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Analysis of pineapple mealybug wilt associated virus -1 and -2 for potential RNA silencing suppressors and pathogenicity factors.

Dey KK, Borth WB, Melzer MJ, Wang ML, Hu JS - Viruses (2015)

Bottom Line: None of the proteins analyzed could interfere with the short distance spread of silencing.We examined the mechanism of systemic suppression activity by investigating the effect of PMWaV-2-encoded p20 and CP proteins on secondary siRNAs.Our results suggest that the PMWaV-2 p20 and CP proteins block the systemic silencing signal by repressing production of secondary siRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Environmental Protection Sciences, University of Hawaii, 3190 Maile Way, Honolulu, HI 96822, USA. kishore@hawaii.edu.

ABSTRACT
Higher plants use RNA silencing to defend against viral infections. As a counter defense, plant viruses have evolved proteins that suppress RNA silencing. Mealybug wilt of pineapple (MWP), an important disease of pineapple, has been associated with at least three distinct viruses, Pineapple mealybug wilt associated virus -1, -2, and -3 (PMWaV-1, -2, and -3). Selected open reading frames (ORFs) of PMWaV-1 and PMWaV-2 were screened for their local and systemic suppressor activities in Agrobacterium-mediated transient assays using green fluorescent protein (GFP) in Nicotiana benthamiana. Results indicate that PMWaV-2 utilizes a multiple-component RNA silencing suppression mechanism. Two proteins, p20 and CP, target both local and systemic silencing in N. benthamiana, while the p22 and CPd proteins target only systemic silencing. In the related virus PMWaV-1, we found that only one of the encoded proteins, p61, had only systemic suppressor activity. Of all the proteins tested from both viruses, only the PMWaV-2 p20 protein suppressed local silencing induced by double-stranded RNA (dsRNA), but only when low levels of inducing dsRNA were used. None of the proteins analyzed could interfere with the short distance spread of silencing. We examined the mechanism of systemic suppression activity by investigating the effect of PMWaV-2-encoded p20 and CP proteins on secondary siRNAs. Our results suggest that the PMWaV-2 p20 and CP proteins block the systemic silencing signal by repressing production of secondary siRNAs. We also demonstrate that the PMWaV-2 p20 and p22 proteins enhanced the pathogenicity of Potato virus X in N. benthamiana.

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Identification of local suppressors in the genome of PMWaV-2. (A) WT. N. benthamiana plants were co-infiltrated with cultures of Agrobacterium carrying 35S-sGFP and Agrobacterium carrying individual constructs. Infiltrated leaves were examined under short-wavelength UV light and photographed with a Nikon 5000 digital camera at 5 days post-infiltration (dpi). Leaves co-infiltrated with 35S-GFP and pBIC-35S-empty vector (EV) or 35S-GFP with Tomato bushy stunt virus (TBSV)-35S p19 were used as negative or positive controls respectively. (A) shows fluorescence produced by the two identified local suppressors, p20 and CP. Northern blots of GFP mRNAs (B) and GFP siRNAs (C) from agroinfiltrated leaves at 5 dpi. Ethidium bromide staining of ribosomal RNA was used to confirm equal loading. Loading of RNAs for GFP siRNAs analysis was estimated by comparison to tRNAs on the same blot.
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viruses-07-00969-f002: Identification of local suppressors in the genome of PMWaV-2. (A) WT. N. benthamiana plants were co-infiltrated with cultures of Agrobacterium carrying 35S-sGFP and Agrobacterium carrying individual constructs. Infiltrated leaves were examined under short-wavelength UV light and photographed with a Nikon 5000 digital camera at 5 days post-infiltration (dpi). Leaves co-infiltrated with 35S-GFP and pBIC-35S-empty vector (EV) or 35S-GFP with Tomato bushy stunt virus (TBSV)-35S p19 were used as negative or positive controls respectively. (A) shows fluorescence produced by the two identified local suppressors, p20 and CP. Northern blots of GFP mRNAs (B) and GFP siRNAs (C) from agroinfiltrated leaves at 5 dpi. Ethidium bromide staining of ribosomal RNA was used to confirm equal loading. Loading of RNAs for GFP siRNAs analysis was estimated by comparison to tRNAs on the same blot.

