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DCL-suppressed Nicotiana benthamiana plants: valuable tools in research and biotechnology.

ABSTRACT: RNA silencing is a universal mechanism involved in development, epigenetic modifications and responses to biotic and abiotic stresses. The major components of this mechanism are Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) proteins. Understanding the role of each component is of great scientific and agronomic importance. Plants, including Nicotiana benthamiana, an important plant model, usually possess four DCL proteins, each of which has a specific role, namely being responsible for the production of an exclusive small RNA population. Here, we used RNA interference (RNAi) technology to target DCL proteins and produced single and combinatorial mutants for DCL. We analysed the phenotype for each DCL knockdown plant, together with the small RNA profile, by next-generation sequencing (NGS). We also investigated transgene expression, as well as viral infections, and were able to show that DCL suppression results in distinct developmental defects, changes in small RNA populations, increases in transgene expression and, finally, higher susceptibility in certain RNA viruses. Therefore, these plants are excellent tools for the following: (i) to study the role of DCL enzymes; (ii) to overexpress proteins of interest; and (iii) to understand the complex relationship between the plant silencing mechanism and biotic or abiotic stresses.

SUBMITTER: Katsarou K

PROVIDER: S-EPMC6637889 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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DCL-suppressed Nicotiana benthamiana plants: valuable tools in research and biotechnology.

Katsarou Konstantina K Mitta Eleni E Bardani Eirini E Oulas Anastasis A Dadami Elena E Kalantidis Kriton K

Molecular plant pathology 20181219 3

RNA silencing is a universal mechanism involved in development, epigenetic modifications and responses to biotic and abiotic stresses. The major components of this mechanism are Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) proteins. Understanding the role of each component is of great scientific and agronomic importance. Plants, including Nicotiana benthamiana, an important plant model, usually possess four DCL proteins, each of which has a specific role, namely being ...[more]

PMID: 30343523

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Project description:Viroids are infectious non-coding RNAs that infect plants. During infection, viroid RNAs are targeted by Dicer-like proteins, generating viroid-derived small RNAs (vd-sRNAs) that can guide the sequence specific cleavage of cognate host mRNAs via an RNA silencing mechanism. To assess the involvement of these pathways in pathogenesis associated with nuclear-replicating viroids, high-throughput sequencing of sRNAs and degradome analysis were carried out on tomato and Nicotiana benthamiana plants infected by potato spindle tuber viroid (PSTVd). Both hosts develop similar stunting and leaf curling symptoms when infected by PSTVd, thus allowing comparative analyses. About one hundred tomato mRNAs potentially targeted for degradation by vd-sRNAs were initially identified. However, data from biological replicates and comparisons between mock and infected samples reduced the number of bona fide targets-i.e., those identified with high confidence in two infected biological replicates but not in the mock controls-to only eight mRNAs that encode proteins involved in development, transcription or defense. Somewhat surprisingly, results of RT-qPCR assays revealed that the accumulation of only four of these mRNAs was inhibited in the PSTVd-infected tomato. When these analyses were extended to mock inoculated and PSTVd-infected N. benthamiana plants, a completely different set of potential mRNA targets was identified. The failure to identify homologous mRNA(s) targeted by PSTVd-sRNA suggests that different pathways could be involved in the elicitation of similar symptoms in these two species. Moreover, no significant modifications in the accumulation of miRNAs and in the cleavage of their targeted mRNAs were detected in the infected tomato plants with respect to the mock controls. Taken together, these data suggest that stunting and leaf curling symptoms induced by PSTVd are elicited by a complex plant response involving multiple mechanisms, with RNA silencing being only one of the possible components.

Dataset Information

DCL-suppressed Nicotiana benthamiana plants: valuable tools in research and biotechnology.

Publications

DCL-suppressed Nicotiana benthamiana plants: valuable tools in research and biotechnology.

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