rgs-CaM Detects and Counteracts Viral RNA Silencing Suppressors in Plant Immune Priming

doi:10.1128/JVI.00761-17

. 2017 Sep 12;91(19):e00761-17.

doi: 10.1128/JVI.00761-17. Print 2017 Oct 1.

rgs-CaM Detects and Counteracts Viral RNA Silencing Suppressors in Plant Immune Priming

Eun Jin Jeon¹, Kazuki Tadamura¹, Taiki Murakami¹, Jun-Ichi Inaba¹, Bo Min Kim¹, Masako Sato², Go Atsumi¹, Kazuyuki Kuchitsu³, Chikara Masuta^{1

2}, Kenji S Nakahara^{4

2}

Affiliations

¹ Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan.
² Research Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan.
³ Department of Applied Biological Science and Research Institute for Science and Technology, Tokyo University of Science, Noda, Chiba, Japan.
⁴ Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan knakahar@res.agr.hokudai.ac.jp.

PMID: 28724770
PMCID: PMC5599751
DOI: 10.1128/JVI.00761-17

rgs-CaM Detects and Counteracts Viral RNA Silencing Suppressors in Plant Immune Priming

Eun Jin Jeon et al. J Virol. 2017.

. 2017 Sep 12;91(19):e00761-17.

doi: 10.1128/JVI.00761-17. Print 2017 Oct 1.

Authors

Eun Jin Jeon¹, Kazuki Tadamura¹, Taiki Murakami¹, Jun-Ichi Inaba¹, Bo Min Kim¹, Masako Sato², Go Atsumi¹, Kazuyuki Kuchitsu³, Chikara Masuta^{1

2}, Kenji S Nakahara^{4

2}

Affiliations

¹ Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan.
² Research Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan.
³ Department of Applied Biological Science and Research Institute for Science and Technology, Tokyo University of Science, Noda, Chiba, Japan.
⁴ Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan knakahar@res.agr.hokudai.ac.jp.

PMID: 28724770
PMCID: PMC5599751
DOI: 10.1128/JVI.00761-17

Abstract

Primary infection of a plant with a pathogen that causes high accumulation of salicylic acid in the plant typically via a hypersensitive response confers enhanced resistance against secondary infection with a broad spectrum of pathogens, including viruses. This phenomenon is called systemic acquired resistance (SAR), which is a plant priming for adaption to repeated biotic stress. However, the molecular mechanisms of SAR-mediated enhanced inhibition, especially of virus infection, remain unclear. Here, we show that SAR against cucumber mosaic virus (CMV) in tobacco plants (Nicotiana tabacum) involves a calmodulin-like protein, rgs-CaM. We previously reported the antiviral function of rgs-CaM, which binds to and directs degradation of viral RNA silencing suppressors (RSSs), including CMV 2b, via autophagy. We found that rgs-CaM-mediated immunity is ineffective against CMV infection in normally growing tobacco plants but is activated as a result of SAR induction via salicylic acid signaling. We then analyzed the effect of overexpression of rgs-CaM on salicylic acid signaling. Overexpressed and ectopically expressed rgs-CaM induced defense reactions, including cell death, generation of reactive oxygen species, and salicylic acid signaling. Further analysis using a combination of the salicylic acid analogue benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and the Ca²⁺ ionophore A23187 revealed that rgs-CaM functions as an immune receptor that induces salicylic acid signaling by simultaneously perceiving both viral RSS and Ca²⁺ influx as infection cues, implying its autoactivation. Thus, secondary infection of SAR-induced tobacco plants with CMV seems to be effectively inhibited through 2b recognition and degradation by rgs-CaM, leading to reinforcement of antiviral RNA silencing and other salicylic acid-mediated antiviral responses.IMPORTANCE Even without an acquired immune system like that in vertebrates, plants show enhanced whole-plant resistance against secondary infection with pathogens; this so-called systemic acquired resistance (SAR) has been known for more than half a century and continues to be extensively studied. SAR-induced plants strongly and rapidly express a number of antibiotics and pathogenesis-related proteins targeted against secondary infection, which can account for enhanced resistance against bacterial and fungal pathogens but are not thought to control viral infection. This study showed that enhanced resistance against cucumber mosaic virus is caused by a tobacco calmodulin-like protein, rgs-CaM, which detects and counteracts the major viral virulence factor (RNA silencing suppressor) after SAR induction. rgs-CaM-mediated SAR illustrates the growth versus defense trade-off in plants, as it targets the major virulence factor only under specific biotic stress conditions, thus avoiding the cost of constitutive activation while reducing the damage from virus infection.

