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Analysis of RNA-mediated virus resistance by NSs and NSm gene sequences from Tomato spotted wilt virus
Plant Science 166 (2004) 771–778
Analysis of RNA-mediated virus resistance by NSs and NSm gene sequences from Tomato spotted wilt virus Shoji Sonoda∗ , Hisaaki Tsumuki Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan Received 18 August 2003; received in revised form 18 November 2003; accepted 18 November 2003
Abstract Nicotiana benthamiana was transformed with the NSs gene sequence of Tomato spotted wilt virus (TSWV). When a non-silenced line showing high levels of the transgene expression was inoculated with Potato virus X (PVX) engineered to contain the NSs gene sequences, reduced transgene mRNA accumulation was observed. RNA silencing was the underlying mechanism for the reduction. Previously, non-transgenic N. benthamiana infected with PVX engineered to contain the nucleocapsid protein (N) gene of TSWV was shown to be resistant to TSWV infection [Plant Sci. 164 (2003) 717]. In the present study, interactions between PVX engineered to contain the NSs gene sequences and TSWV were examined. Plants infected with PVX containing the NSs gene sequences showed a partial virus resistance to TSWV. However, the degree of the resistance was apparently lower than that observed in plants infected with PVX engineered to contain the N gene. Similar low levels of the resistance to TSWV was observed in plants infected with PVX engineered to contain the NSm gene of TSWV. These results suggest that RNA-mediated virus resistance conferred by the NSs and NSm gene sequences of TSWV was less effective to TSWV. Furthermore, it was also suggested that RNA-mediated virus resistance was affected by viral genes or sequences rather than the amount of nucleotide sequence homology. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Cross-protection; RNA silencing; Nicotiana benthamiana; TSWV; Virus resistance
1. Introduction Cross-protection is defined as the ability of one virus to inhibit or prevent infection by a second virus [1]. It has been shown that cross-protection is mediated by viral RNA or viral proteins [2–4]. Ratcliff et al. [2] showed that plants infected with one virus showed resistance against the heterologous virus when both viral genomes shared sequence homology. Similar induction of the virus resistance has been reported in plants infected with certain RNA [5] and DNA viruses [6,7]. The resistance mechanism has been linked with RNA silencing, a homology-dependent RNA degradation mechanism designed to eliminate undesirable RNA in the cytoplasm [8]. RNA silencing is induced by double-stranded RNA, including RNA viruses that replicate via double-stranded RNA intermediates, and is associated with the appearance of specific low-molecular-weight ∗ Corresponding author. Tel.: +81-86-434-1219; fax: +81-86-434-1249. E-mail address: [email protected] (S. Sonoda).
RNA fragments [9]. However, in most cases, the precise mechanism of interaction between the viral genes and genomic sequences which leads to RNA silencing is unknown. Tomato spotted wilt virus (TSWV), a member of the Tospoviridae, infects a wide range of plant species worldwide and causes severe chlorotic, necrotic, and malformation symptoms [10]. The viral genome consists of three single-stranded RNA denoted S, M, and L. The S RNA and the M RNA are characterized by an ambisense gene arrangement and encode nonstructural proteins, NSs and NSm, at the 5 -end in the viral RNA strand, respectively [11,12]. In a previous study, it was shown that the infection of recombinant Potato virus X (PVX) engineered to contain the nucleocapsid protein (N) gene of TSWV caused RNA-mediated suppression of the N transgene mRNA accumulation in transgenic plants and symptom appearance by TSWV in non-transgenic plants [13]. In the present study, the capacity of the NSs gene as inducer and target for RNA silencing was examined using virus-induced gene silencing (VIGS) and RNA-mediated cross-protection.
0168-9452/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2003.11.018
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2. Materials and methods 2.1. Virus constructs TSWV was provided by Dr. S. Tsuda (National Agricultural Research Center, Tsukuba, Japan). Total RNA extraction and first strand cDNA synthesis were described previously [13]. The NSs gene was amplified from the first strand cDNA by PCR using the 5 -and 3 -end primers, 5 -GGGGATCCATGTCTTCAAGTGTTTATGAG-3 and 5 -GGGAGCTCTTATTTTGATCCTGAAGCATG-3 , respectively. The 5 and 3 -end primers were designed to contain BamHI and SacI restriction sites (underlined), respectively. PCR conditions were 30 cycles of 30 s at 94 ◦ C, 1 min at 42 ◦ C and 2 min at 72 ◦ C. However, unexpectedly, the NSs gene lacking the 3 terminal 219 bp was amplified only by the 5 -end primer. The PCR-amplified fragment of the NSs gene (ca. 1.2 kb) was digested with BamHI and cloned into a similarly digested pBluescript (Stratagene, La Jolla, CA, USA) (pBS.NSs-1). The PCR-amplified fragment was also cloned into pGEM-T Easy (Promega, Madison, WI, USA) (pGEM.NSs-1). The EcoRI fragment of pGEM.NSs-1 was cloned into a similarly digested pBluescript (pBS.NSs-2). The SmaI–SalI fragment of pBS.NSs-2 was ligated into the EcoRV and SalI sites of the PVX vector (pP2C2S), a derivative of pGC3 [14], to produce PVX.NSs (Fig. 1). pBS.NSs-1 was digested with HindIII and the resulting fragment of 495 bp was cloned into a similarly digested pBluescript (pBS.NSs-3). The SmaI–SalI fragment of pBS.NSs-3 was ligated into the EcoRV and SalI sites of PVX to produce PVX.NSs-5 (Fig. 1). pBS.NSs-1 was digested with HindIII and the vector containing the 3 portion of the NSs gene sequence was self-ligated (pBS.NSs-4). The HincII–EcoRV fragment of pBS.NSs-4, having a length of 452 bp, was
cloned into the EcoRV site of pBluescript (pBS.NSs-5). The SmaI–SalI fragment of pBS.NSs-5 was ligated into the EcoRV and SalI sites of PVX to produce PVX.NSs-3 (Fig. 1). The 3 -terminal 219 bp of the NSs gene was amplified from the first strand cDNA by PCR using the 5 and 3 -end primers, 5 -CAGTGAAGATATCTGCAAAAGG-3 and 5 -GGGTCGACTTATTTTGATCCTGAAGCATG-3 , respectively. The 5 and 3 -end primers were designed to contain EcoRV and SalI restriction sites (underlined), respectively. The PCR-amplified fragment was digested with EcoRV and SalI and cloned into the EcoRV and SalI sites of PVX to produce PVX.NSs-3 -terminal (Fig. 1). The NSm gene was cloned from the first strand cDNA by PCR using the 5 -and 3 -end primers, 5 -GGGATATCATGTTGACTCTTTTCGGTAAC-3 and 5 -GGGTCGACCTATATTTCATCAAAGGATAAC-3 , respectively. The 5 and 3 -end primers were designed to contain EcoRV and SalI restriction sites (underlined), respectively. The PCR-amplified fragment was digested with EcoRV and SalI and cloned into the EcoRV and SalI sites of PVX (PVX.NSm). 2.2. In vitro transcription and virus inoculation In vitro transcription reactions to produce infectious PVX were described previously [13]. Virus-infected leaf homogenates prepared by grinding systemically infected leaf tissue diluted (1:10, w/v) in 100 mM phosphate buffer (pH 7.0) were used for virus inoculation. Nicotiana benthamiana transformed with the GUS-deleted pBI121 (Clontech, Paro Alto, CA, USA) (GUS-Nb) was used for the analysis of RNA-mediated cross-protection. Virus-infected leaf homogenates were mixed for a final 1:20 dilution of each virus. For single inoculation, leaves were ground at a 1:20 ratio. Inoculated plants were monitored for the appearance of symptoms.
