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1 Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, Singapore 117545, Singapore
2 Functional Genomics Laboratory, Institute of Molecular and Cell Biology, Singapore 138673, Singapore
3 Temasek Life Sciences Laboratory, 1 Research Link, Singapore 117604, Singapore
Correspondence
Sek-Man Wong
dbswsm{at}nus.edu.sg
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A phylogenetic tree based on the Tombusviridae CP sequence is available as supplementary material in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). There are seven open reading frames (ORFs) in the genomic and two subgenomic RNAs. The 5'-proximal p28 ORF and its amber terminator readthrough p81 encode an RNA-dependent RNA polymerase (RdRp), which is involved in virus replication. p23 overlaps p28 and is indispensable for host-specific virus replication, but is unable to suppress gene silencing (Liang et al., 2002b). p27 and its isoforms p25 and p22.5 are located in the 3' region of the genome (unpublished data).
The 38 kDa coat protein (CP) consists of an internal RNA-binding domain (R), a shell-forming domain (S) and a protruding domain (P). The virus has been crystallized and its atomic resolution was determined to 4·5 Å (0·45 nm) (Lee et al., 2003). The virion was reconstructed to about 12 Å (1·2 nm) from cryo-electron microscopy images (Doan et al., 2003). In addition to the role of CP in forming the capsid for HCRSV, CP is believed to play an important role in symptom modulation. Hurtt (1987) reported that the amino acid composition of HCRSV CP was altered and its progeny viruses lost their virulence in kenaf (Hibiscus cannabinus L.) after HCRSV was serially passaged from Hibiscus rosa-sinensis to Chenopodium quinoa. Further experiments showed that eight site-specific amino acid mutations in the CP resulted after serial passage in C. quinoa. The HCRSV mutants M-1+2+3 (which contains three of the eight amino acid mutations) and M-f (which contains all eight amino acid mutations) both demonstrated avirulence in kenaf (Liang et al., 2002a). Thus, co-variation of at least three amino acids at Val49
Ile, Ile95Val and Lys270Arg is sufficient to cause avirulence. These mutations may result in conformational changes and possibly abolish the interaction of CP with host factors.
Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation process against invading viruses and other pathogens. Similar pathways also exist in other organisms: quelling in Neurospora crassa (Cogoni & Macino, 1999) and RNA interference in Caenorhabditis elegans (Fire et al., 1998). To survive, viruses have evolved gene-silencing suppressors, which inhibit the gene-silencing pathway at various steps (Voinnet, 2005). Approximately 30 viral suppressors have been reported among both positive- and negative-strand RNA and DNA viruses, with one silencing suppressor from each of the plant and animal viruses examined. Viruses with large genomes may evolve more sophisticated strategies to evade host gene silencing; for example, Citrus tristeza virus encodes three distinct suppressors that target gene silencing at multiple steps to counter the host antiviral response (Lu et al., 2004). The suppressors identified are functionally diverse, and more information is essential to understand the functional basis of suppression of gene silencing and the adaptation of viruses to hosts on an evolutionary basis. A clear understanding of the molecular basis of gene-silencing suppression is needed and will help us to elucidate the mechanism of gene silencing.
The sequence homology of CPs among HCRSV, Turnip crinkle virus (TCV) and Tomato bushy stunt virus (TBSV) is approximately 30 % at the amino acid level. However, TCV CP has been identified as a strong PTGS suppressor (Qu et al., 2003; Thomas et al., 2003), whereas TBSV CP is not a PTGS suppressor (Silhavy et al., 2002). The TBSV p19 gene encodes a strong PTGS suppressor (Silhavy et al., 2002). Thus, it was of interest to determine whether HCRSV CP is a PTGS suppressor and to compare its similarities and differences with other suppressors.
