Abstract
Chemically induced host resistance is useful phenomenon for comprehending host innate immunity against plant pathogens. To evaluate immune priming against cucumber mosaic virus (CMV) infection, we sprayed benzothiadiazole (BTH), a salicylic acid (SA) analog, on either one-leaf or entire plants of Nicotiana benthamiana (Nb). The chemical immune priming downregulated transcript level of Salicylic acid glucosyltransferase (SAGT) and upregulated that of Pathogenesis-related protein 1a (PR1a) in the treated and untreated upper leaves of the one-leaf-treated Nb. Fluorescent-protein-expressing CMV vectors revealed inhibitory effect on initial infection and delayed systemic infection in the one-leaf-treated Nb inoculated with the virus vectors, and the BTH was more effective than SA. However, the BTH-induced host defensive response was attenuated when wild-type Nb was infected with the virus before BTH spray of one leaf and by constitutive expression of the 2b protein of CMV (2b) in a transgenic Nb line after BTH spray of one leaf. SAGT was downregulated and PR1a upregulated in the upper, middle, and lower leaves when the entire plant was treated with BTH. Viral levels were reduced in BTH-treated middle and -untreated upper leaves, but not in the emerging and uppermost leaves after the whole plant spray. Taken together, these results suggest that the chemical immune priming is more effective than SA to retard systemic infection of CMV. However, the BTH-mediated antiviral responses can be attenuated by prior CMV infection and by constitutive expression of 2b in Nb, and does not efficiently suppress CMV infection in later-emerging leaves.
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Introduction
Plants have developed defense mechanisms to prevent lethal damage caused by numerous pathogens. The use of defense activators to induce immune priming in plants throughout their life, therefore, has been considered a promising strategy to suppress serious disease in production fields. Among such activators, benzothiadiazole (BTH), also known as 1,2,3-benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester or ASM, induces long-lasting host defense responses that are associated with systemically acquired resistance (SAR) (Ryals et al. 1996). After conversion of BTH into acibenzolar catalyzed by Salicylic acid binding protein 2, acibenzolar induces SAR in tobacco (Tripathi et al. 2010). BTH can inhibit plant infection by viruses, but the molecular mechanisms underlying the antiviral effect of BTH remain unknown. We previously found that dipping of leaf into 0.12 mM (25 ppm) BTH downregulated the Salicylic acid glucosyltransferase gene (SAGT) and upregulated the Pathogenesis-related protein 1a gene (PR1a) in the BTH-treated leaf without any phytotoxic effects (Kobayashi et al. 2020). SAGT catalyzes the conversion of salicylic acid (SA) into SA2-O-β-d-glucoside (SAG), which is not required to induce SAR in some plant species including Nicotiana benthamiana (Nb) (Loake and Grant 2007; Pastor et al. 2013). PR1a is a well-known indicator gene for SA-induced SAR.
The plant pathogen cucumber mosaic virus (CMV), the type member of the genus Cucumovirus (family Bromoviridae) (Mochizuki and Ohki 2012; Palukaitis and García-Arenal 2003), is distributed worldwide. CMV RNA2 encodes the 2b protein (2b), which is one of the best-characterized RNA silencing suppressors (RSS) (Ding et al. 1995; Goto et al. 2007; Ji and Ding2001; Nakahara and Masuta 2014). Furthermore, 2b has a critical role in counteracting SA-inducible host resistance and plays an indirect role in facilitating SA biosynthesis in tobacco (Ji and Ding 2001; Lewsey et al. 2010).
To dissect the multiple functions of 2b in plant tissues, Takeshita et al. (2012) developed fluorescent protein-expressing CMV vectors—the 2b-expressing vector CMV-DsRed and 2b-deficient vector CMVΔ2b-DsRed, which helped elucidate that 2b is also associated with virus unloading from the vascular system in systemically infected leaves of Nb. We believe that these CMV vectors can also help to determine the role of 2b in counteracting BTH-mediated antiviral responses.
In the present study, we extended our previous study (Kobayashi et al. 2020) to better understand the immune priming induced after BTH treatment of one or all leaves of Nb plants. To evaluate the induced resistance, we analyzed changes in viral RNA and SAGT and PR1a transcript levels after spraying either one leaf (hereafter “one-leaf” treatment) or all leaves (“whole-plant” treatment) on Nb plants with BTH. Plants were then inoculated with CMV-DsRed or CMVΔ2b-DsRed and observed to monitor virus spread after BTH-induced defensive responses and to elucidate the function of 2b of CMV in the immune priming in one-leaf-treated Nb.
