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. 2018 Jun 26;13(7):e1483673. doi: 10.1080/15592324.2018.1483673

Deletion of TLS polymerases promotes homologous recombination in Arabidopsis

A N Sakamoto a,✉, H Kaya b, M Endo c
PMCID: PMC6128680  PMID: 29944437

ABSTRACT

Unrepaired DNA damage hinders the maintenance of genome integrity because it blocks the catalytic activity of replicase. The stalled replication fork can be processed through either translesion synthesis (TLS) with specific polymerases, or replication using the undamaged template. To investigate how TLS activities are regulated and how the stalled replication fork is processed in plants, reversion frequencies and homologous recombination (HR) frequencies were analyzed using GUS-based substrates. The HR frequencies in plants deficient in DNA polymerase ζ (Pol ζ) or Rev1 were higher than that in wildtype plants under normal conditions, and were significantly increased by ultraviolet light irradiation. Heat shock protein (HSP) 90 is known to be involved in various stress responses. To examine the role of HSP90 in the regulation of damage tolerance, we analyzed reversion frequencies and HR frequencies in plants grown in the presence of a HSP inhibitor, geldanamycin (GDA). Reversion frequency was lower in GDA-treated plants than in mock-treated plants. Though the HR frequency was higher in GDA-treated wildtype plants than in mock-treated plants, no significant difference was detected in Rev1-deficient plants. In yeast, TLS polymerases interacted with each other or with a replication clump component, proliferating cell nuclear antigen (PCNA). HSP90 interacted with REV1 or REV7 in Nicotiana benthamiana cells. These results suggest that HSP90 interacts with TLS polymerase(s), which promotes error-prone TLS in plants.

Keywords: translesion synthesis (TLS), homologous recombination (HR), heat shock protein 90 (HPS90), replication fork, genome stability

Introduction

Maintenance of genome integrity is important for all organisms. Genomic DNA is frequently damaged through cellular physiological activity or by external agents such as UV, ionizing radiation, or chemicals. DNA damage affects gene activities, inhibits DNA replication, induces mutation and jeopardizes genome stability. Organisms have developed various and redundant DNA repair pathways in their ongoing efforts to keep their genomes intact. Unrepaired template DNA damage inhibits the activity of DNA replicase and induces the collapse of the replication fork. Microorganisms and mammalian systems can maintain the replication fork through at least two mechanisms: i) an error-prone mechanism that uses specific polymerases to bypass the damage; and ii) an error-free mechanism that uses the undamaged template to continue the replication.

Specific polymerases that have been reported in plants include DNA polymerase ζ,1 Rev1,2 DNA polymerase η,3 and DNA polymerase κ.4 We previously used uidA-based markers to show that DNA polymerase ζ (Pol ζ) and Rev1 bypass UV- or gamma-induced damage in an error-prone manner.5,6 Several genes involved in the error-free postreplication repair mechanism have also been reported.7,8 AtRAD5a, one of two Arabidopsis homologues of yeast Rad5, is involved in postreplication repair.9,10 Wang et al.11 reported that disruption of both AtREV3, which encodes a catalytic subunit of Pol ζ, and AtRAD5a showed synergistic or additive effect in Arabidopsis root growth when exposed to UV, MMS or crosslink agents. This suggests the presence of multiple damage response pathways, depending on the kind of damage, in plants.12 The rad5a plants failed to induce homologous recombination (HR) events after the treatment with bleomycin.9 Given that AtRAD5a and AtREV3 work via two alternative pathways, disruption of AtREV3 could affect the activity of the error-free postreplication pathway, leading to a change of HR frequency.