Here, we have reported that HCRSV CP could inhibit transiently expressed sense RNA-induced but not dsRNA-induced local and systemic silencing. We propose that HCRSV CP may suppress gene silencing at the initiation step. The changes in the HCRSV CP suppressor function of various CP mutants also may be correlated with avirulence in kenaf. In addition, Potato virus X (PVX) vector-expressed HCRSV CP was able to enhance symptom severity and viral RNA accumulation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was PCR amplified and cloned into pBI121 by replacing the 
-glucuronidase (GUS) sequence between the BamHI and SacI sites to generate pBICP. The third amino acid codon (CAG) of CP was changed to TAG, and the start codon of the overlapping p25 was also mutated to GTG in pBICP to create pBI
CP. Western blots were carried out after transient expression assays to exclude the possibility of reinitiation and contribution from other proteins. pBIp27 contained an insertion of a newly identified overlapping ORF p27 (unpublished data) with the CP third amino acid CAG mutated to TAG. TBSV p19 and Cucumber mosaic virus (CMV) 2b protein were cloned in the same way into pBI121 to generate pBIp19 and pBI2b, respectively. To investigate the contributions of three different CP domains to the suppression function, the R–S domain (nt 2590–3339), the S–P domain (nt 2830–3627) and the P domain (nt 3340–3627) were PCR amplified and inserted into pBI121 by replacing the GUS gene. The resulting constructs were designated pBICP/RS, pBICP/SP and pBICP/P, respectively. To investigate the effects of CP mutations originating from serial passage of HCRSV in its local lesion host, the fragment corresponding to the CP mutants M-1, M-2, M-3, M-1+2, M-1+3, M-2+3 and M-1+2+3 (Liang et al., 2002a) were PCR amplified and cloned into pBI121 to generate pBICPm1, pBICPm2, pBICPm3, pBICPm1+2, pBICPm1+3, pBICPm2+3 and pBICPm1+2+3, respectively. Clones were verified by sequencing. pBICGFP and pBICdsGFP (Takeda et al., 2002) were kindly provided by Professor Kazuyuki Mise (Kyoto University, Japan). For ectopic expression of PVX, the HCRSV CP gene was amplified by PCR and inserted into the pP2C2S vector at the ClaI and SalI sites to form PVX–HCP. PVX–GFP contained the green fluorescence protein (GFP) reporter gene between the two subgenomic RNA promoter sequences before its CP gene. Both the pP2C2S vector and PVX–GFP were kindly provided by Professor David Baulcombe (Sainsbury Laboratory, Norwich, UK).
Agrobacterium co-infiltration and GFP imaging.
Agroinfiltration was carried out as described previously (Li et al., 1999). The Agrobacterium-harbouring constructs described above were mixed in a ratio of 1 : 1 when resuspended in 10 mM MgCl2 buffer with 150 µM acetosyringone to an OD600 of 2·0, kept at room temperature for at least 3 h without shaking and then infiltrated into leaves of 1-month-old seedlings of GFP-transgenic Nicotiana benthamiana (line 16c; kindly provided by Professor David Baulcombe, Sainsbury Laboratory, Norwich, UK). GFP fluorescence was observed under a UV lamp (Vilber Lourmat-6LM, 365 nm tube). Photographs were taken using a Cannon 350D camera with a Hoya K2 yellow filter.
Isolation and detection of high molecular mass (HMM) RNA and small interfering (si)RNA.
The isolation of HMM and low molecular mass (LMM) RNAs was carried out as described previously (Li et al., 1999). To detect HMM RNA, 5 µg total RNA was run on a formaldehyde denaturing gel and transferred to a Hybond-N membrane, followed by detection with a digoxigenin (DIG)-labelled RNA probe. Methylene blue staining of rRNA was carried out as a loading control. To detect siRNA, 5 µg LMM RNA was run on a 15 % acrylamide sequencing gel and detected by an RNA probe randomly labelled with DIG. For verification of equal loading of LMM RNA, the membrane was reprobed with a DIG-labelled 5S rRNA probe. The relative intensity of the RNA band was determined using a Bio-Rad GS800 calibrated densitometer with reference to the pBICGFP negative control.
In vitro transcription and plant inoculation.
The PVX vector-based constructs were linearized by SpeI. After phenol/chloroform extraction, 1 µg of each template was used for in vitro transcription according to the manufacturer's instructions (Ambion mMESSAGE mMACHINE High Yield RNA Transcription kit, Cat. #1344). Newly emerged and fully expanded N. benthamiana leaves of 
4-week-old seedlings were inoculated mechanically with 1 µg of the in vitro transcripts. Photographs were taken at 18 days post-infiltration (p.i.) and samples were collected for total RNA extraction and target gene detection by Northern blot hybridization.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Among these constructs, pBIp19 and pBI2b were used as positive controls, while pBICP and pBIp27 were used as negative controls. Agrobacterium harbouring these constructs were infiltrated into GFP-transgenic N. benthamiana 16c leaves.