Materials and methods
Plants, chemical treatment, and virus
Nb plants were grown in a growth chamber at 25 °C with a 12-h photoperiod. Construction of the 2b GFP-expressing transgenic Nb line was described by Takeshita et al. (2012). For treating one leaf on a plant (n = 5–16), both surfaces of the leaf on the potted plant were sprayed with 0.12 mM BTH (Syngenta Japan K. K., Tokyo, Japan), 0.12 mM SA or water (1 ml per leaf) without a water rinse after treatment. For treating all leaves on a plant (n = 4–12), both surfaces of all leaves on the potted plants were sprayed with 0.12 mM BTH or water (2 ml per plant) without a water rinse after treatment. Wild-type CMV-Y was propagated and purified as described by Suzuki et al. (1991). CMV-DsRed (designated as A1Ds in our previous report; Takeshita et al. 2012) and CMVΔ2b-DsRed (designated as H1Ds in our previous report; Takeshita et al. 2012) were prepared and used to inoculate leaves mechanically as described by Takeshita et al. (2012) (n = 6–16). All statistical analyses were performed using R version 4. 1. 0. (R Core Team 2021).
RNA extraction and RT-qPCR
Total RNA was extracted from individual inoculated (treated) leaves, or from upper, non-inoculated (untreated) leaves for one-leaf-treated Nb plants or from combined leaves from lower, middle, upper, or uppermost positions in whole-plant-treated Nb plants with ISOSPIN Plant RNA (NIPPON GENE, Tokyo, Japan) according to the user manual. We used RT-qPCR to quantify relative accumulation levels of virus RNA and transcript levels of SAGT and PR1a in leaves from Nb plants at 6, 12, and 48 h after one-leaf and at 6, 48, and 72 h after whole-plant BTH treatment essentially as described by Kobayashi et al. (2020). For comparative RT-qPCR targeting of the host gene and CMV RNA, 50 ng of total RNA from Nb, the GoTaq 1-Step RT-qPCR System (Promega, Madison, WI, USA), and the Thermal Cycler Dice RealTime System Single//TP900 (TaKaRa Bio, Kusatsu, Japan) were used according to the manufacturer’s procedures. Transcript levels for each target gene were normalized using L23 from Nb.
Imaging of DsRed2 fluorescence
Images of DsRed2 fluorescence in leaves were acquired using a DsRed filter and MZ10 F modular stereomicroscope (Leica Microsystems, Wetzlar, Germany). Nb plants (n = 6–16) were observed for each inoculum. The numbers of fluorescent spots were counted using MZ10 F. The size of DsRed2 fluorescent spots was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The image data were obtained from each inoculation assay, which was repeated once or twice.
Evaluation of symptom severity
Symptoms on leaves were scored for severity essentially using the methods of Takeshita et al. (2013) (Fig. S1) as 0, no symptoms; 1, vein clearing area on < 50% of leaf area; 2, vein clearing area on > 50% of the leaf area; 3, vein clearing and/or mosaic on entire leaf (Fig. S1). An overall severity score (D) for each plant was then calculated using the individual leaf severity scores as D = (Σdi)/n/3 × 100, where di is severity score of the ith leaf, n is the number of leaves from sector 3 (upper leaves) (n = 3) and from sector 4 (uppermost leaves) (n = 3), and 3 is the highest severity score.
Results and discussion
Kobayashi et al. (2020) reported that when one leaf on the Nb plant was dipped into 0.12 mM BTH, CMV accumulation and virus-induced systemic symptoms were suppressed compared to the control at 5 days post inoculation (dpi). In the present study, we first assessed the effects of this immune priming by BTH on transcript levels of SAGT and PR1a in the BTH-treated and the BTH-untreated upper leaves of Nb plants. For the one-leaf treatment on Nb plants at the 7th–8th leaf stage, the 3rd or 4th true leaf was sprayed with 0.12 mM BTH (or 0.12 mM SA). In the BTH-treated 3rd or 4th leaves and the BTH-untreated 4th–6th or 5th–7th leaves of the one-leaf treated plants, SAGT transcript levels did not differ significantly from those in the water- and SA-treated plants until 48 h post treatment (hpt), when they were significantly lower (Fig. 1). Transcript levels of PR1a in the BTH-treated and the BTH-untreated upper leaves of the one-leaf-treated plants significantly increased compared to levels in the water- and the SA-treated plants at 48 hpt (Fig. 2). In the individual SA-treated leaves, PR1a transcript levels transiently increased until 12 hpt, then were significantly lower by 48 hpt compared to levels in the BTH-treated plants. On the other hand, PR1a transcript levels in the SA-untreated upper leaves did not increase significantly within 48 hpt. Considering our previous results from Nb infected with CMV (Kobayashi et al. 2020), the present results suggest that the BTH spray efficiently primed the host for resistance in the untreated, upper leaves, but SA did not.