The 90-kDa heat shock protein (HSP90) is a well-conserved molecular chaperone that has key roles in the folding and activation of proteins involved in signal transduction and control of cell cycle.13 HSP90 is activated by binding and hydrolyzing ATP. Crystal structure analysis has revealed that HSP90 dimerizes through its C-terminal domains and transiently through its N-terminal ATPase domains when ATP is bound.14 Specific inhibitors such as geldanamycin (GDA) bind to the ATP-binding pocket of HSP90 and inhibit its function. The Arabidopsis genome has four copies of cytosolic HSP90 and three copies of organellar HSP90 genes.13 GDA affects various physiological responses in Arabidopsis, such as disease resistance, circadian clock and brassinosteroid response.15-17 Queitsch et al.18 reported elevated homologous recombination (HR) frequencies after GDA treatment, suggesting the involvement of HSP90 in genome maintenance. It has also been shown that HSP90s directly interact with TLS-type polymerases in mammalian cells.19,20 These results raise the possibility that the HSP90s regulate the activity of TLS polymerases in plants.

In this report, we analyzed HR activity using three types of DNA substrate and showed that the deletion of TLS-type DNA polymerases elevates HR frequencies. Furthermore, GDA reduces mutation frequency but elevates HR frequencies. These results suggest that TLS activities and the error-free postreplication pathway work in parallel, and are at least partly regulated by HSP90.

Results

Detection of homologous recombination by using HR substrates

We used three type of DNA substrate based on β-glucuronidase activity to detect homologous recombination (HR) frequencies (Figure 1A). The tandem repeat substrate (TR; GU-US) has tandemly repeated homologous sequences of Escherichia coli uidA separated with hygromycin resistant (hgt) genes.21 The inverted repeat substrate (IR; U′G′-US) has the 5′ part of uidA inverted and repeated.21 The direct repeat substrate (DR; US-GU) also has tandemly repeated homologous sequences, but the 5′ part of the uidA gene is located downstream of the 3′ part.22 For all substrates, recombination between the homologous overlapping sequences within each construct is expected to restore the activity of uidA. Therefore, each blue GUS+ sector on the somatic tissue can be scored as a HR event.23

Figure 1.

Figure 1.

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Detection of homologous recombination (HR) events using three recombination substrates. (A) Structure of TR (GU-US), IR (U′G′-US) and DR (US-GU) substrates and expected recombination. (B) Relative HR frequency in wildtype and TLS-deficient plants. The TR, IR or DR substrates were introduced into polh-1, rev3-1 or rev1-4 plants. The mutant plants and Columbia (Col) plants were grown side by side and recombination events were scored by detecting blue GUS+ sectors on somatic tissue. The relative HR frequency was shown as a ratio of recombination events in mutant to Col. For each experiment, 70–140 plants were analyzed. The data are an average of at least three independent experiments with SEM. * indicates p < 0.05 in t-test.

To compare HR frequencies in different genetic backgrounds, the substrate lines were crossed to the polymerase-deficient mutant lines rev3-1, polh-1 and rev1-4, and F2 mutant plants and wildtype siblings were selected. The HR frequencies in polymerase-deficient plants and wildtype plants were compared, and the ratios of mutant to wildtype frequencies were calculated. Under normal growth conditions, the number of recombination events on the TR and IR substrates of polh-1 plants, which are deficient in DNA polymerase η, was similar to that of the wildtype plants (Figure 1B). By contrast, rev3-1 plants, which are deficient in DNA polymerase ζ, showed higher, or a trend of higher, recombination frequencies on these substrates. The Rev1-deficient plants rev1-4 also showed high recombination on TR substrates. On the DR substrate, although there were fewer recombination events in polh-1 plants than in wildtype plants, rev3-1 plants showed almost the same recombination frequency as wildtype plants.

When TR-substrate plants were exposed to 500 J/m2 UVC radiation, there were significantly more recombination events in rev3-1 plants than in wildtype siblings (Figure 2A). Similarly, rev1-4 plants had more recombination events than wildtype siblings (Figure 2B). These results indicate that UV damage significantly induces HRs when DNA polymerase ζ and Rev1 are absent. Conversely, polh-1 plants had a similar number of recombination events even after UV-irradiation (Figure 2C), suggesting that the absence of DNA polymerase η does not enhance HR.