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) and the level of GFP fluorescence was about the same as in the pBICGFP+pBIp19-infiltrated leaves (Fig. 2a). In contrast, leaf patches infiltrated with pBICGFP+pBI2b, pBICGFP+pBICP and pBICGFP+pBIp27 (data not shown) showed only light green fluorescence, which was similar to the level of GFP observed on leaves infiltrated with pBICGFP alone (Fig. 2a). At 6 days p.i., a significantly reduced level of GFP fluorescence was observed in leaves infiltrated with pBICGFP compared with 3 days p.i. Red fluorescence at the edge of the infiltrated zones was observed. This observation is consistent with the conclusion that transient expression of GFP mRNA at high levels rapidly triggers PTGS (Voinnet et al., 1999). The pBICGFP+pBICP- and pBICGFP+pBIp27-infiltrated leaves displayed similar levels of GFP fluorescence to the pBICGFP-infiltrated leaves. In contrast, pBICGFP+pBICP- and pBICGFP+pBIp19-infiltrated leaves sustained a strong green fluorescence up to 9 days p.i. (data not shown). These observations suggested that HCRSV CP is a putative PTGS suppressor that is capable of blocking transiently expressed GFP sense RNA-induced PTGS. The ability of HCRSV CP to inhibit the onset of local PTGS was comparable to TBSV p19. The results of pBICGFP+pBIp27-infiltrated leaves showed that p27 was unable to inhibit GFP sense RNA-induced PTGS. Hence, the GFP fluorescence observed on pBICGFP+pBICP-infiltrated leaves could be attributed to CP, rather than the overlapping ORF p27.
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). Agrobacterium co-infiltration assays showed that CP/RS, CP/SP and CP/P all showed a level of GFP fluorescence that was reduced to the same level as the negative controls of GFP alone or GFP+vector at 3 days p.i. (Fig. 2b, GFP and 121, respectively). Under the same experimental conditions, the complete CP showed a threefold increase in GFP mRNA level compared with the domain deletion mutants, indicating that the complete CP is needed for the suppression of sense RNA-induced PTGS.
After the onset of PTGS, the triggered mobile silencing signal is believed to spread systemically and leads to systemic silencing of the endogenous homologous gene (Palauqui et al., 1997; Voinnet & Baulcombe, 1997; Guo & Ding, 2002; Mlotshwa et al., 2002). The systemic leaves of all plants infiltrated with pBICGFP+pBI2b (20/20 16c plants) and pBICGFP+pBICP (30/30 16c plants) exhibited the same green fluorescence under UV light as 16c plants at 9 days p.i. (Fig. 2c). However, plants infiltrated with pBICGFP, pBICGFP+pBICP and pBICGFP+pBICP/RS (a total of 30 16c plants for each infiltration experiment) all developed systemic silencing and red fluorescence was observed along the phloem or over the entire leaf (Fig. 2c). It was apparent that HCRSV CP is capable of inhibiting the onset of systemic silencing. The domain deletions, for example pBICP/RS (Fig. 2c), all developed systemic silencing, suggesting that the complete CP is required to prevent systemic silencing effectively. The molecular basis of the suppression function remains to be elucidated.