To visualize the effects of BTH (or SA) on the spread of CMV in plants, we examined Nb for DsRed2 fluorescence from the CMV vectors, CMV-DsRed (2b-expressing) and CMVΔ2b-DsRed (2b-deficient) (Takeshita et al. 2012). The one-leaf BTH treatment at 2 days before virus inoculation prevented extensive distribution of CMV-DsRed and CMVΔ2b-DsRed within the majority of the inoculated and the upper, non-inoculated leaves at 9 days post inoculation (dpi) although differences between BTH treatment and control checks were less pronounced when inoculated with CMV∆2b-DsRed (Fig. 3a, b). On the other hand, suppression of CMV-DsRed and CMVΔ2b-DsRed spread by prior treatment of Nb with SA was less effective than treatment with BTH at 2 days before inoculation (Fig. 3a, b). There was no large delay at 9 dpi in CMV-DsRed and CMVΔ2b-DsRed infections in the upper, non-inoculated leaves of Nb treated with BTH (or SA) at 2 dpi (Fig. 3a, b). These results suggest that prior treatment of Nb with BTH caused a delay and inhibition of extensive distribution of CMV into the BTH-untreated leaves of the plants. Furthermore, the inhibitory effects of BTH on virus movement in Nb appeared to be more effective than those of exogenous SA.
To elucidate functions of 2b of CMV in the BTH-mediated immune priming, we inoculated transgenic Nb plants that constitutively express 2b of CMV-Y (2b-Nb), with CMV∆2b-DsRed. In this one-leaf BTH (or SA) treatment assay, we observed less suppression of systemic virus movement when BTH was applied 2 days before inoculation of 2b-Nb with CMVΔ2b-DsRed (Fig. 3c). The results suggested that constitutive expression of 2b can attenuate the preinoculation BTH-mediated immune priming in Nb. Our approach in this study for the first time revealed a possible role for the 2b gene of CMV in attenuating even the BTH-responsive resistance, in addition to its function against SA-related resistance reported by Lewsey et al. (2010). However, further analyses of the expression levels of SAGT and PR1a in BTH-treated and non-treated 2b-Nb will be necessary to conclude whether interference with immune priming by 2b, rather than enhancement of the levels of CMV∆2b-DsRed and DsRed2 fluorescent protein by overexpression of 2b, has an important role in the attenuation of the BTH (or SA)-mediated immune priming in Nb or not.
The number and size of fluorescent spots in the inoculated leaves of one-leaf BTH-treated and -untreated Nb were visualized after inoculation with CMV-DsRed and CMVΔ2b-DsRed (Fig. 4). The average number of fluorescent spots from CMV-DsRed (or CMVΔ2b-DsRed) was reduced to less than half of those in BTH-untreated control checks, indicating an inhibitive effect on initial infection (Fig. 4a–c, e–g). However, the average size of spots in the inoculated leaves was equivalent to that in BTH-untreated control checks (Fig. 4d, h). In this assay, single-cell infections with CMV-DsRed or CMVΔ2b-DsRed were not observed in the BTH-treated and -untreated Nb at 2 dpi. These results indicate that there are no significant differences in cell-to-cell movement of CMV-DsRed between the BTH-treated and -untreated Nb. Subsequently, effects of BTH treatment on systemic spread of CMV-DsRed and CMVΔ2b-DsRed were evaluated by the time-course analysis in one-leaf BTH-treated Nb (Figs. 5, S2). Appearance of DsRed2 fluorescence signal from CMV-DsRed in the upper leaves of the one-leaf BTH-treated Nb was significantly delayed compared with that of the BTH-untreated at least until 5 dpi (Fig. 5a–c). The results suggest that prior treatment with BTH retarded long-distance movement rather than cell-to-cell movement of CMV. Meanwhile, the DsRed2 fluorescent signal from CMVΔ2b-DsRed in the 3rd and 4th leaves of the one-leaf BTH-treated Nb was equivalent to that of the BTH-untreated Nb control checks at least until 5 dpi (Fig. 5e, f). The results from CMV-DsRed and CMVΔ2b-DsRed suggest that BTH-mediated resistance slows the systemic spread of CMV-DsRed more than CMV∆2b-DsRed, and that 2b of CMV contributes to BTH-mediated resistance in the context of virus infection.