Figure 2.

Figure 2.

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UV irradiation significantly induces HR events in TLS-deficient plants. The rev3-1 (A) or rev1-4 (B) and polh-1 (C) plants and Columbia (Col) plants carrying a TR (GU-US) substrate were irradiated with 500 J/m2 of UVC and recombination events were detected. For each experiment, 85–130 plants were analyzed. Compared with Col, UV-induced HR events were significantly higher in rev3-1 and rev1-4 but not in polh-1.

Effect of HSP-inhibitors on reversion frequencies

To examine whether TLS activities are regulated by HSP90 in plants, as reported in mammals, we first measured the somatic reversion frequencies in the presence of an HSP90 inhibitor. The reversion marker uidA166G-T, which contains a G-T mutation, is activated when T-G transversion occurs.5,6 The resulting blue GUS+ sector is scored as a reversion event. Columbia and TLS-deficient plants carrying the marker were grown in the presence of 1–2 µM geldanamycin (GDA) or control DMSO and irradiated with UVC at day 14. Plants were harvested one week after UV treatment, stained with X-gluc solution, and then the numbers of GUS+ sectors were counted. The number of blue sectors in GDA-treated plants was significantly lower in wildtype plants. Similar trends were observed in rev1-4 and polh-1 plants (Figure 3). These results showed that TLS activities are upregulated by HSP90 in Arabidopsis, as reported in mammalian cells.19,20

Figure 3.

Figure 3.

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Inhibition of HSP90 reduces the reversion frequencies. Wildtype, rev1-4, or polh-1 plants were grown on media containing geldanamycin (+) or DMSO (-). The relative reversion frequency is shown as (-) is 1. The bars used for data normalization are presented in one figure although their absolute values are different. For each experiment, 40–80 plants were analyzed. The data are an average of two independent experiments with SEM. * indicates p < 0.05 in t-test.

Effect of HSP-inhibitors on homologous recombination

To confirm the effect of HSP90 on HR frequency, and to examine whether the effect is related to TLS activity, wildtype or rev1-4 plants carrying the HR substrates were grown in the presence of 2 mM GDA or DMSO. The two-week-old plants were stained and the numbers of GUS+ sectors were counted. The HR frequency was elevated in wildtype plants carrying TR-type substrates treated with GDA (Figure 4A). Similar results were obtained with IR- or DR-type substrates in wildtype plants (Figure 4C). The GDA treatment did not elevate the HR frequency in rev1-4 or polh-1 plants carrying TR substrates (Figure 4A, B). This indicates that the elevation of HR frequency in GDA-treated plants is caused by the repression of TLS activities.

Figure 4.

Figure 4.

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Inhibition of HSP90 induces the recombination. (A) Effect of HSP90 inhibitors on the HR frequencies in wildtype or rev1-4 plants. Wildtype or rev1-4 plants carrying the TR substrate were grown on medium containing geldanamycin (+) or DMSO (-). The relative reversion frequency is shown as (-) is 1. For each experiment, 70–85 plants were analyzed. The data are shown as an average of at least two independent experiments with SEM. * indicates p < 0.05 in t-test.(B) Effect of HSP90 inhibitors on the HR frequencies in polh-1 plants. polh-1 plants carrying the TR substrate were grown on medium containing geldanamycin (+) or DMSO (-). The relative reversion frequency is shown as (-) is 1. The data are an average of ~80 plants with SEM. (C) Effect of HSP90 inhibitors on the HR frequencies in IR and DR. Wildtype plants were grown on medium containing geldanamycin (+) or DMSO (-). The relative reversion frequency is shown as (-) is 1. The data are an average of ~85 plants with SEM. * indicates p < 0.05 in t-test.

