Research. Summary. Introduction

Barley stripe mosaic virus

infection requires PKA-mediated

phosphorylation of

c

b for suppression of both RNA silencing and

the host cell death response

Xuan Zhang

, Kai Dong

, Kai Xu

, Kun Zhang

, Xuejiao Jin

, Meng Yang

, Yongliang Zhang

, Xianbing Wang

,

Chenggui Han

, Jialin Yu

and Dawei Li

1

State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100193, China;2Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, China

Author for correspondence:

Dawei Li Tel: +86 10 62733326 Email: [email protected] Received:10 November 2017 Accepted:22 January 2018 New Phytologist(2018)218:1570–1585 doi: 10.1111/nph.15065

Key words: Barley stripe mosaic virus

(BSMV), host cell death response,Nicotiana benthamiana, phosphorylation, PKA, RNA silencing, suppression,cb protein.

Summary

TheBarley stripe mosaic virus (BSMV)cb protein is a viral suppressor of RNA silencing (VSR) and symptom determinant. However, it is unclear how post-translational modification affects the different functions ofcb.

Here, we demonstrate thatcb is phosphorylated at Ser-96 by a PKA-like kinasein vivoand in vitro. Mutant viruses containing a nonphosphorylatable substitution (BSMVS96A or

BSMVS96R) exhibited reduced viral accumulation inNicotiana benthamianadue to transient

induction of the cell death response that constrained the virus to necrotic areas. By contrast, a BSMVS96D mutant virus that mimics cb phosphorylation spread similarly to the wild-type

virus.

Furthermore, the S96A mutant had reduced local and systemiccb VSR activity due to hav-ing compromised its bindhav-ing activity to 21-bp dsRNA. However, overexpression of other VSRs in transorin cisfailed to rescue the necrosis induced by BSMVS96A, demonstrating that

sup-pression of cell death bycb phosphorylation is functionally distinct from its RNA silencing sup-pressor activities.

These results provide new insights into the function of cb phosphorylation in regulating RNA silencing and the BSMV-induced host cell death response, and contribute to our under-standing of how the virus optimizes the balance between viral replication and virus survival in the host plants during virus infection.

Introduction

RNA viruses encode a limited number of proteins in their small genomes, but cause tremendous damage to host cells. Viruses inflict damage by generating proteins that often have multiple functions. Numerous studies have shown that post-translational modifications, including phosphorylation (Ubersax & Ferrell, 2007), ubiquitination (Imura et al., 2015; Verchot, 2016), sumoylation (Cheng et al., 2017), glycosylation (Vigerust & Shepherd, 2007), and lipidation (Chianget al., 2013), allow viral proteins to perform different functions during infection.

Phosphorylation is a ubiquitous post-translational modifica-tion in eukaryotic cells that usually activates enzyme activities and induces a cascade of reactions. Protein phosphorylation is a reversible process that requires cooperation between protein kinases and phosphatases to regulate protein function (Friso & van Wijk, 2015). As obligatory parasites, viruses have evolved an ability to use host protein phosphorylation to maximize their

pathogenicity. For example, early studies onTobacco mosaic virus (TMV) showed that its movement protein (MP) is phosphory-lated by a cell wall-associated kinase that facilitates viral cell-to-cell transport through plasmodesmata (Citovsky et al., 1993; Karpova et al., 1999; Waigmann et al., 2000). Phosphorylation of viral movement proteins was then shown to be a common strategy that enables plant viruses to spread (Modenaet al., 2008; Linket al., 2011; Samuilovaet al., 2013; Huet al., 2015). Vari-ous viral proteins with distinct functions are phosphorylated dur-ing viral infection, includdur-ing RNA-dependent RNA polymerase (RdRp) (Kim et al., 2002; Jakubiec et al., 2006; Jakubiec & Jupin, 2007), coat protein (CP) (Ivanovet al., 2003; Hunget al., 2014; Zhaoet al., 2015), and proteins that suppress RNA silenc-ing and symptom development (Lewseyet al., 2009; Shenet al., 2011, 2014; Zhonget al., 2017).

Viral suppressors of RNA silencing (VSRs), including potyvirus HC-pro (Urcuqui-Inchima et al., 2001; Valli et al., 2018), Cucumber mosaic virus(CMV) 2b (Lewseyet al., 2010),

geminivirus bC1 (Cui et al., 2005; Yang et al., 2008; Zhang et al., 2012; Zhong et al., 2017), Barley strip mosaic virus (BSMV) cb (Yelina et al., 2002; Bragg et al., 2004; K. Zhang et al., 2017), and Cauliflower mosaic virus (CaMV) P6 (Love et al., 2012; Schoelz et al., 2016), often have multiple functions in addition to RNA silencing suppression. A common and intriguing feature of viral proteins is their involvement in disease symptom development. For example, transgenic Arabidopsis plants expressing viral proteins 2b (Donget al., 2016), HC-pro (Kasschauet al., 2003), p19 (Dunoyeret al., 2004), or P6 (Zijl-straet al., 1996) show diverse developmental defects that are not apparent in wild-type plants. Furthermore, CMV 2b inhibits the disease recovery of CMV-infected Arabidopsis thaliana ecotype C24 (Lewsey et al., 2009) and participates in viral transient infection in the shoot apical meristem (Sunpapao et al., 2009; X-P. Zhanget al., 2017). By contrast, geminivirusbC1 induces leaf abnormalities during viral infection (Yang et al., 2008). Although different VSRs have evolved to share common bio-chemical properties, such as double-stranded RNA (dsRNA) binding (Meraiet al., 2006), they give rise to viral disease symp-toms through different routes.

BSMV, the type member of the genus Hordeivirus, is a plus-strand RNA virus consisting of RNAa, b and c, which collectively encode seven major proteins (Jackson et al., 2009). The BSMV encoded cb protein is a multifunctional protein involved in RNA silencing suppression, single-stranded RNA (ssRNA) binding, long-distance movement, viral pathogenic-ity, and viral chloroplast-targeted replication (Donald & Jack-son, 1994, 1996; Yelina et al., 2002; Bragg & Jackson, 2004; K. Zhang et al., 2017). However, it remains to be determined if cb is phosphorylated, and if phosphorylation influences the BSMV infection cycle.

In this study, we provide evidence that BSMVcb is phosphory-lated during BSMV infection. We show that Ser-96 of cb is a functional phosphorylation site targeted by PKA-like kinases. A phosphorylation-deficient mutation S96A, but not a phosphory-lation-mimicry mutation S96D, impaired cb’s RNA silencing suppressor activity. Interestingly, mutations at the major phos-phorylation site Ser-96 intensified virus-induced cell death response, effectively restricting virus spread and allowing plants to recover from BSMV infection. Our results show that BSMV opti-mizes the balance between viral replication and virus survival in the host by producing the multifunctionalcb protein, the func-tion of which depends on its phosphorylafunc-tion status at Ser-96.

Materials and Methods

Plant growth conditions

Nicotiana benthamiana Domin plants were grown in a climate chamber with a 14 h : 10 h, light : dark photoperiod at 23–25°C as described previously (Yuan et al., 2011). Barley (Hordeum vulgare L.) (Yangfu 4056) and wheat (Triticum aestivum L.) (Yangmai 158) were grown in a glasshouse until the two-leaf stage and then transferred to a climate chamber after inoculation with BSMV (Leeet al., 2012; Huet al., 2015).

