Acibenzolar-S-methyl inhibits accumulation of replication-associated DNA of tomato yellow leaf curl virus

Abstract

Acibenzolar-S-methyl (ASM), a functional analogue of salicylic acid, induces systemic acquired resistance and restricts infection by several RNA viruses, yet its mechanisms against DNA viruses remain unknown. By examining the accumulation of tomato yellow leaf curl virus (TYLCV) DNA in inoculated leaves of Nicotiana benthamiana, we investigated whether ASM suppresses begomovirus infection. TYLCV was inoculated by agroinfiltration, and virion-sense (VS) and complementary-sense (CS) strands of TYLCV DNA were quantified separately using two-step strand-specific qPCR. ASM pretreatment significantly reduced the total TYLCV DNA accumulation at 1, 2, and 3 days post-inoculation. To determine whether this reduction reflected a direct effect on TYLCV rather than impaired Agrobacterium-mediated T-DNA delivery, we used a replication-defective mutant (TYLCVΔC1) as a delivery control and confirmed that ASM did not affect T-DNA delivery. Both VS and CS strands of wild-type TYLCV were substantially reduced by ASM treatment, providing the first direct evidence that ASM suppresses begomovirus infection by targeting viral DNA accumulation at the replication stage.

Introduction

Plants possess a multi-layered immune system to defend against diverse microbial pathogens. One of the most important components is systemic acquired resistance (SAR), a broad-spectrum, primed immune state activated following pathogen exposure that confers both local and systemic protection against subsequent infections. SAR is predominantly regulated by salicylic acid (SA)-dependent signalling and the downstream defense pathways.¹⁾ Exogenous application of synthetic compounds can artificially induce SA-dependent immune responses similar to those triggered by pathogens; these compounds are collectively referred to as plant activators (PAs).^2–4) Among commercially available PAs, acibenzolar-S-methyl (ASM), a benzothiadiazole S-methyl ester derivative and functional analogue of SA, has been widely used in disease management practices, particularly in Europe and the United States.⁵⁾ ASM shows efficacy in more than 120 pathosystems, highlighting its broad utility as a PA.⁵⁾

Plant viruses remain a major global challenge in agriculture due to the limited availability of effective control strategies. Current management approaches rely mainly on insecticides targeting insect vectors or on the use of resistant cultivars,^6,7) while no chemical treatments directly targeting the virus itself are available. In this context, SA and its analogue ASM are promising candidates because they induce antiviral responses against several RNA viruses, including cucumber mosaic virus (CMV), cucurbit chlorotic yellows virus, Plantago asiatica mosaic virus (PlAMV), tobacco mosaic virus (TMV), and tospoviruses.^8–12) The mechanisms of PA- or SA-induced resistance against RNA viruses have been examined by inoculating plants with green fluorescent protein (GFP)-expressing viruses after PA or SA application. Previous studies have shown that PAs can restrict replication of TMV at the inoculation site, delay vascular loading of CMV, and inhibit both of these steps in PlAMV infection.^11,13–15) In contrast, the mechanisms underlying PA- or SA-mediated resistance against plant DNA viruses remain poorly understood.

Begomoviruses are single-stranded DNA (ssDNA) viruses of the family Geminiviridae and represent economically significant pathogens that pose a major threat to global agriculture.^16,17) Some PAs, as well as the plant hormones SA and jasmonic acid, have been shown to suppress tomato yellow leaf curl virus (TYLCV) accumulation in non-inoculated upper leaves of N. benthamiana and tomato.^18,19) ASM has also been reported to reduce disease severity and viral accumulation of begomoviruses in non-inoculated upper leaves of tomato, cassava, N. benthamiana and pepper.^20–22) However, it remains unclear whether the reductions in disease severity and viral titers observed in ASM-treated plants reflect inhibition of early viral accumulation in the inoculated leaves.

