Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery.

Sweet orange (Citrus sinensis) is one of the most important fruit crops worldwide. Virus infections in this crop can interfere with cellular processes, causing dramatic economic losses. By performing RT-qPCR analyses, we demonstrated that citrus psorosis virus (CPsV)-infected orange plants exhibited higher levels of unprocessed microRNA (miRNA) precursors than healthy plants. This result correlated with the reported reduction of mature miRNAs species. The protein 24K, the CPsV suppressor of RNA silencing (VSR), interacts with miRNA precursors in vivo. Thus, this protein becomes a candidate responsible for the increased accumulation of unprocessed miRNAs. We analyzed 24K RNA-binding and protein-protein interaction domains and described patterns of its subcellular localization. We also showed that 24K colocalizes within nuclear D-bodies with the miRNA biogenesis proteins DICER-LIKE 1 (DCL1), HYPONASTIC LEAVES 1 (HYL1), and SERRATE (SE). According to the results of bimolecular fluorescence complementation and co-immunoprecipitation assays, the 24K protein interacts with HYL1 and SE. Thus, 24K may inhibit miRNA processing in CPsV-infected citrus plants by direct interaction with the miRNA processing complex. This work contributes to the understanding of how a virus can alter the regulatory mechanisms of the host, particularly miRNA biogenesis and function.IMPORTANCESweet oranges can suffer from disease symptoms induced by virus infections, thus resulting in drastic economic losses. In sweet orange plants, CPsV alters the accumulation of some precursors from the regulatory molecules called miRNAs. This alteration leads to a decreased level of mature miRNA species. This misregulation may be due to a direct association of one of the viral proteins (24K) with miRNA precursors. On the other hand, 24K may act with components of the cell miRNA processing machinery through a series of predicted RNA-binding and protein-protein interaction domains.

Citrus Genetic Transformation: An Overview of the Current Strategies and Insights on the New Emerging Technologies.

Citrus are among the most prevailing fruit crops produced worldwide. The implementation of effective and reliable breeding programs is essential for coping with the increasing demands of satisfactory yield and quality of the fruit as well as to deal with the negative impact of fast-spreading diseases. Conventional methods are time-consuming and of difficult application because of inherent factors of citrus biology, such as their prolonged juvenile period and a complex reproductive stage, sometimes presenting infertility, self-incompatibility, parthenocarpy, or polyembryony. Moreover, certain desirable traits are absent from cultivated or wild citrus genotypes. All these features are challenging for the incorporation of the desirable traits. In this regard, genetic engineering technologies offer a series of alternative approaches that allow overcoming the difficulties of conventional breeding programs. This review gives a detailed overview of the currently used strategies for the development of genetically modified citrus. We describe different aspects regarding genotype varieties used, including elite cultivars or extensively used scions and rootstocks. Furthermore, we discuss technical aspects of citrus genetic transformation procedures via Agrobacterium, regular physical methods, and magnetofection. Finally, we describe the selection of explants considering young and mature tissues, protoplast isolation, etc. We also address current protocols and novel approaches for improving the in vitro regeneration process, which is an important bottleneck for citrus genetic transformation. This review also explores alternative emerging transformation strategies applied to citrus species such as transient and tissue localized transformation. New breeding technologies, including cisgenesis, intragenesis, and genome editing by clustered regularly interspaced short palindromic repeats (CRISPR), are also discussed. Other relevant aspects comprising new promoters and reporter genes, marker-free systems, and strategies for induction of early flowering, are also addressed. We provided a future perspective on the use of current and new technologies in citrus and its potential impact on regulatory processes.

Up-regulation of microRNA targets correlates with symptom severity in Citrus sinensis plants infected with two different isolates of citrus psorosis virus.