Plasmid construction

BSMV strain ND18 (Pettyet al., 1989) was used in this study. A QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) was used to generate site-specific mutagenesis plasmids, using the primer pairs presented in Supporting Information Table S1. All mutants described below were verified by DNA sequencing.

Infectious cDNA clones containing various cb mutants for in vitrotranscription were generated in the pT7-c vector (Petty et al., 1989; Leeet al., 2012). Site-directed mutagenesis was used to substitute Ser-96 ofcb protein with either alanine (A), aspartic acid (D), or arginine (R) to generate pT7-c-cbS96A, pT7-c

-cbS96D or pT7-c-cbS96R, respectively. To engineer BSMV

derivatives for agroinfiltration (Yuan et al., 2011), full-length cDNAs were amplified from the pT7-c containing cb mutants described above, and inserted between theStuI andBamHI sites of the pCass-4Rz vector (Annamalai & Rao, 2005), generating pCaBS-c-cbS96A, pCaBS-c-cbS96Dand pCaBS-c-cbS96R.

For transient expression assays, cDNAs of cb or the cb mutants S96A, S96D or S96R were amplified from pT7-cor its derivatives and cloned between theXhoI andBamHI sites of the pGD binary vector (Goodinet al., 2002), fusing the cDNAs with a 3xFlag sequence at their C-terminus.

Virus inoculation

In vitrotranscription followed by viral RNA inoculation was per-formed as previously described (Leeet al., 2012; Huet al., 2015). Briefly, transcripts of RNA a, b and c were mixed at a molar ratio of 1 : 1 : 1, added with equal amounts of FES inoculation buffer (0.06 M potassium phosphate, 0.1 M glycine, 1% ben-tonite, 1% sodium pyrophosphate decahydrate, 1% celite, pH 8.5), and then inoculated onto the leaves of 7- to 10-d-old barley and wheat seedlings. For viral inoculation into N. benthamiana leaves, the pCaBS-a, pCaBS-b and pCaBS-c plasmids or their derivatives were transformed into Agrobacterium tumefaciens strain EHA105 and infiltrated into the leaves of 3- to 4-wk-old plants (Yuanet al., 2011).

Immunoprecipitation ofcb protein and identification of its

phosphorylation site by LC-MS/MS

To enrich forcb protein in viral infection conditions, an infec-tious plasmid pCaBS-c-cb3xFlagencoding a 3xFlag peptide at the

C-terminus of cb was constructed and co-agroinfiltrated with pCaBS-a and pCaBS-b into N. benthamiana leaves. The immunoprecipitation (IP) assay was performed as previously described (Zhaoet al., 2015). The final extracted and purifiedcb proteins were detected by Western blot with anti-Flag antibody (Sigma) or with SDS-PAGE analysis followed by silver staining. Thecb band was sliced from the gel and digested with trypsin at 37°C overnight. The digested peptides were then analyzed by Q-Exactive liquid chromatography tandem mass spectrometry (LC-MS/MS) (Thermo Scientific, Waltham, MA, USA) at the Mass Spectrometry Facility of China Agricultural University.

Expression and purification of recombinantcb proteins

For protein purification, the cb fragment was amplified from pT7-c and cloned into the pET-30a(+) vector (Novagen) that digested with NdeI and XhoI sites to generate the pET-30a-cb plasmid. The pET-30a-cb plasmid or its derivatives were trans-formed intoEscherichia colistrain BL21 (DE3). The recombinant proteins were purified as described previously (Hu et al., 2015; Zhaoet al., 2015). Briefly, the transformed bacteria were resus-pended in T buffer containing 20 mM Tris-HCl (pH 9.0), 500 mM NaCl, 10% glycerol, 0.1% Triton X-100 and 1 mM PMSF, then disrupted by ultrasonication. The supernatant was collected and recombinant proteins were purified using an Ni-NTA agarose affinity column (Bio-Rad). After washing with an increasing concentration gradient of imidazole (20 mM, 60 mM, 100 mM, 200 mM, 300 mM and 400 mM), proteins were eluted in T buffer containing 200 mM imidazole and concentrated with Amicon-Ultra-10 filters (Millipore).

In vitrophosphorylation assays

Soluble protein extracts of healthyN. benthamiana leaves or com-mercial mammalian kinase (PKA) was used forin vitrokinase assays according to previously described protocols (Vijayapalani et al., 2012; Hung et al., 2014; Zhao et al., 2015). Briefly, each 15ll reaction contained 5lgcb protein and either 5lgN. benthamiana soluble protein extracts or 0.5lg commercial mammalian kinase (PKA) (New England Biolabs, Ipswich, MA, USA), reacted in 19kinase reaction buffer with [c-32P] ATP and incubated for 30 min at 30°C. To inhibit the PKA kinase activity, a PKA-specific inhibitor H-89 (Sigma) was added in gradient concentrations (1lM, 5lM and 10lM) to the reaction buffer (Reuveni et al., 2002). After treatment, 59SDS loading buffer was added to termi-nate the reaction and the samples were separated by 15% SDS-PAGE. The gels were dried with a Model 583 Gel Dryer (Bio-Rad) and radioactive signals were visualized by autoradiography.

Trypan blue staining

Trypan blue staining was carried out as previously described with minor modifications (Zhuet al., 2013). The inoculated or systemic infected leaves were cleaned with absolute ethyl alcohol for 2–3 min, and then combined with equal volumes of absolute ethyl alcohol and Trypan blue solution (10 ml lactic acid, 10 ml water phenol, 10 ml glycerol and 10 ml sterile water with 15 mg of Trypan blue). The samples were placed in boiling water for 10–15 min and incu-bated at room temperature for 6–8 h. After destaining two or three times using chloral hydrate (2.5 g ml1), the samples were pho-tographed and analyzed using IMAGEJ software (http://imagej.net/).

Tissue imprinting assay

Tissue imprinting was performed as described previously (Srini-vasan & Tolin, 1992; Andika et al., 2005). Briefly, systemic infected leaves were sandwiched between two pieces of filter paper and pounded with a hammer, then washed with sterile

water containing 2% Triton X-100. The leaves were then blocked for 30 min in blocking buffer (20 mM Tris-HCl (pH 7.5), 0.5 M NaCl (Tris-buffered saline, TBS), 3% bovine serum albumin (BSA)) (Andika et al., 2005), and incubated for 90 min with BSMV CP antibody in TBS buffer with 1% BSA. After washing three times with washing buffer (TBS and 0.05% Tween-20), the filters were incubated for 2 h with goat anti-rabbit IgG conju-gated to alkaline phosphatase (Sigma). The tissue print was detected with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium chloride (NBT) in AP buffer (100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 5 mM MgCl2). Agrobacteriuminfiltration and GFP imaging

VSR detection was performed as previously described (Johansen & Carrington, 2001) with minor modifications reported in recent studies (Dong et al., 2016; K. Zhang et al., 2017; X-P. Zhang et al., 2017); equal volumes of A. tumefaciens cultures (OD600=0.5) harboring plasmids expressing positive sense-GFP

(sGFP) (Bragg & Jackson, 2004) and A. tumefaciens cultures (OD600=0.5) harboring pGD-cb-3xFlag, pGD-S96A-3xFlag,

pGD-S96D-3xFlag, or pGD-S96R-3xFlag expression vectors were mixed and co-infiltrated into the leaves of 4- to 5-wk-old wild-type or GFP-expressing transgenic line 16c (Voinnet & Baulcombe, 1997). The VSR p19 encoded byTomato bushy stunt virus (TBSV) expression plasmid and an empty vector (pGD) were used as the positive or negative controls, respectively. The agroinfiltrated leaves were illuminated under a long-wavelength UV lamp (UVP) and photographed under a yellow filter. In local silencing assays, GFP expression was measured at 3 and 6 d post-infiltration (dpi). In systemic silencing assays, GFP was measured and counted at 10 dpi (Zhuoet al., 2014; Donget al., 2016). All experiments were repeated three times.

