A putative antiviral role of plant cytidine deaminases

Transient overexpression of AtCDAs in Nicotiana bigelovii plants infected with CaMV

AtCDAs cDNAs were cloned under the 35S promoter in a pBIN61 vector³⁹. N. bigelovii plants were inoculated with CaMV virions purified from Brassica rapa plants⁴⁰ previously infected with the clone pCaMVW260⁴¹. Symptomatic leafs were agroinfiltrated³⁹ with one of the nine AtCDAs and with the empty vector pBIN61, each on one half of the leaf. Samples were collected three days post-agroinfiltration.

Inducible co-suppression of multiple AtCDAs by RNAi

The design and cloning of the artificial micro-RNA (amiR) able to simultaneously suppress the expression of AtCDAs 1, 2, 3, 4, 7, and 8 was performed as described in ref. 42. The amiRNA was cloned under the control of Aspergillus nidulans ethanol regulon^43,44 and used to transform A. thaliana by the floral dip method⁴⁵. By doing so, we obtained the transgenic line amiR1-6-3. One-month-old seedlings of transgenic and wild-type A. thaliana were treated with 2% ethanol (or water for the control groups) three times every four days. Three days after the third treatment, plants were inoculated with the infectious clone pCaMVW260 as described in ref. 41. Infections were established by applying 1.31×10¹¹ molecules of pCaMVW260 to each of three leaves per plant. Subsequently, plants were subjected to two additional treatments with 2% ethanol (or water) one and five days post-infection. Finally, samples were taken eight days after inoculation and handled as previously described⁴⁶. For each genotype (transgenic or wild-type) and treatment (ethanol or water) combination, 22 plants were analyzed.

Detection of A/T enriched genomes

CaMV genomic DNA was purified using DNeasy Plant Mini Kit (Qiagen) according to manufacturer’s instructions. For detection of edited genomes 3D-PCR was performed using primers HCa8Fdeg and HCa8Rdeg. PCRs were performed in a Mastercycler^® (Eppendorf) at denaturation temperatures 82.1 °C, 82.9 °C, 83.9 °C, and 85.0 °C. PCR products obtained with the lowest denaturation temperature were cloned in pUC19 vector (Fermentas), transformed in Escherichia coli DH5α and sent to GenoScreen (Lille, France) for sequencing.

RT-qPCR analysis of AtCDA1 mRNA and qPCR analysis of CaMV load in transgenic plants

Total RNA was extracted from A. thaliana plants using the RNeasy^® Plant Mini Kit (Qiagen), according to manufacturer’s instructions. AtCDA1 specific primers qCDA1-F and qCDA1-R were designed using Primer Express software (Applied Biosystems). RT-qPCR reactions were performed using the One Step SYBR PrimeScript RT-PCR Kit II (Takara). Amplification, data acquisition and analysis were carried out using an Applied Biosystems Prism 7500 sequence detection system. All quantifications were performed using the standard curve method. To quantify AtCDA1 mRNA, a full-ORF runoff transcript was synthetized with T7 RNA polymerase (Roche) using as template a PCR product obtained from cloned AtCDA1 and primers T7-CDA1F and qCDA1-R. CaMV qPCR quantitation was performed as described in ref. 46.

Primers

All primers used are listed in Supplementary Table S3.

Effect of AtCDAs overexpression on CaMV mutational spectrum

To test the mutagenic activity of A. thaliana CDAs, nine N. bigelovii plants were inoculated with CaMV. After systemic infection was established, we performed transient AtCDA overexpression experiments. To do so, the same leaf was agroinfiltrated twice; one half of the leaf was infiltrated with one of the nine AtCDA genes and the other half of the leaf was infiltrated with the empty vector. This test was done for all nine AtCDA genes in different plants. The presence of AtCDA mRNAs was verified by RT-PCR from DNase-treated RNA extracts. DNA was extracted from agroinfiltrated areas for 3D-PCR amplification of a 229 bp fragment in the ORF VII of CaMV. 3D-PCR uses a gradient of low denaturation temperatures during PCR to identify the lowest one, which potentially allows differential amplification of A/T rich hypermutated genomes⁴⁷. There were no differences in the lowest denaturation temperature that could result in differential amplification of controls and the AtCDA-agroinfiltrated samples, suggesting that hypermutated genomes should be at low frequency, if present at all.

PCR products obtained at the lowest denaturation temperature were cloned and sequenced. In a preliminary experiment, we sequenced 25 clones from each AtCDA/negative control pair (Supplementary Table S1). At least one G to A transition was detected in clones from areas infiltrated with AtCDA1, AtCDA2 and AtCDA9 genes. For these three genes, we further increased the number of sequenced clones up to 106. The CaMV mutant spectra was significantly different between plants overexpressing AtCDA1 and their respective negative controls (Figure 1a: χ² = 25.760, 7 d.f., P = 0.001). This difference was entirely driven by the 471.43% increase in G to A transitions observed in the plants overexpressing AtCDA1. A thorough inspection of alignments showed that most of the G to A mutations (65.6%) detected in the different samples were located at the nucleotide position 181 (Supplementary Table S1). By contrast, no overall difference existed between the mutant spectra of CaMV populations replicating in plants overexpressing AtCDA2 (Figure 1b: χ² = 8.944, 6 d.f., P = 0.177) or AtCDA9 (Figure 1c: χ² = 6.539, 8 d.f., P = 0.587) and their respective controls. Consistently, the mutant spectra from the three AtCDA-overexpressed samples were significantly heterogeneous (χ² = 41.063, 16 d.f., P = 0.001), again due to the enrichment in G to A transitions observed in the case of AtCDA1. By contrast, the three independent control inoculation experiments showed homogeneous mutant spectra for CaMV (χ² = 14.605, 18 d.f., P = 0.689), undistinguishable from the mutant spectra previously reported for natural isolates of this virus⁴⁸. The consistency of the mutant spectra observed for the three control experiments and for a natural isolate of the virus suggests that in absence of a perturbation such as the overexpression of AtCDA1, the CaMV mutant spectrum is rather stable.

