Generation of virus-resistant potato plants by RNA genome targeting.
Ontology highlight
ABSTRACT:
SUBMITTER: Zhan X
PROVIDER: S-EPMC6686122 | biostudies-literature | 2019 Sep
REPOSITORIES: biostudies-literature
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Project description:Genome editing technology is important for plant science and crop breeding. Genome-edited plants prepared using general CRISPR-Cas9 methods usually contain foreign DNA, which is problematic for the production of genome-edited transgene-free plants for vegetative propagation or highly heterozygous hybrid cultivars. Here, we describe a method for highly efficient targeted mutagenesis in Nicotiana benthamiana through the expression of Cas9 and single-guide (sg)RNA using a potato virus X (PVX) vector. Following Agrobacterium-mediated introduction of virus vector cDNA, >60% of shoots regenerated without antibiotic selection carried targeted mutations, while ?18% of shoots contained T-DNA. The PVX vector was also used to express a base editor consisting of modified Cas9 fused with cytidine deaminase to introduce targeted nucleotide substitution in regenerated shoots. We also report exogenous DNA-free genome editing by mechanical inoculation of virions comprising the PVX vector expressing Cas9. This simple and efficient virus vector-mediated delivery of CRISPR-Cas9 could facilitate transgene-free gene editing in plants.
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Project description:Potato virus Y (PVY) is one of the most important plant viruses affecting potato production. The interactions between potato and PVY are complex and the outcome of the interactions depends on the potato genotype, the PVY strain, and the environmental conditions. A potato cultivar can induce resistance to a specific PVY strain, yet be susceptible to another. How a single potato cultivar responds to PVY in both compatible and incompatible interactions is not clear.In this study, we used RNA-sequencing (RNA-Seq) to investigate and compare the transcriptional changes in leaves of potato upon inoculation with PVY. We used two potato varieties: Premier Russet, which is resistant to the PVY strain O (PVY(O)) but susceptible to the strain NTN (PVY(NTN)), and Russet Burbank, which is susceptible to all PVY strains that have been tested. Leaves were inoculated with PVY(O) or PVY(NTN), and samples were collected 4 and 10 h post inoculation (hpi). A larger number of differentially expressed (DE) genes were found in the compatible reactions compared to the incompatible reaction. For all treatments, the majority of DE genes were down-regulated at 4 hpi and up-regulated at 10 hpi. Gene Ontology enrichment analysis showed enrichment of the biological process GO term "Photosynthesis, light harvesting" specifically in PVY(O)-inoculated Premier Russet leaves, while the GO term "nucleosome assembly" was largely overrepresented in PVY(NTN)-inoculated Premier Russet leaves and PVY(O)-inoculated Russet Burbank leaves but not in PVY(O)-inoculated Premier Russet leaves. Fewer genes were DE over 4-fold in the incompatible reaction compared to the compatible reactions. Amongst these, five genes were DE only in PVY(O)-inoculated Premier Russet leaves, and all five were down-regulated. These genes are predicted to encode for a putative ABC transporter, a MYC2 transcription factor, a VQ-motif containing protein, a non-specific lipid-transfer protein, and a xyloglucan endotransglucosylase-hydroxylase.Our results show that the incompatible and compatible reactions in Premier Russet shared more similarities, in particular during the initial response, than the compatible reactions in the two different hosts. Our results identify potential key processes and genes that determine the fate of the reaction, compatible or incompatible, between PVY and its host.
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Project description:Hepatitis C virus (HCV) infections frequently recur after liver transplantation in patients with HCV-related liver diseases. Approximately 30% of these patients progress to cirrhosis within 5 years after surgery. In this study, we proposed an effective therapeutic strategy to overcome the recurrence of HCV. CRISPR-Cas9 was used to insert an expression cassette encoding an RNA aptamer targeting HCV NS5B replicase as an anti-HCV agent into adeno-associated virus integration site 1 (AAVS1), known as a "safe harbor," in a hepatocellular carcinoma cell line to confer resistance to HCV. The RNA aptamer expression system based on a dihydrofolate reductase minigene was precisely knocked in into AAVS1, leading to the stable expression of aptamer RNA in the developed cell line. HCV replication was effectively inhibited at both the RNA and protein levels in cells transfected with HCV RNA or infected with HCV. RNA immunoprecipitation and competition experiments strongly suggested that this HCV inhibition was due to the RNA aptamer-mediated sequestration of HCV NS5B. No off-target insertion of the RNA aptamer expression construct was observed. The findings suggest that HCV-resistant liver cells produced by genome editing technology could be used as a new alternative in the development of a treatment for HCV-induced liver diseases.
