Virology 425 (2012) 113–121

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The Rice stripe virus pc4 functions in movement and foliar necrosis expression inNicotiana benthamiana

Chao Zhang a, Xinwu Pei a, Zhixing Wang a, Shirong Jia a, Shiwei Guo b, Yongqiang Zhang a,⁎, Weimin Li a,⁎a Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 12 South Zhongguancun Street, Beijing, 100081, PR Chinab Institute of Food Crops, Jiangsu High Quality Rice R & D Center, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, PR China

⁎ Corresponding authors. Fax: +86 10 82106102.E-mail addresses: zhangyongq@sina.com (Y. Zhang)

(W. Li).

0042-6822/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.virol.2012.01.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 October 2011Returned to author for revision20 November 2011Accepted 6 January 2012Available online 2 February 2012

Keywords:RSVpc4Cell-to-cell movementLong-distance movementFoliar necrosisDomain

The Rice stripe virus (RSV) pc4 has been determined as the viral movement protein (MP). In this study, thepc4 gene was cloned into a movement-deficient Tobacco mosaic virus (TMV). The resulting hybrid TMV-pc4, in addition to spreading cell to cell in Nicotiana tabacum, moved systemically and induced foliar necrosisin Nicotiana benthamiana, indicating novel functions of the RSV MP. A systematic alanine-scanning mutagen-esis study established the region K122–D258 of the pc4 substantially associated with cell-to-cell movement,and mutants by replacement of KGR122–124, D135, ED170–171, ER201–202, EFE218–220 or ELD256–258 with ala-nine(s) no longer moved cell to cell. However, only one amino acid group KGR122–124 was linked withlong-distance movement. The region D17–K33 was recognized as a crucial domain for leaf necrosis response,and mutagenesis of DD17–18 or RK32–33 greatly attenuated necrosis. The overall data suggested manifold rolesof the pc4 during the RSV infection in its experimental host N. benthamiana.

© 2012 Elsevier Inc. All rights reserved.

Introduction

Rice stripe virus (RSV) is the typemember of genus Tenuivirus, whichhas not been assigned to any family yet (Fauquet et al., 2005). Othermembers in this genus includeMaize stripe virus (MStV), Rice hoja blancavirus (RHBV), Echinochloa hoja blanca virus (EHBV), Iranian Wheat StripVirus (IWSV) and Urochloa hoja blanca virus (UHBV), and Rice grassystunt virus (RGSV). RSV is transmitted in a circulative persistent mannerby the small brown planthopper (Laodelphax striatellus), and causes asevere disease of rice in East Asian countries. RSV consists of foursingle-stranded genomic RNA segments, denoted as RNA1, 2, 3 and 4in the decreasing size (Toriyama and Watanabe, 1989). The RNA1 is anegative sense and contains one large open reading frame (ORF) thatencodes a putative protein with a molecular weight of 337 kDa, whichwas supposed to be theRNA-dependent RNApolymerase (RdRP) associ-ated with the RSV filamentous ribonucleoprotein (Toriyama et al.,1994). The RNA2–4, however, are all ambisense wherein ORFs are pre-sent at 5′ half of both viral and viral complementary senses. The RNA2encodes two nonstructural proteins p2 and pc2 (Takahashi et al.,1993), the RNA3 encodes one nonstructural protein p3 and a majorstructural protein pc3 (Kakutani et al., 1991), while the RNA4 encodestwo nonstructural proteins, a disease-specific protein (SP) and pc4(Kakutani et al., 1990; Zhu et al., 1992).

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The unusual genome organization and translation strategy haveprevented the development of a cloned genetic system for RSV,making it impossible to directly manipulate the specific genes withinthe context of viral genomes. So far, most investigations have beenrestricted to phylogenetic analysis of different isolates (Kakutaniet al., 1990; Zhu et al., 1992; Wei et al., 2009), and analysis of nucleicacid binding activity and subcellular localization of the RSV-encodedgene product (Liang et al., 2005a, 2005b). Little progress has beenmade in characterizing the biological roles of the seven RSV-encoded proteins until recent years. Using the Potato virus X-derivedvectors, the pc4 has been defined as viral movement protein (MP)(Xiong et al., 2008), while both NS2 and NS3 as gene silencingsuppressors (Du et al., 2011; Xiong et al., 2009).

RSV naturally occurs only in rice, wheat and maize, about 30 spe-cies in the family Gramineae. Recently, the RSV was noticed to be ableto systemically infect Nicotiana benthamiana (Xiong et al., 2008),a widely used experimental host in plant virology (Goodin et al.,2008). Although symptoms caused by RSV in rice vary with theplant cultivars and developmental stage, the typical foliar symptomis chlorotic to yellowish stripes, and in severe infections the leavesdevelop brown to gray necrotic streaks and die (Ishii and Ono,1966). Efforts have been made to determine the symptom determi-nant (Xiong et al., 2009), no progress is available yet.

