected with PSTVd-I at 9, 10, and 11 days postinoculation (dpi), representing very early stages of infection. Each time point included 3 biological replicates, with 1 plant per replicate, totaling 3 plants per time point. RNA blots were performed to detect PSTVd in sample extracts (Fig. 2A). Quantitative analysis revealed a greater than 2.5-fold increase in PSTVd RNA accumulation from 9 to 11 dpi in all 3 replicates (Fig. 2B). Progeny sequences were analyzed by Sanger sequencing, with more than 10 clones for each time point. Across the 3 time points, the proportion of variants remained consistently around 10% (Fig. 2C, Supplementary Table S1). Consequently, it appears that in infected plants, variant content within PSTVd-I quasispecies remains relatively constant over a range of time points and viroid RNA levels.
Figure 2.
Variant content in quasispecies does not increase with increasing PSTVd RNA accumulation. A) Tomato plants were infected with PSTVd-I, and systemic leaves were collected at 9, 10, and 11 dpi. Each time point consisted of 3 biological replicates, with 1 plant per replicate (Rep), totaling 3 plants per time point. RNA blot analysis was conducted to detect PSTVd in sample extracts. The circular form (PSTVd-C), which is the functional form, is shown. Ethidium bromide staining of rRNA was used as the loading control. “P” was a sample known to be positive for PSTVd. “M” represents the mock control. B) RNA blot signals were quantified using Quantity One software. The graphs show PSTVd RNA levels relative to the 9-d time point. C) Progeny sequences in each RNA sample were analyzed by Sanger sequencing, with more than 10 clones analyzed for each time point. The graph shows the percentage of variant clones observed at each time point. Two-tailed t test is used for comparisons between 2 groups. Bars indicate the Se of the means obtained from 3 biological replicates. n.s., not statistically significant.
PSTVd variants accumulate to higher levels in guard cells compared to in whole leaf protoplasts
We theorized that cell-to-cell communication plays a role in shaping PSTVd quasispecies and speculated that analysis of sequence populations in symplastically isolated cells could provide insight into this question. Guard cells in developing epidermal tissues are connected to other cells and sister guard cells through plasmodesmata, enabling PSTVd infection. However, once mature, plasmodesmal connections to other cells and sister guard cells are abolished (Willmer and Sexton 1979), creating a symplastically isolated environment where PSTVd quasispecies can evolve. Loss of guard cell plasmodesmal connections was demonstrated in a previous study, which showed that PSTVd was trapped in mature tomato and tobacco (Nicotiana tabacum) guard cells after microinjection (Ding et al. 1997).
In the present study, in vitro transcripts of PSTVd-I (100 ng per plant) were rub-inoculated to the upper epidermis of 2 fully expanded cotyledons of 3 groups of 1-wk-old tomato (‘Rutger’) plants. Ten plants were included in each group. At 3 wk postinoculation, the second fully expanded compound leaf (from top to bottom) was collected from each plant. This leaf was selected because the epidermal tissues had matured for approximately 1 wk, providing an isolated environment for PSTVd to replicate multiple times and generate variants.
Isolating guard cell protoplasts from mature plants is challenging, and the purity is often low. The methods described by Yao et al. (2018) were used with minor modifications to enrich guard cell or mesophyll cell protoplasts (Fig. 3A, Supplementary Fig. S1, A to D). Guard cell protoplasts are much smaller than those derived from mesophyll cells that make up the vast majority of leaf cells and can easily be distinguished (Supplementary Fig. S1, B and C). Protoplast preparations were examined under a light microscope, and guard cell purity was judged to be greater than 85% in all 3 biological replicates, with mesophyll cell protoplasts and cell debris as the main contaminants. The expression of β-carbonic anhydrases (βCAs) such as βCA1, βCA2, and βCA3 is prominent in guard cells (Hu et al. 2010; Yao et al. 2018), whereas expression of a potassium channel POTASSIUM CHANNEL IN ARABIDOPSIS THALIANA 1 (KAT1) gene is mainly observed in mesophyll cells (Yao et al. 2018). To further validate the purity of enriched guard cell protoplasts, reverse transcription PCR (RT-PCR) was conducted to compare the expression of these markers in protoplast preparations enriched for guard cells or whole leaf protoplasts (mostly mesophyll cells) derived from the same compound leaf. This analysis showed that βCA1, βCA2, and βCA3 were expressed to high levels in guard cell protoplast preparations, while KAT1 was expressed at low levels (Fig. 3B), further confirming the enrichment of guard cell protoplasts.
Figure 3.
Enrichment and confirmation of tomato guard cell protoplasts and analysis of PSTVd quasispecies structure. A) Enrichment of guard cells from PSTVd-I-infected tomato plants. In vitro transcripts (100 ng) of PSTVd-I were rub-inoculated onto 30 1-wk-old tomato plants, which were randomly divided into 3 biological replicates, 10 plants in each. Three weeks later, tomato guard cell protoplasts were enriched from the second fully expanded compound leaf (from top to bottom). Guard cell protoplasts (red arrows) were photographed under a light microscope. Scale bar = 20 μm. B) Analysis of KAT1 and βCA gene expression in guard cell and whole leaf samples. The expression of KAT1, βCA1, βCA2, and βCA3 genes was analyzed by RT-PCR with actin as an endogenous control. C) Analysis of PSTVd-I progeny. Progeny were sequenced from guard cell and whole leaf protoplasts in the 3 biological replicates. Variant U178G/U179G, which is defective only for mesophyll cell entry (Wu et al. 2019), is labeled in red. D) Comparison of variant percentages in guard cell and whole leaf samples. Percentages of mutant progeny (variants) in all 3 biological replicates presented in C) were summed and compared using 2-tailed t test. Numbers of total sequenced (black) and mutant progeny identified (red) are indicated above the columns. Bars indicate the Se of the means of 3 biological replicates. Asterisks indicate significant difference (P < 0.01). E) RNA blot analysis was conducted to detect PSTVd in both guard cell and whole leaf protoplasts of 3 biological replicates (Rep), as described in Fig. 2A. F) The graph shows normalized RNA blot signals quantified using Quantity One software. Bars represent the Se of the means obtained from 3 biological replicates. n.s., not statistically significant.
To analyze PSTVd quasispecies in guard cell and whole leaf protoplasts, 12 or more PSTVd progeny in both types of samples from the 3 biological replicates were cloned and subjected to Sanger sequencing. Progeny sequences are listed in Fig. 3C. Overall, a markedly higher proportion of variants was detected in guard cell protoplasts as compared to in whole leaf samples in all 3 biological replicates (Fig. 3D). Variants comprised nearly a third of the sequenced clones from guard cell protoplasts (14/43; 32.5%), whereas variants accounted for less than 5% of clones from whole leaf protoplasts (2/45; 4.4%). To highlight a specific variant, U178G/U179G, which is defective only for movement from epidermal cells to mesophyll cells but competent to move in the reverse direction (from mesophyll cells to epidermal cells) and between all other cell types (Wu et al. 2019), was detected 3 times in 2 out of 3 replicates. But this variant was not detected in whole leaf samples used in this study or our previous studies (Fig. 3C;Wu et al. 2019, 2020; Wu and Bisaro 2020). Therefore, the U178G/U179G variant can be generated and accumulated in single cells, but cell-to-cell communication may exclude it from PSTVd quasispecies.
To exclude a possible influence of PSTVd RNA accumulation on variant content, RNA blot analysis was conducted to detect PSTVd in both guard cell and whole leaf protoplasts (Fig. 3E). Quantitative analysis indicated no significant differences in PSTVd levels between guard cell and whole leaf samples (Fig. 3F). Therefore, the increased abundance of variants observed in guard cells is not due to greater RNA accumulation.
PSTVd is replicated by host Pol II, which normally transcribes DNA into messenger RNA precursors. To test whether the fidelity of Pol II was altered in guard cells and/or during the preparation of guard cell protoplasts, PCR products of βCA2 and βCA3 were purified, cloned, and Sanger sequenced. A total of 8 clones of βCA2 (328 bp) and 2 clones of βCA3 (433 bp) were sequenced, and no mutations were detected (Supplementary Fig. S2), suggesting that the fidelity of Pol II was not significantly affected in guard cells during experiments.