MAIN CONCLUSION: miRNA targets from Citrus sinensis are predicted and validated using degradome data. They show an up-regulation upon infection with CPsV, with a positive correlation between target expression and symptom severity. Sweet orange (Citrus sinensis) may suffer from disease symptoms induced by virus infections, thus resulting in drastic economic losses. Infection of sweet orange plants with two isolates of citrus psorosis virus (CPsV), expressing different symptomatologies, alters the accumulation of a set of endogenous microRNAs (miRNAs). Here, we predicted ten putative targets from four down-regulated miRNAs: three belonging to the CCAAT-binding transcription factor family (CBFAs); an Ethylene-responsive transcription factor (RAP2-7); an Integrase-type DNA-binding superfamily protein (AP2B); Transport inhibitor response 1 (TIR1); GRR1-like protein 1-related (GRR1); Argonaute 2-related (AGO2), Argonaute 7 (AGO7), and a long non-coding RNA (ncRNA). We validated six of them through analysis of leaf degradome data. Expressions of the validated targets increase in infected samples compared to healthy tissue, showing a more striking up-regulation those samples with higher symptom severity. This study contributes to the understanding of the miRNA-mediated regulation of important transcripts in Citrus sinensis through target validation and shed light in the manner a virus can alter host regulatory mechanisms leading to symptom expression.

ICTV Virus Taxonomy Profile: Ophioviridae.

The Ophioviridae is a family of filamentous plant viruses, with single-stranded negative, and possibly ambisense, RNA genomes of 11.3-12.5 kb divided into 3-4 segments, each encapsidated separately. Virions are naked filamentous nucleocapsids, forming kinked circles of at least two different contour lengths. The sole genus, Ophiovirus, includes seven species. Four ophioviruses are soil-transmitted and their natural hosts include trees, shrubs, vegetables and bulbous or corm-forming ornamentals, both monocots and dicots. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Ophioviridae, which is available at http://www.ictv.global/report/ophioviridae.

Identification and characterization of two RNA silencing suppressors encoded by ophioviruses.

Citrus psorosis virus and Mirafiori lettuce big-vein virus are two members of the genus Ophiovirus, family Ophioviridae. So far, how these viruses can interfere in the antiviral RNA silencing pathway is not known. In this study, using a local GFP silencing assay on Nicotiana benthamiana, the 24K-25K and the movement protein (MP) of both viruses were identified as RNA silencing suppressor proteins. Upon their co-expression with GFP in N. benthamiana 16c plants, the proteins also showed to suppress systemic RNA (GFP) silencing. The MPCPsV and 24KCPsV proteins bind long (114 nucleotides) but not short-interfering (21 nt) dsRNA, and upon transgenic expression, plants showed developmental abnormalities that coincided with an altered miRNA accumulation pattern. Furthermore, both proteins were able to suppress miRNA-induced silencing of a GFP-sensor construct and the co-expression of MPCPsV and 24KCPsV exhibited a stronger effect, suggesting they act at different stages of the RNAi pathway.

Citrus psorosis virus 24K protein interacts with citrus miRNA precursors, affects their processing and subsequent miRNA accumulation and target expression.

Sweet orange (Citrus sinensis), one of the most important fruit crops worldwide, may suffer from disease symptoms induced by virus infections, thus resulting in dramatic economic losses. Here, we show that the infection of sweet orange plants with two isolates of Citrus psorosis virus (CPsV) expressing different symptomatology alters the accumulation of a set of endogenous microRNAs (miRNAs). Within these miRNAs, miR156, miR167 and miR171 were the most down-regulated, with almost a three-fold reduction in infected samples. This down-regulation led to a concomitant up-regulation of some of their targets, such as Squamosa promoter-binding protein-like 9 and 13, as well as Scarecrow-like 6. The processing of miRNA precursors, pre-miR156 and pre-miR171, in sweet orange seems to be affected by the virus. For instance, virus infection increases the level of unprocessed precursors, which is accompanied by a concomitant decrease in mature species accumulation. miR156a primary transcript accumulation remained unaltered, thus strongly suggesting a processing deregulation for this transcript. The co-immunoprecipitation of viral 24K protein with pre-miR156a or pre-miR171a suggests that the alteration in the processing of these precursors might be caused by a direct or indirect interaction with this particular viral protein. This result is also consistent with the nuclear localization of both miRNA precursors and the CPsV 24K protein. This study contributes to the understanding of the manner in which a virus can alter host regulatory mechanisms, particularly miRNA biogenesis and target expression.