Mentions: Seven ORFs from the 3’-terminus of PMWaV-2 (Hsp70, p46, CP, Cpd, p20, p22, and p6) and four ORFs from the 3’-terminus of PMWaV-1 (Hsp70, p61, CP, and p24) (Figure 1) were screened for potential local RNA silencing suppressors in agroinfiltrated N. benthamiana plants at 2, 3, 5, 7, 8, and 12 days post infiltration (dpi). As expected, two to three days following infiltration strong GFP fluorescence was observed in all leaves that had been co-infiltrated with Agrobacterium containing the 35S-empty vector and Agrobacterium containing 35S-sGFP (Figure S1) Similar results were observed in leaves co-infiltrated with Agrobacterium carrying 35S-sGFP and Agrobacterium carrying individual PMWaV-1 and PMWaV-2 ORFs (Figure S1). By 5 to 7 dpi GFP fluorescence declined significantly in those plants infiltrated with 35S-sGFP and 35S-empty vector, becoming almost undetectable by 7 dpi. As expected, co-infiltration with 35S-p19, the well-characterized RNA silencing suppressor of TBSV, elicited strong green fluorescence that persisted for several days. When leaves were infiltrated with a mixture of Agrobacterium suspensions containing both 35S-sGFP and constructs harboring various ORFs from PMWaV-1 and PMWaV-2, only ORFs p20 and CP from PMWaV-2 showed higher fluorescence in majority of the infiltrated leaves than did the empty vector controls at 5 dpi. None of the other ORFs from PMWaV-1 or -2 that were tested suppressed local GFP silencing (Figure 2). The p22 ORF of PMWaV-2, despite failing to suppress GFP local silencing, nonetheless caused the development of necrotic lesions in the infiltrated area of leaves, suggesting a potential role of p22 in pathogenicity that may be independent of silencing suppressor activity (Figure S2). The strong fluorescence observed when 35S-p19 was infiltrated persisted beyond 7 dpi and was maintained until 12 dpi. However when either the p20 or CP ORFs from PMWaV-2 were co-infiltrated with 35S-sGFP, there was a marked reduction of GFP fluorescence by 7 dpi that almost disappeared by 8 dpi (Figure S3). To confirm that the increased fluorescence of GFP was indeed due to the result of suppression of RNA silencing, the steady-state levels of GFP mRNAs were analyzed by Northern blots. The results obtained were consistent with the observations of GFP fluorescence (Figure 2); the GFP mRNA levels were significantly higher in leaves co-infiltrated with the 35S-sGFP and 35S-p20 or 35S-CP than in leaves co-infiltrated with the 35S-sGFP and 35S-empty vector, which showed significantly reduced GFP levels. This result indicates that p20 and CP of PMWaV-2 effectively delay RNA silencing in the transient-expression system. The effect of PMWaV-2 p20 and CP on local GFP silencing was considerably less than that of 35S-p19, as determined by the GFP mRNA levels at 5 dpi. Analysis of GFP siRNAs showed the accumulation of ~21 nt and ~24 nt siRNAs in leaves co-infiltrated with Agrobacterium carrying the 35S-sGFP and the 35S-empty vector, confirming that gene silencing was active in these plants [13,51]. In contrast, GFP-specific siRNAs were remarkably low in leaves co-infiltrated with Agrobacterium carrying PMWaV-2 p20 and were absent in leaves co-infiltrated with the 35S-p19 (positive control) (Figure 2). However, the siRNA levels produced by PMWaV-2 CP infiltrations did not show such a negative correlation; the levels of the GFP siRNAs were comparable to those produced by the negative control.