Experimental design to examine virus accumulation in the inoculated leaves depends on methods of virus inoculation. Because begomoviruses cannot be inoculated mechanically, their experimental infection relies either on insect-mediated transmission or on Agrobacterium-mediated inoculation (agroinfiltration). Insect-mediated transmission is inherently variable in efficiency and reproducibility, making controlled comparisons between experiments difficult. Agroinfiltration offers a reproducible laboratory method for initiating begomovirus infection.²³⁾ However, a critical limitation of this approach is that the viral genomic DNA cannot be distinguished from Agrobacterium-derived T-DNA by conventional quantitative PCR (qPCR). Moreover, SA-mediated host immune responses can influence the efficiency of Agrobacterium-mediated T-DNA delivery, which is a prerequisite for initiating virus infection; this complicates the interpretation of results concerning PA- or SA-mediated restriction of viral DNA accumulation.²⁴⁾

In this study, we used TYLCV, a well-characterized monopartite ssDNA begomovirus, as a model to examine the mechanisms of ASM-mediated resistance against plant DNA viruses. TYLCV infection can be initiated by agroinfiltration in a reproducible manner.²³⁾ In agroinoculated cells, T-DNA carrying the TYLCV genome is delivered as double-stranded DNA (dsDNA), which serves as a template for transcription by the host RNA polymerase II. The critical step of TYLCV replication is mediated by replication-associated protein Rep (C1), which is translated from a viral transcript and initiates viral DNA replication by introducing a site-specific nick in the virion-sense (VS) strand of the double-stranded DNA at the origin of replication. Using the complementary-sense (CS) strand as a template, a new VS strand is subsequently synthesized via rolling circle replication, displacing the parental strand to form new circular genomes.²⁵⁾

Thus, CS DNA accumulation serves as a reliable marker of active viral replication in infected cells.^26–30) Taking advantage of this replication scheme, we developed a replication-defective ΔC1 TYLCV mutant to distinguish the effects of ASM-mediated resistance from those of Agrobacterium-mediated T-DNA delivery, and employed strand-specific two-step qPCR to quantify VS and CS strand accumulation.²⁹⁾ We demonstrate that ASM reduces the accumulation of TYLCV DNA, including both VS and CS strands, independently of T-DNA delivery, indicating that it affects replication-associated processes. This approach provides a mechanistic basis for understanding ASM-mediated suppression of begomovirus infection.

Materials and methods

1. Plant material

Nicotiana benthamiana plants were grown in a controlled growth chamber under long-day conditions (16 hr light/8 hr dark) at 25±2°C and approximately 60% relative humidity. Plants were used 4–5 weeks after sowing.

2. ASM treatment

ASM (50% (w/w) active ingredient (Actigard, Syngenta, Tokyo, Japan)) was dissolved in water containing 0.004% (v/v) Silwet L-77 (Nippon Unicar, Kanagawa, Japan) to make a 1 mM solution, as described previously.¹¹⁾ Control plants were treated with water containing Silwet L-77 alone. ASM solution or water was sprayed onto the third youngest fully expanded leaf of 4–5-week-old N. benthamiana plants 3 days before agroinfiltration.

3. Plasmid construction of replication-defective TYLCV mutant (TYLCVΔC1)

The infectious clone of wild-type tomato yellow leaf curl virus Mild strain (TYLCV-Mld; TYLCV-Mld-[Japan:Takatsuki], GenBank accession AB921568³¹⁾ was used as the parental construct. A TYLCVΔC1 mutant carrying a truncated C1 (Rep) gene (amino acids 1–116) was generated by site-directed mutagenesis. The plasmid pGreen2-p35S-TYLCV-Mld was digested with SalI, and the linearized fragment was purified by phenol–chloroform–isoamyl alcohol extraction followed by ethanol precipitation. The SalI-generated 5′ overhangs were filled in using the Klenow fragment in the presence of dNTPs to introduce a frameshift mutation within the C1 coding region. The fill-in reaction was performed at 37°C for 20 min, followed by heat inactivation at 70°C for 10 min. The resulting blunt-ended fragment was gel-purified and self-ligated using the Ligation-Convenience Kit (Nippon Gene, Tokyo, Japan) to generate pGreen2-p35S-TYLCV-Mld-ΔC1. The construct was transformed into Agrobacterium tumefaciens strain EHA105.