Keywords: RNA interference; RNA silencing suppressor; calmodulin-like protein; cucumber mosaic virus; innate immunity; plant viruses; priming; salicylic acid signaling; systemic acquired resistance.

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Figures

**FIG 1**
Overexpressed and ectopically expressed rgs-CaM elicits immune responses in tobacco, implying a link between rgs-CaM and salicylic acid signaling. (Ai) Transgenic tobacco plants overexpressing rgs-CaM showed phenotypic characteristics indicating activation of immune responses, such as necrosis and dwarfing, at 7 weeks after sowing of transgenic lines 16 (rgs-CaM16) and 23 (rgs-CaM23). (Aii) Within each of these two transgenic lines, the severity of the lesion mimic phenotype was variable. Individual plants from each line are shown in order from mild (1) to severe (6) phenotypes. (Aiii) These individuals were confirmed to have the rgs-CaM transgene by detecting the 35S and rgs-CaM nucleotide sequences by PCR. PCR products amplified from the binary vector pBE2113-rgs-CaM, with which tobacco plants were transformed, with the same primer pairs were loaded as a control (lane C). (B and C) Cell death (B) and generation of reactive oxygen species (ROS) (C) in leaves were compared between transgenic tobacco overexpressing rgs-CaM and the wild type (WT) by Evans blue and 2′,7′-dichlorofluorescein diacetate (H₂DCF) staining, respectively. BF, bright-field images. (Di) Expression of *PR1a*, an indicator of salicylic acid signaling, was investigated by Northern blotting. Samples from seven plants of transgenic line 16 were ordered from left to right by increasing severity of the phenotype. The *PR1a* mRNA level was investigated by Northern blotting. Overexpression of rgs-CaM in these plants was confirmed by Western blotting for its protein and by semiquantitative RT-PCR (sqRT-PCR) for its mRNA. Wild-type (WT) tobacco was used as a control. (Dii) Transgenic line 23, which overexpressed rgs-CaM and showed a phenotype similar to that of line 16, was also shown by Northern blotting to induce *PR1a* expression; as in the case of line 16, expression varied within the line. Coomassie brilliant blue-stained (CBB) and ethidium bromide-stained (*rRNA*) gels are shown as loading controls.

**FIG 2**
Defense responses and salicylic acid signaling were induced by transient expression of rgs-CaM. (A) A PVX vector expressing rgs-CaM (PVX-rgs-CaM), a PVX vector expressing the subgenomic RNA containing the rgs-CaM open reading frame without its initiation codon [PVX-rgs-CaM(−ATG)], and an empty vector (PVX) were inoculated into wild-type tobacco (cv. Xanthi) plants. Inoculated leaves at 7 days postinoculation are shown. Their *PR1a* expression was investigated by real-time PCR. The mRNA levels relative to that of mock-inoculated plants are shown in the bar graph (n = 4). Error bars indicate standard errors (SE). Student's t test was applied to the data; **, P < 0.01. (B) Protoplasts prepared from wild-type tobacco plants were transfected with expression cassettes with the rgs-CaM cDNA and the modified cDNA without the initiation codon [rgs-CaM(−ATG)] and stained with Evans blue. Black bars, 50 μm. The cell death rate (Evans blue-stained cells/total cells) is shown in the bar graph (n = 5). Error bars indicate SE. Student's t test was applied to the data; *, P < 0.05, relative to protoplasts without transfection (Cont). (C) When the protoplasts described in panel B were stained with H₂DCF, protoplasts generating ROS were detected among those transfected with the rgs-CaM expression cassette. Among protoplasts transfected with rgs-CaM(−ATG) or not transfected (Cont), no H₂DCF signal was detected. BF, bright-field images. White bars, 10 μm.