The proportion of variants in quasispecies increases with increasing accumulation of PSTVd in cultured mesophyll cell protoplasts
While we demonstrated greater PSTVd variant accumulation in symplastically isolated guard cells, it is important to note that guard cells and mesophyll cells are distinct entities with unique cellular environments. The impact of these environments on viroid replication remains incompletely understood. Hence, analyzing variant content in isolated mesophyll cells is crucial. While transfected protoplasts might seem an ideal choice for this purpose, stressful transfection conditions could, for example, significantly affect the Pol II transcription machinery responsible for PSTVd replication. Further, in our experience, the original PSTVd sequence is rarely maintained in transfected protoplasts (Wu et al. 2020).
An approach was devised to obtain PSTVd-infected tomato mesophyll cell protoplasts without subjecting them to the stress of transfection. Briefly, tomato plants were inoculated with PSTVd-I. Subsequently, systemic leaves were collected at a very early stage of systemic infection (9 dpi) to prepare mesophyll cell protoplasts, which were cultivated in vitro to allow PSTVd-I to replicate and accumulate mutations. Sampling was conducted on the mesophyll cell protoplasts at 0, 8, 16, and 24 h after the initiation of cell culture for the analysis of PSTVd progeny sequences (Fig. 4A). The occurrence of mutations depends on PSTVd replication, which is reflected in increased PSTVd RNA accumulation. Since PSTVd replication can be influenced by various factors such as initial infection stage, culture conditions, and other unpredictable issues, a total of 9 biological replicates were included, with each replicate consisting of protoplasts from 1 tomato plant. Cell extracts were obtained at several time points, and RNA blots were performed to analyze PSTVd accumulation (Fig. 4B). Quantitative analysis revealed that only replicates 1, 2, and 4 exhibited more than a 10-fold increase in RNA accumulation over 24 h, suggesting the occurrence of PSTVd replication. In these replicates, increased RNA accumulation was evident from 0 to 8 h and from 8 to 16 h, and no further significant increase was observed from 16 to 24 h. This suggests that PSTVd reached its maximum accumulation level at 16 h, or that culture conditions did not permit further replication. Consequently, no further sampling was conducted (Fig. 4C). The morphology of cells in replicates 1, 2, and 4 was monitored throughout the entire experimental procedure, and no significant alterations were observed (Fig. 4D). PSTVd progeny sequences were analyzed by Sanger sequencing, including over 10 clones for each replicate at each time point. Interestingly, a significant increase in variant content was observed, from 5% to 10% to ∼25%, between 0 and 8h. A further increase from ∼25% to 35% occurred between 8 and 16 h, with no additional increase from 16 to 24 h. These increases in variant content corresponded consistently with increasing RNA accumulation (Fig. 4E, Supplementary Table S2). These data indicate that, unlike whole plant infections, greater PSTVd accumulation in isolated mesophyll protoplasts leads to increased variant levels, providing further evidence that cell-to-cell movement constrains the sequence diversity of PSTVd-I quasispecies.
Figure 4.
Analysis of PSTVd progeny in in vitro-cultivated mesophyll cell protoplasts. A) The diagram illustrates an experiment devised to obtain PSTVd-infected tomato mesophyll cell protoplasts without subjecting them to the stress of transfection. Initially, tomato plants were inoculated with PSTVd-I. Subsequently, systemic leaves were harvested at 9 dpi, representing a very early systemic infection stage, for the preparation of mesophyll cell protoplasts. These mesophyll cell protoplasts were cultured in vitro to promote PSTVd-I replication. Sampling was conducted at 0, 8, 16, and 24 h after initiating cell culture to analyze PSTVd progeny sequences. B) Nine biological replicates were included, each consisting of 1 tomato plant. RNA isolation and RNA blot were performed on these samples to analyze PSTVd accumulation (see Fig. 2A for details). C) Quantity One software was used to quantify RNA accumulation levels. D) The morphology of cells in replicates 1, 2, and 4 underwent continuous monitoring throughout the entire experimental procedure. Scale bar = 10 μm. E) PSTVd progeny sequences were examined using Sanger sequencing, with more than 10 clones included for each replicate at every time point. Bars represent the Se of the means derived from 3 biological replicates. Two-tailed t test was used for comparisons between 2 groups. n.s., not statistically significant, *P < 0.05, and **P < 0.01.
Tomato cells can be infected multiple times by PSTVd
Superinfection exclusion has been extensively investigated in virus studies (Perdoncini Carvalho et al. 2022), but its relevance to viroids remains a mystery, despite potential supporting evidence (Serra et al. 2023). To explore the possibility of multiple infections in a single plant cell, coinfection experiments involving PSTVd-I and mutants U177A/A182U and C181G, followed by single-cell sequencing, were conducted. We previously demonstrated that U177A/A182U and C181G exhibit infection rates similar to PSTVd-I (Wu et al. 2019). In this study, single infections with PSTVd-I, U177A/A182U, and C181G, as well as coinfections, were conducted in tomato plants to determine the time of earliest systemic infection. To outline the experimental procedure briefly, tomato plants were inoculated and the last fully expanded systemic leaves collected at 7, 9, 11, 13, 15, 20, 25, and 30 dpi, followed by RNA isolation and RNA blot to detect PSTVd. For each 2 × 2 coinfection experiment, PSTVd-I and/or mutant RNAs were inoculated onto separate leaves. The earliest systemic infection times for PSTVd-I, U177A/A182U, C181G, PSTVd-I + U177A/A182U, PSTVd-I + C181G, and U177A/A182U + C181G were 9, 11, 13, 9, 9, and 11 dpi, respectively (Fig. 5A). Therefore, varying periods are required for PSTVd-I, U177A/A182U, and C181G to reach systemic leaves. RNA blot signals were quantified to determine the time of the maximum accumulation of PSTVd RNA. In these 6 groups, the greatest accumulation was observed at 20, 25, 30, 20, 20, and 25 dpi, respectively (Fig. 5B). In the following experiment, coinfections were repeated, and systemic leaves were collected at 20 dpi (for PSTVd-I + U177A/A182U and PSTVd-I + C181G) and 25 dpi (for U177A/A182U + C181G) to prepare mesophyll cell protoplasts. These intervals, which correspond to the highest PSTVd accumulation levels, were chosen to maximize the number of infected cells. Isolated mesophyll cell protoplasts were then diluted multiple times to achieve a final cell density of approximately 200 cells/mL of cell culture medium, and micrococcal nuclease was added to digest extracellular nucleic acids. Subsequently, the diluted cells were dispensed onto a glass slide, with approximately 5 µL per drop, to maximize the likelihood of each drop containing only a single cell. Each droplet underwent a meticulous examination using a light microscope. Droplets containing only 1 cell were transferred to a new centrifuge tube, followed by RNA isolation using TRIzol reagent. Glycogen was added at a concentration of 0.05 μg/μL to facilitate RNA precipitation, followed by RT-PCR to detect PSTVd (Fig. 5C). A total of 9 cells were included in the RT-PCR analysis for each coinfection. During the preparation of single cells, the droplet without cells was also subjected to RNA isolation to serve as a mock (M) control. A different mock control was included for each coinfection experiment. The positive control was an RNA sample known to be positive for PSTVd. In the PSTVd-I + U177A/A182U, PSTVd-I + C181G, and U177A/A182U + C181G experiments, β-actin was detected in 5/9, 5/9, and 6/9 cells, respectively, suggesting successful RNA isolation and RT-PCR in most cases. PSTVd was detected in 4/9, 3/9, and 3/9 cells, and samples positive for PSTVd were always positive for β-actin, suggesting that not all cells were infected by PSTVd. Mock controls were consistently negative for both β-actin and PSTVd, indicating complete digestion of extracellular nucleic acids by micrococcal nuclease (Fig. 5D). Progeny sequencing was performed on cells positive for PSTVd using Sanger sequencing, and at least 5 clones were sequenced from each cell. In 9/10 PSTVd-positive cells, sequences of both coinoculated RNAs were detected. In 1 out of 3 cells of the PSTVd-I + C181G experiment only, PSTVd-I was detected (Fig. 5E). In addition, new variants were observed in 7/9 cells. While the possibility of simultaneous infection cannot be entirely ruled out, the distinct earliest systemic infection times and the observed coinfection in 9 out of 10 single cells included in this experiment strongly suggest that sequential infection is more likely.