A TMV-based genetic platform has been developed recently andits usefulness has been proven in characterizing the TSWV NSm insymptomatology and movement (Lewandowski and Adkins, 2005;Li et al., 2009) and the Rice ragged stunt virus Pns6 in cell-to-cell

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114 C. Zhang et al. / Virology 425 (2012) 113–121

movement (Wu et al., 2010). In the current study, using the sameTMV-based system (Lewandowski and Adkins, 2005), we demon-strated that the pc4, the previously identified RSV MP (Xiong et al.,2008), not only complemented cell-to-cell movement of a MP- andCP-deficient TMV in Nicotiana tabacum and N. benthamiana, but alsosupported the TMV hybrid long-distance movement as well as in-duced foliar necrosis in N. benthamiana. These findings determinedtwo novel functions associated with the RSV MP, and indicated thatthe pc4 is a multifunctional protein involved in cell-to-cell move-ment, long-distance movement and symptomatology. Using the strat-egy of alanine-substitution mutation, we further characterized theessential pc4 domains required for cell-to-cell movement, long-distance movement and foliar necrosis induction, and the possiblerelationship among the three pc4-mediated biological activities wasdiscussed.

Results

RSV isolate JS caused systemic symptoms of chlorotic mottling inN. benthamiana leaves

It has been reported that RSV systemically infects N. benthamianabut without any symptom description (Xiong et al., 2008). In thisstudy, an RSV isolate JS, which causes typical foliar symptom of chlo-rotic striping in japonica rice Wuyujing No. 3 (Fig. 1A), was collectedfrom Jiangsu, southeast China, and was transmitted to N. benthamianathrough mechanical sap inoculation. After ~14 days post-inoculation(dpi), the inoculated leaves remained asymptomatic, whereas theupper non-inoculated leaves appeared chlorotic mottling (Fig. 1B).The spread of RSV throughout the N. benthamiana plants was con-firmed with standard reverse transcription PCR (RT-PCR) assays fordetection of the p2, p3 and pc4 genes in both inoculated and uppernon-inoculated leaves (data not shown).

The pc4 supported the TMV hybrid cell-to-cell movement in tobacco

The pc4 ORF from the RSV isolate JSwas cloned and sequenced, andwas found to be completely identical at both the nucleotide and aminoacid levels to a RSV pc4 sequences available in Genbank (ABC68339).The pc4 gene was inserted behind the CP sgRNA promoter inpTMVcpGFP, a MP- and CP-deficient TMV vector (Grdzelishvili et al.,2000), generating a hybrid virus TMV-pc4 (Fig. 2A) which encode

Fig. 1. The RSV-infected rice (A) and N. benthamiana (B) present foliar symptoms ofchlorotic/yellowish stripes and chlorotic mottling, respectively.

the native pc4 protein. The TMVcpMP (Fig. 2A), which express thenative TMV MP (Genbank accession number NC_001367) driven bythe CP sgRNA promoter, was also constructed as a control.

The RNA transcripts derived from the pTMV-pc4 were used toinoculated tobacco plants homozygous for the resistance gene N andthe TMV MP [NN-MP(+)] as a positive control for cell-to-cell move-ment. Significant local lesions appeared 3–4 dpi on the inoculatedleaves (Fig. 2B), indicating the viability in plant. The transcriptswere then inoculated in the expended leaves of N. tabacum Xanthinc, and in concert with the previous finding that the pc4 trans-complements the intercellular trafficking of a movement-deficientPVX in N. benthamiana (Xiong et al., 2008), the TMV-pc4 spread cellto cell, thus producing local lesions that were first apparent at4–5 dpi and reached a mean diameter of 1.06 mm at 10 dpi (Fig. 2Band Table 1). Interestingly, on inoculated leaves of susceptible tobac-co Xanthi, the TMV-pc4 did not produce local lesions but inducedtan-colored necrotic spots (Fig. 2C). Similarly, the transcripts fromthe pTMVcpMP induced apparent local lesions in NN-MP(+) and to-bacco Xanthi nc at 4 dpi (Fig. 2B). However, this hybrid virus causedno symptom in inoculated leaves of tobacco Xanthi at 10 dpi(Fig. 2C), though the successful local infection was established asrevealed by the RT-PCR assays (data not shown). These data demon-strated that the TMV-pc4 established local infection in tobacco plants,also suggested a novel role of the pc4 in foliar necrosis development.

The pc4 induced foliar necrosis and supported the TMV hybridlong-distance movement in N. benthamiana

To verify the ability of the pc4 in foliar necrosis expression, theTMV-pc4 was inoculated in N. benthamiana, the experimental hostof RSV (Xiong et al., 2008). At 6–8 dpi, the inoculated leaves ofN. benthamiana appeared chlorotic, which turned tan colored necroticringspots ~10 dpi (Fig. 1C); while at ~8–10 dpi, the expanded uppernon-inoculated leaves began to show deformation that precedednecrosis striping/mottling at ~15 dpi (Fig. 2D). Northern blotting anal-ysis confirmed that the TMV-pc4moved beyond the inoculated leaves(Fig. 2E). The inoculations were repeated four times for a total of 16experimental plants, and the results were reproducible. In contrast,the TMVcpMP established local and systemic infection in N. benthami-ana (Fig. 2E) but caused no visible symptom in inoculated leaves andonly leaf deformation in upper non-inoculated leaves (Fig. 2D).Taken together, these data conclusively indicated that the pc4, theputative RSV MP, is the symptom determinant in N. benthamiana, aswell as has an additional role in long-distance movement in this host.