**FIG 3**
Susceptibility of rgs-CaM knockdown tobacco plants to PVX and salicylic acid signaling in response to PVX infection. (A) PVX was inoculated into rgs-CaM knockdown (IR-rgs-CaM) and wild-type (WT) tobacco plants. Accumulation of PVX CP and rgs-CaM and of PVX genomic and subgenomic RNAs (*gPVX* and *sgPVX*, respectively) was investigated in the inoculated leaves by Western and Northern blotting, respectively, at 1 and 3 days postinoculation (dpi). (B) The same type of inoculation as described for panel A was done with more individual plants (n = 8). Accumulation of PVX genomic RNA was measured by real-time PCR using a pair of primers for amplification of a partial cDNA sequence of viral RNA-dependent RNA polymerase (RdRp). Similarly, accumulation of PVX RNAs, including both genomic and subgenomic RNAs, was measured with a pair of primers for amplification of a partial cDNA of viral coat protein (CP). (C and D) The levels of *PR1a* (C) and *rgs-CaM* (D) mRNA were investigated by real-time PCR (n = 5). mRNA levels relative to those of mock-inoculated plants are shown. Bars indicate SE. Student's t test was applied to the data; *, P < 0.05. Coomassie brilliant blue-stained (CBB) and ethidium bromide-stained (*rRNA*) gels are shown as loading controls of Western and Northern blotting, respectively.

**FIG 4**
Implication of rgs-CaM involvement in salicylic acid signaling in response to infection by CMV. CMV-Y (A and B) and CMV lacking 2b (CMVΔ2b) (C and D) were inoculated into wild-type (WT) and rgs-CaM knockdown (IR-rgs-CaM) tobacco plants, and accumulation of CMV CP, 2b, and rgs-CaM proteins and CMV genomic and subgenomic RNAs (*gCMV* and *sgCMV*, respectively) (A and C) and the *PR1a* (experiment [Exp.] 1) and *rgs-CaM* mRNAs (B and D) were investigated (n = 3) as described for Fig. 3. (B, Exp. 2) The same type of inoculation as used for experiment 1 was done with more individual plants (n = 9), and the *PR1a* mRNA level was investigated. Error bars indicate SE. Student's t test was applied to the data; * and **, P < 0.05 and P < 0.01, respectively. Coomassie brilliant blue-stained (CBB) and ethidium bromide-stained (*rRNA*) gels are shown as loading controls.

**FIG 5**
Model of salicylic acid signaling in response to CMV infection in tobacco plants (A to C) and salicylic acid signaling in response to wounding stress (D and E). (A to C) In this model, rgs-CaM functions as an immune receptor that perceives viral RSS and Ca²⁺. Tobacco plants induce salicylic acid signaling when rgs-CaM perceives both 2b and Ca²⁺ as CMV infection cues in an inoculated leaf (A) but not when rgs-CaM perceives either 2b or Ca²⁺ alone, e.g., in a noninoculated upper leaf (B) or in a leaf inoculated with CMV lacking 2b (CMVΔ2b) (C). (D) Transgenic tobacco plants expressing CMV 2b and ClYVV HC-Pro were microperforated by bundled pins. Immediately after microperforation, cell death (middle panels) and ROS generation (lower panels) were visualized by staining leaves with Evans blue or H₂DCF, respectively. (E) Expression of *PR1a* was investigated by RT-PCR at different time points after microperforation of tobacco leaves.

**FIG 6**
Induction of salicylic acid signaling in viral RNA silencing suppressor (RSS)-expressing tobacco plants with Ca²⁺ influx. (A) A Ca²⁺ ionophore, A23187 (75 μM), was infiltrated into leaves of wild-type (WT) and transgenic tobacco plants expressing 2b, HC-Pro, or CMV CP. At 24 h after infiltration, the mRNA levels of *PR1a* were investigated by Northern blotting. + and −, infiltration of phosphate buffer (PBS) with and without A23187, respectively. (B) Tobacco leaves were infiltrated with A23187. A23187 was dissolved in PBS at the indicated concentrations and used to infiltrate wild-type (WT) and transgenic tobacco expressing RNA silencing suppressors CMV 2b and ClYVV HC-Pro. Photographs were taken 24 h after infiltration with A23187. (Ci and ii) To test whether *PR1a* induction was dependent on Ca²⁺ influx, EGTA (10 mM) was infiltrated along with A23187. *PR1a* and *rgs-CaM* mRNA levels and rgs-CaM protein levels were investigated by Northern and Western blotting, respectively, 1 and 24 h after infiltration. Coomassie brilliant blue-stained (CBB) and ethidium bromide-stained (*rRNA*) gels are shown as loading controls.