Figure 5.
Single-cell analysis of PSTVd progeny following coinfection. A) To explore the possibility of multiple infections within a single plant cell, single inoculations with PSTVd-I, U177A/A182U, and C181G, as well as coinoculations involving 2 of these variants, were conducted to determine the time point of the earliest systemic infection in tomato plants. The last fully expanded systemic leaves were then collected at 7, 9, 11, 13, 15, 20, 25, and 30 dpi, followed by RNA isolation and RNA blot to detect PSTVd. In coinfection experiments, each mutant was inoculated onto separate leaves. Each plant was included for each time point of each coinfection experiment (see Fig. 2A for details). B) RNA blot data were quantified using Quantity One software. C) In a subsequent experiment, coinfections (PSTVd-I + U177A/A182U, PSTVd-I + C181G, and U177A/A182U + C181G) were repeated, and the last fully expanded systemic leaves were collected at 20 dpi (for PSTVd-I + U177A/A182U and PSTVd-I + C181G) and 25 dpi (for U177A/A182U + C181G) to prepare mesophyll cell protoplasts. These isolated cells were then diluted multiple times to reach a final density of about 200 cells per 1 mL of culture medium. Micrococcal nuclease was added for extracellular nucleic acid digestion. Subsequently, the diluted cells were dispensed onto a glass slide (about 5 µL per drop) to maximize the chances of containing only 1 cell per drop. All droplets were carefully checked under a light microscope. The droplets, which contained only 1 cell, were then transferred to a new centrifuge tube for subsequent RNA isolation and RT-PCR analysis to detect PSTVd. D) In every coinfection group, a total of 9 cells were included for RT-PCR detection of PSTVd. While preparing the individual cells, cell-free droplets were also subjected to RNA isolation to serve as a mock (M) control. A distinct M control was incorporated for each coinfection experiment. Subsequently, RT-PCR was conducted to detect PSTVd, using β-actin as an endogenous control. PSTVd-positive cells were labeled in red. E) Progeny sequencing was performed on PSTVd-positive cells using Sanger sequencing, and a minimum of 5 clones underwent sequencing. PSTVd-I, U177A/A182U, C181G, and new mutations were distinguished by color coding: black, red, blue, and purple, respectively.
PSTVd quasispecies contain replication-competent variants with higher fitness compared to the master sequence
Although master sequences appear most frequently in viral quasispecies, they are not necessarily the ones with the highest fitness (Biebricher and Eigen 2006; Domingo and Perales 2019). But whether this is true for viroids is unknown. To address this question, quasispecies originating from PSTVd-I in N. benthamiana plants were analyzed in inoculated local (l) leaves (mixed samples collected at 8 and 10 dpi) and in systemic (s) leaves (mixed samples collected at 14 and 21 dpi) through deep sequencing (Wu et al. 2020). The choice of N. benthamiana plants for subsequent experiments is due to the fact that PSTVd induces disease symptoms in tomatoes. In particular, during the later stages of infection, new leaves seldom develop, making it difficult to perform long-term coinfection experiments. In contrast, PSTVd infection in N. benthamiana plants is asymptomatic, rendering it a more suitable host for this experiment. Two biological replicates (a and b) were performed. Four libraries, including al (local leaves replicate a), as (systemic leaves replicate a), bl (local leaves replicate b), and bs (systemic leaves replicate b), were prepared and sequenced. Raw data processing, including cutoff values for mutation detection, procedures used to exclude sequencing artifacts, and other relevant methods, have been previously described (Wu et al. 2020). Briefly, unit-length cDNA was amplified by PCR using Phusion DNA polymerase, known for its ultralow error rate. Subsequently, the MiSeq platform (Illumina) was employed for paired-end sequencing. This technology enables the sequencing of more than 250 bp from each end, facilitating the generation of paired-end sequences that span the entire PSTVd genomes (359 nt) embedded within the original cDNAs. Mutations in unique sequences were mapped to the secondary structure of PSTVd (Supplementary Fig. S3, A to D). Interestingly, mutations from libraries al and as were almost uniformly distributed across the genome, while mutation hotspots were observed in libraries bl and bs (Supplementary Fig. S3, A to D). Thus, even though plants in biological replicates a and b were cultivated simultaneously under the same conditions, 2 distinct evolutionary patterns were observed. This suggests that the evolution of PSTVd-I quasispecies may be influenced by unpredictable and unknown factors, such as fitness of the initial variants, or differences in microenvironments.
To explore replication-competent variants in PSTVd quasispecies, unique sequences with single and double base substitutions were analyzed to identify prime variants. Only unique sequences with 1 or 2 base substitutions were included because sequences with multiple mutations are difficult to analyze and base insertions and/or deletions usually cause a lethal phenotype. If a mutation was detected both independently and in combination with other mutations in a unique sequence, it was designated as a prime mutation. The variant that only contained this prime mutation was then defined as a prime variant. These prime variants are considered capable of replication as their replication was likely necessary for the occurrence of secondary mutations. Prime mutations and their linked secondary mutations were identified in each library (Supplementary Table S3), and in Fig. 6A, they are presented in light blue rectangles. The number of secondary mutations linked to each prime mutation is indicated in light gray solid circles. However, it should be noted that double mutations may occur during the same replication. Prime mutations identified using this method possess a heightened likelihood of replication, although such replication is not guaranteed with certainty.
Figure 6.
Replication-competent variants in PSTVd quasispecies and analysis of their relative fitness. A) Replication-competent variants in PSTVd quasispecies derived from PSTVd-I. The quasispecies of PSTVd derived from PSTVd-I in N. benthamiana plants were analyzed by deep sequencing in inoculated local leaves (l) and in systemic leaves (s) in 2 biological replicates (a and b). Unique sequences in each of the 4 libraries (al, as, bl, and bs) were identified, and those with single or double base substitutions were selected to identify replication-competent variants. If 1 mutation was detected both independently and in conjunction with secondary mutation(s) in a unique sequence, the mutation was named a prime mutation and the variant carrying only this mutation was designated a prime variant. Prime variants identified from each library are presented in light blue rectangles, and the number of mutations that occurred simultaneously with each prime mutation is indicated in light gray circles. The number of prime variants identified from each library is indicated in parentheses. A total of 12 prime variants (indicated in red) were selected to test function through plant infection experiments, with 10 N. benthamiana plants for each mutant. Relative fitness compared to PSTVd-I (wild-type [WT]), as measured by RNA accumulation, of the 12 selected variants in local B) and systemic C) leaves was assessed. Local and systemic infections were analyzed at 10 and 28 dpi, respectively. The number of plants with positive PSTVd signals (out of 10 inoculated) is indicated above each column. Variants that retained original mutations without acquiring new ones are indicated by red arrows, while variants that failed to infect plants are indicated by solid red triangles. The relative fitness of each variant, as measured by RNA accumulation, was analyzed and compared to PSTVd-I using 2-tailed t test. Error bars indicate Se of the mean. *P < 0.05 and **P < 0.01.
The relative fitness of 12 prime variants, including U18G, A32C, U90C, C113U, A126C, A142C, A142G, A150C, U161C, U177A, U179C, and U191G (indicated in red letters and numbers in Fig. 6A), was tested to assess their replication competence. These were selected to represent prime variants with different numbers of secondary mutations, and their replication competence compared to PSTVd-I was evaluated in plant infection experiments. In vitro transcripts of each variant were generated and rub-inoculated onto the first 2 fully expanded true leaves (a true leaf is any leaf other than a cotyledon) of 2-wk-old N. benthamiana plants, with 10 plants per variant. Accumulation of circular PSTVd (PSTVd-C, the functional genomic form) was analyzed by RNA blot at 14 dpi for local, inoculated leaves (Supplementary Fig. S4) and 28 dpi for systemic leaves (Supplementary Fig. S5). Among the 12 variants tested, 6 (C113U, A126C, A142C, A142G, U161C, and U179C) were found to infect at least 5 out of 10 plants in both local and systemic leaves. These variants also retained the original mutations without acquiring additional mutations, indicating their replication competence (Table 1). The other 6 variants, namely U18G, A32C, U90C, A150C, U177A, and U191G, either failed to infect plants or reverted to the original PSTVd-I sequence and acquired new mutations.