4. Agroinfiltration of TYLCV-Mld and TYLCVΔC1 in N. benthamiana

At 3 days post-treatment, the ASM- or water-pretreated leaves were agroinfiltrated with either wild-type TYLCV-Mld or TYLCVΔC1 with minor modifications to the previously described method.²³⁾ Briefly, A. tumefaciens harboring pGreen2-p35S-TYLCV-Mld or pGreen2-p35S-TYLCV-Mld-∆C1 was cultured overnight in 4 mL of 2× YT liquid medium (16 g tryptone, 10 g yeast extract, 5 g NaCl/1000 mL) containing 100 mg/L kanamycin and 50 mg/L rifampicin in the shaker incubator at 180 rpm at 28°C, collected by centrifugation, and resuspended in agroinfiltration buffer (10 mM MgCl₂, 10 mM MES, 200 µM acetosyringone, pH 5.6) to OD₆₀₀=1.0. After incubation in the dark at room temperature for 2 hr, the bacterial suspension was infiltrated into the entire abaxial surface of the treated leaf using a 1 mL needleless syringe. After inoculation, plants were maintained under the same growth conditions until sampling.

5. DNA extraction and total TYLCV DNA quantification by qPCR

Total genomic DNA was extracted from 100 mg of inoculated leaf tissue collected at 1, 2 and 3 days post-inoculation (dpi) using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Each qPCR reaction was performed with 5 ng/µL total DNA as the template using GoTaq® qPCR Master Mix (Promega, Madison, WI, USA) on a Takara TP970 real-time PCR system. Reactions were prepared in a 20 µL volume containing 2 µL DNA template, 10 µL GoTaq qPCR Master Mix, 1 µL each of forward and reverse primer (10 µM each), and 6 µL nuclease-free water. Relative DNA accumulation of TYLCV-Mld and TYLCVΔC1 was quantified using TYLCV-specific primers (Supplementary Table S1) and normalized to the N. benthamiana reference gene NbITS. Cycling conditions were 95°C for 2 min, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. Amplification specificity was confirmed by melting curve analysis.

6. Preparation of single-stranded DNA for strand-specific qPCR

Strand-specific single-stranded DNA (ssDNA) standards were prepared following a previously described method with modifications. The full-length monomeric genome of TYLCV-Mld was amplified from pGreen2-p35S-TYLCV-Mld using the primer sets shown in Supplementary Table S1, and cloned into the pUC118 and pUC119, which carry oppositely oriented f1 phage origins, to generate virion-sense (pUC118-TYLCVvs) and complementary-sense (pUC119-TYLCVcs) constructs, respectively. These constructs were transformed into Escherichia coli JM109 (Takara Bio, Kusatsu, Japan). To extract ssDNA, E. coli JM109 transformants harboring each construct were superinfected with M13KO7 helper phage (New England Biolabs, Ipswich, MA, USA) to enable phagemid ssDNA secretion. ssDNA was recovered from the culture supernatant and verified by agarose gel electrophoresis as described previously.²⁶⁾ Absolute quantification of VS and CS strands was performed by two-step qPCR, in which the first step used strand-specific primer and T4 DNA polymerase, with minor modifications to the published protocol.²⁹⁾ Each reaction used 70 ng of ssDNA template in a 9 µL volume containing 1.3 µL ssDNA template, 1 µL reaction buffer, 1.7 µL dNTP mixture, 1 µL of 1 mM strand-specific tagged primer (TAG-OCS or TAG-OVS), and 4 µL nuclease-free water (primers listed in Supplementary Table S1). The mixture was heated at 95°C for 10 min, then 1 µL T4 DNA polymerase was added and incubated at 37°C for 30 min. Tag-extended ssDNA products were purified by ethanol precipitation, and serial dilutions were prepared for use as standard curves in strand-specific qPCR.