Fig. 1. Schematic representation of TSWV NSs gene sequences introduced in the PVX vector, together with the structure of the ambisense TSWV RNA. DNA probes used in this study were also shown above the ambisense TSWV RNA.
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2.3. Plant transformation N. benthamiana was transformed with the NSs gene sequence. The BamHI–EcoRV fragment of pBS.NSs-1 was ligated into the SmaI and BamHI sites of pGEM3Z f(+) (Promega) (pGEM.NSs-2). The BamHI–SacI fragment of pGEM.NSs-2 was inserted between the 35S cauliflower mosaic virus promoter and the nopaline synthase terminator sequences of the binary plasmid, pBI121, after the GUS gene had been removed. The resulting plasmid was used for transformation using Agrobacterium tumefaciens LBA4404 (Clontech) [15]. 2.4. RNA gel blot analysis Total RNA was extracted from leaf tissues, as described by Sonoda et al. [16]. Total RNA was size-fractionated on a 1.2% agarose gel containing 0.66 M formaldehyde and transferred to Biodyne PLUS membrane (Pall Corporation, Ann Arbor, MI, USA). The blot was hybridized with a random primed 32 P-labeled fragment containing the NSs gene coding sequences. RNA gel blot analysis of 21–25 nucleotide RNA was performed essentially as described by Hamilton and Baulcombe [9]. Strand-specific RNA probes were generated by in vitro RNA transcription from pBS.NSs-3 and pBS.NSs-4 in the presence of 32 P-␣-UTP. 2.5. ELISA ELISA analysis [17] with a TSWV monoclonal antibody (Japan Plant Protection Association, Ushiku, Japan) was used to detect TSWV. A result was recorded as positive when
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the A405 value was two-fold higher than that of the negative control.
3. Results 3.1. VIGS of TSWV NSs transgene mRNA by PVX containing the NSs gene sequences N. benthamiana was transformed with the NSs gene sequence and 10 transgenic lines were regenerated in the presence of kanamycin. Among 10 transgenic lines regenerated, line NSs2 showing high levels of the transgene expression was used for further analysis (data not shown). T1 plants of line NSs2 were susceptible to PVX containing the NSs gene sequences and developed typical PVX symptoms (vein clearing and chlorotic mosaic) as was the case in the control plants carrying the GUS-deleted pBI121 (Clontech) (GUS-Nb) (data not shown). When plants of line NSs2 were inoculated with TSWV, a 1–4-day delay in symptom appearance relative to the control plants was observed, but a high proportion of the plants was eventually infected with TSWV (data not shown). To examine VIGS of the NSs transgene mRNA by PVX containing the transgene sequences, T1 plants of line NSs2 were inoculated with one of three PVX derivatives, PVX.NSs-5 , PVX.NSs-3 and PVX (Fig. 1). The accumulation of the NSs transgene mRNA in plants of line NSs2 was determined by RNA gel blot analysis using the 5 or 3 -NSs specific probe to discriminate the transgene mRNA from the viral RNA (Fig. 1). In plants infected with PVX.NSs-5 or PVX.NSs-3 , reduced transgene mRNA accumulation was observed at 14 days post-inoculation (dpi) (Fig. 2A). On the other hand, detectable levels of the transgene mRNA
Fig. 2. Analysis of the NSs-related RNA in virus-infected or non-infected transgenic N. benthamiana line NSs2. Total RNA (20 g) (A) and small RNA fraction (20 g) (B) samples were subjected to RNA gel blot analysis. A 32 P-␣-dCTP labeled NSs-5 or NSs-3 probe generated by random priming was used as the probe for mRNA gel blot analysis, while a 32 P-␣-UTP labeled in vitro transcripts corresponding to the antisense strand of NSs-5 or NSs-3 probe was used in RNA gel blot analysis of siRNA. The photographs of the ethidium bromide-stained RNA gel before transfer are shown as the loading control in (A).