Phylogenetic analysis of the putative tenuiviral MPs identifiescommonalities for mutagenesis

Besides the RSV pc4, the RGSV pc6 has also been determined asa tenuiviral MP (Hiraguri et al., 2011). Both proteins only share 24%sequence identify but do have a few common domains (Fig. 3). Todate, the MPs of the other five recognized tenuivirus species, includ-ing MStV, RHBV, EHBV, IWSV and UHBV, are still unknown. However,amino acid sequence comparisons revealed that the RSV pc4 shared aconsiderably high degree of similarity with the pc4 orthologs of thesefive tenuivirus species (Fig. 3). Additionally, beyond the conservationacross the primary sequences, all these tenuiviral pc4 proteins shareda similar secondary structure (data not shown) as predicated previ-ously (Melcher, 2000), suggesting the role of the five putative pc4orthologs in the tenuivirus cell-to-cell movement.

To determine functional domain(s) of the RSV pc4 protein requiredfor movement and foliar necrosis development, twelve clusters ofcharged amino acid(s) conserved within identical or highly similarregions of the tenuiviral pc4 proteins were selected for alanine-substitution mutagenesis in the RSV isolate used in this study(Fig. 3). The resulting mutants were sequenced to confirm that the

Fig. 2. Cell-to-cell movement, long-distance movement and foliar symptom expression of the TMV-pc4. (A) Genome organization of TMV, TMV-pc4 and TMVcpMP. Black box belowline represents the TMV CP subgenomic promoter. (B) Local lesions on inoculated leaves of NN-MP(+) and Xanthi nc tobacco plants induced by the TMV-pc4 at 4 and 10 dpi,respectively, and by the TMVcpMP at 4 dpi. (C) TMV-pc4 caused necrotic spots on inoculated leaves of Xanthi tobacco plants (10 dpi), whereas TMVcpMP induced no any localsymptoms. (D) TMV-pc4 induced necrotic ringspots on inoculated leaves (10 dpi) and deformation and necrosis striping/mottling on upper non-inoculated leaves (15 dpi) of N.benthamiana, whereas TMVcpMP induced no local symptoms (10 dpi) and caused systemic symptoms of only leaf deformation (15 dpi). (E) Northern blotting analysis of totalRNA extracted from the inoculated leaves and upper non-inoculated leaves of N. benthamiana at 6 dpi and 10 dpi, respectively. Bands corresponding to genomic and subgenomicRNAs are marked. The 18S and 28S rRNAs are shown to indicate the relative equivalency of samples loaded.

Table 1Summary of the characteristics of alanine-substituted mutants of the pc4 in plants.

Mutanta Local infection Systemic infection Symptoms in NB-MP (+)

Xanthi ncb N. benthamianac N. benthamianad NB-MP(+)d Locale Systemice

wt pc4TMV-pc4 1.06±0.22 + 16/16 16/16 NR Severe N

Alanine mutantsA5 0.99±0.15 + 10/10 12/12 NR Severe NA17–18 − + 0/10 12/12 Mild NR Mild NA32–33 0.34±0.012 + 0/10 12/12 Mild NR⁎ Mild NA77–79 − + 0/10 12/12 NR NA86–87 1.18±0.26 + 10/10 12/12 NR Severe NA122–124 − − 0/10 0/12 NR NoneA135 − − 0/10 12/12 NR NA170–171 − − 0/10 12/12 NR NA201–202 − − 0/10 12/12 NR NA218–220 − − 0/10 12/12 NR NA256–258 − − 0/10 12/12 NR NA276–277 0.56±0.10 + 10/10 12/12 NR Severe N

a RNA transcripts of corresponding mutants were used to inoculate as described in Materials and methods. Successful infection of plants was determined by symptom appearanceand by Northern blotting analysis using RNA from the inoculated and systemic leaves. Results of at least three separate experiments were combined.

b Infectivity on N. tabacum Xanthi nc (Xanthi nc). Mean size of lesions on the inoculated leaves was measured 10 dpi (diameter in mm±SD of at least 50 measurements). −, nolesions developed.

c Local infection of N. benthamiana at 10 dpi. At least 10 plants were used for each mutant. The infection status of the inoculated leaves was determined by symptom appearanceand Northern blotting analysis of extracted RNA. +, symptom appeared and viral RNA detected; −, no symptom and no viral RNA accumulated.

d Systemic infection of N. benthamiana (20 dpi) and TMV MP-transgenic N. benthamiana [NB-MP(+)] (15 dpi) showed as ‘number of plants systemically infected/numberof plants inoculated’.

e Local and systemic symptoms of the inoculated NB-MP(+) plants at 10 and 15 dpi, respectively. Due to lack of systemic infection, the TMV-A122–124 induce no systemicsymptom in NB-MP(+) plants. NR, necrotic ringspot; N, necrosis.

