RNA silencing (RNAi) induced by virus-derived double-stranded RNA (dsRNA), which is in a sense regarded as a pathogen-associated molecular pattern (PAMP) of viruses, is a general plant defense mechanism. To counteract this defense, plant viruses express RNA silencing suppressors (RSSs), many of which bind to dsRNA and attenuate RNAi. We showed that the tobacco calmodulin-like protein, rgs-CaM, counterattacked viral RSSs by binding to their dsRNA-binding domains and sequestering them from inhibiting RNAi. Autophagy-like protein degradation seemed to operate to degrade RSSs with the sacrifice of rgs-CaM. These RSSs could thus be regarded as secondary viral PAMPs. This study uncovered a unique defense system in which an rgs-CaM–mediated countermeasure against viral RSSs enhanced host antiviral RNAi in tobacco.
SummaryA highly infectious cDNA clone of clover yellow vein virus (pClYVV) was tested as a viral vector, especially for legume species. The genes for green¯uorescent protein (GFP) and soybean glutamine synthetase (GS) were inserted between the genes for P1 and HC-Pro on pClYVV to create three recombinant plasmids: pClYVV-GFP, pClYVV-GFP-GS, and pClYVV-GFP:GS. In the former two constructs all the junctions between the inserted proteins contained the sequences of protease cleavage recognition sites, whereas the third construct expressed a fusion of GFP and GS. Western blot analyses showed that GFP and GS appeared to have been precisely excised from the viral polyprotein with the viral proteases (P1 and NIa). Under UV irradiation, green¯uorescence was detected in infected broad bean, kidney bean, and soybean plants. The stability of the constructs in the symptomatic tissues was con®rmed by RT±PCR and Western blot analyses. The plants expressing GS together with GFP became tolerant to the herbicide glufosinate, and¯owered early. As the GS gene, one of the nodulin genes for nitrogen ®xation, is expressed in legume species, this system will be useful for examining the function of genes important to legume plants.
We examined whether the expression of wheat catalase (EC 1.11.1.6) cDNA in transgenic rice (Oryza sativa L.) could enhance tolerance against low temperature injury. Transgenic rice plants expressing wheat CAT protein showed an increase of activities in leaves at 25°C, 2‐ to 5‐fold that in non‐transgenic rice. At 5°C, catalase activities were about 4–15 times higher than those in non‐transgenic rice were. A comparison of damage observed in leaves as they withered due to chilling at 5°C showed that transgenic rice displayed an increased capability to resist low temperature stress. The exposure of these plants to low temperature at 5°C for 8 days resulted in decreased catalase activities in leaves at 25°C, but the transgenic plants indicated 4 times higher residual catalase activities than those of non‐transgenic ones. The concentration of H2O2 in leaves was kept lower in transgenic rice than that of the control plants during the 8 days chilling. These results suggest that the improved tolerance against low temperature stress in genetically engineered rice plants be attributed to the effective detoxification of H2O2 by the enhanced catalase activities.
We have developed Cucumber mosaic virus (CMV) as a plant virus vector especially for production of pharmaceutical proteins. The CMV vector is a vector modifiable for different host plants and does not require further engineering steps. CMV contains three genomic RNA molecules (RNAs 1-3) necessary for infectivity. With this system, instead of creating different vector constructs for each plant we use, we take advantage of the formation of pseudrecombinants between two CMV isolates by simply reassembling a vector construct (RNA 2 base) and an RNA molecule containing the host determinant (mostly RNA 3). In this study, the gene for acidic fibroblast growth factor (aFGF), one of the human cytokines, was cloned under the control of the subgenomic promoter for RNA 4A of the CMV-based vector, C2-H1. Infected Nicotiana benthamiana plants produced aFGF at levels up to 5-8% of the total soluble protein. The tobacco-produced aFGF was purified, and its biological activity was confirmed. Using this system, which provides a versatile and viable strategy for production of therapeutic proteins in plants, we also demonstrated a high level of aFGF in Glycine max (soybean) and Arabidopsis thaliana.
We report here the production of transgenic potato plants expressing the major capsid protein VP6 of bovine group A rotavirus (GAR). Transgenic plants under the control of a cauliflower mosaic virus 35S promoter, or a modified promoter linked to the tobacco mosaic virus 5'-untranslated sequence were positive for GAR antigens by ELISA. The expressed protein was consistent in size with VP6 of GAR by Western blot assay. The presence of the VP6 gene and its transcript was detected by PCR and RT-PCR. Adult BALB/c mice were immunized intraperitoneally with concentrated transgenic potato extracts emulsified in Freund's adjuvant. Sera collected after immunization showed the anti-VP6 response in ELISA and Western blot assay. These results suggest that the immunogenic VP6 protein expressed in plants could be useful for the preparation of diagnostic reagents.
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