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accumulated in plants infected with PVX at 14 dpi, as was the case in the non-inoculated healthy plants (Fig. 2A). Two independent experiments showed similar results. To examine whether the reduced transgene mRNA accumulation was attributable to RNA silencing, the small RNA fraction was extracted from plants infected with PVX.NSs-5 , PVX.NSs-3 or PVX and analyzed by RNA gel blot analysis using sense and antisense RNA probes specific for the 5 or 3 -NSs sequence. Transgene-derived small RNA (small interfering RNA, siRNA), characteristic of RNA silencing, were detected in plants that had been infected with PVX.NSs-5 or PVX.NSs-3 , but not in the PVX-infected and non-inoculated control plants (data for an antisense probe shown in Fig. 2B). These results suggest that PVX containing the NSs gene sequences were able to initiate VIGS of the homologous transgene mRNA. 3.2. Interactions between TSWV and PVX containing the NSs gene sequences in vector-transformed N. benthamiana The interactions between PVX and TSWV were analyzed in plants of GUS-Nb using five PVX derivatives, PVX.NSs-5 , PVX.NSs-3 , PVX.NSs-terminal, PVX.NSs or PVX (Fig. 1). The interactions between PVX and TSWV were reported in the previous study [13]. Similar results were observed in the present study. Briefly, the newly emerged leaves of plants infected with PVX developed typical PVX symptoms. The plants infected with TSWV generally showed a 2–5-day delay in symptom development compared to those with PVX. TSWV-infected plants initially exhibited vein clearing and severe mottling followed by necrosis that sometimes prevented sampling by 14 dpi. On the plants infected with PVX and TSWV, symptoms of both viruses were observed (Table 1, data not shown). The newly emerged leaves of plants infected with PVX.NSs-5 developed typical PVX symptoms (Table 1, data not shown). Symptom expression by TSWV in plants simultaneously inoculated with TSWV and PVX.NSs-5 was delayed by 1–7 days as compared with those inoculated with TSWV alone (data not shown), indicating interference by PVX.NSs-5 with TSWV infection. However, a high proportion of plants eventually developed both PVX and TSWV symptoms (Table 1, data not shown). ELISA analysis showed the presence of TSWV in some plants exhibiting no visible necrotic symptom (Table 1). Similar low levels of interference were observed in plants simultaneously inoculated with TSWV and either of two PVX derivatives, PVX.NSs-3 and PVX.NSs-3 -terminal (Table 1, data not shown). RNA gel blot analysis confirmed the presence of both PVX and TSWV RNA in the uninoculated upper leaves of the co-infected plants (data for PVX.NSs-5 and PVX.NSs-3 shown in Fig. 3). PVX.NSs showed little interference against TSWV infection (Table 1, data not shown). In the case of PVX.NSs, the viral progeny was a
Table 1 Interference by PVX containing the NSs or NSm gene against TSWV infection in vector-transformed Nicotiana benthamiana Inocula
Number of infected plants PVX
TSWV
PVX.NSs-5
PVX.NSs-5 and TSWV TSWV
8/8 13/13 –
– 8/13 (11/13) 8/8 (8/8)
PVX.NSs-3 PVX.NSs-3 and TSWV TSWV
8/8 13/13 –
– 7/13 (11/13) 7/8 (7/8)
PVX.NSs-3 -terminal PVX.NSs-3 -terminal and TSWV TSWV
8/8 10/10 –
– 7/10 (8/10) 10/10 (10/10)
PVX.NSs PVX.NSs and TSWV TSWV
8/8 16/16 –
– 16/16 (16/16) 10/10 (10/10)
PVX.NSm PVX.NSm and TSWV TSWV
10/10 18/18 –
– 14/18 (14/18) 10/10 (10/10)
PVX PVX and TSWV TSWV
8/8 10/10 –
– 10/10 (10/10) 10/10 (10/10)
The values show the number of plants with symptoms over the total number tested for virus resistance at 14 dpi. Results of enzyme-linked immunosorbent assay (ELISA) are shown in parentheses. Combined results of two independent experiments are presented.
mixture of populations that contained the full-length inserts or had partially or totally lost the inserted sequences (data not shown). These results suggest that there was RNA-mediated cross-protection against TSWV by PVX containing the NSs gene sequences, but the protection was less effective than that conferred by PVX containing the N gene [13]. To examine the effects of lag period on RNA-mediated cross-protection against TSWV by PVX.NSs-5 or PVX. NSs-3 , TSWV was inoculated on the upper leaves of plants at 14 dpi with PVX.NSs-5 or PVX.NSs-3 . All five plants infected with PVX.NSs-5 or PVX.NSs-3 developed TSWV symptoms, as was the case of eight non-inoculated healthy plants of the same age (data not shown). The presence of TSWV was confirmed by ELISA (data not shown). Thus, the lag period did not improve the protection level of plants from TSWV infection. 3.3. Interactions between TSWV and PVX containing the NSm gene sequence in vector-transformed N. benthamiana The interactions between PVX and TSWV were analyzed in plants of GUS-Nb using PVX.NSm engineered to contain another non-structural gene of TSWV, NSm. The newly emerged leaves of plants infected with PVX.NSm developed typical PVX symptoms (Table 1, data not shown). Symptom expression by TSWV in plants simultaneously inoculated with TSWV and PVX.NSm was delayed by 1–7
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Fig. 3. RNA gel blot analysis for detection of TSWV and PVX derivatives engineered to contain TSWV NSs or NSm gene sequences. Total RNA isolated from the vector-transformed N. benthamiana plants infected with PVX derivatives indicated, TSWV or both viruses were subjected to denaturing gel electrophoresis, transferred to nylon membrane and hybridized with a 32 P-␣-dCTP labeled NSs-5 , NSs-3 or NSm probe generated by random priming. The photographs of the ethidium bromide-stained RNA gel before transfer are shown as the loading control.
days as compared with those inoculated with TSWV alone (data not shown), indicating an interference by PVX.NSm against TSWV infection. However, a high proportion of plants eventually developed PVX and TSWV symptoms (Table 1, data not shown). RNA gel blot analysis confirmed the presence of PVX and TSWV RNA in the uninoculated upper leaves of the co-infected plants (Fig. 3). These results suggest that RNA-mediated cross-protection conferred not only by the NSs gene sequences but also by the NSm gene was less effective for the elimination of TSWV. 3.4. Interactions between TSWV and PVX containing the NSs gene sequences in transgenic N. benthamiana expressing the NSs gene sequence To examine the effects of the transgene mRNA on the interactions between TSWV and PVX.NSs-5 or PVX.NSs-3 , both viruses were simultaneously inoculated on plants of line NSs2. As mentioned above, line NSs2 showed only a low resistance against TSWV. However, since no siRNA was detected in the non-inoculated NSs2 plants (Fig. 2B), RNA silencing may not be involved in the resistance. The
newly emerged leaves of line NSs2 plants infected with PVX.NSs-5 showed typical symptoms by PVX (Table 2, data not shown). Symptom expression by TSWV in plants simultaneously inoculated with TSWV and PVX.NSs-5 was delayed by 1–7 days as compared with those inoculated Table 2 Interference by PVX containing the NSs gene sequences against TSWV infection in transgenic Nicotiana benthamiana line NSs2 Inocula
Number of infected plants PVX
TSWV
PVX.NSs-5
PVX.NSs-5 and TSWV TSWV
6/6 11/11 –
– 2/11 (2/11) 9/10 (9/10)
PVX.NSs-3 PVX.NSs-3 and TSWV TSWV
10/10 18/18 –
– 8/18 (8/18) 12/15 (12/15)
The values show the number of plants with symptoms over the total number tested for virus resistance at 14 dpi. Results of enzyme-linked immunosorbent assay (ELISA) are shown in parentheses. Combined results of two and three independent experiments are presented for inoculations using PVX.NSs-5 and PVX.NSs-3 , respectively.