Electrophoretic mobility shift assays

Electrophoretic mobility shift assays (EMSAs) were carried out as previously described (Duanet al., 2012; Donget al., 2016). The 21-bp duplex siRNA probes were synthesized by two ssRNA oligos (siGFP-1: 50-GUCACUACUAUGGGUUAUGAG-30and siGFP-2: 50-CAUAACCCAUAGUAGUGACUG-30) (Donget al., 2016). The two ssRNA oligos were radiolabeled with [c-32P] ATP, com-bined in equimolar concentrations, and annealed by heating at 99°C for 5 min, followed by free-cooling to room temperature and syn-thesis of the 21-bp dsRNA. The 21-bp dsRNA was incubated with

cb protein or its mutants for 40 min at room temperature and the protein–RNA complexes were separated on a 6% native polyacry-lamide gel (Duanet al., 2012). The gel was dried using a Model 583 Gel Dryer (Bio-Rad) and the protein–RNA complexes were detected by autoradiography with a storage phosphor screen (GE Healthcare, Chicago, IL, USA).

Bimolecular fluorescence complementation

For bimolecular fluorescence complementation (BiFC) assays, the cDNAs ofcb, S96A, S96D, and S96R were amplified from

pT7-c or its derivatives and cloned into the pSPYNE-35S or pSPYCE-35S vectors (Walteret al., 2004) between theXbaI and BamHI sites. All the BiFC plasmids were transformed into A. tumefaciensstrain EHA105. The infiltration mixtures were at a concentration of 0.8 at OD600and composed of 0.3 YFPN, 0.3

YFPC, and 0.2 TBSV p19. The co-infiltrated N. benthamiana leaves were observed under an Olympus confocal FV1000 micro-scope at 3 dpi.

Yeast two-hybrid assay

Yeast two-hybrid assays were performed as previously described (K. Zhanget al., 2017).

RNA extraction, Northern blot analysis, and real-time quantitative PCR

RNA extraction and Northern blot analysis were performed as described previously (K. Zhanget al., 2017). Briefly, total RNAs were extracted and evaluated by a NanoDrop ND-1000 (Thermo-Fisher Scientific). To detect the RNAaof BSMV, 3lg of total RNA was separated on a 1.2% agarose gel containing formaldehyde. The RNAs were transferred onto Hybond-N+ nylon membranes, fixed by UV cross-linking, and stained with methylene blue solution (0.04% methylene blue, 0.5 M NaOAc). The nylon membranes were hybridized using a [c-32P] UTP-labeled RNAa-specific probe and autoradiographed using a stor-age phosphor screen (GE healthcare) for 4–6 h.

For real-time quantitative PCR (RT-qPCR) analysis, cDNA was synthesized from 5lg total RNA (DNase-treated) using an oligo-dT primer and M-MLV reverse transcriptase (Promega). The gene fragments were amplified using 29SsoFastTM

EvaGreen Supermix (Bio-Rad) and the primers shown in Table S2. The Elongation factor 1a (EF1a) gene served as the internal control (Liuet al., 2012) and data were analyzed using CFX MANGEsoftware (Bio-Rad).

Results

The BSMVcb protein is phosphorylatedin planta

BSMVcb is a multifunctional protein that regulates many steps of the virus infection process (Jacksonet al., 2009). It is unclear how one viral protein mediates these multiple steps. Since phos-phorylation is a key protein post-translational modification pro-cess and is essential for protein enzymatic functions, we analyzed whether cb is phosphorylated in BSMV-infected plant cells. A BSMV derivative (BSMVcb-3xFlag) in whichcb was tagged with

3xFlag at its C-terminus was used to infectN. benthamianaleaves via agroinfiltration. Total proteins were extracted from infiltrated leaves at 3 dpi and then subjected to Western blot analysis using anti-phosphoserine or anti-Flag antibodies. A distinct protein band of c. 17 kDa was detected by both antibodies, indicating that the BSMVcb protein might be phosphorylated during virus infection (Fig. 1a). We further purifiedcb-3xFlag from the leaves of BSMVcb-3xFlag infected plants by Flag affinity purification

(Fig. 1b, upper panel). The purified proteins were analyzed by Q-Exactive liquid chromatography-tandem mass spectrometry (LC-MS/MS). Eighteen potential phosphorylation sites in cb were identified (Fig. 1b, lower panel). Five of these potential sites with the highest scores (Y89, S96, S136, S137 and S141) were chosen for further testing of their roles in BSMV infectivity. We substituted these potential phosphorylation sites with alanine in BSMV infectious cDNA clones, resulting in four BSMV deriva-tives withcb mutations: BSMVY89A, BSMVS96A, BSMVS136/137A

and BSMVS141A. We then tested their influence on BSMV

pathogenicity in barley leaves. Among these mutants, BSMVS96A

elicited bleached symptoms in systemic infection leaves, which was different from the typical chlorotic stripes and mosaic symp-toms infected by wild-type (WT) BSMV (Fig. 1d). Viral accumu-lation of BSMVS96Aon systemic leaves wasc. 50% less than that

of WT BSMV when assessed using an enzyme-linked immunosorbent assay (ELISA) against BSMV virions, suggesting that phosphorylation of Ser-96 promotes BSMV pathogenicity. Through LC-MS/MS (Fig. 1c) and reverse-genetics (Fig. 1d) analyses, we showed that Ser-96 is a potential phosphorylation site ofcbin plantaand plays an important role in BSMV infec-tion and its induced symptoms.