Interaction of REV1, POLH and DNA polymerase ζ in yeast cells

Cells use the TLS polymerase instead of the replication-type DNA polymerase (replicase) when a replication fork encounters DNA damage and stalls. This switch from replicase to TLS polymerase is performed through the protein-protein interaction of monoubiquitinated proliferating cell nuclear antigen (PCNA) and TLS-type polymerases, and also among the plural TLS-type polymerases.24–26 Arabidopsis DNA polymerase η has been shown to interact with AtPCNA1 and AtPCNA2 in yeast.27 The AtREV1 protein contains the ubiquitin binding motifs (UBMs), a conserved motif seen in proteins interacting with monoubiquitinated PCNA,28 suggesting that AtREV1 may also interact with PCNA.

To examine whether AtREV1 actually interacts with PCNA and other TLS polymerases, we performed two-hybrid assays in yeast (Y2H). We found that AtREV1 interacted with PCNA2 in yeast (Figure 5A). AtREV1 also interacted with AtPOLH and AtREV7, a regulatory subunit of DNA polymerase ζ (Figure 5B).

Figure 5.

Figure 5.

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Interaction of TLS polymerase and AtPCNA2 in yeast cells. (A) Interaction of AtPCNA2 and AtREV1 and AtPOLH in yeast. Full-length ORF of AtREV1, AtPOLH and AtPCNA2 were subcloned into bait or prey plasmids, which express the Bait/DNA-binding domain (DBD)- or the Prey/transcription activation domain (AD)-fusion protein in yeast. The interaction between the bait and the prey was investigated through cell growth on non-selective media (NSM) or selective media (SM). – means no insert.(B) Interaction of AtREV1, AtPOLH, and AtREV7 in yeast. Full-length ORF of AtREV1, AtPOLH and AtREV7 were subcloned into bait or prey plasmids and the interaction was investigated on selective media (SM).(C) Detection of the interaction domain of AtREV1 with AtPOLH or AtREV7. The REV1 protein contains three conserved motifs: BRCT domain at the N-terminal, UmuC domain at the center and ubiquitin-binding motifs (UBM) at the C-terminal. Full-length or truncated ORF of AtREV1 was subcloned into the prey plasmid and introduced into yeast with a bait plasmid expressing a AtREV7 or AtPOLH-fusion protein. The interaction between the bait and the prey was investigated through growth on selective media (SM). AtREV1 interacted with AtREV7, a regulatory subunit of AtPolζ, at its C-terminal. The AtREV1 also interacted with POLH, though no obvious interaction domain was determined.

To determine the interaction domain(s) of AtREV1, we prepared three types of truncated AtREV1 proteins and examined their interactions with AtREV7 or AtPOLH. AtREV1 interacted with AtREV7 with its C-terminal domain (Figure 5C). By contrast, all truncated AtREV1 forms did not interact with AtPOLH, suggesting that more than one domain might be required for AtREV1 to interact with AtPOLH (Figure 5C). We hypothesize that AtREV1 interacts with PCNAs as well as AtPol η and AtPol ζ, then the complex recruits the appropriate polymerase to the replication site.

Interaction of REV1 and HSP90 in Nicotiana benthamiana cells

It has been shown that TLS polymerases interact directly with HSP90 in mammalian cells.19,20 To test if similar interactions happen among plant proteins, we subcloned the cDNA of a cytosolic/nucleic copy of Arabidopsis thaliana HSP90 (AtHSP90-2) for bimolecular fluorescence complementation (BiFC) assay using Nicotiana benthamiana cells. The N-terminal and C-terminal domains of yellow fluorescent protein (YFP) fused to AtREV1, AtREV7 or AtHSP90-2 were transiently expressed in N. benthamiana and the fluorescent signal of reconstituted YFP was detected under fluorescent microscope.