Table 1.
Summary of local and systemic infection rates and progeny sequences of 12 prime variants
| Local replication | Systemic trafficking | |||
|---|---|---|---|---|
| Variants | Infection rate | Progeny sequences | Infection rate | Progeny sequences |
| U18G | 10/10 | 2 PSTVd-I; 1 U252C | 1/10 | 3 PSTVd-I |
| A32C | 0/10 | NA | 1/10 | 2 PSTVd-I |
| U90C | 0/10 | NA | 0/10 | NA |
| C113U | 10/10 | 2 C113U | 5/10 | 3 C113U |
| A126C | 10/10 | 1 A126C | 10/10 | 3 A126C |
| A142C | 10/10 | 2 A142C | 8/10 | 3 A142C |
| A142G | 10/10 | 2 A142G | 9/10 | 2 A142G |
| A150C | 0/10 | NA | 0/10 | NA |
| U161C | 10/10 | 2 U161C | 8/10 | 3 U161C |
| U177A | 10/10 | 1 PSTVd-I | 3/10 | 1 Δ99A; 1 PSTVd-I |
| U179C | 10/10 | 1 U179C | 10/10 | 3 U179C |
| U191G | 0/10 | NA | 2/10 | 2 PSTVd-I |
The in vitro transcripts of 12 selected prime variants were rub-inoculated onto the surface of the first 2 fully expanded true leaves of N. benthamiana plants, with 300 ng per plant and 10 plants per variant. Local and systemic infections were evaluated by RNA blot at 10 and 28 dpi, respectively, and the infection rate (number infected of 10 inoculated) is indicated. Tissue samples infected by each variant were mixed, and progeny were cloned by RT-PCR followed by Sanger sequencing. The number of sequenced clones is indicated, and original retained mutations are in bold.
NA, none available.
The RNA accumulation levels of each variant and PSTVd-I in both local (Fig. 6B) and systemic (Fig. 6C) leaves were measured and compared to that of PSTVd-I. The results showed that some independently infectious variants, such as U161C, may exhibit higher fitness (i.e. ability to replicate and/or spread) than PSTVd-I in both local and systemic leaves. These findings suggest that, under the conditions of this test, PSTVd quasispecies may harbor variants with superior fitness compared to the master sequence. Additional factors, such as genome interactions and cell-to-cell communication, could contribute to the maintenance of the dominant PSTVd-I master sequence. However, relative fitness analysis should be further confirmed by coinfection experiments using PSTVd-I and the related variants. Furthermore, viroid RNA accumulation may vary based on the timing of sampling. Therefore, monitoring viroid accumulation at multiple time points is also crucial for a comprehensive assessment of fitness.
Initial population composition affects the sequence population of PSTVd quasispecies
Intrapopulation interactions within viral quasispecies have been shown to play a role in shaping the evolution of viral quasispecies. Interactions can have several outcomes. Cooperative interactions among variants favor their replication, while competitive or interfering interactions can compromise population viability (Lázaro 2020). The presence of error thresholds suggests that the master sequence can help stabilize variant populations at or near the maximum mutation rate (Eigen and Schuster 1977; Swetina and Schuster 1982; Fornés et al. 2019). Upon violations of error thresholds caused by mutagenic agents, antiviral inhibitors, or polymerase mutations, the master sequence may be lost, leading to shifts in the sequence population and possibly extinction (Bull et al. 2007; Perales et al. 2011; Van Slyke et al. 2015). However, alterations in error rates are rare in nature. Another and perhaps more frequent cause of population shifts is coinfection of a host by multiple viral quasispecies with different master sequences. When this occurs, population shift could occur at the initiation of infection (Schröter et al. 2003). However, there is limited understanding regarding the influence of the initial viral sequence population on quasispecies structure, especially the generation and accumulation of novel variants.
Coinfection experiments were performed to test the role of initial sequence composition in the evolution of PSTVd quasispecies. Briefly, the first 2 fully expanded true leaves of 2-wk-old N. benthamiana plants were coinoculated with in vitro transcripts of PSTVd-I and the 12 prime variants, 264 ng per plant. Coinoculation was performed using 2 strategies. The first involved an inoculum containing 240 ng in vitro transcripts of PSTVd-I and 2 ng in vitro transcripts of each prime variant (24 ng in total) for each N. benthamiana plant to mimic the natural structure of a master sequence–derived PSTVd quasispecies, i.e. the master sequence occupies about 90% of the sequence population. For convenience, this coinfection strategy was named PSTVd-I(10)/Var(1). To mimic coinfection with multiple variants, each plant was inoculated with 24 ng in vitro transcripts of PSTVd-I and 20 ng of each of the 12 variants (240 ng in total). This coinfection strategy was named PSTVd-I(1)/Var(10). Experiments were performed with 3 plants included in each biological replicate, and 3 biological replicates were conducted for each strategy. Using mixed samples from 3 plants, local (inoculated leaf) infection was assessed at 5 and 10 dpi (L 5 dpi and L 10 dpi), and systemic infection was analyzed at 14, 60, and 100 dpi (S 14 dpi, S 60 dpi, and S 100 dpi). Inoculation and sampling strategies are illustrated in Fig. 7A. The circular form of PSTVd (PSTVd-C), the functional genomic form, was detected by RNA blot at each time point, with ribosomal RNA (rRNA) included as a loading control (Fig. 7B). RNA accumulation levels were quantified and compared between the 2 groups at each time point. The results showed that accumulation of PSTVd RNA was significantly higher in the PSTVd-I(10)/Var(1) group compared to the PSTVd-I(1)/Var(10) group at L 5 dpi and S 14 dpi. However, no significant differences were observed at other time points (Fig. 7C). So, presence of the PSTVd-I master sequence (PSTVd-I(10)/Var(1)) appeared to be an advantage during the initiation of local replication (L 5 dpi) and systemic trafficking (L 14 dpi). But this was later compensated in both local and systemic leaves, and initial sequence composition did not have a significant effect on overall PSTVd RNA accumulation in the long term.
Figure 7.
Analysis of PSTVd quasispecies established with and without a master sequence. A) Two strategies of coinoculation. In vitro transcripts of PSTVd-I and 12 selected prime variants were coinoculated onto the first 2 fully expanded true leaves of 2-wk-old N. benthamiana plants, 264 ng per plant. In the first strategy, each plant was inoculated with 240 ng PSTVd-I in vitro transcripts and 2 ng in vitro transcripts of each variant (24 ng in total) to mimic the structure of a PSTVd quasispecies derived from a master sequence in nature. This strategy was designated PSTVd-I(10)/Var(1). In the second strategy, each plant was inoculated with 24 ng PSTVd-I in vitro transcripts and 20 ng in vitro transcripts of each variant (240 ng in total) to mimic a coinfection of multiple variants. This strategy was named PSTVd-I(1)/Var(10). Three biological replicates were included for each strategy, with 3 plants for each replicate. Three biological replicates, each with 3 plants, were sampled repeatedly at each time point. Samples were collected from locally inoculated leaves (L) at 5 and 10 dpi and from the top 2 fully expanded systemic leaves (S) at 14, 60, and 100 dpi. B) Analysis of PSTVd RNA accumulation in collected samples (see Fig. 2A for details). C) Quantification of PSTVd RNA accumulation. RNA blot signals were quantified using Quantity One software and compared between PSTVd-I(10)/Var(1) and PSTVd-I(1)/Var(10) groups at each time point using two-tailed t test. Bars indicate the Se of the mean of 3 biological replicates. *P < 0.05 and **P < 0.01. D) Analysis of PSTVd quasispecies sequence populations. PSTVd progeny in collected samples were sequenced, and the percentages with additional mutation(s) were calculated and compared between the 2 groups at each time point using the χ2 test. Bars indicate the Se of the mean of 3 biological replicates. *P < 0.05 and **P < 0.01. E) Percentages of PSTVd-I progeny in PSTVd-I(10)/Var(1) group were calculated and presented. Bars indicate the Se of the mean of 3 biological replicates. F) Analysis of the origins (parents) of progeny with additional mutation(s). The numbers of sequenced parent clones (black) and mutant progeny clones (red) linked to each parent are indicated above the bars.