PKA-like kinase contributes to the phosphorylation ofcb at

Ser-96

To verifycb phosphorylationin vitro, we purified recombinant

cb-6xHis protein from E. coli and subjected it to an in vitro phosphorylation assay using N. benthamiana soluble protein extracts (NbEX) as a kinase source. We found thatcb was phos-phorylated byNbEX (Fig. 2a). However, mutation of Ser-96 to alanine (A) significantly reducedin vitrophosphorylation of cb (Fig. 2b), suggesting that Ser-96 is a major phosphorylation site for cb. Based on a phosphorylation specificity analysis of pro-teins (Ubersax & Ferrell, 2007), as well as predictions from the online servers KinasePhos (http://kinasephos2.mbc.nctu.edu.tw) and NetPhos 3.1 (http://www.cbs.dtu.dk/services/NetPhos/) (Fig. S1), the protein motif 92LLKRSEQEL100 containing Ser-96 is found to be very similar to the substrate recognition site of cAMP-dependent protein kinase (PKA) (Ubersax & Ferrell, 2007). Thus, we evaluated in vitro cb phosphorylation in the presence of the PKA inhibitor H-89 (Reuveni et al., 2002) and found that H-89 inhibited the phosphorylation ofcb byNbEX (Fig. 2c). This suggests that protein kinases belonging to the PKA family were responsible for cb phosphorylation. Indeed, when PKA was added to recombinantcbin vitro,cb was phos-phorylated (Fig. 2d). The phosphorylation level ofcb was much reduced when Ser-96 was mutated to a nonphosphorylatable ala-nine (A) or a positively charged argiala-nine (R). Taken together, these results show that Ser-96 is a major phosphorylation site of

cb and is possibly a target of a kinase from the PKA family. Additionally, these results showed that 5lM H-89 reduced PKA-driven cb phosphorylation to c. 9% of the control levels (Fig. 2e).

However, it was unexpectedly observed that the S96D mutant displayed phosphorylation levels similar to WTcb (Fig. 2D), as

0.0 0.5 1.0 1.5 2.0

Barley systemic leaves infected by:

Mr BSMV BSMVγb-3xFlag 25 kDa

LC-MS/MS results

:

MMATFSCVCCGTLTTSAYCGK

R

CERKHV

Y

S

ETRNKRLEL

Y

KK

Y

LLEPQKCALNGIVGH

S

CGMPC

S

IAEEACDQLPIVSRFCGQKHAD

L

Y

D

S

LLKR

S

EQELLLEFLQKKMQELKL

S

HIVKMAKLE

S

EVNAIRK

S

VA

SS

FED

S

VG

CDDS

SS

V

S

K

A

∗∗

Fig. 1Identification of the phosphorylated sites ofcb that affectBarley stripe mosaic virus(BSMV) infection. (a)In vivophosphorylation of the BSMVcb protein. Total proteins were extracted from recombinant BSMVcb-3xFlagvirus infected leaves and analyzed by Western blot using anti-phosphoserine

(Millipore) or anti-Flag antibodies (Sigma). Non-inoculated mock was designed as a negative control. (b) Upper panel, silver staining ofcb protein immunoprecipitated using anti-Flag beads from BSMV or BSMVcb-3xFlaginfectedNicotiana benthamianaleaf tissues. Thecb band (arrowhead) was

analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) (top panel). Lower panel, LC-MS/MS results; allcb amino acids were identified by LC-MS/MS except one arginine residue (in blue). Potential phosphorylation sites are shown in red. The five potential sites with the highest scores (underlined) were chosen for further research. (c) MS spectrum of the phosphorylated peptides96_{pSEQELLLEFLQKK}108_{containing the Ser-96}

phosphorylated residue. The absence of phosphoric acid (98 Da) on the y3 ion fragment strongly demonstrates that Ser-96 is a phosphorylation site within thecb protein. (d) Systemic symptoms in barley (Yangfu 4056) after inoculation of BSMV or potential phosphorylation deficient mutants. The plants were photographed at 10 dpi (upper panel). Upper uninoculated leaves were harvested and subjected to CP ELISA to evaluate the accumulation of viruses (lower panel). Error bars indicateSE from three independent experiments (Student’st-test:**,P<0.01).

aspartic acid should not be phosphorylated by PKA. To further investigate the surprising behavior of the S96D mutant, we incu-bated S96D protein purified from E. coli with PKA kinase in vitroand then analyzed its phosphorylation status by LC-MS/ MS. These results showed that S133, S136 and S137 were phos-phorylated by PKA kinase (Fig. S2a). The S133 residue in the

130_IRKS133 _{site is also a conserved PKA phosphorylation motif}

(Ubersax & Ferrell, 2007). To investigate whether these potential phosphorylation sites on the S96D mutant protein were indeed phosphorylated, the double mutant protein S96D/S133A and triple mutant protein S96D/S136A/S137A were purified from E. coli and subjected to an in vitro phosphorylation assay with PKA kinase. This assay showed that S96D/S133A had negligible levels of phosphorylation (Fig. S2b, lane 6). By contrast, the phosphorylation level of the triple mutant S96D/S136A/S137A was comparable to that of S96D (Fig. S2b, lane 7). Thus, Ser-133 is a secondary phosphorylation site ofcb and its phosphory-lation is coupled with the phosphoryphosphory-lation of Ser-96. Unlike the

S96A mutation, which led to reduced systemic viral infection (Fig. 1d), the phosphorylation of Ser-133 did not have a discernible effect on viral infection (Fig. S2c,d).

cb phosphorylation inhibited cell death caused by BSMV

and promoted pathogenicity inN. benthamiana

As the BSMVS96A mutation elicited bleached symptoms and

reduced BSMV accumulation in barley (Fig. 1d), we next studied how these symptom changes related to BSMV pathogenicity. We inoculated BSMV or its cb Ser-96 variants (BSMVS96A,

BSMVS96Dand BSMVS96R) intoN. benthamianausing

agroinfil-tration. At 7 dpi, we observed that BSMVS96A and BSMVS96R

caused an enhanced cell death response at the infiltrated leaf area (Fig. 3a, upper panel). Trypan blue selectively stains dead tissues and was used to indicate the level of cell death induced by viral infections. These results showed that in the infiltrated leaf area, BSMVS96A and BSMVS96R induced higher levels of cell death

γb NbEX – + +– ++ CBB γb NbEX wt + S96A + – + CBB 100 30.6 ± 4.3 0 % 0 1 5 10 γb+PKA 100 68 ± 14 9 ± 3 3 ± 1 % 0 1 5 10 γb+NbEX H-89 (μM) Substrate PKA + γb – – γb + S96+A S96D+ S96R+ 0 0 100 33 ± 9 105 ± 11 24 ± 18 % H-89 (μM) 100 69 ± 3 54 ± 10 39 ± 8 % CBB CBB CBB (a) (c) (e) (b) (d)

Fig. 2A PKA-like kinase phosphorylatescb protein at Ser-96 site. (a)In vitrophosphorylation of His-tagged recombinantcb protein purified from

Escherichia coli. The total soluble proteins extracted from healthyNicotiana benthamianaleaves (NbEX) were used as a kinase resource. A reaction containing kinase andcb protein produced an autoradiography band, and reactions lackingcb protein or kinase served as negative controls. Coomassie brilliant blue (CBB)-stainedcb loadings are shown (bottom). (b)In vitrophosphorylation ofcb and S96A mutant byNbEX. A reaction withoutcb protein was used as a negative control. The autoradiography bands (top) and CBB-stained loading controls (bottom) are shown. IMAGEJ software was used to quantify the autoradiography bands. Values are the meanSD from three independent experiments. (d) Phosphorylation ofcb and its mutant proteins by PKA kinase. The autoradiography bands representing phosphorylation levels are shown in the top panel and the CBB loading controls are in the bottom panel. Error bars indicateSE from three independent experiments. (c, e) Effects of the PKA-specific inhibitor H-89 oncb phosphorylation mediated by (c)

NbEX or (e) PKA kinase. Samples were immersed in buffers lacking or containing 1lM, 5lM, and 10lM of H-89. Autoradiography bands and CBB loading controls are shown in the top and bottom panels, respectively. Error values indicateSE from three independent experiments.