The BiFC signal showed that HSP is self-assembled in the cytoplasm (Figure 6C). The AtREV1 and AtREV7 proteins interacted in the nucleus (Figure 6A). The AtREV1-m1 protein, which lacks the C-terminal domain, did not interact with AtREV7 (Figure 6D), as seen in yeast cells. Interaction between HSP90 and AtREV1 was weakly detected in the nucleus (Figure 6B, 6G). The AtREV1-m1 and AtREV1-m3 proteins interacted with HSP90 in the nucleus with stronger intensity (Figure 6E, 6I). These results suggest that HSP90 interacts with multiple regions of AtREV1. By contrast, the signals for interaction between HSP90 and AtREV7 were detected in both the cytoplasm and the nucleus (Figure 6F). Taken together, these results indicate that HSP90 interacts with TLS polymerases in plant cells, possibly to regulate TLS activities. In addition, the AtREV7 showed self-assembled signal in the cytoplasm near the membrane as a particle like-structure (Figure 6H).

Figure 6.

Figure 6.

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Interaction of TLS polymerase and HSP90 in Nicotiana benthamiana cells. Pairwise combination of the N-terminal (YN) and C-terminal (YC) domains of yellow fluorescent protein (YFP) fused to AtREV1, AtREV1-m1, AtREV1-m3, AtREV7 or AtHSP90-2 were expressed in N. benthamiana leaf cells and the reconstructed YFP signal was detected under fluorescent microscope. (A) REV7-YN and REV1-YC, (B) HSP-YN and REV1-YC, (C) HSP-YN and HSP-YC, (D) REV7-YN and REV1-m1-YC, (E) HSP-YN and REV1-m1-YC, (F) HSP-YN and REV7-YC, (G) REV1-YN and HSP-YC, (H) REV7-YN and REV7-YC, (I) REV1-m3-YN and HSP-YC. bar: 100 µm.

Discussion

Accurate replication of genomic DNA is the most important task in maintaining genome integrity. However, genomic DNA sustains various forms of damage caused by internal and external agents. The damaged DNA cannot fit the catalytic sites of replicases, and the replication fork stalls. Studies within microorganisms and mammals have revealed several pathways for resolving the stalled replication fork. Translesion synthesis is one such pathway, in which specific polymerases with relaxed catalytic pocket structure replace the replicase and bypass the damage. The switching from the replicase to the TLS-type polymerase is thought to be managed through the monoubiquitination of the proliferating cell nuclear antigen (PCNA).24–28 After the bypass of the damaged site, the TLS-type polymerase is replaced with the replicase to complete the replication.29 Alternatively, through a second pathway, the stalled replication end can be elongated using the undamaged DNA strand. This pathway is thought to work by polyubiquitination of PCNA involving all three of Ubc13, Mms2 and Rad5.30

AtRAD5a, one of two Arabidopsis homologues of yeast Rad5, is reported to be involved in the mono- and polyubiquitination of PCNA.10 Wang et al.11 reported that disruption of AtREV3 and AtRAD5a showed a synergistic or additive effect in Arabidopsis root growth when exposed to UV, MMS or crosslink agents. Chen et al.9 reported that the disruption of AtRAD5a failed to induce HR events after bleomycin treatment. Therefore, the elevation of HR activities in AtPol ζ- and AtRev1-deficient plants in our study could have been due to the activation of a RAD5-dependent postreplication pathway. Mannuss et al.31 reported that AtRAD5A is involved in synthesis-dependent strand annealing (SDSA), but not single-strand annealing (SSA), using recombination substrates with inducible site-specific double strand breaks (DSBs). Thus, stalled replication in TLS-deficient lines leads to DSB formation, which might induce SDSA in plants. Recently, Fan et al. showed a direct interaction between yRad5 and yREV1 and proposed that yeast Rad5 might be recruited to the damage site through its affinity with the 3′-OH group at the end of the single stranded DNA.32 However, our unpublished results did not detect interaction between AtRAD5A and AtREV1 (data not shown). In addition, in vitro synthesized AtRAD5A interacts with several kinds of branched DNA regardless of the presence or absence of the 3′-OH end.33 Thus, further analysis is necessary to clarify the mechanism of DNA damage tolerance in plants.