After focusing on one-leaf BTH treatment to analyze the chemical immune priming in Nb, we needed to evaluate the effects of whole-plant treatments with BTH on chemical immune priming as would be used in practice. To determine whether the SAGT and PR1a expression profiles at 6, 48, and 72 h after the whole-plant treatment with BTH are similar to those after the one-leaf treatment (Figs. 6, 7), we used Nb at the 9th–11th leaf stage and defined four leaf sectors on the plants to sample leaves based on the number of leaves on the plant: (1) lower (e.g., 2nd–4th or 3rd–5th leaves), (2) middle (e.g., 5th –7th, 6th–8th leaves), (3) upper (e.g., 8th–10th, 9th–11th leaves consist of young leaves), and (4) uppermost (e.g., 11th–13th, 12th–14th leaves consist of emerging leaves including a terminal bud at the time point of BTH treatment). Transcript levels of SAGT in the BTH-treated Nb were greatly downregulated in the lower, middle, and upper leaves after 48 hpt (Fig. 6d–i). In contrast, PR1a transcripts in the lower, middle, and upper leaves of the BTH-treated Nb plants had higher levels than in the corresponding positions of the water-treated after 48 hpt (Fig. 7d–i). The results suggested that the induction of immune priming by BTH was similar irrespective of leaf position. Nor did the BTH-treated and -untreated Nb plants, regardless of treatment method, differ significantly in fresh mass (Fig. S3), suggesting that one spray of BTH at 0.12 mM onto the whole plant does not cause any severe developmental alterations in the plants.
To examine the suppressive effect of the whole-plant treatment with BTH on viral diseases, the middle leaves (leaf sector 2) of Nb were inoculated with CMV-Y at 2 days post BTH treatment. Whole-plant treatment significantly reduced symptom severity through at least 9 dpi (11 days post BTH treatment) on the upper leaves (leaf sector 3) that were well expanded during the BTH spray (Fig. 8a). However, symptoms were not suppressed on the uppermost leaves (the leaf sector 4) that developed after whole-plant BTH treatment (Fig. 8b). In addition, levels of CMV-Y after the whole-plant BTH treatment were significantly lower in the middle (inoculated) and upper leaves (leaf sectors 2 and 3), but not in the uppermost leaves (leaf sector 4) (Fig. 9a–c). The results suggest that whole-plant BTH treatment can suppress long-distance movement of CMV-Y, whereas BTH does not efficiently induce resistance against CMV-Y in later-emerging leaves. Moreover, the results indicate the possibility that SAGT and PR1a can serve as negative and positive markers of this immune priming, respectively. Our group (Takeshita et al. 2013) showed that spraying 0.12 mM BTH on all leaves of melon seedlings at the 1st to 2nd leaf stage induces antiviral host responses against cucurbit chlorotic yellows virus in potted melon without any progressive phytotoxicity. Foliar application (50 or 100 mg/l: approximately 0.12 mM or 0.24 mM) of BTH at weekly intervals significantly reduced rose rosette disease caused by rose rosette emaravirus without negatively affecting flowering or plant growth (Babu et al. 2022). Furthermore, Doungous et al. (2021) suggested that BTH foliar sprays induce resistance against African cassava mosaic virus and East African cassava mosaic Cameroon virus in Nb and their whitefly vector. Considering the previous and present results from our group and others, multiple spraying onto whole plants with BTH are effective for long-lasting disease suppression.
Using the two CMV vectors, CMV-DsRed and CMVΔ2b-DsRed, we visualized the inhibitory effect of BTH on virus spread in the BTH-treated Nb plants. In particular, prior treatment with BTH resulted in considerable restriction of systemic virus infection (Fig. 3a, b). Matsuo et al. (2019) reported suppression of viral RNA replication of plantago asiatica mosaic virus (PlAMV) by BTH treatment in protoplasts. Subsequently, Novianti et al. (2021) visualized that loading of GFP-expressing PlAMV into vascular tissues in inoculated leaves was restricted after pretreatment with BTH. Chivasa et al. (1997) reported that SA treatment can induce resistance to tobacco mosaic virus accumulation in inoculated leaf tissue of tobacco. Furthermore, Naylor et al. (1998) suggested that a delay in development of systemic symptoms in tobacco by SA treatment before inoculation with CMV is due to inhibition of long-distance CMV movement rather than inhibition of CMV replication. Taking these reports into account, restriction of unidentified steps in initial infection and in long-distance movement of CMV might lead to the suppression and delay of loading of virus into vascular systems in the inoculated leaves treated with BTH.