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Fig. 4. RNA gel blot analysis for detection of TSWV and PVX derivatives engineered to contain TSWV NSs gene sequences. Total RNA isolated from plants of transgenic N. benthamiana line NSs2 infected with PVX derivatives indicated, TSWV or both viruses were subjected to denaturing gel electrophoresis, transferred to nylon membrane and hybridized with a 32 P-␣-dCTP labeled NSs-5 or NSs-3 probe generated by random priming. The photographs of the ethidium bromide-stained RNA gel before transfer are shown as the loading control.
with TSWV alone (data not shown). However, no complete virus resistance against TSWV was observed in two independent experiments (Table 2). Similar results were observed in plants simultaneously inoculated with PVX.NSs-3 and TSWV (Table 2). RNA gel blot analysis confirmed the presence of PVX and TSWV RNA in the uninoculated upper leaves of the co-infected plants (Fig. 4). ELISA analysis was also carried out to confirm the presence or absence of TSWV (Table 2).
4. Discussion Plant endogenous genes and transgenes have been targeted for RNA silencing induced by the infection with virus engineered to contain homologous sequences (the VIGS system) [18,19]. In the previous study, it was shown that TSWV N transgene mRNA was silenced by PVX engineered to contain the N gene sequences [13]. In the present study, VIGS of the NSs transgene mRNA was examined using PVX engineered to contain the homologous sequences. In the upper leaves of the infected plants, accumulation of the NSs transgene mRNA was strongly reduced (Fig. 2A). The reduction of the NSs transgene mRNA was dependent on sequence homology between the NSs transgene mRNA and sequences inserted into the PVX, as inoculation with PVX did not affect the NSs transgene mRNA level (Fig. 2A). Furthermore, siRNA, hallmarks of RNA silencing [9], were found in plants showing decreased levels of the NSs transgene mRNA (Fig. 2B). These results suggest that the reduction of the NSs transgene mRNA was associated with RNA silencing. In the previous study, simultaneous inoculations of plants with TSWV and PVX engineered to contain TSWV N gene sequences were shown to lead to a degradation of TSWV RNA and no TSWV was detected in the upper leaves [13]. In the present study, plants inoculated with TSWV and PVX engineered to contain TSWV NSs gene sequences showed partial resistance (a delay in symptom appearance) against
TSWV. However, a high proportion of the plants was eventually infected with TSWV. Similar results were observed in inoculations of plants with TSWV and PVX engineered to contain TSWV NSm gene sequence. Jan et al. [20] proposed that RNA-mediated virus resistance and RNA silencing can be uncoupled if the virus sequence is below a minimum size. The lengths of the inserts analyzed in the present study ranged from 219 bp of PVX.NSs-3 -terminal to ca. 1.2 kb of PVX.NSs (Fig. 1). Since the N gene segments of TSWV inserted into PVX (185–204 bp) were shown to be able to induce RNA silencing and eliminate TSWV [13], the idea that the lengths of the NSs gene sequences inserted into PVX were not sufficient to target and eliminate TSWV RNA seems to be unlikely in our case. These results suggest that RNA-mediated virus resistance is not merely defined by the amount of sequence homology. PVX engineered to contain the 5 or 3 -portion of the NSs gene sequences (PVX.NSs-5 and PVX.NSs-3 ) were able to induce RNA silencing and degrade the NSs transgene mRNA (Fig. 2). In addition to the NSs gene, the NSm transgene was also shown to confer virus resistance as a result of RNA silencing [21,22]. Therefore, it is apparent that the failure of PVX engineered to contain the NSs or NSm gene sequences to eliminate TSWV was not due to an inability to induce RNA silencing. Szittya et al. [23] demonstrated that different regions of Cymbidium ringspot tombusvirus genome showed varied target activities. It is possible that the NSs and NSm gene sequences themselves could be resistant to RNA silencing. However, the data showing that the NSs transgene mRNA was degraded by VIGS-mediated RNA silencing does not support the idea (Fig. 2). The reason why the less effective RNA-mediated virus resistance to TSWV was observed in the present study currently remains obscure. One possibility is that the NSs and NSm gene sequences in TSWV are protected from degradation by sequestration in replication or translation complexes, virions, or other virus-infection specific structures in the cytoplasm. Another possibility is that the degree of
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VIGS-mediated RNA silencing induced by the NSs or NSm gene sequences is lower compared to that induced by the N gene. These two possibilities are not mutually exclusive and could be cooperatively involved. It has been shown that transgenic virus resistance conferred by RNA silencing is strongly enhanced by transcription of a fragment of viral sequence from the host genome [24]. The VIGS-mediated RNA silencing to the homologous endogene or transgene mRNA was also shown to enhance the degradation of the replicating viral RNA [25,26]. To test whether the RNA degradation mechanism by VIGS was enhanced by the transgene, transgenic plants expressing the NSs transgene were simultaneously inoculated with TSWV and PVX engineered to contain the sequences homologous to the transgene (PVX.NSs-5 and PVX.NSs-3 ). However, the simultaneous inoculations did not completely block TSWV infectivity and the infected plants showed systemic symptoms of both viruses. These results suggest that the NSs transgene and/or its transcript has little effect, if any, in the enhancement of RNA silencing. Numerous viral genes and sequences were shown to be able to confer virus resistance to plants [27]. TSWV N gene has been used as the source of pathogen-derived resistance (PDR) [28,29]. The NSm gene was also shown to be effective for PDR [21,22]. However, in the present study, it was shown that RNA-mediated virus resistance using the NSs and NSm gene sequences of TSWV was less effective for TSWV relative to that using the N gene. Careful examination of viral genes and sequences may be necessary for developing RNA-mediated virus resistant plants.
Acknowledgements This study was supported in part by The Sanyo Broadcasting Foundation for Scientific Research and The Ohara Foundation for Agricultural Research. The authors thank Dr. D.C. Baulcombe (The Sainsbury Laboratory, Norwich, UK) for providing the PVX vector; Dr. S. Tsuda (National Agricultural Research Center, Tsukuba, Japan) for providing Tomato spotted wilt virus; Dr. I.B. Andika (Okayama University, Kurashiki, Japan) for advice on detection of small RNA; Dr. M. Nishiguchi (Ehime University, Matsuyama, Japan) for reading the manuscript.
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[26] J.-P. Hiriart, E.-M. Aro, K. Lehto, Dynamics of the VIGS-mediated chimeric silencing of the Nicotiana benthamiana ChlH gene and of the tobacco mosaic virus vector, Mol. Plant-Microbe Interact. 16 (2003) 99–106. [27] R.N. Beachy, Mechanisms and applications of pathogen-derived resistance in transgenic plants, Curr. Opin. Biotechnol. 8 (1997) 215–220.