Figure 1. Number of mutations in CaMV genomes isolated from plant tissues agroinfiltrated with different AtCDAs.

(a) AtCDA1, (b) AtCDA2 and (c) AtCDA9. The pBIN61 empty vector was agroinfiltrated in the same leaves than their corresponding AtCDAs (mock). For each sample 20,034 nucleotides were sequenced.

We conclude that overexpressing the AtCDA1 gene results in a significant shift in CaMV genome composition towards G to A mutations, as expected from cytidine deaminase hypermutagenic activity.

Effect of suppressing AtCDA expression on the viral load and mutational spectrum of CaMV

To test the effects of suppressing the expression of AtCDA on viral accumulation we produced a transgenic line of A. thaliana Col-0, named amiR1-6-3. This line was stably transformed with an amiR, controlled by the A. nidulans ethanol regulon to achieve ethanol-triggered RNAi-mediated simultaneous suppression of AtCDAs 1, 2, 3, 4, 7, and 8 expression. Transgenic and wild-type plants were subjected to periodical treatment with 2% ethanol (or water for the control groups). Subsequently, plants were inoculated with the infectious clone pCaMVW260 that expresses the genome of CaMV. Samples were taken eight days after inoculation and AtCDA1 mRNA and CaMV viral load were quantified by real time RT-qPCR and qPCR, respectively, in the same samples. For each genotype and/or treatment, 22 plants were analyzed.

The expression of AtCDA1 mRNA depended on the plant genotype (Figure 2a; GLM: χ² = 28.085, 1 d.f., P < 0.001) as well as on the interaction of plant genotype and treatment (χ² = 26.037, 1 d.f., P < 0.001), suggesting a differential accumulation of AtCDA1 mRNA on each plant genotype depending on the amiR1-6-3 induction state. Ethanol treatment reduced the amount of AtCDA1 mRNA by 24.01% in transgenic plants, proving that triggering the expression of the amiR1-6-3 significantly and efficiently silences the expression of AtCDA1. Unexpectedly, the effect was the opposite in wild-type plants, for which we observed 23.76% increase in AtCDA1 mRNA accumulation (Figure 2a) upon treatment with ethanol. This increase in expression of AtCDA1 in wild-type plants after ethanol treatment and the underlying mechanisms certainly deserve to be investigated further. However, for the purpose of this study, its relevance is that it may increase the number of G to A mutations in the CaMV genome, thus making the antiviral effect stronger to some extent.

Figure 2. Accumulation of AtCDA1 mRNA molecules and CaMV genomes.

(a) Number of AtCDA1 mRNA molecules/80 ng total RNA quantified by RT-qPCR using the standard curve method for absolute quantification. (b) Number of CaMV genomes/80 ng total DNA. For each block of plants (wild-type and amiR1-6-3), values were normalized to the average number of genomes estimated in the corresponding water-treated (control) plants.

More interestingly, the relative accumulation of CaMV in ethanol-treated plants was significantly different, depending on the plant genotype being infected (Figure 2b; Mann-Whitney U test, P = 0.002): silencing the AtCDA1 gene bolstered CaMV accumulation to 103.10% compared to the accumulation observed in wild-type plants. Furthermore, there was a significant negative correlation between the number of molecules of AtCDA1 mRNA and viral load (partial correlation coefficient controlling for treatment: r = –0.299, 86 d.f., P = 0.005).

Given the significant increase of viral load in plants with lower levels of AtCDA1 mRNA, we sought the molecular signature of deamination in transgenic plants. For this, we selected three biological replicates from each treatment group (ethanol or control) and sequenced between 39–45 clones of the CaMV fragment from each replicate. As shown in Figure 3, silencing of the AtCDA1 gene affects the composition of CaMV mutant spectrum by reducing the number of G to A transitions by 69.23%. Nevertheless, overall, both mutational spectra were not significantly different (Figure 3: χ² = 9.108, 6 d.f., P = 0.168), prompting caution against making a definite conclusion on the role of deamination in the observed increase in CaMV accumulation.

Figure 3. Number of mutations found pooling the CaMV sequences from ethanol-treated and control amiR1-6-3 plants (3 replicates).

The number of nucleotides sequenced was 23,436 for control and 24,003 for ethanol-treated plants. Ethanol-treated plants turn on the expression of amiR1-6-3 that was designed to silence the expression of the AtCDA1 gene.

We conclude that suppressing the expression of the AtCDAs 1, 2, 3, 4, 7, and 8 significantly reduces the accumulation of CaMV. However, the characterization of the mutant spectrum of the same CaMV populations provides no strong enough support to the cytidine deamination hypothesis.