To determine whether the altered GFP fluorescence resulted in changes in the steady-state level of GFP mRNA, total RNAs were extracted from infiltrated leaves at 3 days p.i. The GFP mRNA from pBICGFP+pBICP-infiltrated leaves (Fig. 2d, lane 2) showed a threefold increase when compared with leaves infiltrated with pBICGFP+pBICP and pBICGFP+pBIp27, respectively (Fig. 2d, lanes 3 and 4). This was in agreement with the observation of a strong GFP fluorescence at the infiltrated zones (Fig. 2b, CP). In comparison, a detectable level of GFP siRNA accumulated in leaves infiltrated with pBICGFP+pBICP (Fig. 2d, lane 2), and high levels of GFP siRNAs were detected in the pBICGFP+pBICP and pBICGFP+pBIp27-infiltrated leaves (Fig. 2d, lanes 3 and 4), as in the pBICGFP-infiltrated leaves (Fig. 2d, lane 1). This result is different from that reported for TCV CP, which showed that GFP-specific siRNAs were absent in TCV CP-infiltrated leaves (Qu et al., 2003). The difference may have resulted from: (i) different viral suppressors; (ii) different agroinfiltration vectors; or (iii) variable test environmental conditions. The mRNA of CP and p27 could not suppress PTGS. These results were consistent with the findings of TCV CP as a gene-silencing suppressor (Qu et al., 2003). The three CP domain deletions pBICP/P, pBICP/SP and pBICP/RS (Fig. 2d, lanes 5–7) accumulated GFP mRNA to a low level, but the GFP siRNA reached a similar high level to the pBICGFP+pBICP-infiltrated leaves (Fig. 2d, lane 3). This result was in agreement with the observation of reduced GFP fluorescence of Agrobacterium-infiltrated 16c leaves. None of the CP mutants could suppress sense RNA-induced PTGS, indicating that the complete CP is essential for suppression of PTGS.
Avirulent mutant CPm1+2+3 is not able to suppress PTGS
Serial passage of HCRSV in its local lesion host leads to specific amino acid mutations (Liang et al., 2002a). To test whether the amino acid changes had an effect on the CP suppression function, different combinations of the CP mutants were made (Fig. 3) and tested by the agroinfiltration system.
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). The double-mutant pBICPm1+3 and triple-mutant pBICPm1+2+3 showed GFP fluorescence that was reduced to the same level as the GFP-infiltrated negative control (Fig. 4a). Among all these mutants, only the triple mutant pBICPm1+2+3 (28/30 16c plants) failed to suppress systemic silencing (data not shown). Northern blot hybridization analysis showed that mutants pBICPm1, pBICPm2, pBICPm3, pBICPm1+2 and pBICPm2+3 were able to inhibit GFP mRNA degradation induced by the transiently expressed GFP sense RNA to a similar level as the complete CP (Fig. 4b, lanes 2–7). In contrast, mutants pBICPm1+3 and pBICPm1+2+3 both showed decreased levels of GFP mRNA and increased GFP siRNA, especially the 21 nt species (Fig. 4b, lanes 8 and 9). The long and short GFP siRNAs accumulated to similar levels in plants agroinfiltrated with pBICPm1+2+3 or the pBICGFP negative control (Fig. 4b, lanes 9 and 1, respectively). To exclude the possibility of differential accumulation levels of mutated CP protein, co-infiltrated leaves were collected at 3 days p.i. and Western blot analysis was carried out to detect their level of CP using HCRSV CP polyclonal antibody (Fig. 4c). All CP mutants accumulated CP to the same level as the wt CP control. The amino acid mutations may contribute to the structural changes of the CP and render avirulence in kenaf. There seemed to be a correlation between failure of the mutant protein CPm1+2+3 to inhibit the onset of systemic silencing and impaired movement of mutant virus M-1+2+3 in kenaf. In addition, mutant M-1+2+3 is able to replicate efficiently and to assemble into virions in kenaf protoplasts (Liang et al., 2002a).
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; Kasschau & Carrington, 1998; Li et al., 1999; Voinnet et al., 1999). HCRSV CP is likely to be a symptom determinant during infection (Liang et al., 2002a). To investigate the effect of HCRSV CP on PVX infection, the CP gene was cloned into the pP2C2S (PVX) vector (Fig. 5a) and in vitro transcripts were used to inoculate 16c and wt N. benthamiana.
