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Yicheng Yu Zhiyuan Pan Xiao Wang Xiaofeng Bian Weichi Wang Qiang Liang Meng Kou Hongtao Ji Yanjuan Li Daifu Ma Zongyun Li Jian Sun 《Molecular Plant Pathology》2022,23(1):104-117
Sweet potato (Ipomoea batatas) is one of the most important crops in the world, and its production rate is mainly decreased by the sweet potato virus disease (SPVD) caused by the co-infection of sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus. However, methods for improving SPVD resistance have not been established. Thus, this study aimed to enhance SPVD resistance by targeting one of its important pathogenesis-related factors (i.e., SPCSV-RNase3) by using the CRISPR-Cas13 technique. First, the RNA targeting activity of four CRISPR-Cas13 variants were compared using a transient expression system in Nicotiana benthamiana. LwaCas13a and RfxCas13d had more efficient RNA and RNA virus targeting activity than PspCas13b and LshCas13a. Driven by the pCmYLCV promoter for the expression of gRNAs, RfxCas13d exhibited higher RNA targeting activity than that driven by the pAtU6 promoter. Furthermore, the targeting of SPCSV-RNase3 using the LwaCas13a system inhibited its RNA silencing suppressor activity and recovered the RNA silencing activity in N. benthamiana leaf cells. Compared with the wild type, transgenic N. benthamiana plants carrying an RNase3-targeted LwaCas13a system exhibited enhanced resistance against turnip mosaic virus TuMV-GFP and cucumber mosaic virus CMV-RNase3 co-infection. Moreover, transgenic sweet potato plants carrying an RNase3-targeted RfxCas13d system exhibited substantially improved SPVD resistance. This method may contribute to the development of SPVD immune germplasm and the enhancement of sweet potato production in SPVD-prevalent regions. 相似文献
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Huimin Xu Houri Khalilian Mohamed Eweida Sam Squire Mounir G. Abouhaidar 《Plant cell reports》1995,15(1-2):91-96
The genes for the capsid protein (CP) and the 8K movement protein of PVX were introduced into potato (Solanum tuberosum L.) and expressed under the control of CaMV 35S promoter using a binary vector andAgrobacterium tumefaciens. Four commercial potato cultivars (Russet Burbank, Shepody, Desirée and Bintje) have been efficiently transformed. Eleven independent transgenic clones, with CP expression levels higher than 0.05% of the soluble leaf proteins, were analyzed for resistance to inoculation with PVX (5 and 50µg/ml). The resistance of the transgenic plants to PVX was observed with the lower titer of virus inoculation (5 µg/ml) but not with higher titer (50 µg/ml). A significant reduction in the accumulation of virus in the inoculated transgenic potato plants has been observed under greenhouse and field conditions. Furthermore, the CP gene is very stable and is transferred to new plants originated from stem cuttings or from tubers. The transgenic plants appeared to be phenotypically identical to the nontransformed controls.Abbreviations BAP
benzyl-aminopurine
- BCIP
5-bromo-4-chloro-3-indolylphosphate p-Toluidine salt
- CaMV
cauliflower mosaic virus
- CP
capsid protein
- GA3
gibberellic acid
- Kbp
kilobase pair
- NAA
naphthalene acetic acid
- NBT
nitroblue tetrazolium chloride
- NOS
nopaline synthase
- NPT II
neomycin phosphotransferase II
- PMSF
phenyl methyl sulfonyl fluoride
- PVX
potato virus X
- PVY
potato virus Y 相似文献
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The C2 protein of tomato leaf curl Taiwan virus is a pathogenicity determinant that interferes with expression of host genes encoding chromomethylases
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Yu‐Ching Tu Wen‐Shi Tsai Jyuan‐Yu Wei Kai‐Ya Chang Chang‐Ching Tien Hui‐Yu Hsiao Shih‐Feng Fu 《Physiologia plantarum》2017,161(4):515-531
Tomato (Solanum lycopersicum) is one of the most important crops worldwide and is severely affected by geminiviruses. Tomato leaf curl Taiwan virus (ToLCTWV), belonging to the geminiviruses, was isolated in Taiwan and causes tremendous crop loss. The geminivirus‐encoded C2 proteins are crucial for a successful interaction between the virus and host plants. However, the exact functions of the viral C2 protein of ToLCTWV have not been investigated. We analyzed the molecular function(s) of the C2 protein by transient or stable expression in tomato cv. Micro‐Tom and Nicotiana benthamiana. Severe stunting of tomato and N. benthamiana plants infected with ToLCTWV was observed. Expression of ToLCTWV C2‐green fluorescent protein (GFP) fusion protein was predominately located in the nucleus and contributed to activation of a coat protein promoter. Notably, the C2‐GFP fluorescence was distributed in nuclear aggregates. Tomato and N. benthamiana plants inoculated with potato virus X (PVX)‐C2 displayed chlorotic lesions and stunted growth. PVX‐C2 elicited hypersensitive responses accompanied by production of reactive oxygen species in N. benthamiana plants, which suggests that the viral C2 was a potential recognition target to induce host‐defense responses. In tomato and N. benthamiana, ToLCTWV C2 was found to interfere with expression of genes encoding chromomethylases. N. benthamiana plants with suppressed NbCMT3–2 expression were more susceptible to ToLCTWV infection. Transgenic N. benthamiana plants expressing the C2 protein showed decreased expression of the NbCMT3–2 gene and pNbCMT3–2::GUS (β‐glucuronidase) promoter activity. C2 protein is an important pathogenicity determinant of ToLCTWV and interferes with host components involved in DNA methylation. 相似文献
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X. -G. Li J. Yang R. Wang X. -F. Tang J. -J. Meng H. -J. Qin X. Liu F. Guo S. -B. Wan 《Photosynthetica》2013,51(3):387-394