**FIG 7**
*PR1a* induction depends on rgs-CaM. (A) Wild-type (WT) and transgenic tobacco expressing RNA silencing suppressors CMV 2b and ClYVV HC-Pro were inoculated with a PVX empty vector (PVX) and a PVX vector expressing the *rgs-CaM* ORF sequence lacking the initiation codon as a means of inducing VIGS of rgs-CaM [VIGS(rgs-CaM)]. These inoculated leaves were infiltrated with A23187 (+) or buffer alone (−) 3 days after inoculation with PVX. The levels of *PR1a* mRNA, PVX CP, and *rgs-CaM* mRNA were investigated by Northern blotting, Western blotting, and semiquantitative RT-PCR, respectively, 24 h after infiltration with A23187. Samples were also prepared from plants that were inoculated with buffer but not infiltrated (Mock). (B) WT and transgenic tobacco plants expressing salicylate hydroxylase (NahG), which antagonizes salicylic acid signaling, were inoculated with PVX and CMVΔ2b and infiltrated with A23187 at 3 days postinoculation. The levels of *PR1a* mRNA and viral CPs were investigated by Northern and Western blotting, respectively, 24 h after infiltration with A23187. Samples were also prepared from buffer-inoculated plants without infiltration (Mock). Coomassie brilliant blue-stained (CBB) and ethidium bromide-stained (*rRNA*) gels are shown as loading controls.

**FIG 8**
Enhanced resistance against CMV-Y in SAR-induced tobacco plants depends on rgs-CaM. (Ai) Comparison of symptoms (yellowing) on noninoculated upper leaves of tobacco plants inoculated with CMV-Y. CMV-Y was inoculated into wild-type (WT) and rgs-CaM knockdown (IR-rgs-CaM) tobacco plants 7 weeks after sowing. The photograph was taken at 16 days postinoculation (dpi) with CMV-Y. All of the rgs-CaM knockdown tobacco plants that were inoculated with CMV-Y developed systemic symptoms on their leaves, but wild-type tobacco plants did not express symptoms. (Aii) The difference in susceptibility between wild-type and rgs-CaM knockdown plants was confirmed by detecting CMV CP in noninoculated upper leaves of these plants by Western blotting. (Aiii) The mRNA level of *rgs-CaM* relative to that of mock-inoculated wild-type plants was investigated by real-time PCR and is shown in the bar graph (n = 3). Error bars indicate SE. Student's t test was applied to the data; *, P < 0.05. (Bi) Five days after SAR induction by treatment with benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), WT and IR-rgs-CaM tobacco plants were inoculated with CMV-Y. Control plants (Cont) were treated with a solution containing 1.4% (vol/vol) acetone and 0.2% Tween 20 (the solution used to dissolve BTH). Symptoms on upper leaves were photographed 30 dpi. (Bii and iii) CMV CP and 2b proteins were detected by Western blotting. CMV genomic and subgenomic RNAs (*gCMV* and *sgCMV*, respectively), rgs-CaM, and *PR1a* mRNA were detected by Northern blotting. Coomassie brilliant blue-stained (CBB) and ethidium bromide-stained gels are shown as loading controls. (C) Experiments similar to those shown in panel B were done with CMVΔ2b. (D) PVX CP accumulation in plants inoculated with PVX 5 days after BTH treatment. Accumulation of PVX CP was detected in inoculated and noninoculated upper leaves by Western blotting. CBB-stained gels are shown as loading controls. Control samples were prepared from buffer-inoculated plants (Mock).

**FIG 9**
Degradation of CMV 2b is enhanced by benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) in transgenic BY2 cultured tobacco cells expressing 2b. Transgenic BY2 cultured cells expressing 2b were treated with BTH by adding it to the medium at a final concentration of 10 μM with or without an inhibitor, concanamycin A (concA) at 0.1 μM (A) or E64d at 10 μM (B). The CMV 2b and rgs-CaM proteins were detected by immune staining using specific fluorescent secondary antibodies 1 h after treatment with BTH with or without an inhibitor. Nuclei were visualized by DAPI staining. Differential interference contrast (DIC) images are also shown. White bars, 25 μm.