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Project description:Plant-virus interactions are greatly influenced by environmental factors such as temperatures. In virus-infected plants, enhanced temperature is frequently associated with more severe symptoms and higher virus content. However, the mechanisms involved in controlling the temperature regulation of plant-virus interactions are poorly characterised. To elucidate these further, we analysed the responses of potato plants cv Chicago to infection by potato virus Y (PVY) at normal (22 °C) and elevated temperature (28 °C), the latter of which is known to significantly increase plant susceptibility to PVY. Using RNAseq analysis, we showed that single and combined PVY and heat-stress treatments caused dramatic changes in gene expression, affecting the transcription of both protein-coding and non-coding RNAs. Among the newly identified genes responsive to PVY infection, we found genes encoding enzymes involved in the catalysis of polyamine formation and poly ADP-ribosylation. We also identified a range of novel non-coding RNAs which were differentially produced in response to single or combined PVY and heat stress, that consisted of antisense RNAs and RNAs with miRNA binding sites. Finally, to gain more insights into the potential role of alternative splicing and epitranscriptomic RNA methylation during combined stress conditions, direct RNA nanopore sequencing was performed. Our findings offer insights for future studies of functional links between virus infections and transcriptome reprogramming, RNA methylation and alternative splicing.
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Project description:Viruses classified in the order Mononegavirales have a single nonsegmented RNA molecule as the genome and employ similar strategies for genome replication and gene expression. Infectious particles of Measles virus (MeV), a member of the family Paramyxoviridae in the order Mononegavirales, with two or three RNA genome segments (2 seg- or 3 seg-MeV) were generated using a highly efficient reverse genetics system. All RNA segments of the viruses were designed to have authentic 3' and 5' self-complementary termini, similar to those of negative-stranded RNA viruses that intrinsically have multiple RNA genome segments. The 2 seg- and 3 seg-MeV were viable and replicated well in cultured cells. 3 seg-MeV could accommodate up to six additional transcriptional units, five of which were shown to be capable of expressing foreign proteins efficiently. These data indicate that the MeV genome can be segmented, providing an experimental insight into the divergence of the negative-stranded RNA viruses with nonsegmented or segmented RNA genomes. They also illustrate a new strategy to develop mononegavirus-derived vectors harboring multiple additional transcriptional units.
| S-EPMC1472037 | biostudies-literature
Project description:Potato virus S (PVS) often causes significant losses in potato production in potato-growing countries. In this study, the ordinary strain of PVS (PVSO) was purified from PVS-infected potato plants and used as the immunogen to produce hybridomas secreting monoclonal antibodies (MAbs). Five highly specific and sensitive murine MAbs (1A3, 16C10, 18A9, 20B12, and 22H4) against PVS were prepared using conventional hybridoma technology. Using these MAbs, tissue print-enzyme-linked immunosorbent assay (ELISA), dot-ELISA, and double-antibody sandwich (DAS)-ELISA were developed for sensitive and specific detection of PVS infection in potato plants. The results of sensitivity assays revealed that PVS could be reliably detected in PVS-infected leaf crude extracts diluted at 1:10 240 and 1:163 840 (w/v, g/ml) in phosphate buffer saline (PBS) by dot-ELISA and DAS-ELISA, respectively. Twenty-two samples collected from potato fields in Yunnan Province, China were tested for PVS infection using the serological assays we had developed, and 14 of them were found to be positive. This indicates that PVS is now prevalent in potato fields in Yunnan Province.
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Project description:In the field, plants are challenged by more than one biotic stressor at the same time. In this study, the molecular interactions between potato (Solanum tuberosum L.), Colorado potato beetle (Leptinotarsa decemlineata Say; CPB) and Potato virus Y(NTN) (PVY(NTN) ) were investigated through analyses of gene expression in the potato leaves and the gut of the CPB larvae, and of the release of potato volatile compounds. CPB larval growth was enhanced when reared on secondary PVY(NTN) -infected plants, which was linked to decreased accumulation of transcripts associated with the antinutritional properties of potato. In PVY(NTN) -infected plants, ethylene signalling pathway induction and induction of auxin response transcription factors were attenuated, while no differences were observed in jasmonic acid (JA) signalling pathway. Similarly to rearing on virus-infected plants, CPB larvae gained more weight when reared on plants silenced in JA receptor gene (coi1). Although herbivore-induced defence mechanism is regulated predominantly by JA, response in coi1-silenced plants only partially corresponded to the one observed in PVY(NTN) -infected plants, confirming the role of other plant hormones in modulating this response. The release of β-barbatene and benzyl alcohol was different in healthy and PVY(NTN) -infected plants before CPB larvae infestation, implicating the importance of PVY(NTN) infection in plant communication with its environment. This was reflected in gene expression profiles of neighbouring plants showing different degree of defence response. This study thus contributes to our understanding of plant responses in agro-ecosystems.