[28] J.J.L. Gielen, P. de Haan, A.J. Kool, D. Peters, M.Q.J.M. van Grinsven, R.W. Goldbach, Engineered resistance to tomato spotted wilt virus, a negative-strand RNA virus, Bio/Technology 9 (1991) 1363–1367. [29] D.J. MacKenzie, P.J. Ellis, Resistance to tomato spotted wilt virus infection in transgenic tobacco expressing the viral nucleocapsid gene, Mol. Plant-Microbe Interact. 5 (1992) 34–40.
Analysis of RNA-mediated virus resistance by NSs and NSm gene sequences from Tomato spotted wilt virus Shoji Sonoda∗ , Hisaaki Tsumuki Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan Received 18 August 2003; received in revised form 18 November 2003; accepted 18 November 2003
Abstract Nicotiana benthamiana was transformed with the NSs gene sequence of Tomato spotted wilt virus (TSWV). When a non-silenced line showing high levels of the transgene expression was inoculated with Potato virus X (PVX) engineered to contain the NSs gene sequences, reduced transgene mRNA accumulation was observed. RNA silencing was the underlying mechanism for the reduction. Previously, non-transgenic N. benthamiana infected with PVX engineered to contain the nucleocapsid protein (N) gene of TSWV was shown to be resistant to TSWV infection [Plant Sci. 164 (2003) 717]. In the present study, interactions between PVX engineered to contain the NSs gene sequences and TSWV were examined. Plants infected with PVX containing the NSs gene sequences showed a partial virus resistance to TSWV. However, the degree of the resistance was apparently lower than that observed in plants infected with PVX engineered to contain the N gene. Similar low levels of the resistance to TSWV was observed in plants infected with PVX engineered to contain the NSm gene of TSWV. These results suggest that RNA-mediated virus resistance conferred by the NSs and NSm gene sequences of TSWV was less effective to TSWV. Furthermore, it was also suggested that RNA-mediated virus resistance was affected by viral genes or sequences rather than the amount of nucleotide sequence homology. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Cross-protection; RNA silencing; Nicotiana benthamiana; TSWV; Virus resistance
1. Introduction Cross-protection is defined as the ability of one virus to inhibit or prevent infection by a second virus [1]. It has been shown that cross-protection is mediated by viral RNA or viral proteins [2–4]. Ratcliff et al. [2] showed that plants infected with one virus showed resistance against the heterologous virus when both viral genomes shared sequence homology. Similar induction of the virus resistance has been reported in plants infected with certain RNA [5] and DNA viruses [6,7]. The resistance mechanism has been linked with RNA silencing, a homology-dependent RNA degradation mechanism designed to eliminate undesirable RNA in the cytoplasm [8]. RNA silencing is induced by double-stranded RNA, including RNA viruses that replicate via double-stranded RNA intermediates, and is associated with the appearance of specific low-molecular-weight ∗ Corresponding author. Tel.: +81-86-434-1219; fax: +81-86-434-1249. E-mail address: [email protected] (S. Sonoda).
RNA fragments [9]. However, in most cases, the precise mechanism of interaction between the viral genes and genomic sequences which leads to RNA silencing is unknown. Tomato spotted wilt virus (TSWV), a member of the Tospoviridae, infects a wide range of plant species worldwide and causes severe chlorotic, necrotic, and malformation symptoms [10]. The viral genome consists of three single-stranded RNA denoted S, M, and L. The S RNA and the M RNA are characterized by an ambisense gene arrangement and encode nonstructural proteins, NSs and NSm, at the 5 -end in the viral RNA strand, respectively [11,12]. In a previous study, it was shown that the infection of recombinant Potato virus X (PVX) engineered to contain the nucleocapsid protein (N) gene of TSWV caused RNA-mediated suppression of the N transgene mRNA accumulation in transgenic plants and symptom appearance by TSWV in non-transgenic plants [13]. In the present study, the capacity of the NSs gene as inducer and target for RNA silencing was examined using virus-induced gene silencing (VIGS) and RNA-mediated cross-protection.
0168-9452/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2003.11.018
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2. Materials and methods 2.1. Virus constructs TSWV was provided by Dr. S. Tsuda (National Agricultural Research Center, Tsukuba, Japan). Total RNA extraction and first strand cDNA synthesis were described previously [13]. The NSs gene was amplified from the first strand cDNA by PCR using the 5 -and 3 -end primers, 5 -GGGGATCCATGTCTTCAAGTGTTTATGAG-3 and 5 -GGGAGCTCTTATTTTGATCCTGAAGCATG-3 , respectively. The 5 and 3 -end primers were designed to contain BamHI and SacI restriction sites (underlined), respectively. PCR conditions were 30 cycles of 30 s at 94 ◦ C, 1 min at 42 ◦ C and 2 min at 72 ◦ C. However, unexpectedly, the NSs gene lacking the 3 terminal 219 bp was amplified only by the 5 -end primer. The PCR-amplified fragment of the NSs gene (ca. 1.2 kb) was digested with BamHI and cloned into a similarly digested pBluescript (Stratagene, La Jolla, CA, USA) (pBS.NSs-1). The PCR-amplified fragment was also cloned into pGEM-T Easy (Promega, Madison, WI, USA) (pGEM.NSs-1). The EcoRI fragment of pGEM.NSs-1 was cloned into a similarly digested pBluescript (pBS.NSs-2). The SmaI–SalI fragment of pBS.NSs-2 was ligated into the EcoRV and SalI sites of the PVX vector (pP2C2S), a derivative of pGC3 [14], to produce PVX.NSs (Fig. 1). pBS.NSs-1 was digested with HindIII and the resulting fragment of 495 bp was cloned into a similarly digested pBluescript (pBS.NSs-3). The SmaI–SalI fragment of pBS.NSs-3 was ligated into the EcoRV and SalI sites of PVX to produce PVX.NSs-5 (Fig. 1). pBS.NSs-1 was digested with HindIII and the vector containing the 3 portion of the NSs gene sequence was self-ligated (pBS.NSs-4). The HincII–EcoRV fragment of pBS.NSs-4, having a length of 452 bp, was
cloned into the EcoRV site of pBluescript (pBS.NSs-5). The SmaI–SalI fragment of pBS.NSs-5 was ligated into the EcoRV and SalI sites of PVX to produce PVX.NSs-3 (Fig. 1). The 3 -terminal 219 bp of the NSs gene was amplified from the first strand cDNA by PCR using the 5 and 3 -end primers, 5 -CAGTGAAGATATCTGCAAAAGG-3 and 5 -GGGTCGACTTATTTTGATCCTGAAGCATG-3 , respectively. The 5 and 3 -end primers were designed to contain EcoRV and SalI restriction sites (underlined), respectively. The PCR-amplified fragment was digested with EcoRV and SalI and cloned into the EcoRV and SalI sites of PVX to produce PVX.NSs-3 -terminal (Fig. 1). The NSm gene was cloned from the first strand cDNA by PCR using the 5 -and 3 -end primers, 5 -GGGATATCATGTTGACTCTTTTCGGTAAC-3 and 5 -GGGTCGACCTATATTTCATCAAAGGATAAC-3 , respectively. The 5 and 3 -end primers were designed to contain EcoRV and SalI restriction sites (underlined), respectively. The PCR-amplified fragment was digested with EcoRV and SalI and cloned into the EcoRV and SalI sites of PVX (PVX.NSm). 2.2. In vitro transcription and virus inoculation In vitro transcription reactions to produce infectious PVX were described previously [13]. Virus-infected leaf homogenates prepared by grinding systemically infected leaf tissue diluted (1:10, w/v) in 100 mM phosphate buffer (pH 7.0) were used for virus inoculation. Nicotiana benthamiana transformed with the GUS-deleted pBI121 (Clontech, Paro Alto, CA, USA) (GUS-Nb) was used for the analysis of RNA-mediated cross-protection. Virus-infected leaf homogenates were mixed for a final 1:20 dilution of each virus. For single inoculation, leaves were ground at a 1:20 ratio. Inoculated plants were monitored for the appearance of symptoms.