Nevertheless, significant effects were observed on the generation of new variants. A considerable number of progeny from samples collected at each time point were subjected to Sanger sequencing (Table 2, Supplementary Table S4), and the proportion that acquired novel mutations was determined for both groups (Fig. 7D). In the PSTVd-I(10)/Var(1) group, the proportion of variant progeny ranged from 4.76% (L 10 dpi) to 10.53% (L 5 dpi) of the entire sequence population. In the PSTVd-I (1)/Var(10) group, the proportion of variant progeny was much greater, with additional mutation(s) ranging from 25.00% (S 60 dpi) to 48.78% (S 14 dpi). A greater percentage of variants was observed in the PSTVd-I(1)/Var(10) group compared to the PSTVd-I(10)/Var(1) group at each time point, with differences achieving statistical significance at L 10 dpi, S 14 dpi, and S 100 dpi (Fig. 7D). Interestingly, in the PSTVd-I(1)/Var(10) group, new variants accumulated rapidly in inoculated leaves and reached a peak in systemically infected leaves at 14 dpi, after which the number of variants declined. This suggests that in the absence of a true master sequence (i.e. PSTVd-I was not dominant in terms of inoculum proportion), a surge of variants appeared in inoculated leaves and during the early stages of systemic infection, after which many were eliminated and the population stabilized over time.
Table 2.
Summary of progeny sequences in N. benthamiana plants with 2 coinfection strategies
| Time points | Groups | No. of clones | No. of original genomes | No. of novel variant(s) | Percent novel variant(s) |
|---|---|---|---|---|---|
| L 5 dpi | PSTVd-I(10)/Var(1) | 19 | 17 | 2 | 10.53 |
| PSTVd-I (1)/Var(10) | 23 | 17 | 6 | 26.09 | |
| L 10 dpi | PSTVd-I(10)/Var(1) | 21 | 20 | 1 | 4.76 |
| PSTVd-I (1)/Var(10) | 28 | 17 | 11 | 39.29 | |
| S 14 dpi | PSTVd-I(10)/Var(1) | 22 | 20 | 2 | 9.09 |
| PSTVd-I (1)/Var(10) | 41 | 21 | 20 | 48.78 | |
| S 60 dpi | PSTVd-I(10)/Var(1) | 28 | 26 | 2 | 7.14 |
| PSTVd-I (1)/Var(10) | 20 | 15 | 5 | 25.00 | |
| S 100 dpi | PSTVd-I(10)/Var(1) | 23 | 21 | 2 | 8.70 |
| PSTVd-I (1)/Var(10) | 24 | 16 | 8 | 33.33 |
Two inoculation strategies were used in coinfection experiments: PSTVd-I(10)/Var(1) and PSTVd-I (1)/Var(10) (see text). Each strategy was conducted in 3 biological replicates, and 3 plants were included in each biological replicate. Local infection (L) was evaluated at 5 and 10 dpi and systemic infection (S) at 14, 60, and 100 dpi. RT-PCR was performed to clone progeny. The number of clones sequenced, original genomes, and novel variants (genomes with additional mutation(s)) are indicated. The percentages of variants were calculated for each treatment.
The fate of the prime variants was also impacted by the sequence composition of the inoculum. Among the 12 prime variants, only A142G was detected once in the PSTVd-I(10)/Var(1) group at S 60 dpi, and none of the other 11 variants were detected (Supplementary Table S4). In PSTVd-I(1)/Var(10) group, C113U, A126C, A142G, and U161C, which can infect plants independently without acquiring additional mutations (Fig. 6, B and C), were detected. Another functional variant, A142C, was observed twice, but with the presence of additional mutations (Δ123A/A142C/A291G and Δ124A/A142C). Although U179C can also infect plants without acquiring additional mutations and its fitness is comparable to that of PSTVd-I, it was not detected. The other 6 prime variants, which failed to infect plants or maintain the introduced mutations in single-infection experiments, were also not detected (Supplementary Table S4). Thus, in the PSTVd-I(1)/Var(10) group, 4 of the 6 prime variants capable of independently infecting plants (C113U, A126C, A142G, and U161C) came to dominate the population over time.
Following the fate of the original PSTVd master sequence provides another useful perspective. In the PSTVd-I(10)/Var(1) group, PSTVd-I remained the dominant sequence under all conditions and time points (104/112 [93%] of total clones sequenced). In contrast, in the PSTVd-I(1)/Var(10) group, PSTVd-I was absent from progeny of replicate 3 and represented less than 20% (19/112) of total sequenced clones (Supplementary Table S4). However, it does not rule out the possibility that certain variant progeny with a single mutation in replicate 3 of the PSTVd-I(1)/Var(10) group originated from PSTVd-I. Its proportion among progeny was highest during early local replication (L 5 dpi) and systemic infection (S 14 dpi; Fig. 7E). This suggests that, in the context of quasispecies, PSTVd-I may be superior to other variants in the initiation of local infection, which requires replication and cell-to-cell movement, and initiation of systemic infection, which additionally requires long-distance trafficking. After the establishment of infection, other variants may outcompete PSTVd-I through their higher fitness, cooperation, and/or ability to more effectively interact with host factors involved in other infection processes.
Taken together, the results of this study confirm that PSTVd-I remains the dominant master sequence (∼90% of the sequence population) when it similarly dominates the inoculum. They further indicate that reducing the proportion of the PSTVd-I master sequence (to 10%) led to the accumulation of some prime variants included in the inoculum and enhanced the emergence of novel variants. At the same time, overall PSTVd RNA accumulation was not significantly impacted in the long term. Thus, initial sequence composition plays a significant role in shaping the evolution of the PSTVd sequence population.
Finally, the origins of mutant progeny were analyzed by inspecting the 12 prime variants (parents) and derived sequences. In cases where prime mutations were absent, PSTVd-I was considered as the parent. Progeny retaining the sequences of PSTVd-I and 12 prime variants without acquiring additional mutations were also counted. The ratios of parent clones to clones of mutant progeny are presented (Fig. 7F, Supplementary Table S5). In the PSTVd-I(10)/Var(1) group, a total of 104 PSTVd-I clones were obtained, while only 8 clones of variants derived from PSTVd-I were detected, further emphasizing the dominance of the master sequence. In the PSTVd-I(1)/Var(10) group, 19 PSTVd-I clones and 19 clones of variants derived from PSTVd-I were detected. For C113U, A126C, A142G, U161C, and A142C, the ratios of parent clones to mutated progeny clones were 14/8, 11/9, 25/5, 17/7, and 0/2, respectively. In all cases, the ratio of mutant progeny clones to their parent clones in the PSTVd-I(1)/Var(10) group was higher than that in the PSTVd-I(10)/Var(1) group. This analysis confirms that initial sequence composition enhances the accumulation of additional mutations derived from different variants in the same quasispecies. A possible explanation is that different variants may differently interact with host factors, such as replication factors and plasmodesmal factors to facilitate the generation and/or accumulation of certain variants. However, it is known that several prime variants can revert to PSTVd-I in plant infection assays (Table 1). Therefore, the number of progeny that originated from PSTVd-I in both groups is likely overestimated. Nevertheless, our findings suggest that coinfection with multiple variants could expedite the evolution of viroids, and potentially viruses, by facilitating the accumulation of new mutations within quasispecies.
Single cells were isolated from systemic leaves collected from the PSTVdI(1)/Var(10) group at 60 dpi in the exact same manner as detailed in Fig. 5C. The S 60 dpi point was chosen as it corresponds to the peak of viroid RNA accumulation (Fig. 7C). Nine cells were selected for analysis, but only 3 tested positive for PSTVd (Fig. 8A). Progeny sequences were subjected to Sanger sequencing, with 6 clones analyzed for each cell. Intriguingly, it was observed that sequencing only 6 clones could detect 3 to 5 of the original sequences, including PSTVd-I and 4 of the 12 variants (Fig. 8B), providing further evidence that plant cells can indeed be repeatedly infected by PSTVd.
Figure 8.
Single-cell sequencing analysis of PSTVd progeny from systemic leaves of N. benthamiana plants inoculated with PSTVd-I(1)/Var(10). A) Mesophyll cell protoplasts were prepared, and single cells were isolated as previously described in Fig. 5C. The presence of PSTVd in isolated cells was confirmed through RT-PCR with β-actin serving as an endogenous control. B) Progeny sequencing was carried out on PSTVd-positive cells using Sanger sequencing, with 6 clones sequenced for each cell. Newly identified mutations are highlighted in red.