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). However, PVX–HCP induced more severe symptoms than PVX, producing necrotic local lesions in the systemic leaves (Fig. 5b). Similar observations were reported recently for the soil-borne wheat mosaic virus 19 kDa protein when expressed in the PVX vector (Te et al., 2005). However, these symptoms were less severe than those described for PVX–TCV CP, which was introduced into the plant by agroinfiltration and caused partial and complete necrosis of the whole plant by 6 and 10 days p.i., respectively (Thomas et al., 2003). To check the effect of HCRSV CP on viral RNA accumulation, PVX–GFP and PVX–HCP were inoculated onto wt and 16c N. benthamiana. Northern blot analysis with a GFP sequence-specific probe showed that PVX–GFP inoculated onto wt N. benthamiana accumulated PVX RNA to a much higher level than in the 16c plants (Fig. 5c, lanes 1 and 2). However, the PVX–GFP and PVX–HCP co-inoculated 16c plants accumulated PVX RNA to a higher level than PVX–GFP in the wt and 16c plants (Fig. 5c, lanes 1–3). The enhanced accumulation and symptom severity of PVX thus may be attributed to a synergistic reaction, which has been associated with an increased suppression of PTGS in plants (Pruss et al., 1997).
HCRSV CP fails to suppress dsGFP-induced local and systemic PTGS
To test whether HCRSV CP could suppress dsGFP-induced PTGS, GFP-transgenic 16c N. benthamiana were co-infiltrated with an Agrobacterium mixture containing plasmids pBICdsGFP+pBICP, pBICdsGFP+pBICP or pBICdsGFP+pBICPm1+2+3. Plants infiltrated with pBICGFP and pBICGFP+pBICP were used as controls.
At 6 days p.i., pBICdsGFP+pBICP-infiltrated leaves displayed no green fluorescence, as with pBICdsGFP-, pBICdsGFP+pBICP- and pBICdsGFP+pBICPm1+2+3-infiltrated leaves (Fig. 6a). In contrast, parallel control leaves infiltrated with pBICGFP+pBICP showed a strong GFP fluorescence (Fig. 6a). Systemic silencing was detected in all combinations of co-infiltration with pBICdsGFP (20 16c plants each), but not in the pBICGFP+pBICP co-infiltrated plants (20/20 16c plants for each infiltration) (Fig. 6b), indicating that CP failed to inhibit the dsGFP-induced systemic silencing signal. Similarly, CPm1+2+3 could not inhibit the mobile silencing signal induced by dsGFP (Fig. 6b). Molecular analysis showed that all of the dsGFP co-infiltrated leaves accumulated GFP siRNA to the same high level as pBICdsGFP-infiltrated leaves. However, the level of GFP mRNA accumulation was negligible (Fig. 6c, lanes 4 and 5). These results indicated that HCRSV CP could not inhibit the degradation of the infiltrated dsGFP inducer and the endogenous GFP transgene mRNA. These results were consistent with the low level of GFP fluorescence and systemic silencing observed (Fig. 6a and b).
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, lane 7) and, correspondingly, GFP siRNA accumulated to a high level, as in the pBICdsGFP- or pBICdsGFP+pBICP-infiltrated leaves (Fig. 6c, lanes 6 and 8, respectively), this result confirmed that HCRSV CP could not inhibit the degradation of the dsGFP inducers of PTGS. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Hannon, 2002). Although intense research has been invested since its discovery in 1990 (Napoli et al., 1990; van der Krol et al., 1990) and substantial progress has been made, the detailed processes of gene silencing are still largely unclear. Differences have been found between PTGS in plants and RNA interference in other organisms. The discovery of microRNA and trans-acting siRNA has revealed that the gene-silencing regulation network is even more complicated (Peragine et al., 2004; Allen et al., 2005). Virus-encoded suppressors provide useful tools for dissection of the PTGS pathway. They also help us to understand the viral infection process and host-symptom development. Most studies have focused on the identification of viral silencing suppressors from different virus families and on proteins with different functions in virus infection. However, little attention has been given to comparisons of these proteins on a phylogenetic and evolutionary basis. We constructed a phylogenetic tree using CP amino acid sequences from viruses of the family Tombusviridae (see Supplementary Figure, available in JGV Online). It is a common feature of viruses belonging to the family Tombusviridae that the CPs contain three structural and functional domains. Since TBSV CP is not a PTGS suppressor (Voinnet et al., 1999; Silhavy et al., 2002), it would be interesting to know whether other tombusvirus CPs also possess a suppression function or have evolved other viral proteins to function as suppressors. The results of this study have shown that, although HCRSV CP and TCV CP are structurally and functionally similar, their gene-silencing suppression mechanisms are different.