Fig. 1. Schematic representation of TSWV NSs gene sequences introduced in the PVX vector, together with the structure of the ambisense TSWV RNA. DNA probes used in this study were also shown above the ambisense TSWV RNA.
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2.3. Plant transformation N. benthamiana was transformed with the NSs gene sequence. The BamHI–EcoRV fragment of pBS.NSs-1 was ligated into the SmaI and BamHI sites of pGEM3Z f(+) (Promega) (pGEM.NSs-2). The BamHI–SacI fragment of pGEM.NSs-2 was inserted between the 35S cauliflower mosaic virus promoter and the nopaline synthase terminator sequences of the binary plasmid, pBI121, after the GUS gene had been removed. The resulting plasmid was used for transformation using Agrobacterium tumefaciens LBA4404 (Clontech) [15]. 2.4. RNA gel blot analysis Total RNA was extracted from leaf tissues, as described by Sonoda et al. [16]. Total RNA was size-fractionated on a 1.2% agarose gel containing 0.66 M formaldehyde and transferred to Biodyne PLUS membrane (Pall Corporation, Ann Arbor, MI, USA). The blot was hybridized with a random primed 32 P-labeled fragment containing the NSs gene coding sequences. RNA gel blot analysis of 21–25 nucleotide RNA was performed essentially as described by Hamilton and Baulcombe [9]. Strand-specific RNA probes were generated by in vitro RNA transcription from pBS.NSs-3 and pBS.NSs-4 in the presence of 32 P-␣-UTP. 2.5. ELISA ELISA analysis [17] with a TSWV monoclonal antibody (Japan Plant Protection Association, Ushiku, Japan) was used to detect TSWV. A result was recorded as positive when
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the A405 value was two-fold higher than that of the negative control.
3. Results 3.1. VIGS of TSWV NSs transgene mRNA by PVX containing the NSs gene sequences N. benthamiana was transformed with the NSs gene sequence and 10 transgenic lines were regenerated in the presence of kanamycin. Among 10 transgenic lines regenerated, line NSs2 showing high levels of the transgene expression was used for further analysis (data not shown). T1 plants of line NSs2 were susceptible to PVX containing the NSs gene sequences and developed typical PVX symptoms (vein clearing and chlorotic mosaic) as was the case in the control plants carrying the GUS-deleted pBI121 (Clontech) (GUS-Nb) (data not shown). When plants of line NSs2 were inoculated with TSWV, a 1–4-day delay in symptom appearance relative to the control plants was observed, but a high proportion of the plants was eventually infected with TSWV (data not shown). To examine VIGS of the NSs transgene mRNA by PVX containing the transgene sequences, T1 plants of line NSs2 were inoculated with one of three PVX derivatives, PVX.NSs-5 , PVX.NSs-3 and PVX (Fig. 1). The accumulation of the NSs transgene mRNA in plants of line NSs2 was determined by RNA gel blot analysis using the 5 or 3 -NSs specific probe to discriminate the transgene mRNA from the viral RNA (Fig. 1). In plants infected with PVX.NSs-5 or PVX.NSs-3 , reduced transgene mRNA accumulation was observed at 14 days post-inoculation (dpi) (Fig. 2A). On the other hand, detectable levels of the transgene mRNA
Fig. 2. Analysis of the NSs-related RNA in virus-infected or non-infected transgenic N. benthamiana line NSs2. Total RNA (20 g) (A) and small RNA fraction (20 g) (B) samples were subjected to RNA gel blot analysis. A 32 P-␣-dCTP labeled NSs-5 or NSs-3 probe generated by random priming was used as the probe for mRNA gel blot analysis, while a 32 P-␣-UTP labeled in vitro transcripts corresponding to the antisense strand of NSs-5 or NSs-3 probe was used in RNA gel blot analysis of siRNA. The photographs of the ethidium bromide-stained RNA gel before transfer are shown as the loading control in (A).