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image of Fig.�2

Fig. 3. Alignment of tenuiviral pc4 proteins reveals conserved amino acids targeted inRSVpc4 formutagenesis. Positions of theRSVpc4 alanine-substitutedmutants (A5, A17–18, A32–33,A77–79, A86–87, A122–124, A135, A170–171, A201–202, A218–220, A256–258 and A276–277) are indicated above the sequences. Aspartate residue of the conserved ‘D’motif found in‘30K-superfamily’movement proteins are indicated below the sequenceswith an asterisk. The alignmentwas generated using the Clustal-X program, andGenbank accession numbers forthe pc4 proteins of the different viruses aligned are RSV, ABC68339;Maize stripe virus (MStV), AAB22542; Rice hoja blanca virus (RHBV), AAB69770; Echinochloa hoja blanca virus (EHBV),AAC38000; Iranian Wheat Strip Virus (IWSV), AAP82278; Urochloa hoja blanca virus (UHBV), AAB58303; and Rice grassy stunt virus (RGSV), BAA86112.

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intended pc4mutagenesis was successful and to confirm that no addi-tional mutations were introduced. All mutants were individuallycloned into pTMVcpGFP (Grdzelishvili et al., 2000), as described previ-ously (Lewandowski and Adkins, 2005).

To test for infectivity of the TMV-based hybrids encoding mutantpc4 proteins, the RNA transcripts were generated and independentlyinoculated in the expended leaves of NN-MP(+) plants. As expected,all of hybrids induced local lesions on inoculated leaves 3–4 dpi, sim-ilar to the behaviors of the parent hybrid TMV-pc4 in the NN-MP(+)plants (Fig. 2B).

Domains of the pc4 for cell-to-cell movement

The previous report (Xiong et al., 2008) with our current data con-clusively determined the role of the pc4 in cell-to-cell movement. Todefine the domain(s) essential for this biological activity, the RNAtranscripts derived from the twelve alanine mutants as well aspTMV-pc4 were inoculated in Xanthi nc tobacco plants. Only fourmutants, A5, A32–33, A86–87, and A276–277 retained cell-to-cellmovement as evidenced by induction of local lesions at 4–6 dpi, sim-ilar to TMV-pc4 (Table 1). At 10 dpi, local lesions induced by mutantA5 reached a mean diameter of 0.99 mm, while the local lesions bymutant A86–87 were even larger (1.18 mm mean diameter) thanthose by TMV-pc4 (1.06 mm mean diameter). However, mutantsA32–33 and A276–277 induced only tiny lesions with a mean diame-ter of 0.34 mm and 0.56 mm, respectively, much smaller comparedwith those of TMV-pc4, indicating that replacement of RK32–33 orRK276–277 with alanines impaired but did not abolish cell-to-cellmovement. No noticeable lesions were induced by the other eightmutants A17–18, A77–79, A122–124, A135, A170–171, A201–202,A218–220 and A256–258 (Table 1).

Consistent with their behaviors in Xanthi nc tobacco, mutants A5,A32–33, A86–87 and A276–277 also spread from cell to cell in N.benthamiana. Notably, mutants A17–18 and A77–79, which did notinduce local lesions in Xanthi nc plants, apparently moved from cellto cell in N. benthamiana (Fig. 4A, and Table 1). The typical symptomson the leaves inoculated with TMV-pc4 and mutants varied at 10 dpi.Mutants A5, A86–87 and A276–277 induced apparent symptoms ofnecrotic ringspot like those of TMV-pc4, whereas A32–33 causedsmall faint necrotic ringspots, and A17–18 and A77–79 induced tinynecrotic spots (Fig. 4A). Consistently, Northern blot analysis showedthat mutants A5, A86–87 and A276–277 accumulated levels of viralRNA comparable to that of TMV-pc4 in inoculated leaves at 10 dpi(Fig. 4B, upper panel, lanes 1, 2, 6 and 7), while the accumulationlevels of mutants A32–33, especially A17–18 and A77–79 RNA weregreatly reduced (Fig. 4B, upper panel, lanes 3–5). No viral RNA wasdetectable in the asymptomatic N. benthamiana leaves inoculatedwith the other six mutants A122–124, A135, A170–171, A201–202,A218–220 and A256–258, all of which failed to induce local lesionsin Xanthi nc tobacco.