**FIG 10**
Reduction of ClYVV HC-Pro accumulation in transgenic tobacco plants expressing HC-Pro (A) and schematic models of detection and counteraction of viral RSSs by rgs-CaM (B). (A, left) Four leaves (numbered 1 to 4) of individual transgenic plants expressing HC-Pro were treated with benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH). A23187 in PBS was infiltrated into one half of a leaf 1 day after BTH treatment; the other half was infiltrated with buffer (PBS). (A, right) The HC-Pro and rgs-CaM proteins were detected by Western blotting. Values under the HC-Pro panel are band intensities of samples from the leaf part infiltrated with A23187 relative to that without A23187 in the same leaf (leaves 1 to 4). (B, left) In normally growing tobacco plants, the rgs-CaM-mediated defense system does not inhibit CMV infection but induces salicylic acid (SA) signaling via perception of CMV 2b and Ca²⁺ as CMV infection cues. Blue arrowheads indicate bands detected by anti-rgs-CaM antibody. (B, center) When the phase of rgs-CaM is changed by SAR induction, subsequent CMV infection is inhibited by rgs-CaM-mediated anti-RSS defense reactions. rgs-CaM directs degradation of RSS (CMV 2b) via autophagy, resulting in reinforcement of antiviral RNA silencing in addition to SA-mediated antiviral immunity. (B, right) When plants are infected with PVX or CMVΔ2b and Ca²⁺ influx is artificially induced with A23187, SA signaling is induced, probably via perception by rgs-CaM of Ca²⁺ and viral proteins other than RSS or host intermediate proteins that are induced by virus infection.

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References

1. Savvides A, Ali S, Tester M, Fotopoulos V. 2016. Chemical priming of plants against multiple abiotic stresses: mission possible? Trends Plant Sci 21:329–340. doi:10.1016/j.tplants.2015.11.003. - DOI - PubMed
1. Fu ZQ, Dong X. 2013. Systemic acquired resistance: turning local infection into global defense. Annu Rev Plant Biol 64:839–863. doi:10.1146/annurev-arplant-042811-105606. - DOI - PubMed
1. Gilpatrick JD, Weintraub M. 1952. An unusual type of protection with the carnation mosaic virus. Science 115:701–702. doi:10.1126/science.115.3000.701. - DOI - PubMed
1. Chester KS. 1933. The problem of acquired physiological immunity in plants. Q Rev Biol 8:275–324. doi:10.1086/394440. - DOI
1. Gao QM, Zhu S, Kachroo P, Kachroo A. 2015. Signal regulators of systemic acquired resistance. Front Plant Sci 6:228. doi:10.3389/fpls.2015.00228. - DOI - PMC - PubMed

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[1] Savvides A, Ali S, Tester M, Fotopoulos V. 2016. Chemical priming of plants against multiple abiotic stresses: mission possible? Trends Plant Sci 21:329–340. doi:10.1016/j.tplants.2015.11.003. - DOI - PubMed

[2] Savvides A, Ali S, Tester M, Fotopoulos V. 2016. Chemical priming of plants against multiple abiotic stresses: mission possible? Trends Plant Sci 21:329–340. doi:10.1016/j.tplants.2015.11.003. - DOI - PubMed

[3] Fu ZQ, Dong X. 2013. Systemic acquired resistance: turning local infection into global defense. Annu Rev Plant Biol 64:839–863. doi:10.1146/annurev-arplant-042811-105606. - DOI - PubMed

[4] Fu ZQ, Dong X. 2013. Systemic acquired resistance: turning local infection into global defense. Annu Rev Plant Biol 64:839–863. doi:10.1146/annurev-arplant-042811-105606. - DOI - PubMed

[5] Gilpatrick JD, Weintraub M. 1952. An unusual type of protection with the carnation mosaic virus. Science 115:701–702. doi:10.1126/science.115.3000.701. - DOI - PubMed

[6] Gilpatrick JD, Weintraub M. 1952. An unusual type of protection with the carnation mosaic virus. Science 115:701–702. doi:10.1126/science.115.3000.701. - DOI - PubMed

[7] Chester KS. 1933. The problem of acquired physiological immunity in plants. Q Rev Biol 8:275–324. doi:10.1086/394440. - DOI

[8] Chester KS. 1933. The problem of acquired physiological immunity in plants. Q Rev Biol 8:275–324. doi:10.1086/394440. - DOI

[9] Gao QM, Zhu S, Kachroo P, Kachroo A. 2015. Signal regulators of systemic acquired resistance. Front Plant Sci 6:228. doi:10.3389/fpls.2015.00228. - DOI - PMC - PubMed

[10] Gao QM, Zhu S, Kachroo P, Kachroo A. 2015. Signal regulators of systemic acquired resistance. Front Plant Sci 6:228. doi:10.3389/fpls.2015.00228. - DOI - PMC - PubMed

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rgs-CaM Detects and Counteracts Viral RNA Silencing Suppressors in Plant Immune Priming

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rgs-CaM Detects and Counteracts Viral RNA Silencing Suppressors in Plant Immune Priming

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