Pyrococcus furiosus is a hyperthermophilic archaeon. Its ribulose-1,5-bisphosphate carboxylase/oxygenase (PfRubisco) has only large subunit (L). PfRubisco has a novel (L2)5, decameric structure and it possesses higher carboxylase activity and thermotolerance. To assess the potential functionality of PfRubisco in higher plants under high-temperature stress, PfRubisco coding sequence was transiently expressed in Nicotiana benthamiana by Pea early browning virus mediated ectopic expression. The transgenic PfRubisco plants produced chlorotic yellow stripes in their leaves. Relative to the control leaves, those with yellow stripes exhibited decreased net photosynthetic rate and chlorophyll content, altered chloroplast ultrastructure, and more severe photoinhibition of both photosystem I and II. We concluded that the ectopic expression of PfRubisco might disrupt the chloroplast development and function in N. benthamiana. The potential cause of the disruption was discussed. 相似文献
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Expression of the rabies virus nucleoprotein in plants at high-levels and evaluation of immune responses in mice 总被引:1,自引:0,他引:1
Perea Arango I Loza Rubio E Rojas Anaya E Olivera Flores T Gonzalez de la Vara L Gómez Lim MA 《Plant cell reports》2008,27(4):677-685
Transgenic plants have been employed successfully as a low-cost system for the production of therapeutically valuable proteins
including antibodies, antigens and hormones. Here, we report expression of a full-length nucleoprotein gene of rabies virus
in transgenic tomato plants. The nucleoprotein was also transiently expressed in Nicotiana benthamiana plants by agroinfiltration. In both cases, the nucleoprotein was expressed at high levels, 1–5% of total soluble protein
in tomato and 45% in N. benthamiana. Previously, only epitopes of the nucleoprotein had been expressed in plants. The presence and expression of the transgene
was verified by PCR, Southern, northern and western blots. Mice were immunized both intraperitoneally (i.p.) and orally with
tomato protein extracts containing the N protein induced the production of antibodies. The antibody titer of mice immunized
i.p., was at least four times higher than that of mice immunized orally. These results were reflected in the challenge experiments
where i.p.-immunized mice were partially protected against a peripheral virus challenge whereas orally immunized mice were
not. This protection was comparable to that obtained in previous experiments employing different expression systems. Work
is in progress to express both G and N proteins in transgenic plants and evaluate protection in mice. 相似文献
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Improved in Planta Expression of the Human Islet Autoantigen Glutamic Acid Decarboxylase (GAD65) 总被引:3,自引:0,他引:3
Avesani L Falorni A Tornielli GB Marusic C Porceddu A Polverari A Faleri C Calcinaro F Pezzotti M 《Transgenic research》2003,12(2):203-212
The smaller isoform of the enzyme glutamic acid decarboxylase (GAD65) is a major islet autoantigen in autoimmune type 1 diabetes mellitus (T1DM). Transgenic plants expressing human GAD65 (hGAD65) are a potential means of direct oral administration of the islet autoantigen in order to induce tolerance and prevent clinical onset of disease. We have previously reported the successful generation of transgenic tobacco and carrot that express immunoreactive, full-length hGAD65. In the present study, we tested the hypothesis that the expression levels of recombinant hGAD65 in transgenic plants can be increased by targeting the enzyme to the plant cell cytosol and by mediating expression through the potato virus X (PVX) vector. By substituting the NH2-terminal region of hGAD65 with a homologous region of rat GAD67, a chimeric GAD671-87/GAD6588-585 molecule was expressed in transgenic tobacco plants. Immunolocalization analysis showed that immunoreactive GAD67/65 was found in the plant cell cytosol. By using a radio-immuno assay with human serum from a GAD65 autoantibody-positive T1DM patient, the highest expression level of the recombinant GAD67/65 protein was estimated to be 0.19% of the total soluble protein, compared to only 0.04% of wild-type hGAD65. Transient expression of wild-type, full-length hGAD65 in N. benthamiana mediated by PVX infection was associated with expression levels of immunoreactive protein as high as 2.2% of total soluble protein. This substantial improvement of the expression of hGAD65 in plants paves the way for immunoprevention studies of oral administration of GAD65-containing transgenic plant material in animal models of spontaneous autoimmune diabetes. 相似文献
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Insect resistance of transgenic plants that express modified Bacillus thuringiensis cryIA(b) and cryIC genes: a resistance management strategy 总被引:7,自引:0,他引:7
Theo van der Salm Dirk Bosch Guy Honée Lanxiang Feng Ellie Munsterman Petra Bakker Willem J. Stiekema Bert Visser 《Plant molecular biology》1994,26(1):51-59
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Zhong Huang Qiang Chen Brooke Hjelm Charles Arntzen Hugh Mason 《Biotechnology and bioengineering》2009,103(4):706-714
Recombinant virus‐like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low‐level antigen accumulation and long‐time frame to produce transgenic plants are the two major roadblocks in the practical development of plant‐based VLP production. In this article, we describe the optimization of geminivirus‐derived DNA replicon vectors for rapid, high‐yield plant‐based production of VLPs. Co‐delivery of bean yellow dwarf virus (BeYDV)‐derived vector and Rep/RepA‐supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 5 days. Co‐expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built‐in Rep/RepA cassette without P19 drove protein expression at similar levels as the three‐component system. These results demonstrate the advantages of fast and high‐level production of VLP‐based vaccines using the BeYDV‐derived DNA replicon system for transient expression in plants. Biotechnol. Bioeng. 2009;103: 706–714. © 2009 Wiley Periodicals, Inc. 相似文献