Failure to move from cell to cell in inoculated leaves of Xanthi nctobacco and/or N. benthamiana plants implied that mutagenesis dis-rupted functional domain(s) by replacement of one or more essentialamino acids. Six mutants were completely abolished cell-to-cellmovement in both Nicotiana spp., indicating that the charged aminoacids (shown in bold) of KGR122–124, D135, ED170–171, ER201–202,EFE218–220 and ELD256–258 were essential for cell-to-cell movement.Mutants A17–18, A32–33 and A77–79 induced tiny or even no visiblelocal lesions in Xanthi nc tobacco, and accumulated at significantlow levels in N. benthamiana, suggesting the importance of DD17–18,RK32–33 and KQD77–79 for cell-to-cell movement. Taken together,the behaviors of alanine-substituted mutants indicated the essential

image of Fig.�3

Fig. 4. Characterization of the alanine-substituted pc4 mutants in N. benthamiana. (A) Local symptoms induced by the indicated pc4 mutants at 10 dpi. Faint necrotic ringspots/spotsinduced by mutants A17–18, A32–33 and A77–79 are indicated with arrows. (B) Northern blotting analysis of total RNA from leaves of N. benthamiana inoculated with the indicatedmutants. The inoculated and upper non-inoculated leaves were collected at 10 dpi and 15 dpi, respectively. Bands corresponding to genomic and subgenomic RNAs are indicated.The 18S and 28S rRNAs are shown to indicate the relative equivalency of samples loaded.

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role of the central portion and its immediately adjacent region towardC-terminus, whereas both the extreme N- and C-terminus had no orlittle effect.

Domains of the pc4 for long-distance movement in N. benthamiana

The wt pc4 supported long-distance movement of TMV-basedhybrids in N. benthamiana (Figs. 2D and E). To determine whetherthe alanine substitutions affected long distance movement, N.benthamiana plants inoculated with transcripts of the twelvealanine-substituted mutants were observed up to 20 dpi for systemicsymptom development. When no symptom appeared, Northern blot-ting analysis was performed to assay for the presence of virus.MutantsA5, A86–87, and A276–277 moved similarly to TMV-pc4 onto uppernon-inoculated leaves of all plants at 15 dpi (Table 1 and Fig. 4B, lowpanel, lanes 1, 2, 6 and 7), whereas none of A17–18, A32–33 andA77–79 moved long distance (Table 1 and Fig. 4B, low panel, lanes3–5) though competent for cell-to-cell movement. None of themutants (A122–124, A135, A170–171, A201–202, A218–220 andA256–258) deficient in cell-to-cell movement was detected in uppernon-inoculated leaves of N. benthamiana (Table 1).

To discriminate between the effects of mutations on long-distancemovement from their effects on cell-to-cell movement, the alanine-substituted mutants were further inoculated onto the TMV MP trans-genic N. benthamiana [NB-MP(+)] (Table 1). Symptoms were evalu-ated on all plants and total RNA was extracted from upper leavesfor Northern blot analysis at 15 dpi to diminish the possibility ofconfusing the role that the mutated pc4 plays in facilitating long-distance movement with that of the transgenically-expressed TMVMP (Li et al., 2009).

In the presence of transgenic expression of the TMV MP, thetwelve alanine mutants efficiently moved from cell to cell as the

TMV-pc4 (Table 1 and Fig. 5C, upper panel, lanes 1–13). Long-distance movement of only one mutant A122–124, which was absentof cell-to-cell movement in N. benthamiana, was not detected in theupper non-inoculated leaves of any NB-MP(+) plant at 15 dpi(Table 1 and Fig. 5C, low panel, lane 7) or selected plants retainedfor 20 dpi, indicating the essential role of amino acids KGR122–124for both long-distance movement and cell-to-cell movement. On thecontrary, the other eleven alanine-substituted mutants significantlyaccumulated in the upper non-inoculated leaves of all inoculatedNB-MP(+) plants at 10 dpi, similar with TMV-pc4 (Table 1 andFig. 5C, low panel, lanes 1–6 and 8–12). Long-distance movementprofiles of mutants A17–18, A32–33 and A77–79 with impaired cell-to-cell movement, and mutants A135, A170–171, A201–202, A218–220 and A256–258 defective for cell-to-cell movement were obvious-ly different when in wt and TMV MP-transgenic plants, indicatingthat the effects of mutations on long-distance movement were inde-pendent of cell-to-cell movement.

Domains of the pc4 involved in symptom development

The domain(s) involved in pc4-mediated symptom developmentwere also identified by comparing symptoms induced by TMV-pc4and the alanine mutants on NB-MP(+) plants (Figs. 5A and B). Inasymptomatic leaves, infection was confirmed by RNA hybridization.As a negative control, the TMVcpMP was also used to inoculate theNB-MP(+) plants. Consistent with the observations from N.benthamiana (Fig. 2D), no symptoms appeared on the inoculatedleaves of NB-MP(+) at 10 dpi, while the upper non-inoculated leavesshowed significant deformation but without necrosis expression at15 dpi (Supplementary Fig. 1).

Twelve alanine mutants induced two distinguishable responses onthe inoculated leaves at 10 dpi. Mutants A5, A77–79, A86–87, A122–

image of Fig.�4

Fig. 5. Characterization of the pc4 and alanine-substituted pc4 mutants in NB-MP(+) plants. (A) Typical symptoms induced by the pc4 and indicated pc4 mutants on inoculatedleaves at 10 dpi. Faint necrotic ringspots induced by mutants A17–18 and A32–33 are indicated with arrows. (B) Typical symptoms induced by the indicated pc4 mutants onupper non-inoculated leaves at 15 dpi. Mild necrosis induced by mutants A17–18 and A32–33 are indicated with arrows. No systemic symptoms were observed with A122–124at 15 dpi. (C) Northern blotting analysis of the RNA accumulation of TMV-pc4 and indicated mutants in NB-MP(+) plants. The inoculated leaves and upper non-inoculated leaveswere collected at 10 dpi and 15 dpi, respectively. Bands corresponding to genomic and subgenomic RNAs are marked. The 18S and 28S rRNAs are shown to indicate the relativeequivalency of samples loaded.