α-CP

1

2

3

4

5

6

7

8

BSMV

BSMV

BSMV

BSMV

Infiltrated leaves

Systemic

leaves

Systemic

leaves

New shoots and whole plant

BSMVS96R BSMVS96D

Systemic

leaves

Systemic leaves

BSMV

BSMV

BSMV

BSMV

∗∗∗

∗

∗

∗

Fig. 3Different disease symptoms were elicited byBarley stripe mosaic virus(BSMV) and mutant viruses inNicotiana benthamiana. (a) Macroscopic phenotype (upper panel) and Trypan blue staining (lower panel) ofN. benthamianaleaves infected by BSMV or the indicated mutant viruses. Inoculated leaves were photographed at 7 d post infiltration (dpi). (b) Systemic symptoms ofN. benthamianaplants infected with BSMV and BSMVS96A, BSMVS96Dor

BSMVS96Rmutant viruses. Higher magnifications of the boxed regions are shown on the right. Pictures were imaged at 10 dpi. (c) Macroscopic phenotypes

(first and third rows) and Trypan blue staining (second row) or tissue imprinting (fourth row) of systemic leaves infected with BSMV or mutant viruses. Photographs were taken at 10 dpi. (d) RT-qPCR analysis ofPR1,TGA4,PDF1.2andMYC2transcripts in BSMV or BSMS96Ainfected systemic leaves at

10 dpi. Relative mRNA levels were normalized against that of theEF1Atranscript. Values are meanSD (Student’st-test:*,P<0.05;***,P<0.001). (e) Symptoms of wild-type (WT) BSMV, BSMVS96A, BSMVS96Dor BSMVS96RinfectedN. benthamianaat 30 dpi (top and middle rows). Western blot analysis

than WT BSMV or cb mimic-phosphorylation mutant BSMVS96D (Fig. 3a, lower panel). Inoculated BSMV derivatives

caused notable symptoms in young systemic leaves at early stages of infection (10 dpi) (Fig. 3b). We performed a tissue imprinting assay using antibodies against BSMV CP to evaluate virus distri-bution and accumulation. We found that WT BSMV or the BSMVS96D mutant spread to the whole leaf area, albeit at low

levels. However, BSMVS96Aor BSMVS96Rmutants accumulated

aggressively in a relatively confined area close to the petiole (Fig. 3c, lower panel) where the level of cell death was also high (Fig. 3c, upper panel). In contrast to the BSMVS96A mutant,

nonphosphorylatable mutants of the secondary phosphorylation sites (i.e. BSMVS133Aor BSMVS96D/S133A) did not induce a

sev-ere cell death response or symptom changes (Fig. S3).

We then measured the transcript levels of several defense-related genes in leaf areas systemically infected with BSMV or BSMVS96A. Salicylic acid (SA)-mediated defense marker genes,

including PR-1 and TGA4 (Clarkeet al., 2000; Alazem & Lin, 2015), were markedly upregulated in the BSMVS96A-infected leaf

area compared to that infected by WT BSMV (Fig. 3d). Thus, the SA-mediated defense mechanism might play a role in the cell death responses against BSMVS96A. By contrast, the jasmonic

acid (JA)-mediated defense genes MYC2 and PDF1.2 (Clarke et al., 2000) were transcriptionally suppressed by BSMVS96A

infection (Fig. 3d).

At the late stage of infection (30 dpi), newly emerging systemic leaves of N. benthamiana plants infected with BSMVS96A or

BSMVS96Rmutants had no detectable viral CP (Fig. 3e, lanes 3,

4 and 7, 8), suggesting that these plants were recovering from the infection. WT BSMV or BSMVS96Dinfected plants still had high

levels of virus in systemic leaves (Fig. 3e, lanes 1, 2 and 5, 6) and showed a stunted growth phenotype (Fig. 3e, upper panel), indi-cating thatcb phosphorylation plays an important role in main-taining BSMV pathogenicity and symptom development.

Together, these results suggest that phosphorylation ofcb pro-tein at Ser-96 inhibits the early cell death response to BSMV infection, which is likely induced by the SA-mediated plant defense mechanism. Furthermore,cb appears to promote BSMV pathogenicity at a relatively late stage of viral infection.

cb phosphorylation inhibited the bleached phenotype

caused by BSMV in monocots and promoted viral accumulation

To evaluate systemic infection of BSMV or itscb Ser-96 mutants on monocots natural hosts, barley or wheat leaves were co-inoculated within vitrotranscripts of RNAa,bandc. Barley and wheat plants had similar BSMV symptoms on systemic leaves at 10 dpi. As expected, WT BSMV or BSMVS96D caused mosaic

symptoms of faint yellow streaks running along the parallel veins (Fig. 4a,b), whereas leaves inoculated with cb phosphorylation-deficient mutant viruses BSMVS96Aor BSMVS96Rshowed severe

symptoms, including white stripes and necrosis along systemic leaves (Fig. 4a,b, upper panels) and severe cell death (Fig. 4a,b, lower panels). Systemic infected leaves were subjected to ELISA with antibodies against BSMV CP to evaluate viral accumulation.

ELISA showed that the levels of BSMVS96Aand BSMVS96Rwere c. 50% lower than those of WT BSMV and BSMVS96D(Fig. 4c,

d). These results suggest that the phosphorylation ofcb at Ser-96 inhibits cell death-like symptoms and promotes viral accumula-tion in monocots.

cb phosphorylation at Ser-96 is required for its VSR activity

To determine if the phosphorylation ofcb at Ser-96 affects its VSR activity (Yelina et al., 2002; Bragg & Jackson, 2004), we examined the ability of WT cb and its mutant forms (S96A, S96D, and S96R) to suppress positive-sense GFP (sGFP)-induced RNA silencing (Dong et al., 2016; K. Zhang et al., 2017). At 3 dpi, GFP fluorescence nearly disappeared without co-infiltration with a VSR (Fig. 5a, empty vector) and both GFP protein and GFP mRNA were detected at very low levels (Fig. 5b). In plant leaves co-infiltrated with cb or its S96D mutant, TBSV p19 (positive control) respectively, we detected bright GFP fluorescence (Fig. 5a), high GFP protein levels, and high GFP mRNA levels (Fig. 5b), demonstrating that RNA silencing induced by sGFP overexpression was inhibited by these proteins. However, phosphorylation-deficientcb mutants (S96A and S96R) could not effectively suppress sGFP-induced RNA silencing, indicated by the low levels of green fluorescence under UV light (Fig. 5a), as well as decreased GFP protein or GFP mRNA levels (Fig. 5b). These results suggest that phosphoryla-tion of cb at Ser-96 is required for efficient VSR activity. At 6 dpi, the protein accumulation of GFP, S96A and S96R all decreased when sGFP was co-infiltrated with S96A or S96R, pos-sibly due to reduced VSR activity in the S96A and S96R muta-tion (Fig. 5a,b). Consistent with its nonfuncmuta-tional effect on viral infection (Fig. S3), phosphorylation ofcb Ser-133 did not affect its VSR activity (Fig. S4).