Agroinfiltration assays showed that HCRSV CP was able to suppress transiently expressed sense RNA-induced local PTGS. The suppression of local PTGS was as effective as with TBSV p19 (Fig. 2a). In addition, HCRSV CP could prevent the onset of systemic silencing induced by GFP sense RNA. Under our experimental conditions, HCRSV CP could inhibit the degradation of GFP mRNA, and greatly reduced but detectable levels of siRNAs were present in the LMM RNAs of the infiltrated leaves (Fig. 2d, lane 2). Each of the CP domain deletion mutants lost the ability to suppress sense RNA-induced local and systemic PTGS (Fig. 2b). Large domain deletions of the TCV CP also abolish suppression activity (Choi et al., 2004). These results may be explained by interactions of CP with other elements in the PTGS pathway. The absence of functional domains or structural changes may abolish these interactions. It is possible that the suppression mechanisms of these two CPs overlap in certain steps. Investigation of the absolute requirements for complete CPs of HCRSV and TCV to suppress PTGS will further dissect the suppression mechanism.
When inoculated onto the plants, HCRSV CP expressed from the PVX genome was able to enhance symptom severity. This is consistent with the effects of other silencing suppressors in similar viral vectors (Voinnet et al., 1999). An enhanced accumulation of PVX RNA was also observed (Fig. 5c). These findings indicate that HCRSV CP functions as a virulence factor and a suppressor of gene silencing. This may also explain the synergism in symptom expression resulting from co-infection of the two viruses. Plants inoculated with PVX–HCP RNA transcripts showed necrotic local lesions, while the agroinfiltrated PVX–TCV CP induced much more severe symptoms, which led to necrosis of the whole plant (Thomas et al., 2003).
Co-infiltration of pBICP+pBICdsGFP could not enhance the accumulation of GFP mRNA (Fig. 6c) in 16c plants. GFP siRNA in pBICdsGFP+pBICP co-infiltrated leaves (Fig. 6c, lanes 4 and 7) reached the same level of accumulation as in leaves infiltrated with pBICdsGFP (Fig. 6c, lanes 3 and 6), pBICdsGFP+pBICP (Fig. 6c, lanes 5 and 8) and pBICdsGFP+pBICPm1+2+3 (data not shown), suggesting that the steps involving generation of the dsGFP-induced GFP siRNAs and the RNA-induced silencing complex-guided mRNA cleavage were not blocked. These results indicated that HCRSV CP could not inhibit PTGS induced by dsRNA. TCV can suppress sense RNA-induced and dsRNA-induced PTGS and is suggested to suppress PTGS, possibly by interfering with the function of the Dicer-like RNase in planta (Qi et al., 2004). These results suggest that TCV CP and HCRSV CP may suppress PTGS at distinct steps. HCRSV CP could suppress sense RNA-induced but not dsRNA-induced PTGS. Therefore, CP should suppress PTGS at the initiation step, possibly acting at or before the dsRNA generation step. Further analysis of CP-interacting proteins in the host will help to clarify their exact involvement in the PTGS suppression pathways.
The CP mutants from serial passages showed reduced or complete loss of suppression activity. The accumulation of short 21 nt GFP siRNAs was greatly enhanced by pBICPm1+3 and pBICPm1+2+3, especially by pBICPm1+2+3. As the 21–22 nt siRNAs correlate with mRNA degradation, enhanced short siRNAs led to a decrease in GFP mRNA in the infiltrated leaves (Fig. 4b, lanes 8 and 9). Since the 25 nt siRNA is implicated in systemic silencing and is dispensable for sequence-specific mRNA degradation (Hamilton et al., 2002), the significantly increased accumulation of the long siRNA by pBICPm1+2+3 (Fig. 4b, lane 9) may lead to systemic silencing in the triple mutant-infiltrated plants. Thus, the failure of HCRSV CPm1+2+3 to block the long siRNA accumulation may contribute to the avirulence of mutant M-1+2+3 (Liang et al., 2002a) in kenaf. Knowing how the CP interferes with the PTGS pathway at the molecular and protein levels will help us to understand the changes in suppression ability resulting from amino acid mutations in HCRSV CP.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 2 October 2005;
accepted 24 October 2005.
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