118 C. Zhang et al. / Virology 425 (2012) 113–121

124, A135, A170–171, A201–202, A218–220, A256–258 and A276–277 induced local symptoms of necrotic ringspots/striping that werecomparable to the typical symptoms caused by TMV-pc4 (Fig. 4A),indicating that the substituted amino acids in these mutants had littleor no effect on local symptom development. In contrast, mutantsA17–18 and A32–33 induced only mild necrotic ringspots, and

sometimes A32–33 induced even no symptoms in inoculated leaves(Fig. 5A).

The systemic symptoms that developed at 20 dpi in NB-MP(+)plants inoculated with transcripts were also evaluated. Of themutants which produced local symptoms comparable to TMV-pc4,mutants A5, A86–87 and A276–277 consistently induced systemic

image of Fig.�5

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symptoms of leaf deformation following necrotic striping and conflu-ent necrosis similar to TMV-pc4 (Fig. 5B), whereas mutants A77–79,A135, A170–171, A201–202, A218–220 and A256–258 induced leafdeformation that preceded only necrotic striping but no confluentnecrosis (Fig. 5B). Consistent with the mild symptoms observed ininoculated leaves (Fig. 5A), mutants A17–18 and A32–33 inducedsystemic leaf deformation and mild or even no necrosis. Systemicsymptoms were absent in NB-MP(+) plants inoculated with mutantA122–124 (Fig. 5B), which failed to move beyond the inoculatedleaves (Fig. 5C, low panel, lane 7). Notably, the attenuated systemicsymptoms of mutants A77–79, A135, A170–171, A201–202, A218–220 and A256–258 as well as the significantly ameliorated systemicsymptoms of mutants A17–18 and A32–33 were not due to the re-duced long-distance movement, as evidence that all these alaninemutants established systemic infection as efficiently as TMV-pc4 inNB-MP(+) plants (Fig. 5C, low panel, lanes 1, 3–6 and 8–11).

Combined, these data indicated that the amino acids R5, RK86–87

and KK276–277 had little or no effect on both local and systemicsymptom development, whereas the amino acids KQD77–79, D135,ED170–171, ER201–202, EFE218–220 and ELD256–258 were involved insymptom severity in systemic leaves but were not related to localsymptom expression. Two clusters of amino acids, DD17–18 andRK32–33, were crucial for necrosis development in both inoculatedand upper non-inoculated leaves, suggesting the N-terminal regionD17–K33 as a putative domain play a fundamental role in foliar necro-sis induction.

Discussion

Owing to lack of a reverse genetic system for RSV, the biologicalfunctions of the seven RSV-encoded proteins are far less known todate. The pc4 is one of the non-structural proteins of RSV, and hasbeen recently defined as the viral MP due to trans-complementationof a movement-deficient PVX in N. benthamiana (Xiong et al., 2008).In the current study, we showed that the pc4, when expressedthrough the MP- and CP-deficient TMV vector, induced apparentnecrosis in both inoculated and upper non-inoculated leaves inN. benthamiana, indicating that the RSV MP also functions as theviral symptom determinant. Possibly due to the timing and levels ofthe pc4 protein synthesis that occur during a natural infection couldnot be duplicated with the TMV-based expression system, the foliarsymptoms induced by the heterologously expressed pc4 were some-what different with those caused by the wt RSV in N. benthamiana.

In further pursuit of the pc4-induced symptom expression, usinga systemic alanine-scanning mutagenesis, we identified the region(D17–K33) near the N-terminus of pc4 as a putative domain critical

Fig. 6. Overview of the characteristics of the alanine-substituted pc4 mutants related to cell-to-the bottom represents the pc4 from the N to C termini. The Kyte–Doolittle hydropathy plot ofhydrophilic regions (H105–D170). Vertical dashed lines in the hydropathy plot correspond to twith alanines. The crucial amino acids involved in the pc4-mediated cell-to-cell movement, lo

for leaf necrosis induction (Fig. 6). Replacement of amino acidsDD17–18 or RK32–33 within this region with alanines greatly attenuat-ed foliar necrosis expression, indicating the role of these residues inthe phenotypic symptom response. Though mechanisms of viralsymptom development are still largely unknown for most viruses(Culver and Padmanabhan, 2007), it is conceivable that the foliarnecrosis observed in this study is derived from specific interactionsbetween the pc4 and host component(s) yet unknown. Accordingly,the mutation introduced into mutant A17–18 or A32–33 might alterthe interaction between the pc4 and host component(s), therebyresulting in the greatly reduced foliar symptoms.