Next, we tested whether cb phosphorylation on Ser-96 affected the systemic movement of RNA silencing signals. WT

cb or its Ser-96 mutants were co-infiltrated with the positive-sense GFP (sGFP) expression vector in leaves of GFP-transgenic N. benthamiana line 16c (Voinnet & Baulcombe, 1997). Co-infiltration of a vector expressing TBSV p19 or empty vector pGD was used as the positive or negative control, respectively. Three independent experiments were performed to see if GFP silencing could be transferred from infiltrated leaves to systemic leaves. As expected, transient expression of TBSV p19 suppressed systemic movement of silencing signals the most effectively (Fig. 5c), which is in agreement with previous reports (Lakatos et al., 2004; Donget al., 2016). WTcb or S96D mutant inhib-ited systemic GFP silencing in more than half of the tested plants, but both the S96A and S96R mutants showed significantly reduced abilities to suppress systemic GFP silencing (Fig. 5c). Based on the above results, we concluded thatcb phosphoryla-tion at Ser-96 is required to suppress local RNA silencing as well as the systemic movement of silencing signals.

It has been proposed that dsRNA binding is a general func-tional strategy for VSRs, including BSMV cb (Merai et al., 2006). We therefore explored the effects of phosphorylation of

was first subjected toin vitro phosphorylation, with or without the addition of PKA in the presence of ATP. Then, EMSA was carried out to test the abilities of the nonphosphorylated or phos-phorylated cb to bind the radiolabeled 21-bp dsRNA. In the presence of 0.05lgll1PKA, the His-taggedcb protein bound to the 21-bp dsRNA more efficiently (Fig. 5d,e) and the binding capacities increased approximately two- to three-fold at each cb concentration (Fig. 5e). By contrast, there were no detectable shifts in bands in the PKA-only negative control (Fig. 5d). These results indicated that PKA-mediated phosphorylation enhanced

cb’s binding kinetics to 21-bp dsRNA and contributed to its VSR activities.

The S96A mutation decreases viral replication, but does not

affectcb self-interaction

To test whether the severe cell death induced by BSMVS96A

(Fig. 3a) is due to high levels of BSMV replication, we compared the replication levels of WT BSMV and BSMVS96A in N. benthamiana. Total proteins and RNA extracted from infil-trated leaves at 3 dpi were analyzed by Western blot and

Northern blot, respectively. Viral CP, as well as triple gene block protein 1 (TGB1), andcb proteins, were detected respectively by Western blot analysis, and RNAawas detected by Northern blots (K. Zhang et al., 2017). These results showed that BSMVS96A

exhibited a lower abundance of viral proteins and RNAs in N. benthamiana leaves (Fig. 6a). To directly visualize the viral accumulation, we then inoculatedN. benthamiana leaves with a BSMV derivative BSMVcb-GFP in which GFP was fused to the

C-terminus of cb (K. Zhanget al., 2017). These results showed that both GFP fluorescence and viral RNAs were reduced when Ser-96 was mutated to A or R, while the mimic-phosphorylated S96D mutant could partially rescue the viral replication level to that of BSMVcb-GFP(Fig. 6b,c). These reductions in viral RNA

or protein accumulation levels are likely due to loss ofcb VSR activities. Similar to the infection in dicot plants, significantly reduced viral accumulation was also detected in barley (Fig. S5a) and wheat (Fig. S5b) infected with BSMVS96A and BSMVS96R.

Thus, severe cell death caused by loss ofcb phosphorylation was not due to enhanced viral replication.

Previous studies have shown that the coiled-coil region (CC, aa 95–140) within the C-terminus of cb can self-interact to

Barley systemic leaves infected by: Wheat systemic leaves infected by:

A405 A405 0.0 0.5 1.0 1.5 1 2 3 4 5 Mock BSMV BSMVS96ABSMVS96DBSMVS96R ∗∗ ∗ 0.0 0.5 1.0 1.5 1 2 3 4 5 Mock BSMV BSMVS96ABSMVS96DBSMVS96R ∗ ∗∗ (a) (c) (b) (d)

Fig. 4Systemic symptoms induced in barley or wheat byBarley stripe mosaic virus

(BSMV) or its mutant viruses and viral accumulations. Systemic symptoms (upper row) and cell death response evaluated by Trypan blue staining (lower row) elicited in (a) barley (Yangfu 4056) or (b) wheat (Yangmai 158) by BSMV or phosphorylation related mutant viruses BSMVS96A, BSMVS96D

or BSMVS96R. Systemic leaves were

photographed at 10 dpi (top). Bars, 0.5 cm. Upper leaf tissues were harvested and subjected to ELISA to determine the accumulation of virus in (c) barley and (d) wheat. Error bars indicateSE from three independent experiments (Student’st-test:

EV p19 γb S96A S96D S96R α-GFP α-Flag CBB GFP mRNA rRNA γb (μg) 0.05 0.1 0.2 0.3 0.4 0.5 0.05 0.1 0.2 0.3 0.4 0.5 Shifted Free γb γb + PKA

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Fig. 5Phosphorylation ofcb is critical for its local and systemic RNA silencing suppression activity. (a) InfiltratedNicotiana benthamianaleaves

coexpressing sGFP andcb or its mutants S96A, S96D and S96R were imaged under long-wave UV light at 3 d post-infiltration (dpi) or 6 dpi. TBSV p19 and empty vector (pGD) were used as the positive and negative controls, respectively. (b) Western blot (upper panels) and Northern blot (lower panels) of GFP protein and GFP mRNA, respectively, from co-infiltrated leaves at 3 dpi (left) and 6 dpi (right). In the Western blot, anti-GFP and anti-Flag antibodies were used to detect the accumulation of GFP andcb variants. Coomassie brilliant blue (CBB)-stained total proteins were shown as loading controls. In the Northern blot analysis, methylene blue-stained rRNAs were used as loading controls. (c) The suppression of systemic silencing of GFP mRNA in GFP transgenicN. benthamiana16c bycb and its mutants S96A, S96D and S96R. Photographs were taken at 10 dpi (upper panel). The ratio shows the number of systemic silenced plants out of the total number of infiltrated plants. The silencing efficiency was scored in three independent experiments (lower panel). (d) Comparative EMSA was performed with an increasing amount (0.05–0.5lg) of recombinantcb protein. The 21-bp dsRNAs were labeled with [c-32_P]

ATP. The reaction containing PKA only was used as a negative control. (e) The relative intensity of each shifted band was quantified using QUANTITYONE software. Error bars representSE from three independent experiments (Student’st-test:*,P<0.05).

form homologous oligomerizations, which are required for VSR activity (Bragg & Jackson, 2004). To test the effects of PKA-mediated phosphorylation oncb self-interaction, we per-formed a yeast two-hybrid study. As shown in Fig. S6(a), S96A, S96D or S96R mutations did not disrupt cb self-interaction. To confirm this result, we carried out a bimolecular fluores-cence complementation (BiFC) assay in which cb or Ser-96 mutants were fused with the N-terminal (YFPN) or C-terminal (YFPC) halves of yellow fluorescence protein (YFP), respec-tively, and coexpressed combinations of these in plant leaf epi-dermal cells. YFP signals were detected in homologous combinations (Fig. S6b), suggesting that the self-interaction was not compromised by the disruption ofcb phosphorylation. Furthermore, to detect the multiple oligomerization species of

cb protein (Bragg & Jackson, 2004), a 3xFlag tag was fused to the C-terminus of WT cb or its phosphorylation mutants. Transiently expressed proteins were harvested and subjected to Western blot with anti-Flag antibody. These results showed that all of the mutants multimerized in vivo, depicted by the higher molecular bands corresponding to the size ofcb dimers or oligomers (Bragg & Jackson, 2004) (Fig. S6c). The above

results indicate that the PKA-mediated phosphorylation ofcb on Ser-96 had no apparent effect oncb self-interaction.