We used three uidA-based substrates to investigate HR activity: a tandem repeat type (TR; 1406), an inverted repeat type (IR; 1415) and a direct repeat type (DR; IC9). Recombination events in all three substrates were uniformly higher in TLS-deficient plants than in wildtype plants, which raises the possibility that the HR-active condition is induced in the plant independently of substrate structure. By contrast, Schuermann et al.34 reported that recombination events with IR-type substrates, but not with TR- or DR-type, were significantly elevated in polδ1-1 hemizygous plants. They hypothesized that overcrowded ssDNA on the lagging strand induces strand breaks or aberrant hairpin structures, which lead to high HR events on the IR-type substrate. Thus, the mechanism of HR induction in the TLS-deficient line could be different from that in polδ1-1.

Sekimoto et al.19 and Pozo et al.20 showed that human HSP90 regulates family Y DNA polymerases in mammalian cells. Human Pol η was co-immunoprecipitated with HSP90. Co-immunoprecipitation was diminished by the pretreatment of cells with 17-AAG, an HSP inhibitor.18 Pol η-proficient cells showed reduced UV-induced supF mutation in the presence of HSP90, but Pol η-knockdown cells did not.19 Furthermore, Pozo et al.20 showed that hHSP90 also interacts with hREV1 and that 17-AAG treatment diminished REV1-dependent mutation when hPol η was absent. The fact that the absence of HSP90 induces ATP-dependent protein degradation of Pol η and REV1 suggests that HSP90 works in the refolding of family Y polymerases, which promotes their interaction with Ub-PCNA to perform translesion synthesis. Contrasting with results in mammals, our results showed that treatment with an HSP90 inhibitor reduces mutation frequencies in wildtype plants and shows a similar trend in polh-1, and rev1-4 plants. This suggests that HSP90 mainly interacts with the error-prone TLS polymerases in Arabidopsis. Conversely, GDA treatment enhances the HR frequencies of wildtype plants.18 The elevation of HR frequencies is not obvious in rev1-4 and polh-1 plants. Therefore, inactivation of TLS polymerase by GDA might induce HR activities in Arabidopsis.

BiFC analysis showed that HSP90 interacts with AtREV1 and AtREV7, suggesting the possibility that HSP90 regulates not only Rev1 but also DNA polymerase ζ. While the AtREV1–HSP90 interaction was only detected in the nucleus, the AtREV7–HSP90 interaction was detected in both the cytosol and the nucleus (Figure 6F). The REV7 protein is a member of a group of HORMA domain-containing proteins, which form various homo- or hetero-multimers.35 It has been shown that the human REV3 has two REV7-interacting motifs and Pol ζ presents as a REV3-REV7-REV7 trimer.36 In plant REV3s, the conserved REV7-interacting sequence has only been found at one location (supplemental figure S1). Thus, the conformation of active DNA polymerase ζ in plants is not clear, but it is possible that AtPol ζ is present as a multimer containing multiple AtREV7 copies or unidentified FORMA-containing protein(s). The results of our BiFC assay suggests that the interaction of AtREV7 with AtREV1 and/or HSP90 regulates the activity of AtPol ζ in the nucleus. It is not clear whether the AtREV7-HSP90 complex in the cytosol is just in transit to the nuclei or if it has an as-yet-unknown function in the cytosol.