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accumulated in plants infected with PVX at 14 dpi, as was the case in the non-inoculated healthy plants (Fig. 2A). Two independent experiments showed similar results. To examine whether the reduced transgene mRNA accumulation was attributable to RNA silencing, the small RNA fraction was extracted from plants infected with PVX.NSs-5 , PVX.NSs-3 or PVX and analyzed by RNA gel blot analysis using sense and antisense RNA probes specific for the 5 or 3 -NSs sequence. Transgene-derived small RNA (small interfering RNA, siRNA), characteristic of RNA silencing, were detected in plants that had been infected with PVX.NSs-5 or PVX.NSs-3 , but not in the PVX-infected and non-inoculated control plants (data for an antisense probe shown in Fig. 2B). These results suggest that PVX containing the NSs gene sequences were able to initiate VIGS of the homologous transgene mRNA. 3.2. Interactions between TSWV and PVX containing the NSs gene sequences in vector-transformed N. benthamiana The interactions between PVX and TSWV were analyzed in plants of GUS-Nb using five PVX derivatives, PVX.NSs-5 , PVX.NSs-3 , PVX.NSs-terminal, PVX.NSs or PVX (Fig. 1). The interactions between PVX and TSWV were reported in the previous study [13]. Similar results were observed in the present study. Briefly, the newly emerged leaves of plants infected with PVX developed typical PVX symptoms. The plants infected with TSWV generally showed a 2–5-day delay in symptom development compared to those with PVX. TSWV-infected plants initially exhibited vein clearing and severe mottling followed by necrosis that sometimes prevented sampling by 14 dpi. On the plants infected with PVX and TSWV, symptoms of both viruses were observed (Table 1, data not shown). The newly emerged leaves of plants infected with PVX.NSs-5 developed typical PVX symptoms (Table 1, data not shown). Symptom expression by TSWV in plants simultaneously inoculated with TSWV and PVX.NSs-5 was delayed by 1–7 days as compared with those inoculated with TSWV alone (data not shown), indicating interference by PVX.NSs-5 with TSWV infection. However, a high proportion of plants eventually developed both PVX and TSWV symptoms (Table 1, data not shown). ELISA analysis showed the presence of TSWV in some plants exhibiting no visible necrotic symptom (Table 1). Similar low levels of interference were observed in plants simultaneously inoculated with TSWV and either of two PVX derivatives, PVX.NSs-3 and PVX.NSs-3 -terminal (Table 1, data not shown). RNA gel blot analysis confirmed the presence of both PVX and TSWV RNA in the uninoculated upper leaves of the co-infected plants (data for PVX.NSs-5 and PVX.NSs-3 shown in Fig. 3). PVX.NSs showed little interference against TSWV infection (Table 1, data not shown). In the case of PVX.NSs, the viral progeny was a
Table 1 Interference by PVX containing the NSs or NSm gene against TSWV infection in vector-transformed Nicotiana benthamiana Inocula
Number of infected plants PVX
TSWV
PVX.NSs-5
PVX.NSs-5 and TSWV TSWV
8/8 13/13 –
– 8/13 (11/13) 8/8 (8/8)
PVX.NSs-3 PVX.NSs-3 and TSWV TSWV
8/8 13/13 –
– 7/13 (11/13) 7/8 (7/8)
PVX.NSs-3 -terminal PVX.NSs-3 -terminal and TSWV TSWV
8/8 10/10 –
– 7/10 (8/10) 10/10 (10/10)
PVX.NSs PVX.NSs and TSWV TSWV
8/8 16/16 –
– 16/16 (16/16) 10/10 (10/10)
PVX.NSm PVX.NSm and TSWV TSWV
10/10 18/18 –
– 14/18 (14/18) 10/10 (10/10)
PVX PVX and TSWV TSWV
8/8 10/10 –
– 10/10 (10/10) 10/10 (10/10)
The values show the number of plants with symptoms over the total number tested for virus resistance at 14 dpi. Results of enzyme-linked immunosorbent assay (ELISA) are shown in parentheses. Combined results of two independent experiments are presented.
mixture of populations that contained the full-length inserts or had partially or totally lost the inserted sequences (data not shown). These results suggest that there was RNA-mediated cross-protection against TSWV by PVX containing the NSs gene sequences, but the protection was less effective than that conferred by PVX containing the N gene [13]. To examine the effects of lag period on RNA-mediated cross-protection against TSWV by PVX.NSs-5 or PVX. NSs-3 , TSWV was inoculated on the upper leaves of plants at 14 dpi with PVX.NSs-5 or PVX.NSs-3 . All five plants infected with PVX.NSs-5 or PVX.NSs-3 developed TSWV symptoms, as was the case of eight non-inoculated healthy plants of the same age (data not shown). The presence of TSWV was confirmed by ELISA (data not shown). Thus, the lag period did not improve the protection level of plants from TSWV infection. 3.3. Interactions between TSWV and PVX containing the NSm gene sequence in vector-transformed N. benthamiana The interactions between PVX and TSWV were analyzed in plants of GUS-Nb using PVX.NSm engineered to contain another non-structural gene of TSWV, NSm. The newly emerged leaves of plants infected with PVX.NSm developed typical PVX symptoms (Table 1, data not shown). Symptom expression by TSWV in plants simultaneously inoculated with TSWV and PVX.NSm was delayed by 1–7
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Fig. 3. RNA gel blot analysis for detection of TSWV and PVX derivatives engineered to contain TSWV NSs or NSm gene sequences. Total RNA isolated from the vector-transformed N. benthamiana plants infected with PVX derivatives indicated, TSWV or both viruses were subjected to denaturing gel electrophoresis, transferred to nylon membrane and hybridized with a 32 P-␣-dCTP labeled NSs-5 , NSs-3 or NSm probe generated by random priming. The photographs of the ethidium bromide-stained RNA gel before transfer are shown as the loading control.
days as compared with those inoculated with TSWV alone (data not shown), indicating an interference by PVX.NSm against TSWV infection. However, a high proportion of plants eventually developed PVX and TSWV symptoms (Table 1, data not shown). RNA gel blot analysis confirmed the presence of PVX and TSWV RNA in the uninoculated upper leaves of the co-infected plants (Fig. 3). These results suggest that RNA-mediated cross-protection conferred not only by the NSs gene sequences but also by the NSm gene was less effective for the elimination of TSWV. 3.4. Interactions between TSWV and PVX containing the NSs gene sequences in transgenic N. benthamiana expressing the NSs gene sequence To examine the effects of the transgene mRNA on the interactions between TSWV and PVX.NSs-5 or PVX.NSs-3 , both viruses were simultaneously inoculated on plants of line NSs2. As mentioned above, line NSs2 showed only a low resistance against TSWV. However, since no siRNA was detected in the non-inoculated NSs2 plants (Fig. 2B), RNA silencing may not be involved in the resistance. The
newly emerged leaves of line NSs2 plants infected with PVX.NSs-5 showed typical symptoms by PVX (Table 2, data not shown). Symptom expression by TSWV in plants simultaneously inoculated with TSWV and PVX.NSs-5 was delayed by 1–7 days as compared with those inoculated Table 2 Interference by PVX containing the NSs gene sequences against TSWV infection in transgenic Nicotiana benthamiana line NSs2 Inocula
Number of infected plants PVX
TSWV
PVX.NSs-5
PVX.NSs-5 and TSWV TSWV
6/6 11/11 –
– 2/11 (2/11) 9/10 (9/10)
PVX.NSs-3 PVX.NSs-3 and TSWV TSWV
10/10 18/18 –
– 8/18 (8/18) 12/15 (12/15)
The values show the number of plants with symptoms over the total number tested for virus resistance at 14 dpi. Results of enzyme-linked immunosorbent assay (ELISA) are shown in parentheses. Combined results of two and three independent experiments are presented for inoculations using PVX.NSs-5 and PVX.NSs-3 , respectively.