Suppression of BSMV-induced cell death response bycb is

functionally distinct from its VSR activities

Since the PKA-mediated phosphorylation of cb on Ser-96 is related to both viral suppression of cell death and viral suppression of RNA silencing, we examined whether these two processes were functionally related. We infectedN. benthamianaleaves with WT BSMV or acb phosphorylation deficient mutant (BSMVS96A) via

agroinfiltration, together with an empty vector orcb protein tran-sient expression vector. Cell death levels were four- to five-fold greater in BSMVS96A-infected leaves than in WT BSMV-infected

leaves (Fig. 7a,b). Overexpression of WT cb or S133A mutant, but not S96A, greatly reduced cell death in plant leaves infected with BSMVS96A(Figs 7a,b, S7a,b). Since S96A could not suppress

RNA silencing, we wondered if the overexpression of other VSRs could suppress the BSMVS96Ainduced cell death response. We

chose four well-studied VSRs, including TBSV p19 (Lakatos et al., 2004), Tobacco etch virus (TEV) HC-pro (Valli et al.,

0 20 40 60 80 100 120 α-CP α-TGB1 α-γb rRNA RNA α W e stern blot Northern blot 100 76 5 95 2 74 3 % α-GFP CBB

N. benthamianainfected by:

RNAγ-GFP sgRNAγ-GFP Relative intensity of GFP fluorescence CBB rRNA -BSMV_γb-GFP BSMVS96R-GFP BSMVS96D-GFP BSMV_S96A-GFP 100 51 4 70 5 44 8 % 100 31 6 60 5 30 2 % *** * *** 100% 28 4 % 77 11 % 26 3 % (a) (b) (c)

Fig. 6Accumulation ofBarley stripe mosaic virus(BSMV) or mutant viruses in inoculated

Nicotiana benthamianaleaves. (a) Western blot and Northern blot analysis of total protein and RNA samples extracted from infiltrated leaves at 3 d post-infiltration (dpi). Anti-TGB1, anti-CP, and anti-cb antibodies were used to detect viral protein

accumulation. RNAastrand-specific RNA probe was used to determinate viral RNA accumulation. Coomassie brilliant blue (CBB)-stained total proteins or methylene blue-stained rRNA were used as loading controls. RNAaaccumulations were quantified by IMAGEJ software. Error bars indicateSE. (b) GFP fluorescence in infiltratedN. benthamianaleaves at 3 dpi was analyzed by confocal laser scanning microscopy (CLSM) (left panel). Bars, 100lm. Relative intensity of GFP fluorescence was quantified by IMAGEJ software (right panel). Error bars representSE from three independent experiments (Student’st-test:*,P<0.05;

***,P<0.001). (c) Western blot (upper panel) and Northern blot (lower panel) analysis of GFP or viral RNA accumulation. Accumulation levels of GFP protein or sgRNAc-GFP were quantified using IMAGEJ software. TheSE was from three independent experiments. CBB-stained total proteins and methylene blue-stained rRNA served as loading controls.

BSMV

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0

2

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6

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N. benthamiana

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Fig. 7cb suppression ofBarley stripe mosaic virus(BSMV)-induced cell death response is functionally distinct from its VSR activities. (a) Trypan blue staining of BSMV or BSMVS96Ainoculated leaves after co-infiltration with empty pGD vector (EV) or pGD-cb, respectively. The images were taken at 7 d

post-infiltration (dpi). (c) Trypan blue staining of leaves from nontransgenic orcb, HC-pro or p19 transgenicNicotiana benthamianaplants infected with BSMV or mutant virus BSMVS96A, BSMVS96Dor BSMVS96R, respectively (from top to bottom). Photographed were taken at 7 dpi. (e) Trypan blue staining

of BSMV, BSMVS96Aand BSMVDcb-P19inoculated leaves at 7 dpi (upper panel). Systemic infected leaves were photographed at 10 dpi (lower panel). (b, d,

f) Intensity quantification of Trypan blue staining from (a), (c) and (e), respectively, using IMAGEJ software. Three independent experiments were analyzed in each graph. Error bars indicateSE (Student’st-test:*,P<0.05;**,P<0.01). Bars: (a, c, e upper panel) 1 cm; (e lower panel) 5 cm.

2017),Cucumber mosaic virus(CMV) 2b (Duanet al., 2012), and Turnip crinkle virus (TCV) p38 (Thomas et al., 2003). These VSRs were overexpressed with WT BSMV or BSMVS96A

inocu-lated via agroinfiltration. Trypan blue staining of the infected leaf area at 7 dpi showed that the transient overexpression of other VSRs could not compromise the cell death response caused by the BSMVS96Asingle mutation (Fig. S8a,b). We next investigated if

transgenic expression of these VSRs could recover cell death response caused by cb phosphorylation deficient mutants. WT BSMV, BSMVS96A, BSMVS96D or BSMVS96R were inoculated

into transgenic N. benthamiana leaves stably expressing BSMV

cb, TBSV p19 or TEV HC-pro as previously described (K. Zhang et al., 2017). At 7 dpi, the level of cell death response was evalu-ated via Trypan blue staining. High levels of cell death were not observed in all plant leaves infected with BSMV or BSMVS96D

(Fig. 7c,d). However, in leaves infected with BSMVS96A or

BSMVS96R, severe cell death response could only be suppressed

by the transgenic expression of BSMV cb (Fig. 7c,d). We also tested TBSV p19 expression from BSMV. In this case, the open reading frame ofcb in RNAcwas replaced with that of TBSV p19, generating a recombinant virus BSMVDcb-p19 (K. Zhang et al., 2017). Like BSMVS96A, infection of BSMVDcb-p19induced

similarly high levels of cell death response compared to WT BSMV infected leaves (Fig. 7e,f).

Thus, overexpression of other VSRs transiently, transgenically, or from the BSMV genome, all failed to suppress BSMVS96A

induced cell death response, suggesting that inhibition of cell death response by phosphorylated cb is not due to its RNA silencing suppressor activity. This study demonstrated that BSMV cb suppressed virus-induced cell death response to facilitate virus survival in both dicot and monocot plants. Furthermore, it showed that this function is regulated by the PKA-mediated phosphorylation ofcb protein at its Ser-96 site.