Analysis of pineapple mealybug wilt associated virus -1 and -2 for potential RNA silencing suppressors and pathogenicity factors.

Dey KK, Borth WB, Melzer MJ, Wang ML, Hu JS - Viruses (2015)

Identification of local suppressors in the genome of PMWaV-2. (A) WT. N. benthamiana plants were co-infiltrated with cultures of Agrobacterium carrying 35S-sGFP and Agrobacterium carrying individual constructs. Infiltrated leaves were examined under short-wavelength UV light and photographed with a Nikon 5000 digital camera at 5 days post-infiltration (dpi). Leaves co-infiltrated with 35S-GFP and pBIC-35S-empty vector (EV) or 35S-GFP with Tomato bushy stunt virus (TBSV)-35S p19 were used as negative or positive controls respectively. (A) shows fluorescence produced by the two identified local suppressors, p20 and CP. Northern blots of GFP mRNAs (B) and GFP siRNAs (C) from agroinfiltrated leaves at 5 dpi. Ethidium bromide staining of ribosomal RNA was used to confirm equal loading. Loading of RNAs for GFP siRNAs analysis was estimated by comparison to tRNAs on the same blot.
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Related In: Results  -  Collection

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viruses-07-00969-f002: Identification of local suppressors in the genome of PMWaV-2. (A) WT. N. benthamiana plants were co-infiltrated with cultures of Agrobacterium carrying 35S-sGFP and Agrobacterium carrying individual constructs. Infiltrated leaves were examined under short-wavelength UV light and photographed with a Nikon 5000 digital camera at 5 days post-infiltration (dpi). Leaves co-infiltrated with 35S-GFP and pBIC-35S-empty vector (EV) or 35S-GFP with Tomato bushy stunt virus (TBSV)-35S p19 were used as negative or positive controls respectively. (A) shows fluorescence produced by the two identified local suppressors, p20 and CP. Northern blots of GFP mRNAs (B) and GFP siRNAs (C) from agroinfiltrated leaves at 5 dpi. Ethidium bromide staining of ribosomal RNA was used to confirm equal loading. Loading of RNAs for GFP siRNAs analysis was estimated by comparison to tRNAs on the same blot.
Mentions: Seven ORFs from the 3’-terminus of PMWaV-2 (Hsp70, p46, CP, Cpd, p20, p22, and p6) and four ORFs from the 3’-terminus of PMWaV-1 (Hsp70, p61, CP, and p24) (Figure 1) were screened for potential local RNA silencing suppressors in agroinfiltrated N. benthamiana plants at 2, 3, 5, 7, 8, and 12 days post infiltration (dpi). As expected, two to three days following infiltration strong GFP fluorescence was observed in all leaves that had been co-infiltrated with Agrobacterium containing the 35S-empty vector and Agrobacterium containing 35S-sGFP (Figure S1) Similar results were observed in leaves co-infiltrated with Agrobacterium carrying 35S-sGFP and Agrobacterium carrying individual PMWaV-1 and PMWaV-2 ORFs (Figure S1). By 5 to 7 dpi GFP fluorescence declined significantly in those plants infiltrated with 35S-sGFP and 35S-empty vector, becoming almost undetectable by 7 dpi. As expected, co-infiltration with 35S-p19, the well-characterized RNA silencing suppressor of TBSV, elicited strong green fluorescence that persisted for several days. When leaves were infiltrated with a mixture of Agrobacterium suspensions containing both 35S-sGFP and constructs harboring various ORFs from PMWaV-1 and PMWaV-2, only ORFs p20 and CP from PMWaV-2 showed higher fluorescence in majority of the infiltrated leaves than did the empty vector controls at 5 dpi. None of the other ORFs from PMWaV-1 or -2 that were tested suppressed local GFP silencing (Figure 2). The p22 ORF of PMWaV-2, despite failing to suppress GFP local silencing, nonetheless caused the development of necrotic lesions in the infiltrated area of leaves, suggesting a potential role of p22 in pathogenicity that may be independent of silencing suppressor activity (Figure S2). The strong fluorescence observed when 35S-p19 was infiltrated persisted beyond 7 dpi and was maintained until 12 dpi. However when either the p20 or CP ORFs from PMWaV-2 were co-infiltrated with 35S-sGFP, there was a marked reduction of GFP fluorescence by 7 dpi that almost disappeared by 8 dpi (Figure S3). To confirm that the increased fluorescence of GFP was indeed due to the result of suppression of RNA silencing, the steady-state levels of GFP mRNAs were analyzed by Northern blots. The results obtained were consistent with the observations of GFP fluorescence (Figure 2); the GFP mRNA levels were significantly higher in leaves co-infiltrated with the 35S-sGFP and 35S-p20 or 35S-CP than in leaves co-infiltrated with the 35S-sGFP and 35S-empty vector, which showed significantly reduced GFP levels. This result indicates that p20 and CP of PMWaV-2 effectively delay RNA silencing in the transient-expression system. The effect of PMWaV-2 p20 and CP on local GFP silencing was considerably less than that of 35S-p19, as determined by the GFP mRNA levels at 5 dpi. Analysis of GFP siRNAs showed the accumulation of ~21 nt and ~24 nt siRNAs in leaves co-infiltrated with Agrobacterium carrying the 35S-sGFP and the 35S-empty vector, confirming that gene silencing was active in these plants [13,51]. In contrast, GFP-specific siRNAs were remarkably low in leaves co-infiltrated with Agrobacterium carrying PMWaV-2 p20 and were absent in leaves co-infiltrated with the 35S-p19 (positive control) (Figure 2). However, the siRNA levels produced by PMWaV-2 CP infiltrations did not show such a negative correlation; the levels of the GFP siRNAs were comparable to those produced by the negative control.