To further investigate the influence of the master sequence presence or absence on the accumulation of novel variants within PSTVd quasispecies, 3 distinct coinfection experiments were conducted: the PSTVd-I group, involving the exclusive introduction of PSTVd-I without variants; the PSTVd-I(1)/Var(1) group, where PSTVd-I was mixed with its variants in a 1:1 ratio (132 ng PSTVd-I and 11 ng of each variant); and the Var group, employing only the variants, with 22 ng of each variant. These experiments were designed to further examine how the PSTVd-I master sequence affects the accumulation of novel variants within the quasispecies. Samples were collected at L 5 dpi and S 14 dpi, and progeny were subjected to Sanger sequencing analysis. At least 10 clones were sequenced for each sample. In the PSTVd-I group, the proportion of variants remained below 10% at both time points (∼6% to 9%). In contrast, the PSTVd-I(1)/Var(1) group exhibited a somewhat higher variant content, with new variants ranging from ∼16% at L 5 dpi to ∼22% at S 14 dpi. Original variants present in the inoculum that did not acquire additional mutations comprised ∼10% of the total at S 14 dpi. Nevertheless, PSTVd-I remained the dominant sequence, comprising more than 80% of the sequence population at L 5 dpi and ∼70% at S 14 dpi. Remarkably, the Var group exhibited a significantly increased new variant content, surpassing 25% at L 5 dpi and reaching 50% at S 14 dpi. Of the original 12 variants present in the inoculum, only 4 were detected without additional mutations. C113U and U161C each comprised ∼30% of the population at S 24 dpi, with A142G and A126C accounting for ∼25% and 15%, respectively (Fig. 9A, Supplementary Table S6).
Figure 9.
Analysis of PSTVd quasispecies established in N. benthamiana plants with PSTVd-I, PSTVd-I(1)/Var(1), and Var coinfection strategies. A) Analysis of sequence populations of PSTVd quasispecies (see Fig. 7D for details). Two-tailed t test was used for comparisons between 2 groups. Bars indicate the Se of the mean of 3 biological replicates. Compared with PSTVd-I group, *P < 0.05; compared with PSTVd-I group, **P < 0.01; compared with PSTVd-I(1)/Var(1) group, ##P < 0.01; compared with PSTVd-I(1)/Var(1) group, ###P < 0.001. B) Analysis of the origins (parents) of progeny with additional mutation(s) (see Fig. 7D for details).
As before (Fig. 6), an analysis of mutant progeny originating from the 12 prime variants was performed. In instances where multiple prime mutations were observed within the same sequence, the downstream mutation was arbitrarily identified as novel. For example, in the case of mutant A142G/U161C/Δ55A, A142G (where both A142G and U161C are prime variants) was recognized as the prime mutation, while U161C and Δ55A were classified as novel mutations (Fig. 9B, Supplementary Table S7). Over both time points (L 5 and S 14 dpi), a total of 64 PSTVd-I clones were obtained in the PSTVd-I inoculum group, with only 5 clones of variants derived from PSTVd-I detected. In the PSTVd-I(1)/Var(1) group, there were 62 PSTVd-I clones and 12 clones of variants derived from PSTVd-I. For variants present in the inoculum, including C113U, A142G, U161C, and A142C, the ratios of parent clones to mutated progeny clones were 2/4, 2/0, and 1/0, respectively. A126C was not detected in the PSTVd-I(1)/Var(1) group. Within the Var group, the ratios of parent clones to mutated progeny clones were 13/12, 6/4, 14/9, and 12/5 for C113U, A126C, A142G, and U161C, respectively. In most cases, higher ratios of mutant progeny clones to their parent clones were observed in the coinfection group, especially within the Var group. These findings provide further confirmation of the pivotal role played by the initial sequence composition in influencing the accumulation of novel variants within the PSTVd quasispecies.
Discussion
The present study analyzed the constraints on the intricate dynamics of quasispecies in viroids, utilizing PSTVd as a model. Quasispecies, defined as a population of diverse yet related sequences centered around a dominant sequence, have long been recognized as pivotal players in the evolution of RNA viruses and viroids. Employing a variety of approaches, our research demonstrates that the interconnected environment of plant cells plays a crucial role in constraining the sequence diversity within the PSTVd quasispecies. Furthermore, we reveal that a single cell can be infected multiple times by PSTVd, allowing multiple variants to coexist within the same cells. Intriguingly, our findings highlight that the initial sequence composition, specifically the presence of a dominant sequence, significantly influences the accumulation of novel variants.
Plasmodesmata are small channels that interconnect plant cells. These channels traverse cell walls and membranes, establishing a continuous network for the movement of various molecules. Plasmodesmata play a pivotal role in processes such as nutrient transport, developmental signaling, stress responses, and the spread of pathogens within a plant, including the cell-to-cell movement of viruses and PSTVd (Dolja et al. 2020; Petit et al. 2020). To our knowledge, no previous study has investigated virus or viroid quasispecies in symplastically isolated guard cells. Our findings show that there is a significantly greater accumulation of variants in PSTVd populations from guard cell protoplasts compared to whole leaf protoplasts. In addition to guard cells, we examined the accumulation of variants in isolated mesophyll cells with cellular environments distinct from guard cells. This analysis was carried out in conjunction with extended in vitro cell culture and the measurement of PSTVd RNA accumulation. Interestingly, we consistently observed an increased accumulation of variants during the replication of PSTVd in these isolated cells. These results support our hypothesis that cell-to-cell communication plays a critical role in excluding certain variants from PSTVd quasispecies. As plants undergo growth, the continuous development of new leaves results in the creation of numerous uninfected cells, providing fresh opportunities for viroid spread. Even within infected leaves, PSTVd is present in only a subset of cells, as we have previously observed by whole-mount in situ hybridization (Wu et al. 2019, 2020). Thus, variants with restricted movement capacity are selectively filtered out, whereas those with high movement capacity, likely representing the wild type, can infect a larger number of new cells, establishing dominance throughout the leaf or the entire plant. Another potential mechanism is that variants with higher replicative capacity might reach plasmodesmata earlier, displacing those that can more efficiently traverse these structures. Moreover, plasmodesmal transport is also necessary for movement of signaling molecules and, therefore, altered plant immunity in mature guard cells may also contribute to the increased accumulation of variants in PSTVd quasispecies (Brunkard and Zambryski 2017; Cheval et al. 2020). Since our experiments do not distinguish between these possibilities, we favor the term cell-to-cell communication. Our findings suggest that plasmodesmata may similarly regulate the cell-to-cell movement of endogenous RNAs, influencing gene expression and various other biological processes.
Superinfection exclusion, a phenomenon observed when an existing viral infection prevents a secondary infection by the same or closely related virus, has been well documented in various viral contexts (Perdoncini Carvalho et al. 2022). Coinfecting viroids have also been reported to interact with each other. For instance, mild strains of PSTVd, chrysanthemum chlorotic mottle viroid (CChMVd), and peach latent mosaic viroid (PLMVd) are recognized for their ability to suppress the infection caused by cognate severe strains. This interaction often leads to infected plants exhibiting only mild symptoms (Horst 1975; Desvignes 1976; Khoury et al. 1988; Duran-Vila and Semancik 1990; Semancik et al. 1992). However, its potential relevance to viroids at the single-cell level remains uncertain, even in light of supporting evidence (Serra et al. 2023). It is widely accepted that specific viral proteins mediate superinfection exclusion at the cellular level (Folimonova 2012; Perdoncini Carvalho et al. 2022). For instance, the superinfection exclusion of citrus tristeza virus (CTV) between isolates of the same strain is contingent upon the production of a particular viral protein, the p33 protein. The absence of a functional p33 protein entirely eliminates the virus's ability to prevent superinfection by the same or a closely related virus (Folimonova 2012). However, viroids do not encode proteins, and as a result, this phenomenon was not observed in the case of PSTVd infection in the present study. Consequently, variants generated during PSTVd infection can coexist in the same cell. Although superinfection exclusion is typically observed in experiments involving sequential infections, the present study utilized PSTVd-I and variants with different times of earliest systemic infection, resembling a scenario of sequential infection in systemic leaves. However, while PSTVd-I reached systemic leaves more rapidly in single infections than either of variants tested, we cannot rule out the possibility that coinfection might impact the timing of systemic spread.
