Trade-off between local replication and long-distance dissemination during experimental evolution of a satellite RNA

Abstract

Satellite RNAs (satRNAs) are molecular parasites that depend on their non-homologous helper viruses (HVs) for essential biological functions. While there are multiple molecular and phylogenetic studies on satRNAs, there is no experimental evolution study on how satRNAs may evolve in common infection conditions. In this study, we serially passaged the Bamboo mosaic virus (BaMV) associated-satRNA (satBaMV) under conditions in which satBaMV either coinfects an uninfected host plant, Nicotiana benthamiana, with BaMV or superinfects a transgenic N. benthamiana expressing the full-length BaMV genome. Single-nucleotide polymorphisms (SNPs) of satBaMV populations were analyzed by deep sequencing. Forty-eight SNPs were identified across four different experimental treatments. Most SNPs are treatment-specific, and some are also ephemeral. However, mutations at positions 30, 34, 63, and 82, all located at the 5' untranslated region (UTR), are universal in all treatments. These universal SNPs are configured into several haplotypes and follow different population dynamics. We constructed isogenic satBaMV strains only differing at positions 30 and 82 and conducted competition experiments in protoplasts and host plants. We found that the haplotype that reached high frequency in protoplasts and inoculation leaves also exhibited poor dissemination to systemic leaves and vice versa, thus suggesting an apparent trade-off between local replication and long-distance dissemination. We posit that the trade-off is likely caused by antagonistic pleiotropy at the 5' UTR. Our findings revealed a hitherto under-explored connection between satRNA genome replication and movement within a host plant. The significance of such a connection during satRNA evolution warrants a more thorough investigation.

Keywords: antagonistic pleiotropy; bamboo mosaic virus (BaMV); experimental evolution; satBaMV; satellite RNA (satRNA); serial passage.

Figure 1

Experimental design for serial passage experiments. N. benthamiana plants were first infected by agroinfiltration. The subsequent passages were conducted by mechanical inoculation of tissue sap rubbed onto new plants. Sampling and passaging were performed in the inoculation leaf (IL) at 10 days interval and the sixth systemic leaves (SL) at 30 days interval. Ten passages were conducted at the indicated time points (DPA, days post agroinfiltration; DPI, days post sap inoculation). Passages for (A) coinfection and (B) superinfection conditions are shown. The satBaMV populations were analyzed through Illumina deep-sequencing. Star symbols indicate the ILs of agroinfiltration or sap inoculation; green leaves the sampled ILs; grey leaves the sampled SLs.

Figure 2

Accumulation of BSF4 satBaMV in BaMV-transgenic (Line 27–17) and wild type (WT) N. benthamiana plants. (A) Phenotypes in whole plants (a and c) and fully expanded leaves (b and d) of 22-day-old Line 27–17 (a and b) and WT (c and d) plants. (B) The similar symptoms of Line 27–17 and BaMV-infected WT plants. The 22-day-old WT plants were agroinfiltrated with Agrobacterium carrying pKB. BaMV symptoms at 14 days post agroinfiltration (DPA) are shown. The photographs were taken for whole plants (a and c) and systemic leaves (b and d), respectively. (C) Accumulation of BaMV and BSF4 in WT and transgenic line 27–17 plants. The WT and transgenic plants were agroinfiltrated, as described previously. The BaMV and satBaMV accumulated in the inoculation leaves (IL) were assayed at 10 DPA by RNA blots with DIG-labeled probes against the BaMV CP gene or satBaMV genome, respectively. “▬”, non-infiltrated mock plants; Kn, Agrobacterium containing expression vector pKn as control; BSF4, Agrobacterium containing satBaMV infectious cDNA clone, pKF4; BaMV, Agrobacterium containing BaMV infectious cDNA clone, pKB. Horizontal bracket symbols indicate two independent results. Different treatments were also indicated by the labeled number 1–9 below the RNA panels. Bars in (A) and (B) equal 2 cm.

Figure 3

Changes of SNP frequencies in the satBaMV 5′ UTR. SNPs at four notable polymorphic sites (positions 30, 34, 63, and 82) in the 5′ UTR were plotted for the Co-IL, Co-SL, Su-IL, and Su-SL SPEs. Only frequencies ≥0.01 are identified as SNPs. This figure is a graphical representation of part of the data in the Supplementary Table S2. Frequencies lower than 0.01 were plotted for graphing purpose.

Figure 4

Summary distribution of satBaMV SNPs among serial passage experiments. Only mutant frequencies ≥0.01 are identified as single-nucleotide polymorphisms (SNPs). Each of the 48 SNPs is placed in the serial passage experiment from which it was found. Black font indicates SNPs at the 5′ UTR, blue the P20 gene, and red the 3′ UTR. Boldface font indicates mutation resulting in amino acid replacement. See Supplementary Table S2 for details.

Figure 5

Summary distribution of satBaMV 5′ UTR haplotypes among serial passage experiments. Only haplotype frequencies ≥0.01 are shown. Each of the 15 mutant haplotypes is placed in the serial passage experiment from which it was found. See Supplementary Table S3 for details.

Figure 6

Population dynamics of satBaMV 5′ UTR haplotypes among serial passage experiments. Haplotype frequency is plotted against the passage number. Blue curves show the haplotypes with a decreasing trend during the passages, dashed red curves show the rise-and-fall pattern, and black curves show an increasing trend (see text for details). Thick blue curves show the frequencies of the ancestral BSF4, and blue symbols indicate the passage numbers sampled. Although Figure 3 only listed the haplotypes with frequencies ≥0.01, this figure shows the frequencies <0.01 to demonstrate the changes in haplotype frequencies.

Figure 7

Competition of isogenic 5′ UTR haplotypes. (A) The secondary structures of 5′ UTR of satBaMV (Chen et al., 2012). The significant mutation sites after passaging and the substituted nucleotides are labeled in red. (B) Competition of satBaMV haplotypes in N. benthamiana protoplasts. An equal ratio of the listed five haplotypes was co-transfected with pBaORF1, which provides BaMV’s RdRp for genome replication. The satBaMV progenies were sampled 48 h after transfection. (C) Competition of satBaMV haplotypes in inoculation leaf (IL) and systemic leaf (SL). The listed five satBaMV haplotypes were co-inoculated with BaMV to N. benthamiana plants through agro-infiltration. Plant tissues were collected at 10 DPA for IL and 30 DPA for SL. For all competition experiments, the frequency of each satBaMV haplotype was enumerated by cloning and sequencing (see main text). Error bars indicate the 95% confidence intervals of the frequency estimates. The blue horizontal line at 0.2 shows the expected frequency when no competitive difference exists.