Discussion

RNA binding proteins involved in mRNA editing or degrada-tion are often phosphorylated (Thapar, 2015). Phosphoryladegrada-tion affects the kinetics of their RNA binding capacity and thus regu-lates cellular functions. VSRs are a group of viral proteins that can bind to dsRNAs (Meraiet al., 2006) and could be regulated by phosphorylation. A recent report on geminivirus demon-strated that phosphorylation of the viral protein bC1 destroyed its gene silencing suppression activities at both transcriptional and post-transcriptional levels, as well as attenuating its symp-tom-determinant functions (Zhong et al., 2017). Deletion of a putative phosphorylation sequence or the mutation of two serine sites within this sequence in CMV 2b greatly weakened CMV-induced symptoms (Lucy et al., 2000). A recent study showed that the phosphorylation of CMV 2b regulated its subcellular localization to influence the VSR activity of 2b protein (Nemes et al., 2017). Using LC-MS/MS, we showed that the Ser-96 site incb is phosphorylated by a PKA-like protein kinase and found that its phosphorylation is related to BSMV induced symptoms. We discovered that, unlike geminivirus bC1, phosphorylation on Ser-96 is required for BSMV cb suppression of RNA

silencing. We also identified Ser-133 as a secondary phosphory-lation site whose phosphoryphosphory-lation depends on phosphoryphosphory-lation of Ser-96. However, our data suggest that phosphorylation of Ser-133 does not have a detectable function during viral infec-tion (Figs S2–S4, S7).

In vitro kinetics studies also suggest that phosphorylation of cb increased its dsRNA binding by two- to three-fold (Fig. 5e). The N-terminus ofcb contains three motifs, namely C1, BM and C2. C1 and C2 are two cysteine clusters consisting of zinc finger-like motifs (Bragget al., 2004). Between C1 and C2 is a basic motif (BM) that has RNA binding activity (Donald & Jackson, 1996). The C-terminal region ofcb (aa 95–140) contains the Ser-96 phosphoryla-tion site and has six predicted heptad repeat coiled-coil structures that participate in homologous interactions, which is essential for RNA silencing suppression (Bragg & Jackson, 2004). In our study, the phosphorylation-deficient mutants S96A and S96R attenuated the local and systemic VSR activity ofcb (Fig. 5a,c), but maintained an ability to form dimers and oligomers (Fig. S6c). These results indicate that homologous interactions are necessary but not sufficient for RNA silencing suppression. However, phosphorylation of Ser-96 increased its RNA binding activity, which is mediated by the N-terminal half ofcb. In numerous RNA binding proteins, phosphory-lation induces a global conformational change that allosterically alters their RNA-binding functions (Thapar, 2015). Alternatively, phos-phorylation can occur in the RNA binding domains. Our study showed that Ser-96 is located outside the known RNA binding domain, suggesting that the phosphorylation of Ser-96 influencescb RNA binding activity in an allosteric manner, possibly via phospho-rylation-induced protein conformational changes.

Numerous studies have shown that in addition to RNA silenc-ing suppression, VSRs usually play important roles in determin-ing virus-induced symptom developments (Yang et al., 2008; Lewsey et al., 2009; Zhang et al., 2012). However, the mecha-nisms of symptom modulation by most VSRs are not fully understood. Whether VSRs evolved with totally different mecha-nisms or they share some common features for symptom modula-tion is not known. In a previous study, the Arabidopsis thaliana ecotype C24 recovered from disease after being infected with a mutant CMV unable to express 2b, the VSR of CMV (Lewsey et al., 2009). Interestingly, in our study, we also observed a recov-ery phenotype of BSMV infected N. benthamiana plants when Ser-96 of cb was mutated to a nonphosphorylatable alanine (BSMVS96A) (Fig. 3e). We found at the early stage of BSMV

infection that BSMVS96Adid not replicate to a higher level than

WT BSMV. However, in the long term, survival of the plant was favored, as new shoots of BSMVS96A-infected N. benthamiana

plants were shown to be virus-free (Fig. 3e). This symptom mod-ulation function conferred by phosphorylated cb seems to be independent of its VSR activity, since the transient, transgenic or virus-driven expression of other VSRs could not suppress BSMVS96Ainduced cell death response (Figs 7, S8). However,in trans expression of WT cb could complement the loss-of-function phenotype of BSMVS96A. Thus, we conclude thatcb’s

function of symptom modulation is independent of its VSR activity and this novel function is regulated by phosphorylation at its Ser-96 site.

Plant cell death is an innate immune response that prevents pathogens from surviving and spreading into healthy tissues (Coll et al., 2011; Honget al., 2017). To infect a host cell successfully, pathogens have evolved different strategies to evade cell death responses. Bacterial or fungal pathogens, such as Pseudomonas syringae or Phytophthora infestans, evolved type III effectors or effector protein Suppressor of Necrosis 1 (SNE1) to suppress the cell death response in plant cells (Jamiret al., 2004; Kelleyet al., 2010). However, viral proteins, like TMV p50 (Padgett et al., 1997), TCV p38 (Dempsey et al., 1997), Potato virus X(PVX) p25 (Aguilaret al., 2015) and CMV 2b (Inabaet al., 2011), often induce a hypersensitive response or cell death response, as they can be recognized by plant encoded resistance (R) genes or other defense-related proteins. However, it is not known whether viral pathogens act like bacteria or fungi and antagonize or mute severe plant cell death responses. In our study, by observing necrosis levels or using Trypan blue staining to measure cell death under viral infection whencb is expressedin trans,in cis, or within the viral genome, we showed that the phosphorylated version of BSMVcb suppresses plant cell death responses (Fig. 7a,b). This function ofcb favors long-term viral survival and pathogenicity, similar to the role played by type III effectors or effector proteins involved in bacterial or fungal infections.

Overall, we show that successful viral infection by BSMV relies on a balance between maximum viral replication and maximum viral survival (i.e. less viral replication), and that such a balance is achieved through phosphorylation of the BSMV-encoded multi-functional protein cb. This work demonstrates a novel role for VSR in virus–host interactions and contributes to our under-standing of complex viral counter-host mechanisms.

Acknowledgements

We thank Dr Andrew O. Jackson (University of California at Berkeley, USA), Dr Huiqiang Lou (China Agricultural Univer-sity), and members of the Li lab for useful and crucial discussions. We would also like to thank Dr Yau-Heiu Hsu (National Chung Hsing University) for providing the detailed protocol forin vitro phosphorylation assays and Dr Zhen Li (Mass Spectrometry Facil-ity, China Agricultural University) for technical assistance with LC-MS/MS. We are very grateful to Drs Feng Qu and Xiaofeng Zhang (Ohio State University, USA) and Dr Bryce Falk (Univer-sity of California at Davis, USA) who provided p19 and HC-pro transgenicN. benthamianaseeds, respectively. This work was sup-ported by the National Natural Science Foundation of China (31570143 and 31270184) and the Innovative project of SKLAB (2017SKLAB1-6) to D.L., the Fundamental Research Funds for the Central Universities (2017SY003) to Y.Z., and the startup fund provided by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions to K.X.

Author contributions

D.L. and X.Z. designed the research. X.Z., K.D., K.Z., X.J. and M.Y. performed the experiments. X.Z., K.D., K.X. and D.L. analyzed the data. X.Z., D.L. and K.X. wrote and revised

the manuscript. Y.Z., X.W., C.H. and J.Y. contributed through discussions.

ORCID

Dawei Li

X

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Supporting Information

Additional Supporting Information may be found online in the Supporting Information tab for this article:

Fig. S1Prediction of candidate kinase for the phosphorylation of

cb at Ser-96 by the online servers Ne