It is also worth noting that the process of superinfection exclusion may differ for viroids of the Avsunviroidae and Pospiviroidae families. Members of the Pospiviroidae replicate and localize in the nucleus, causing nonspecific and systemic symptoms. In contrast, viroids of the Avsunviroidae induce local and specific symptoms through the generation of viroid-derived small RNAs (vd-siRNAs; Flores et al. 2020; Navarro et al. 2021; Serra et al. 2023). Therefore, conclusions regarding superinfection exclusion drawn from PSTVd cannot be generalized to all viroids. For instance, in the case of CChMVd, a chloroplastic viroid from the Avsunviroidae family, segregation of symptomatic and nonsymptomatic variants was observed in infected chrysanthemum (Dendranthema × grandiflorum) plants. Thus, variant populations of CChMVd with varying pathogenicity demonstrated the ability to colonize different leaf sections and exclude other variants from those sections (Serra et al. 2023). Future studies are needed to explore the distinct mechanisms that mediate superinfection exclusion in viroids belonging to these 2 families. Moreover, it has been documented that the occurrence of multiple infections by viruses, whether closely or distantly related, is constrained within a short time frame (Beperet et al. 2014). The applicability of this temporal limitation to PSTVd and other viroids is yet to be investigated.
In coinfection experiments utilizing variants identified from PSTVd quasispecies, we demonstrated that the presence of a master sequence is critical for maintaining a stable sequence structure. When a master sequence is present in the PSTVd quasispecies, it can more easily dominate most cells compared to the variants derived from it. This dominance leads to a stable sequence structure. However, in the absence of a master sequence, variants have the opportunity to interact with cellular machinery, potentially facilitating the emergence and movement of novel variants. As a result, novel variants accumulate. Viroids are simpler than viruses and do not encode proteins, suggesting that they may have different fitness constraints and evolve differently. Additionally, viroids behave differently than viruses because they must rely entirely on the host cell machinery for replication, spread, and transmission, while virus movement and replication are largely accomplished by viral proteins. Thus, functional viral proteins can support nonfunctional variants in trans for replication, cell-to-cell movement, and long-distance trafficking. However, emerging evidence has shown that cis-acting RNA sequence and structure elements, which cannot be complemented by functional sister genomes, also play critical roles in virus movement and packaging of genomic RNA into virions (Turner et al. 1988; Chen et al. 2017, 2020). Plasmodesmal gates may also interact with viral RNA structure elements to shape quasispecies structure. Thus, our study is likely relevant to the evolution of not only viroid but also plant virus quasispecies. By coinfecting with other variants of the same virus, quasispecies evolution may be manipulated as a complementary approach to improve the outcomes of antiviral approaches. However, before this can be realized, further research on the effects of cell-to-cell movement and initial sequence population on viroid and viral quasispecies structure is needed.
In conclusion, our study effectively demonstrates that cell-to-cell communication mediated by plasmodesmata and the initial sequence composition of PSTVd quasispecies are important constraints on the PSTVd sequence population. These 2 constraints function together with other factors, such as biased replication and degradation, RNA structure, error thresholds, immune escape, and fitness landscape, to maintain a stable structure of PSTVd quasispecies. This study provides insights into the role of plasmodesmata in regulating RNA movement and its impact on the evolution of viroids.
Materials and methods
PSTVd variants and in vitro transcription
PSTVd-I and variants were created by site-directed mutagenesis using pRZ6-2 plasmid (containing T7 promoter) expressing PSTVd-I cDNA as a template (Owens et al. 1995). The plasmid was a gift from Dr. Robert Owens. The plasmid was linearized using Hind III (Cat # R3104, NEB) and used as a template for in vitro transcription with T7 MEGAscript Kit (Cat # AM1334, Thermo Fisher Scientific) to produce in vitro transcripts of both PSTVd-I and variants. DNase I digestion was used to remove template DNA, and RNA purification was performed using MEGAclear Kit (Cat # AM1908, Thermo Fisher Scientific).
To prepare antisense PSTVd-I riboprobes labeled with digoxigenin (DIG), pInter(-) plasmid expressing antisense PSTVd-I was linearized using SpeI (Cat # R3133, NEB; Qi and Ding 2002). DIG RNA Labeling Mix (Cat # 11745832910, Roche) was then used to prepare in vitro transcripts of antisense PSTVd-I riboprobes. After template digestion with DNase I, riboprobes were purified using Sephadex G-25 columns (Cat # 17003201, GE Healthcare Life Sciences).
Plants, inoculation, protoplasts, and sampling
Tomato (S. lycopersicum ‘Rutger’) and N. benthamiana plants were grown in the growth chamber of Ningbo University or the Biotechnology Support Facility at The Ohio State University (14/10-h light/dark cycle, 28 °C, 5,000 lx [Lumilux Cool White lm]). In all cases, inoculation was performed on the 2 dusted cotyledons of 1-wk-old tomato plants or the 2 true leaves of the 2-wk-old N. benthamiana plants.
To investigate whether the proportion of variants within the PSTVd-I quasispecies is associated with infection duration, systemic leaves were collected from tomato plants infected with PSTVd-I (100 ng in vitro transcripts per plant) at 9, 10, and 11 dpi, representing very early stages of infection. Each time point comprised 3 biological replicates, with one plant per replicate, resulting in a total of 3 plants per time point.
To prepare tomato guard cell and mesophyll cell protoplasts for comparison of variant content between guard cells and whole leaves, the second fully expanded compound leaf was collected from 10 PSTVd-I-infected plants of each of the 3 biological replicates at 3 weeks post-inoculation (wpi). Each plant was inoculated with 100 ng in vitro transcripts. Then the methods described by Yao et al. (2018) were followed to isolate guard cell and mesophyll cell protoplasts with minor modifications. The main differences were as follows: cellulose “Onozuka” R-10 (Yakult Pharmaceutical Industry Co., Ltd) instead of “Onozuka” RS was used in enzyme solution 2 digestion, and digestion time was extended from 3.5 to 5 h to release guard cell protoplasts from epidermal tissues. Enriched protoplasts were checked under a light microscope to analyze cell purity.
For the examination of PSTVd progeny within in vitro-cultivated mesophyll cell protoplasts, tomato plants were initially inoculated with PSTVd-I (100 ng in vitro transcripts per plant). Subsequently, the last fully expanded systemic leaves were collected at 9 dpi, corresponding to a very early stage of systemic infection, for the isolation of mesophyll cell protoplasts. These protoplasts were cultured in vitro in WI buffer (0.5 M mannitol, 20 mM KCl, and 20 mM MES, pH 5.7) to facilitate PSTVd-I replication. Sampling occurred at 0, 8, 16, and 24 h post initiation of cell culture to examine PSTVd progeny sequences. Nine biological replicates were incorporated, with each replicate comprising protoplasts from an individual tomato plant.
To conduct coinfections of PSTVd-I + U177A/A182U, PSTVd-I + C181G, and U177A/A182U + C181G, each of the 2 cotyledons of tomato plants was inoculated with 50 ng in vitro transcripts of either PSTVd or the respective mutant. The last fully expanded systemic leaves were collected at 7, 9, 11, 13, 15, 20, 25, and 30 dpi. Each time point in each coinfection experiment included 1 plant. To obtain individual cells for progeny analysis, the last fully expanded systemic leaves were harvested from a single tomato plant at 20 dpi for PSTVd-I + U177A/A182U and PSTVd-I + C181G and at 25 dpi for U177A/A182U + C181G, in order to prepare mesophyll cell protoplasts. The isolation of single cells after coinfection with PSTVd(1)/Var(10) was performed at 60 dpi. These isolated mesophyll cell protoplasts were subsequently subjected to multiple dilution steps until a final cell density of approximately 200 cells per 1 mL of cell culture medium was achieved. To remove extracellular nucleic acids, micrococcal nuclease (Beyotime, Shanghai, China) was added at a concentration of 100 units/μL. The diluted cell suspensions were then carefully dispensed onto glass slides, with approximately 5 µL per droplet, to maximize the probability of each droplet containing only a single cell. Each droplet was meticulously examined under a light microscope, and droplets containing only 1 cell were transferred to new centrifuge tubes.