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Fig. 4. RNA gel blot analysis for detection of TSWV and PVX derivatives engineered to contain TSWV NSs gene sequences. Total RNA isolated from plants of transgenic N. benthamiana line NSs2 infected with PVX derivatives indicated, TSWV or both viruses were subjected to denaturing gel electrophoresis, transferred to nylon membrane and hybridized with a 32 P-␣-dCTP labeled NSs-5 or NSs-3 probe generated by random priming. The photographs of the ethidium bromide-stained RNA gel before transfer are shown as the loading control.
with TSWV alone (data not shown). However, no complete virus resistance against TSWV was observed in two independent experiments (Table 2). Similar results were observed in plants simultaneously inoculated with PVX.NSs-3 and TSWV (Table 2). RNA gel blot analysis confirmed the presence of PVX and TSWV RNA in the uninoculated upper leaves of the co-infected plants (Fig. 4). ELISA analysis was also carried out to confirm the presence or absence of TSWV (Table 2).
4. Discussion Plant endogenous genes and transgenes have been targeted for RNA silencing induced by the infection with virus engineered to contain homologous sequences (the VIGS system) [18,19]. In the previous study, it was shown that TSWV N transgene mRNA was silenced by PVX engineered to contain the N gene sequences [13]. In the present study, VIGS of the NSs transgene mRNA was examined using PVX engineered to contain the homologous sequences. In the upper leaves of the infected plants, accumulation of the NSs transgene mRNA was strongly reduced (Fig. 2A). The reduction of the NSs transgene mRNA was dependent on sequence homology between the NSs transgene mRNA and sequences inserted into the PVX, as inoculation with PVX did not affect the NSs transgene mRNA level (Fig. 2A). Furthermore, siRNA, hallmarks of RNA silencing [9], were found in plants showing decreased levels of the NSs transgene mRNA (Fig. 2B). These results suggest that the reduction of the NSs transgene mRNA was associated with RNA silencing. In the previous study, simultaneous inoculations of plants with TSWV and PVX engineered to contain TSWV N gene sequences were shown to lead to a degradation of TSWV RNA and no TSWV was detected in the upper leaves [13]. In the present study, plants inoculated with TSWV and PVX engineered to contain TSWV NSs gene sequences showed partial resistance (a delay in symptom appearance) against
TSWV. However, a high proportion of the plants was eventually infected with TSWV. Similar results were observed in inoculations of plants with TSWV and PVX engineered to contain TSWV NSm gene sequence. Jan et al. [20] proposed that RNA-mediated virus resistance and RNA silencing can be uncoupled if the virus sequence is below a minimum size. The lengths of the inserts analyzed in the present study ranged from 219 bp of PVX.NSs-3 -terminal to ca. 1.2 kb of PVX.NSs (Fig. 1). Since the N gene segments of TSWV inserted into PVX (185–204 bp) were shown to be able to induce RNA silencing and eliminate TSWV [13], the idea that the lengths of the NSs gene sequences inserted into PVX were not sufficient to target and eliminate TSWV RNA seems to be unlikely in our case. These results suggest that RNA-mediated virus resistance is not merely defined by the amount of sequence homology. PVX engineered to contain the 5 or 3 -portion of the NSs gene sequences (PVX.NSs-5 and PVX.NSs-3 ) were able to induce RNA silencing and degrade the NSs transgene mRNA (Fig. 2). In addition to the NSs gene, the NSm transgene was also shown to confer virus resistance as a result of RNA silencing [21,22]. Therefore, it is apparent that the failure of PVX engineered to contain the NSs or NSm gene sequences to eliminate TSWV was not due to an inability to induce RNA silencing. Szittya et al. [23] demonstrated that different regions of Cymbidium ringspot tombusvirus genome showed varied target activities. It is possible that the NSs and NSm gene sequences themselves could be resistant to RNA silencing. However, the data showing that the NSs transgene mRNA was degraded by VIGS-mediated RNA silencing does not support the idea (Fig. 2). The reason why the less effective RNA-mediated virus resistance to TSWV was observed in the present study currently remains obscure. One possibility is that the NSs and NSm gene sequences in TSWV are protected from degradation by sequestration in replication or translation complexes, virions, or other virus-infection specific structures in the cytoplasm. Another possibility is that the degree of
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VIGS-mediated RNA silencing induced by the NSs or NSm gene sequences is lower compared to that induced by the N gene. These two possibilities are not mutually exclusive and could be cooperatively involved. It has been shown that transgenic virus resistance conferred by RNA silencing is strongly enhanced by transcription of a fragment of viral sequence from the host genome [24]. The VIGS-mediated RNA silencing to the homologous endogene or transgene mRNA was also shown to enhance the degradation of the replicating viral RNA [25,26]. To test whether the RNA degradation mechanism by VIGS was enhanced by the transgene, transgenic plants expressing the NSs transgene were simultaneously inoculated with TSWV and PVX engineered to contain the sequences homologous to the transgene (PVX.NSs-5 and PVX.NSs-3 ). However, the simultaneous inoculations did not completely block TSWV infectivity and the infected plants showed systemic symptoms of both viruses. These results suggest that the NSs transgene and/or its transcript has little effect, if any, in the enhancement of RNA silencing. Numerous viral genes and sequences were shown to be able to confer virus resistance to plants [27]. TSWV N gene has been used as the source of pathogen-derived resistance (PDR) [28,29]. The NSm gene was also shown to be effective for PDR [21,22]. However, in the present study, it was shown that RNA-mediated virus resistance using the NSs and NSm gene sequences of TSWV was less effective for TSWV relative to that using the N gene. Careful examination of viral genes and sequences may be necessary for developing RNA-mediated virus resistant plants.
Acknowledgements This study was supported in part by The Sanyo Broadcasting Foundation for Scientific Research and The Ohara Foundation for Agricultural Research. The authors thank Dr. D.C. Baulcombe (The Sainsbury Laboratory, Norwich, UK) for providing the PVX vector; Dr. S. Tsuda (National Agricultural Research Center, Tsukuba, Japan) for providing Tomato spotted wilt virus; Dr. I.B. Andika (Okayama University, Kurashiki, Japan) for advice on detection of small RNA; Dr. M. Nishiguchi (Ehime University, Matsuyama, Japan) for reading the manuscript.
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