Lewsey et al. (2010) showed that the levels of CMV and SA induced by CMV∆2b were much lower than those by CMV in Arabidopsis thaliana and that CMV 2b facilitated the elicitation of SA biosynthesis. In addition, Zhou et al. (2014) identified the 2b domains required for SA priming, SA evasion, RNA silencing suppression, and nucleolar localization and found that the PR1 accumulation was elevated in tobacco leaves inoculated with CMV, but not with CMV∆2b at 4 dpi. For the BTH effect on SA levels, Kobayashi et al. (2020) demonstrated that BTH greatly downregulated SAGT resulting in a decrease in SAG, while it upregulated PR1a in tobacco, possibly through an increase of SA. Based on this previous information, we propose an explanation for the observed difference in the infection dynamics between CMV-DsRed and CMV∆2b-DsRed. We assume that 2b of CMV-DsRed stimulated SA biosynthesis, which leads to further enhancement of the SA-mediated resistance when BTH was applied, and that the virus is eventually restricted in upper leaves at the step of viral loading into vascular tissues, resulting in a delay in viral long-distance movement (Fig. 5a–c). On the other hand, CMV∆2b-DsRed did not induce the SA-mediated resistance due to decreased SA, and thus it may have been less restricted during the initial infection (Fig. 4e–h). Although the virus does not resist host RNA silencing without its RSS 2b, it can eventually move into vascular tissues and the upper leaves of the [one-leaf BTH-treated] Nb without any significant delay (Fig. 5e, f). However, the spread of both viruses in the nonvascular tissues of the upper leaves is still considerably restricted by the BTH-mediated systemic immune priming, leading to the induction of the resistance pathway downstream of SA accumulation (Friedrich et al. 1996; Lawton et al. 1996). We therefore presume that after the BTH-immune priming decays at a later infection stage, CMV-DsRed but not CMV∆2b-DsRed can extensively spread into newly developing leaves because 2b functions as an RSS (Nakahara and Masuta 2014) to enhance viral unloading into nonvascular tissues in systemic leaves (Takeshita et al. 2012).
Further microscopic observations of plant in which virus movement is suppressed will lead to a better understanding of the mode of action of the immune priming by BTH. In addition, serial foliar applications and soil irrigation with BTH are necessary to evaluate the practical efficacy of the chemical immune priming and any side effects on plant growth.
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Acknowledgements
We thank Syngenta Japan K. K. for providing BTH for the present work. This work was partially supported by JSPS KAKENHI grant number JP18K05655 and JP21J15403.
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10327_2022_1113_MOESM1_ESM.pptx
Supplementary file1 Scoring systemic symptoms and representative leaves of Nicotiana benthamiana treated with BTH before inoculation with CMV-Y. 0, no symptoms; 1, vein clearing area < 50% of leaf area; 2, vein clearing area > 50%; 3, vein clearing and/or mosaic on entire leaf (PPTX 1256 KB)
10327_2022_1113_MOESM2_ESM.pptx
Supplementary file2 DsRed2 fluorescence signal in upper leaves of one-leaf BTH-treated Nicotiana benthamiana. The plants were inoculated with a DsRed2-expressing CMV vector before BTH-treatment. a Representative fluorescence signal initially appeared on an upper, non-inoculated leaf of Nb inoculated with CMV-DsRed and b the same leaf imaged with bright-field optics (PPTX 1919 KB)
10327_2022_1113_MOESM3_ESM.pptx
Supplementary file3 Fresh biomass of Nicotiana benthamiana (Nb) at 14 days after a whole-plant treatment and b one-leaf treatment with 0.12 mM BTH. Error bars at top of histobars are standard errors. Means did not differ significantly between the BTH-treated and water-treated control plants in either treatment in Student’s t-test (p < 0.05, n = 6) (PPTX 94 KB)
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Kobayashi, Y., Hyodo, A., Kuwata, S. et al. Benzothiadiazole is superior to salicylic acid for immune priming in Nicotiana benthamiana, and the 2b protein of cucumber mosaic virus affects viral infection dynamics in the artificially induced resistance. J Gen Plant Pathol 89, 109–121 (2023). https://doi.org/10.1007/s10327-022-01113-1
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DOI: https://doi.org/10.1007/s10327-022-01113-1