To perform infection analysis of the 12 prime mutants and the following coinfection assays, in vitro transcripts of PSTVd-I and its variants were inoculated onto the surfaces of the first 2 true leaves of N. benthamiana plants that had been dusted with carborundum. In single-infection experiments, each N. benthamiana was inoculated with 300 ng in vitro transcripts. In coinfection experiments, 5 different inoculation strategies were used. In the first strategy, an inoculum containing 240 ng of PSTVd-I in vitro transcripts and 2 ng of in vitro transcripts of each variant (24 ng in total) was used for inoculation. This strategy was named PSTVd-I(10)/Var(1). In contrast, the PSTVd-I(1)/Var(10) strategy involved an inoculum containing 24 ng of PSTVd-I in vitro transcripts and 20 ng of in vitro transcripts of each variant (240 ng in total) for each plant. Similarly, the PSTVd-I strategy utilized an inoculum comprising 264 ng of PSTVd-I in vitro transcripts. In contrast, the Var strategy employed an inoculum consisting of 22 ng of in vitro transcripts for each variant (a total of 264 ng) per plant. For the PSTVd-I(1)/Var(1) strategy, the inoculum consisted of 132 ng of PSTVd-I in vitro transcripts and 11 ng of in vitro transcripts for each variant (a total of 132 ng) per plant. Three biological replicates, each with 3 plants, were sampled repeatedly at each time point.
RNA preparation and RNA blot
TRIzol Reagent (Thermo Fisher Scientific) was used to prepare RNA samples. To determine the earliest infection time after single infections with PSTVd-I, U177A/A182U, and C181G, as well as coinfections involving 2 of these variants, the last fully expanded systemic leaves were collected at 7, 9, 11, 13, 15, 20, 25, and 30 dpi. PSTVd infection was detected using RNA blot following the methods described by Zhong et al. (2006). Quantity One software (BioRad) was used to quantify signals.
Deep sequencing PSTVd quasispecies derived from PSTVd-I master sequence
The method of tissue collection for deep sequencing has been described previously (Wu et al. 2020). Briefly, local (l) leaves were collected at 8 and 10 dpi to prepare pooled samples. Pooled systemic samples were also prepared using systemic (s) leaves collected at 14 and 21 dpi. Two biological replicates a and b were included. Unique sequences were isolated from al (local leaves replicate a), as (systemic leaves replicate a), bl (local leaves replicate b), and bs (systemic leaves replicate b) libraries. Mutations of unique sequences were identified through comparisons to PSTVd-I sequence. Mutations were mapped to the secondary structure of PSTVd-I. To identify replication-competent prime variants, unique sequences with 1 or 2 base substitutions were used because sequences with multiple mutations are hard to analyze and base insertions and/or deletions are usually lethal. A mutation was considered to be a replication-competent prime mutation if it was detected as a standalone mutation and also detected simultaneously with other mutations, which were considered as secondary mutations. The rationale behind this criterion is that the occurrence of secondary mutations necessitates the replication of prime mutants. The variant that carries the identified prime mutation was designated as the prime variant.
RT-PCR and PSTVd progeny sequencing
RNA samples were isolated and used as templates for cDNA synthesis through RT using the SuperScript Reverse Transcriptase IV kit (Thermo Fisher Scientific). Primers used in RTs were PSTVd-R (5′-TGAAGCGCTCCTCCGAGCC-3′) for PSTVd progeny sequencing and oligo(dT) for RT-PCR analysis of the expression of tomato KAT1, βCA1, βCA2, and βCA3 genes. PCR reactions were conducted using Phusion High-Fidelity DNA Polymerase (NEB). Sequences of primers used in PCRs were as follows: 5′GGATCCCCGGGGAAACC-3′ (forward) and PSTVd-R (reverse) for PSTVd; 5′-GGAATCAGTTGCCTCCAAGA-3′ (forward) and 5′-GCTGTGGTCTCCCACATAAA-3′ (reverse) for KAT1; 5′-CCTCTTTCTCCCTTAGCTTCATC-3′ (forward) and 5′-GTGGACCCATCATCAGGAATAG-3′ (reverse) for βCA1; 5′-CAGTGCTTGTGGAGGTATCAA-3′ (forward) and 5′-TACGGAAAGAGGAGGAGAAAGA-3′ (reverse) for βCA2; 5′-TTGTTTCCCTCCAGAACCTTATC-3′ (forward) and 5′-GCCTTGATACCTCCACAACTAC-3′ (reverse) for βCA3; and 5′-ACCCTGTTCTCCTGACTG-3′ (forward) and 5′-GCTCCTAGCGGTTTCAAGT-3′ (reverse) for actin. PCR products were subjected to 1% agarose gel electrophoresis. After purification, progeny sequencing was performed using TOPO TA Cloning Kit (Thermo Fisher Scientific).
Statistical analysis
Comparisons of 2 groups were performed using 2-tailed t test. Means were considered significantly different based on a threshold values of P < 0.05, P < 0.01, and P < 0.001, as indicated by *, ** (or ##), and *** (or ###), respectively. Details are shown in Supplementary Data Set 1.
Accession numbers
The original deep sequencing data for this study are available on the NCBI website under the BioProject accession PRJNA1050043.
Supplementary Material
Contributor Information
Jian Wu, State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agroproducts, Institute of Plant Virology, Ningbo University, Ningbo 315211, China; Key Laboratory of Biotechnology in Plant Protection of MARA and Zhejiang Province, Institute of Plant Virology, Ningbo University, Ningbo 315211, China; Department of Molecular Genetics, Center for Applied Plant Sciences, Center for RNA Biology, and Infectious Diseases Institute, The Ohio State University, Columbus, OH 43210, USA.
David M Bisaro, Department of Molecular Genetics, Center for Applied Plant Sciences, Center for RNA Biology, and Infectious Diseases Institute, The Ohio State University, Columbus, OH 43210, USA.
Author contributions
J.W. and D.M.B. conceived the project. J.W. designed the studies and experiments. J.W. performed the experiments. J.W. and D.M.B. analyzed the data and wrote the manuscript.
Supplementary data
The following materials are available in the online version of this article.
Supplementary Figure S1. Preparation of tomato guard cell protoplasts.
Supplementary Figure S2. Sequence alignment of βCA2 and βCA3 partial gene clones.
Supplementary Figure S3. Distribution of unique sequences across the PSTVd genome.
Supplementary Figure S4. Accumulation of PSTVd-I and 12 selected prime mutants in local leaves of infected N. benthamiana plants.
Supplementary Figure S5. Accumulation of PSTVd-I and 12 selected prime variants in systemic leaves of infected N. benthamiana plants.
Supplementary Table S1. Progeny sequences from systemic leaves of PSTVd-I-infected tomato plants at 9, 10, and 11 dpi.
Supplementary Table S2. Progeny sequences from in vitro-cultivated tomato mesophyll cell protoplasts.
Supplementary Table S3. Prime mutations and their linked secondary mutations identified from al, as, bl, and bs libraries.
Supplementary Table S4. Progeny sequences from N. benthamiana plants with PSTVd-I(10)/Var(1) and PSTVd-I(1)/Var(10) coinfection strategies.
Supplementary Table S5. Origins of progeny with additional mutations from N. benthamiana plants with PSTVd-I(10)/Var(1) and PSTVd-I(1)/Var(10) coinfection strategies.
Supplementary Table S6. Progeny sequences from N. benthamiana plants with PSTVd-I, PSTVd-I(1)/Var(1), and Var coinfection strategies.
Supplementary Table S7. Origins of progeny with additional mutations identified from N. benthamiana plants with PSTVd-I, PSTVd-I(1)/Var(1), and Var coinfection strategies.
Supplementary Data Set 1. Details of statistical analysis.
Funding
This work was supported by a grant from the National Natural Science Foundation of China to J.W. (32272483).
Data availability
The raw deep sequencing data for this study is available on the NCBI website under the BioProject accession PRJNA1050043.
Dive Curated Terms
The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The raw deep sequencing data for this study is available on the NCBI website under the BioProject accession PRJNA1050043.