7. Strand-specific quantification of VS and CS ssDNA from the leaf sample

Strand-specific qPCR was performed to quantify VS and CS ssDNA of TYLCV-Mld and TYLCVΔC1 in inoculated leaves. For leaf samples, 25 ng of total DNA was used in the T4 DNA polymerase-mediated TAG extension step. Primers used are listed in Supplementary Table S1. Absolute copy numbers of VS and CS strands were calculated from the standard curves of pUC118-TYLCVvs and pUC119-TYLCVcs, respectively, as described previously,³²⁾ and expressed as log₁₀-transformed values prior to statistical analysis.

8. Statistical analysis

Statistical analyses were performed in R (version 4.4.0) using linear mixed-effects models implemented in the lme4 package. Data from three independent experiments (four plants each) were analyzed with experiment as a random factor and water and ASM treatment as a fixed factor. VS and CS copy numbers were log₁₀-transformed prior to analysis, whereas total TYLCV accumulation was analyzed on the normalized scale after verification of model assumptions. Significance testing was performed using the lmerTest package, and pairwise comparisons were conducted using the emmeans package. Fig. 2d and 2e were analyzed by Welch’s t-test. p<0.05 was considered statistically significant.

Fig. 2. Construction and characterization of the replication-defective TYLCVΔC1 mutant. (a) Schematic representation of the genomic organization of wild-type (WT) TYLCV-Mld and its mutant TYLCVΔC1. The TYLCVΔC1 was generated by filling in the SalI-linearized plasmid with Klenow fragment to introduce a frameshift within the C1-coding region, resulting in premature termination after amino acid 116 of C1. (b) Relative total TYLCV DNA accumulation in the inoculated leaves of plants infiltrated with TYLCV-Mld and TYLCVΔC1 at 3 dpi, quantified by qPCR and normalized to NbITS. Individual data points from three independent experiments (each with four biological replicates) are shown in different colors. Asterisks indicate significant differences between TYLCV-Mld and TYLCVΔC1 (*** p<0.001). (c) Representative symptoms of N. benthamiana plants agroinfiltrated with TYLCV-Mld and TYLCVΔC1 at 15 dpi. (d, e) Absolute quantification of VS (d) and CS (e) strand copy numbers of TYLCV-Mld (orange circles) and TYLCVΔC1 (blue triangles) in inoculated leaves at 1, 2, and 3 dpi. Values are expressed as log₁₀ (Number of molecules per ng of total DNA). Data represent four biological replicates from one independent experiment. Asterisks indicate significant differences between TYLCV-Mld and TYLCVΔC1 at each time point (*** p<0.001; ** p<0.01; * p<0.05).

Results

1. ASM pretreatment reduces total TYLCV DNA accumulation in agroinfiltrated leaves of Nicotiana benthamiana

To investigate the effect of ASM (1 mM) pretreatment on TYLCV infection, we agroinfiltrated a TYLCV infectious clone²³⁾ into ASM-treated or water-treated control leaves at 3 days after treatment and quantified relative total TYLCV DNA accumulation by qPCR at 1, 2, and 3 days post-inoculation (dpi). ASM-treated leaves showed significantly lower accumulation of total TYLCV DNA than water-treated controls at all three time points (Fig. 1a–c). ASM pretreatment reduced total TYLCV DNA accumulation by approximately 60% at 1 dpi and by approximately 90% at both 2 dpi and 3 dpi. Although viral DNA levels varied among the three independent experiments, the degree of ASM-mediated suppression was consistent and statistically significant at each time point. These results indicate that ASM effectively suppresses TYLCV accumulation in leaves inoculated by agroinfiltration.

Fig. 1. Acibenzolar-S-methyl (ASM) reduces total tomato yellow leaf curl virus (TYLCV) DNA accumulation in inoculated leaves at 1–3 days post-inoculation (dpi). Relative accumulation of total TYLCV-Mld DNA in the inoculated leaves of Nicotiana benthamiana at (a) 1 dpi, (b) 2 dpi, and (c) 3 dpi, quantified by quantitative PCR and normalized to the N. benthamiana reference gene NbITS. Individual data points from three independent experiments (each with four biological replicates) are shown in different colors. Asterisks indicate significant differences between the water-treated control and ASM treatments (*** p<0.001).