Based on our data and published research in other organisms, we propose a model for regulation of TLS activity (Figure 7). When replication machinery encounters DNA damage, the replicase cannot bypass the damage and the replication fork stalls. The stalled replication fork puts the stability of the genome at risk because it is prone to collapse, leading to DNA strand breaks. To avoid collapse, the replication fork can be processed through two pathways: translesion synthesis (TLS) or synthesis with the intact template (template switch). Interactions among TLS-type polymerases and monoubiquitinated PCNA recruits an appropriate polymerase into the replication fork to bypass the damage. HSP90 interacts with TLS-type polymerases in the nuclei and promotes their activity. When TLS is deficient or reduced by depletion of HSP90, polyubiqitination of PCNA leads to a template switch, involving homologous recombination.

Figure 7.

Figure 7.

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A model for TLS and template switch to overcome the stalled replication fork. When disrupted, the replication fork can take two pathways: mutagenic synthesis by specific polymerases (TLS) or accurate synthesis using an intact template (template switch). Interactions among TLS-type polymerases and PCNA are important for the recruitment of an appropriate polymerase(s) to the replication fork. HSP90 interacts with TLS-type polymerases in the nucleus and regulates their activity. When TLS is deficient or reduced by depletion of HSP90, polyubiquitination of PCNA leads to template switch, involving homologous recombination.

Materials and methods

Plant materials

Arabidopsis (Arabidopsis thaliana) Columbia was used as the wildtype in this study. The rev3-11 and rev1-4 (supplemental figure S2) lines were obtained by carbon ion beam irradiation at Takasaki Ion Accelerator for Advanced Radiation Application (TIARA), QST. The polh-1 (SALK_129731)27 line was provided by Arabidopsis Biological Resource Center (ABRC). The mutant lines of rev3-1, rev1-4, polh-1 were crossed with uidA166G-T,37 1406 and 1415,21 or IC9.22 From the F2 plants, the test lines carrying a homozygous polymerase mutation and a homozygous substrate gene were selected by PCR and resistance to glufosinate ammonium (WAKO, 079–05371) or hygromycin (WAKO, 085–06153). Multiple control (REV3, REV1, or POLH) lines were selected from the siblings of the test lines.

Growth conditions

To detect recombination frequency, the plants were grown on rockwool (Grodan, TACO BLOCK 23/28) with 0.1% (v/v) commercial nutrient, or combined B5 agar plates [0.5× Gamborg’s B5 medium salt mixture (Wako, 399–00621), 0.1% (v/v) commercial nutrient (Hyponex Japan, 6–10-5 undiluted solution), 1% sucrose, 0.7% agar].38 To detect mutation frequency, the test line and control line were grown on MS plates [1× Murashige and Skoog plant salt mixture (Wako, 392–00591), 1× Gamborg’s B5 vitamin mix (Sigma-Aldrich, G1019), 0.5 gL−1 MES, 1% (w/v) sucrose, 0.7% (w/v) agar]. Plants were grown under 16-h light and 8-h dark cycles at ~70 mmol m–2 s–1 and 23 °C in a growth chamber.

UV irradiation

12–14-day-old seedlings were irradiated with UVC light supplied by a UV lamp (CSL-30C; UVP). The plants were grown for another 7–8 days under the same growth conditions before GUS staining.

GDA treatment

Test plants were sown on plates containing 1–2 µM of geldanamycin (GDA) and kept at 4 °C in the dark. Plants were moved to 23°C in a growth chamber and were grown as described above.

GUS staining

Aerial parts of plants were recovered and vacuum infiltrated twice for 20 min with X-gluc solution [100 mM sodium phosphate buffer (pH 7.2), 0.1% (v/v) Triton X-100, 0.05% sodium azide, 0.5 mM each of potassium hexacyanoferrate (III) and potassium hexacyanoferrate (II) trihydrate, and 1.08 mM 5-bromo-4-chloro-3-indolyl-b-D-glucuronic acid cyclohexylammonium salt (Biosynth, B-7300)]. Following vacuum infiltration, plants were incubated at 37 °C for 2 d in the dark and then bleached with 70% (v/v) ethanol. The number of blue sectors on each plant was determined visually using a stereomicroscope. For each experiment, 40–140 plants were analyzed.

