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Generation of virus-resistant potato plants by RNA genome targeting.

ABSTRACT: CRISPR/Cas systems provide bacteria and archaea with molecular immunity against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a is an RNA-targeting CRISPR effector that provides protection against RNA phages. Here we report the repurposing of CRISPR/Cas13a to protect potato plants from a eukaryotic virus, Potato virus Y (PVY). Transgenic potato lines expressing Cas13a/sgRNA (small guide RNA) constructs showed suppressed PVY accumulation and disease symptoms. The levels of viral resistance correlated with the expression levels of the Cas13a/sgRNA construct in the plants. Our data further demonstrate that appropriately designed sgRNAs can specifically interfere with multiple PVY strains, while having no effect on unrelated viruses such as PVA or Potato virus S. Our findings provide a novel and highly efficient strategy for engineering crops with resistances to viral diseases.

SUBMITTER: Zhan X

PROVIDER: S-EPMC6686122 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Generation of virus-resistant potato plants by RNA genome targeting.

Zhan Xiaohui X Zhang Fengjuan F Zhong Ziyang Z Chen Ruhao R Wang Yong Y Chang Ling L Bock Ralph R Nie Bihua B Zhang Jiang J

Plant biotechnology journal 20190308 9

CRISPR/Cas systems provide bacteria and archaea with molecular immunity against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a is an RNA-targeting CRISPR effector that provides protection against RNA phages. Here we report the repurposing of CRISPR/Cas13a to protect potato plants from a eukaryotic virus, Potato virus Y (PVY). Transgenic potato lines expressing Cas13a/sgRNA (small guide RNA) constructs showed suppressed PVY accumulation and disease symptoms. ...[more]

PMID: 30803101

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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.

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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Generation of virus-resistant potato plants by RNA genome targeting.

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Generation of virus-resistant potato plants by RNA genome targeting.

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