2. A replication-defective TYLCVΔC1 mutant fails to accumulate in agroinoculated leaves of Nicotiana benthamiana

Because TYLCV infection in the above experiments was initiated by agroinfiltration, the total TYLCV DNA detected by qPCR includes not only viral DNA produced during virus replication but also T-DNA delivered by Agrobacterium. A previous study demonstrated that SA-mediated defense responses inhibit T-DNA delivery by Agrobacterium,³³⁾ raising the possibility that the ASM-mediated reduction in TYLCV DNA accumulation may partly reflect impaired T-DNA delivery rather than restricted viral infection. To distinguish between these possibilities, we constructed a replication-defective TYLCV mutant, TYLCVΔC1 (Fig. 2a), to serve as a baseline for estimating T-DNA delivery efficiency. This mutant carries a frameshift mutation within the Rep (C1) coding sequence, resulting in a truncated protein comprising only its N-terminal 116 amino acids, which is predicted to abolish replication initiation activity.

At 3 dpi, total TYLCV DNA accumulation in TYLCVΔC1-infiltrated leaves was approximately 97% lower than in leaves inoculated with wild-type TYLCV-Mld (Fig. 2b), confirming that the frameshift mutation effectively abolished Rep function. This marked reduction in viral DNA was consistent with phenotypic observations at 15 dpi: plants inoculated with TYLCV-Mld developed characteristic disease symptoms of leaf curling and deformation in upper, uninoculated leaves, whereas plants inoculated with TYLCVΔC1 remained symptomless (Fig. 2c).

To further verify the replication deficiency of TYLCVΔC1, we quantified virion-sense (VS) and complementary-sense (CS) DNA strands by strand-specific qPCR. During TYLCV replication, the VS single-stranded DNA genome is converted to a replicative-form (RF) double-stranded DNA intermediate by forming CS from which new VS is produced. Both VS and CS strand levels of TYLCVΔC1 were significantly lower than those of TYLCV-Mld at all time points: VS was reduced by approximately 95.5%, 98.0%, and 98.8%, and CS by 82.1%, 96.2%, and 99.7% at 1, 2, and 3 dpi, respectively (Fig. 2d and 2e). Because the sole sequence difference between TYLCV-Mld and TYLCVΔC1 is a four-nucleotide insertion that would not be expected to affect T-DNA delivery, the substantially lower VS and CS levels of TYLCVΔC1 throughout the experiment confirm its replication deficiency. Together, these results establish TYLCVΔC1 as a replication-defective mutant suitable for separating the effects of ASM at the viral replication stage from its effects on T-DNA delivery.

3. ASM pretreatment inhibits TYLCV DNA accumulation independently of T-DNA delivery

To determine whether ASM-mediated suppression of TYLCV DNA accumulation reflects an effect on viral replication rather than T-DNA delivery, we analysed VS and CS strand accumulation of TYLCV-Mld and TYLCVΔC1 in leaves pre-treated with ASM or water. In TYLCV-Mld-infected plants, ASM significantly reduced both VS and CS strand levels relative to the water-treated control at 3 dpi (Fig. 3a and 3b), consistent with the significant reduction in total TYLCV DNA observed at the same time point (Fig. 1c). In contrast, ASM did not alter total TYLCV DNA accumulation in TYLCVΔC1-inoculated plants at 3 dpi (Fig. 3c). Strand-specific qPCR likewise showed no significant differences in VS or CS DNA levels between ASM-treated and control plants inoculated with TYLCVΔC1 (Fig. 3d and 3e). The absence of any ASM effect on TYLCVΔC1 accumulation, combined with the significant suppression of both VS and CS strands of TYLCV-Mld, demonstrates that ASM directly suppresses TYLCV DNA accumulation at the replication stage.