Previous computer analysis predicated that the RSV pc4 belongs tothe ‘30K superfamily’ of viral MPs (Melcher, 2000). This has been cor-roborated by a recent study, which showed that the pc4 is capableof complementing intercellular trafficking of a movement-deficientPVX in N. benthamiana, and has characteristics of plant virus MPs,such as association with plasmodesmata and RNA binding activity(Xiong et al., 2008). In the current study, we demonstrated thatpc4 could also rescue the cell-to-cell movement of the MP- and CP-deficient TMV hybrid in both N. benthamiana and N. tabacum, provid-ing additional evidence for the pc4 as a viral MP.

Alanine-substitution mutations within the pc4 showed that re-placement of amino acids KGR122–124, D135, ED170–171, ER201–202,EFE218–220 or ELD256–258 with alanines eliminated cell-to-cell move-ment of the pc4, indicating that the region (K122–D258) encompassingthe central portion and its immediately adjacent region towardC-terminus substantially associated with cell-to-cell movement(Fig. 6). Computer analysis with Kyte–Doolittle hydrophobic scalepredicated that the central portion of the pc4 (H105–D170) containeda remarkable alternation of hydrophobic and hydrophilic residues(Fig. 6), a type feature of the ‘30K superfamily’ viral MPs (Bertenset al., 2000). Mutations have been made in the alternating hydropho-bic and hydrophilic regions of TMV, Cauliflower mosaic virus (CaMV),Cowpea mosaic virus (CPMV) and TSWV MPs. In most of thesecases, the altered MPs no longer supported cell-to-cell movement(Thomas and Maule, 1995; Kahn et al., 1998; Bertens et al., 2000; Liet al., 2009), like the behaviors of mutants A122–124, A135, andA170–171 in this study.

The D motif is one of the most conserved motifs within the ‘30Ksuperfamily’ of viral MPs (Mushegian and Koonin, 1993; Melcher,2000). A previous investigation (An et al., 2003) as well as sequencecomparison of tenuiviral pc4 proteins in this study (Fig. 3) suggestedthat amino acid D135 within the pc4 was the putative D motif of thisprotein. Knockout of the D motif often renders the viral MPs disabledin cell-to-cell movement, as evidence from the altered MPs of CaMV(Thomas and Maule, 1995), CPMV (Bertens et al., 2000) and TSWV

cell movement, long-distancemovement and foliar symptom expression. The gray bar onthe pc4 is shown, and a hatched box indicates the putative alternating hydrophobic andhe numbers within the gray bar, indicating the positions of the amino acids substitutedng-distance movement and foliar symptom expression are marked with ‘+’.

image of Fig.�6

120 C. Zhang et al. / Virology 425 (2012) 113–121

(Li et al., 2009). In this study, the RSV pc4 carrying a mutation in theputative D motif (mutant A135) also did not move from cell to cell,supporting the essential role of the D motif in cell-to-cell movement.

The ‘30K superfamily’ members of viral MPs that support long-distance movement in N. benthamiana have been reported previouslywith several phylogenetically distinct viruses, including TMV (Knappet al., 2001), Red clover necrotic mosaic virus (RCNMV; Wang et al.,1998) and TSWV (Lewandowski and Adkins, 2005; Li et al., 2009).Our current study showed that the pc4 alone could support the CP-deficient TMV-based hybrid spread systemically in N. benthamiana,indicating that the RSV MP also functioned as a long-distance move-ment factor at least in the aforementioned host species.

Inoculation of the alanine-scanning mutants on N. benthamianaand NB-MP(+) plants identified that only the amino acids KGR122–124(Fig. 6), which played an essential role in cell-to-cell movement,linked with long-distance movement. In contrast, the other aminoacids D135, ED170–171, ER201–202, EFE218–220, and ELD256–258,essential for cell-to-cell movement, had little effect on long-distancemovement, indicating the separated genetic controls for cell-to-celland long-distance movement within the pc4. The similar conclusionhas been drawn from the Tobacco etch virus CP (Dolja et al., 1994,1995), the RCNMV MP (Wang et al., 1998) and the TSWV NSm (Liet al., 2009), each of which plays a role in both cell-to-cell and long-distance movement but with uncorrelated functional domains. Themost likely explanation for these observations was that cell-to-celland long-distance movement are two connected yet distinct trans-port pathways, both of which involve different types of plant cellsand structures, and require assistance from different host compo-nents as well (Carrington et al., 1996; Waigmann et al., 2004).

Combined, the experimental data in the current study demon-strated that the pc4 functioned in cell-to-cell movement, long-distance movement as well as foliar symptom expression with thedissectible functional domains, suggesting the manifold roles of thepc4 during the life cycle of RSV. To date, with the exception of theRSV pc4, six distinct tenuiviral pc4 orthologs have been determined.The RSV pc4 is the unique one, the biological functions of whichwere investigated. It merits further investigation whether theseroles as well as the related functional domains acquired from theRSV pc4 represent general properties for the tenuiviral pc4 proteins.