Bottom Line: None of the proteins analyzed could interfere with the short distance spread of silencing.We examined the mechanism of systemic suppression activity by investigating the effect of PMWaV-2-encoded p20 and CP proteins on secondary siRNAs.Our results suggest that the PMWaV-2 p20 and CP proteins block the systemic silencing signal by repressing production of secondary siRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Environmental Protection Sciences, University of Hawaii, 3190 Maile Way, Honolulu, HI 96822, USA. kishore@hawaii.edu.

ABSTRACT
Higher plants use RNA silencing to defend against viral infections. As a counter defense, plant viruses have evolved proteins that suppress RNA silencing. Mealybug wilt of pineapple (MWP), an important disease of pineapple, has been associated with at least three distinct viruses, Pineapple mealybug wilt associated virus -1, -2, and -3 (PMWaV-1, -2, and -3). Selected open reading frames (ORFs) of PMWaV-1 and PMWaV-2 were screened for their local and systemic suppressor activities in Agrobacterium-mediated transient assays using green fluorescent protein (GFP) in Nicotiana benthamiana. Results indicate that PMWaV-2 utilizes a multiple-component RNA silencing suppression mechanism. Two proteins, p20 and CP, target both local and systemic silencing in N. benthamiana, while the p22 and CPd proteins target only systemic silencing. In the related virus PMWaV-1, we found that only one of the encoded proteins, p61, had only systemic suppressor activity. Of all the proteins tested from both viruses, only the PMWaV-2 p20 protein suppressed local silencing induced by double-stranded RNA (dsRNA), but only when low levels of inducing dsRNA were used. None of the proteins analyzed could interfere with the short distance spread of silencing. We examined the mechanism of systemic suppression activity by investigating the effect of PMWaV-2-encoded p20 and CP proteins on secondary siRNAs. Our results suggest that the PMWaV-2 p20 and CP proteins block the systemic silencing signal by repressing production of secondary siRNAs. We also demonstrate that the PMWaV-2 p20 and p22 proteins enhanced the pathogenicity of Potato virus X in N. benthamiana.

Show MeSH
Related in: MedlinePlus