Fig. 3. ASM reduces VS and CS strand accumulation of TYLCV-Mld but not of TYLCVΔC1. Absolute numbers of molecules of virion-sense (VS) and complementary-sense (CS) strands of TYLCV-Mld (a, b) and TYLCVΔC1 (d, e), and relative total TYLCV DNA accumulation of TYLCVΔC1 (c), in inoculated leaves at 3 dpi from water-treated control and ASM-treated plants. (a) log₁₀ (Number of VS molecules) and (b) log₁₀ (Number of CS molecules) of TYLCV-Mld. (c) Relative total TYLCV DNA accumulation of TYLCVΔC1, quantified by standard qPCR and normalized to NbITS. (d) log₁₀ (Number of VS molecules) and (e) log₁₀ (Number of CS molecules) of TYLCVΔC1. VS and CS strand copy numbers in (a), (b), (d), and (e) were quantified by two-step strand-specific qPCR and expressed as log₁₀ (Number of molecules per ng of total DNA). Individual data points from three independent experiments (each with four biological replicates) are shown in different colors. Asterisks indicate significant differences between control and ASM treatments (*** p<0.001; ns, non-significant).

Discussion

We demonstrated that application of ASM significantly reduces TYLCV DNA accumulation in inoculated leaves, providing the first direct evidence that ASM targets early begomovirus infection. Although several previous studies have reported that ASM reduces disease severity and viral titers in non-inoculated upper leaves of begomovirus–infected plants,^20–22) none of these studies addressed which viral infection stage was targeted by ASM-mediated antiviral responses. In this study, we demonstrated that this suppression operates at the viral replication stage using strand-specific quantification of VS and CS DNA. To assess the potential impact of ASM treatment on Agrobacterium-mediated T-DNA delivery, we developed a replication-defective TYLCV mutant (TYLCVΔC1) as a control and examined early-stage viral accumulation. ASM treatment did not reduce total viral DNA accumulation or VS and CS DNA levels in TYLCVΔC1-inoculated leaves, confirming that the suppression of viral replication is due to an antiviral effect of ASM rather than impairment of T-DNA delivery by A. tumefaciens. These findings highlight viral replication as a key target of ASM-mediated defense against TYLCV.

Current findings raise the question of whether ASM-induced suppression of TYLCV can be sustained through later stages of infection. ASM has been reported to confer durable antiviral protection in several plant–virus systems through both local and systemic antiviral responses; for example, ASM remarkably suppresses tomato mosaic virus accumulation in Nicotiana tabacum inoculated leaves from 4 hr to 9 days post-inoculation, even when viral inoculation is performed at 7 days after ASM treatment, indicating a sustained local priming effect.³⁴⁾ Similarly, BTH/ASM reduces pepper golden mosaic virus (PepGMV) accumulation in both inoculated and systemic leaves of pepper (Capsicum annuum) at 10 dpi, demonstrating suppression of viral replication and systemic movement.²²⁾ Notably, this protective effect persists when PepGMV is inoculated at 10 days post-treatment of ASM,²²⁾ suggesting prolonged antiviral activity. ASM also restricts replication of PlAMV at the inoculation site and inhibits its loading into vascular tissues of N. benthamiana,^11,15) further demonstrating that ASM can suppress both local replication and systemic spread across different virus–host systems. To determine whether ASM mediates protection in TYLCV-infected N. benthamiana in the non-inoculated upper leaf by suppressing systemic movement, we investigated symptomatology at later stages of infection. ASM-treated plants remained completely symptomless in upper, non-inoculated leaves at 11 dpi, whereas water-treated plants developed typical TYLCV symptoms, including leaf curling, chlorosis, and stunting (Supplementary Fig. S1). The complete absence of symptoms in ASM-treated plants at 11 dpi indicates that ASM-mediated protection extends beyond the early replication stage, although whether this reflects suppressed systemic movement, reduced replication in systemic tissues, or both, remains to be determined by direct quantification of viral DNA in systemic leaves at extended time points. However, our interpretation is further supported by field evidence: a previous study demonstrated that spraying ASM (Bion 50WP) three times at 10-day intervals significantly reduced TYLCV disease severity in susceptible tomato (Solanum lycopersicum cv. Rambo) under both greenhouse and open-field conditions with high whitefly pressure.²⁰⁾ This sustained suppression under field conditions suggests that ASM-mediated antiviral effects maintain efficacy over extended periods. Taken together, the antiviral effects of ASM are not limited to the inoculated leaves and highlight their potential for suppressing systemic infection as a reliable, sustained component for managing TYLCV under field conditions.