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Project description:The potato/tomato psyllid, Bactericera cockerelli (B. cockerelli), is an important plant pest and the vector of the phloem-limited bacterium Candidatus Liberibacter psyllaurous (solanacearum), which is associated with the zebra chip disease of potatoes. Previously, we reported induction of RNA interference effects in B. cockerelli via in vitro-prepared dsRNA/siRNAs after intrathoracic injection, and after feeding of artificial diets containing these effector RNAs. In order to deliver RNAi effectors via plant hosts and to rapidly identify effective target sequences in plant-feeding B. cockerelli, here we developed a plant virus vector-based in planta system for evaluating candidate sequences. We show that recombinant Tobacco mosaic virus (TMV) containing B. cockerelli sequences can efficiently infect and generate small interfering RNAs in tomato (Solanum lycopersicum), tomatillo (Physalis philadelphica) and tobacco (Nicotiana tabacum) plants, and more importantly delivery of interfering sequences via TMV induces RNAi effects, as measured by actin and V-ATPase mRNA reductions, in B. cockerelli feeding on these plants. RNAi effects were primarily detected in the B. cockerelli guts. In contrast to our results with TMV, recombinant Potato virus X (PVX) and Tobacco rattle virus (TRV) did not give robust infections in all plants and did not induce detectable RNAi effects in B. cockerelli. The greatest RNA interference effects were observed when B. cockerelli nymphs were allowed to feed on leaf discs collected from inoculated or lower expanded leaves from corresponding TMV-infected plants. Tomatillo plants infected with recombinant TMV containing B. cockerelli actin or V-ATPase sequences also showed phenotypic effects resulting in decreased B. cockerelli progeny production as compared to plants infected by recombinant TMV containing GFP. These results showed that RNAi effects can be achieved in plants against the phloem feeder, B. cockerelli, and the TMV-plant system will provide a faster and more convenient method for screening of suitable RNAi target sequences in planta.
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Project description:Global threats of ssDNA geminivirus and ss(-)RNA tospovirus on crops necessitate the development of transgenic resistance. Here, we constructed a two-T DNA vector carrying a hairpin of the intergenic region (IGR) of Ageratum yellow vein virus (AYVV), residing in an intron inserted in an untranslatable nucleocapsid protein (NP) fragment of Melon yellow spot virus (MYSV). Transgenic tobacco lines highly resistant to AYVV and MYSV were generated. Accumulation of 24-nt siRNA, higher methylation levels on the IGR promoters of the transgene, and suppression of IGR promoter activity of invading AYVV indicate that AYVV resistance is mediated by transcriptional gene silencing. Lack of NP transcript and accumulation of corresponding siRNAs indicate that MYSV resistance is mediated through post-transcriptional gene silencing. Marker-free progenies with concurrent resistance to both AYVV and MYSV, stably inherited as dominant nuclear traits, were obtained. Hence, we provide a novel way for concurrent control of noxious DNA and RNA viruses with less biosafety concerns.
| S-EPMC4101524 | biostudies-literature
Project description:BackgroundViruses are the major threat to commercial potato (Solanum tuberosum) production worldwide. Because viral genomes only encode a small number of proteins, all stages of viral infection rely on interactions between viral proteins and host factors. Previously, we presented a list of the most important candidate genes involved in potato plants' defense response to viruses that are significantly activated in resistant cultivars. Isolated from this list, Aspartic Protease Inhibitor 5 (API5) is a critical host regulatory component of plant defense responses against pathogens. The purpose of this study is to determine the role of StAPI5 in defense of potato against potato virus Y and potato virus A, as well as its ability to confer virus resistance in a transgenic susceptible cultivar of potato (Desiree). Potato plants were transformed with Agrobacterium tumefaciens via a construct encoding the potato StAPI5 gene under the control of the Cauliflower mosaic virus (CaMV) 35S promoter.ResultsTransgenic plants overexpressing StAPI5 exhibited comparable virus resistance to non-transgenic control plants, indicating that StAPI5 functions in gene regulation during virus resistance. The endogenous StAPI5 and CaMV 35S promoter regions shared nine transcription factor binding sites. Additionally, the net photosynthetic rate, stomatal conductivity, and maximum photochemical efficiency of photosystem II were significantly higher in virus-infected transgenic plants than in wild-type plants.ConclusionOverall, these findings indicate that StAPI5 may be a viable candidate gene for engineering plant disease resistance to viruses that inhibit disease development.
| S-EPMC9097181 | biostudies-literature