Materials and methods

Plant growth conditions

The N. benthamiana, Xanthi nc tobacco, Xanthi tobacco, the TMVMP-transgenic NB-MP(+) and NN-MP(+) plants were all grownand maintained in a greenhouse at 25 °C. Virus-inoculated plantswere also maintained under greenhouse conditions.

Plasmid construction

The pc4 gene was cloned from the RSV isolate by using Pfu DNApolymerase and a specific primer pair (Supplementary Table 1).According to a previously described procedure (Lewandowski andAdkins, 2005), the resulting PCR fragments that represent the pc4ORF were treated with EcoRV and XhoI, then were inserted into theEcoRV/XhoI treated pTMVcpGFP (Grdzelishvili et al., 2000) to gener-ate pTMV-pc4 (Fig. 2A). With the same strategy, the TMV MP genederived from the TMV U1 strain (Dawson et al., 1986) was ligatedwith the EcoRV/XhoI treated pTMVcpGFP, resulting in pTMVcpMP(Fig. 2A).

To define the functional domains within the pc4, twelve alanine-substitution mutants were generated with the primers listed inSupplementary Table 1, and were digested with EcoRV and XhoI,and ligated into EcoRV/XhoI-digested pTMVcpGFP as previously de-scribed (Lewandowski and Adkins, 2005). Notably, the mutations in

pTMV-A5 and pTMV-A276–277 were introduced by direct PCRamplification, whereas the mutations in pTMV-A17–18, pTMV-A32–33, pTMV-A77–79, pTMV-A86–87, pTMV-A122–124, pTMV-A135,pTMV-A170–171, pTMV-A201–202, pTMV-A218–220 or pTMV-A256–258 were introduced by using overlapping PCR, respectively (Ho et al.,1989).

Plant inoculation

For infection of plants with viral vectors, infectious RNA tran-scripts were produced from KpnI-linearized plasmids with T7 RNApolymerase (Lewandowski and Dawson, 1998), and were inoculatedin two upper expanded leaves of ~6 week-old plants as previouslydescribed (Dawson et al., 1986).

For plant sap inoculation experiments, infectious sap was pre-pared with 1 g of fresh RSV-infected rice leaves that were maceratedin 5 mL of inoculation buffer (50 mM KH2P04, pH 7.0, 1% Celite), andwas mechanically inoculated in ~6 week-old N. benthamiana leavesdusted with carborundum powder (600 mesh).

Northern blotting analysis

Equivalent amounts (5 μg) of total RNA (quantified using a Nano-Drop spectrophotometer; NanoDrop Technologies, Wilmington, DE)extracted from leaves were analyzed by Northern blotting using aTMV 3′UTR-specific probe (Lewandowski and Dawson, 1998).

Computer analysis of the RSV pc4 protein

The tenuiviral pc4 protein sequenceswere alignedwith ClustalX 1.83(Thompson et al., 1997) and viewed with GeneDoc (Nicholas andNicholas, 1997). Hydrophobicity was analyzed with Kyte–Doolittlehydrophobic scale (http://www.vivo.colostate.edu/molkit/hydropathy/index.html) (Kyte and Doolittle, 1982).

Supplementary materials related to this article can be found on-line at doi:10.1016/j.virol.2012.01.007.

Acknowledgments

We thankDr. Dennis J. Lewandowski, Department of Plant Pathology,Ohio State University, Columbus, OH, USA, and Dr. Scott Adkins, UnitedStates Department of Agriculture, Agricultural Research Service, FortPierce FL, USA for supplying the TMVcpGFP vector and seeds ofNN-MP(+) and NT-MP(+).We are also grateful to Dr. William Dawson,University of Florida, Citrus Research and Education Center, Lake Alfred,FL, USA for providing the cDNA clone of TMV U1, Dr. Deom C. Michael,Department of Plant Pathology, University of Georgia, Athens, GA, USA,for granting us permission to use the NB-MP(+). This research was sup-ported by the National Natural Science Foundation of China (grant no.31170140), the National Major Special Project of China on NewVarietiesCultivation for Transgenic Organisms (grant nos. 2008ZX08001-002 and2008ZX08004-004), and the Special Fund for Agro-scientific Research inthe Public Interest (grant no. 201003031).

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http://dx.doi.org/10.1111/j.1364-3703.2011.00716.x

The Rice stripe virus pc4 functions in movement and foliar necrosis expression in Nicotiana benthamianaIntroductionResultsRSV isolate JS caused systemic symptoms of chlorotic mottling in N. benthamiana leavesThe pc4 supported the TMV hybrid cell-to-cell movement in tobaccoThe pc4 induced foliar necrosis and supported the TMV hybrid long-distance movement in N. benthamianaPhylogenetic analysis of the putative tenuiviral MPs identifies commonalities for mutagenesisDomains of the pc4 for cell-to-cell movementDomains of the pc4 for long-distance movement in N. benthamianaDomains of the pc4 involved in symptom development

DiscussionMaterials and methodsPlant growth conditionsPlasmid constructionPlant inoculationNorthern blotting analysisComputer analysis of the RSV pc4 protein

AcknowledgmentsReferences