Yeast two hybrid (Y2H) assay

Interactions between TLS polymerases and PCNA were examined using the Pro-Quest two-hybrid system (Thermo Fisher Scientific, PQ1000101). The cDNA of AtPCNA2 was a gift from W. Strzalka.10 Full-length ORF of AtREV1 and AtREV7,2 AtPOLH27 and AtPCNA2 were amplified with specific primers (supplemental table S1) and cloned into pENTR/D-TOPO. To prepare AREV1-m1, -m2 and -m3, the pENTR-REV1 was digested with Hind III (Thermo Fisher Scientific, ER0501); Xho I (Roche, 10899194001) and Sal I (Takara, 1080A); and Avr II (New England Biolabs, R0174) and Xba I (Takara, 1093A), respectively. Then, the individual large fragments were self-ligated. The cDNAs were then transferred to pDEST22 or pDEST32 using Gateway LR Clonase II (Thermo Fisher Scientific, 11791020) to prepare bait and prey plasmids. Yeast strain MaV203 was transformed with a pairwise combination of bait and prey plasmids, and screened on SC-Leu-Trp medium according to the manufacturer’s instructions. To detect the interaction between the two fusion proteins, the yeast cells were grown in liquid medium for 20 h, and resuspended in water to an absorbance at 600 nm of approximately 0.2. About 5 µL of the original suspension and 10× to 100× dilutions were placed on selection medium [SC-Leu-Trp-His supplemented with 7.5–60 mM 3-amino 1,2 4-triazole (Sigma-Aldrich, A8056)] and on non-selection medium (SC-Leu-Trp) and then incubated at 30 °C for 3 days.

Bimolecular fluorescence complementation (BiFC) assay

The BiFC vectors pGTQL1211YN or pGTQL1221YC were gifts from Yuhai Cui (Addgene plasmid #61704 and #6170539; The cDNA of HSP90-2 (At5g56030) was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT/AMED, Japan. Full-length ORF or partial-length of AtREV1, AtREV7and AtPCNA2 were amplified with a forward primer and a reverse primer, with no stop codon (supplemental table S1), and subcloned in to the BiFC vectors by LR Clonase II (Invitrogen, 11791100). Rhizobium radiobacter GV3101 was transformed with the individual BiFC plasmids by electroporation and screened with 25 mgL−1 kanamycin, 10 mg L−1gentamycin and 25 mg L−1 rifampicin. The clones carrying pGTQL1211YN-derived plasmid and pGTQL1211YC-derived plasmid were grown in liquid medium to absorbance at 600 nm of 1.0 to 1.2. Cells were suspended in infiltration buffer (10 mM MgCl2, 100 µg mL−1 acetosyringone) and incubated for 8 h at 22 °C in the dark. Aliquots of each construct were mixed with an equal amount of R. radiobacter suspension. The Nicotiana benthamiana plants were grown at 21 °C, with 16-h light and 8-h dark cycles until they were five to six weeks old. The R. radiobacter suspension was infiltrated into the N. benthamiana leaf with a syringe. The plants were kept at 21 °C, with 16-h light and 8-h dark cycles for two days. A sector of infected leaf tissue was excised and the transient expression of YFP was observed under fluorescent microscopy (Leica, DM4500B). The BiFC signals with either or both of pGTQL1211YN and pGTQL1221YC (vectors without insert) was used to normalize the fluorescent intensity (data not shown).

Funding Statement

This work was partially supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant number 24241028 to A.N.S. and M.E. and Grant number 25440147 to A.N.S.

Acknowledgments

We would like to thank to Seiichi Toki for his critical reading of the manuscript. We also thank to Tatsuya Uchida, Mutsumi Akita and Nobuko Murakami, for their technical assistance. We thank Ann Seward, ELS, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplemental Material

Supplemental data for this article can be accessed on the publisher's website.

Supplemental Material

References

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