TYLCV infects plants via whitefly feeding under field conditions, whereas inoculation sites differ from those of agroinfiltration-based infection. Whiteflies transmit the virus directly to phloem sieve elements⁷⁾ while agroinfiltration primarily delivers T-DNA to mesophyll cells,³⁵⁾ although phloem parenchyma cells within the infiltrated tissue are also spatially accessible to Agrobacterium during pressure infiltration. Consistent with this, CS strand DNA was detectable as early as 1 dpi in our experiment, indicating that TYLCV replication initiates rapidly in the infiltrated tissue including potentially in phloem-associated cells. Furthermore, the molecular mechanism by which ASM suppresses viral DNA accumulation likely operates through nuclear epigenetic pathways, as discussed below, which are not expected to be intrinsically cell-type-specific. Together, these considerations support the mechanistic relevance of our agroinfiltration-based findings to natural whitefly-mediated infection. Complementary studies using whitefly-mediated transmission would further determine whether ASM exerts comparable antiviral effects on TYLCV accumulation under natural infection conditions.

The underlying mechanism of suppressing TYLCV accumulation in the inoculated leaves yet remains to be elucidated. Begomoviruses replicate in the nucleus, and their double-stranded replicative-form DNA is assembled with host histones into nucleosome-containing minichromosomes,^36,37) making the viral genome directly susceptible to host epigenetic silencing machinery. RNA-directed DNA methylation (RdDM) is an established host defense against begomovirus minichromosomes, as methylation-deficient Arabidopsis mutants display markedly enhanced susceptibility to geminivirus infection³⁸⁾; whether ASM can promote RdDM-mediated silencing specifically at viral DNA remains an open question. SA signaling also induces the formation of the HAC–NPR1–TGA histone acetyltransferase complex, which drives large-scale histone acetylation at defense gene loci,³⁹⁾ potentially redistributing histone acetyltransferase activity away from the begomovirus minichromosome and thereby shifting it toward a repressive chromatin state. Consistent with the importance of the epigenetic state of the viral minichromosome during infection, the TYLCV V2 protein suppresses transcriptional gene silencing by interacting with histone deacetylase HDA6 and competing with the maintenance methyltransferase MET1 for HDA6 binding, thereby reducing cytosine methylation of the viral genome.⁴⁰⁾ Whether ASM-induced changes in the epigenetic state of the TYLCV minichromosome account for the suppression of viral DNA accumulation observed here warrants direct investigation in future studies.

Conclusion

This study demonstrates that ASM-mediated suppression of TYLCV infection operates by reducing viral DNA accumulation during the replication stage, as evidenced by suppression of complementary-sense and virion-sense DNA synthesis. The combination of strand-specific quantification and a replication-defective mutant provided a mechanistic experimental framework for distinguishing replication-dependent accumulation from Agrobacterium-mediated T-DNA delivery. This approach may also serve as a generalizable strategy for investigating plant activator-mediated resistance against other begomoviruses.

Conflict of interest

The authors declare no competing interests.

Author Contributions

Conceptualization, K.K.; methodology, N.A., K.S., and K.K.; validation, H.S. and K.K.; investigation, N.A., K.S., and K.K.; resources, K.K.; original writing, N.A. and K.K.; writing—review and editing, H.S., K.S., M.N.I., and K.K.; visualization, N.A. and M.N.I.; supervision, K.K.; project administration, K.K.; funding acquisition, T.A. and K.K. All authors have read and agreed to the published version of the manuscript.

Electronic supplementary materials

The online version of this article contains supplementary materials (Supplementary Table S1 and Supplementary Figure S1), which are available at https://www.jstage.jst.go.jp/browse/jpestics/.