Effect of Grafting on Viral Resistance of Non-transgenic Plum Scion Combined With Transgenic PPV-Resistant Rootstock

In stone fruit trees, resistance to Plum pox virus (PPV) can be achieved through the specific degradation of viral RNA by the mechanism of RNA interference (RNAi). Transgenic virus-resistant plants, however, raise serious biosafety concerns due to the insertion and expression of hairpin constructs that usually contain various selective foreign genes. Since a mature stone tree represents a combination of scion and rootstock, grafting commercial varieties onto transgenic virus-tolerant rootstocks is a possible approach to mitigate biosafety problems. The present study was aimed at answering the following question: To what extent are molecular RNAi silencing signals transmitted across graft junctions in transgrafted plum trees and how much does it affect PPV resistance in genetically modified (GM)/non-transgenic (NT) counterparts? Two combinations, NT:GM and GM:NT (scion:rootstock), were studied, with an emphasis on the first transgrafting scenario. Viral inoculation was carried out on either the scion or the rootstock. The interspecific rootstock “Elita” [(Prunus pumila L. × P. salicina Lindl.) × (P. cerasifera Ehrh.)] was combined with cv. “Startovaya” (Prunus domestica L.) as a scion. Transgenic plum lines of both cultivars were transformed with a PPV-coat protein (CP)-derived intron-separate hairpin-RNA construct and displayed substantial viral resistance. High-throughput sequence data of small RNA (sRNA) pools indicated that the accumulation of construct-specific small interfering RNA (siRNA) in transgenic plum rootstock reached over 2%. The elevated siRNA level enabled the resistance to PPV and blocked the movement of the virus through the GM tissues into the NT partner when the transgenic tissues were inoculated. At the same time, the mobile siRNA signal was not moved from the GM rootstock to the target NT tissue to a level sufficient to trigger silencing of PPV transcripts and provide reliable viral resistance. The lack of mobility of transgene-derived siRNA molecules was accompanied by the transfer of various endogenous rootstock-specific sRNAs into the NT scion, indicating the exceptional transitivity failure of the studied RNAi signal. The results presented here indicate that transgrafting in woody fruit trees remains an unpredictable practice and needs further in-depth examination to deliver molecular silencing signals.

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Plum Pox Virus Coat Protein Gene Intron-hairpin-RNA (ihpRNA) Constructs Provide Resistance to Plum Pox Virus in Nicotiana benthamiana and Prunus domestica

Journal of the American Society for Horticultural Science ◽

10.21273/jashs.132.6.850 ◽

2007 ◽

Vol 132 (6) ◽

pp. 850-858 ◽

Cited By ~ 54

Author(s):

Jean-Michel Hily ◽

Michel Ravelonandro ◽

Vern Damsteegt ◽

Carole Bassett ◽

Cesar Petri ◽

...

Keyword(s):

Coat Protein ◽

Gene Silencing ◽

Nicotiana Benthamiana ◽

High Capacity ◽

Plum Pox Virus ◽

Prunus Domestica ◽

Hairpin Rna ◽

Woody Perennial ◽

Virus Coat ◽

Interfering Rna

Constructs with self-complementary sequences separated by an intron produce “hairpin” RNA [intron-hairpin-RNA (ihpRNA)] structures that efficiently elicit posttranscriptional gene silencing (PTGS). In the current study, the authors use this technology to confer resistance to plum pox virus (PPV) in herbaceous and woody perennial plants by silencing the PPV–coat protein (CP) gene. The authors confirmed the high capacity of ihpRNA constructs for inducing RNA silencing in Nicotiana benthamiana Domin., as more than 75% of the transformants displayed PTGS as evaluated by specific small interfering RNA (siRNA) production. The authors demonstrated that ihpRNA constructs provided PPV resistance, and they found a correlation between the length of the PPV sequence introduced in the ihpRNA constructs and the frequency of transgenic-resistant plants. Plants transformed with the full-length sequence produced a higher percentage of resistant lines. The authors further demonstrated for the first time that ihpRNA technology is applicable to a woody perennial species. A transgenic plum (Prunus domestica L.) PPV-CP ihpRNA line showed gene silencing characteristics (hypermethylation of the transgene sequence and specific siRNA production) and resistance to PPV infection 16 months after inoculation.

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In Vivo Transfer of Small Interfering RNA or Small Hairpin RNA Targeting Glomeruli

Methods in Molecular Biology - Kidney Research ◽

10.1007/978-1-59745-352-3_18 ◽

2008 ◽

pp. 251-263 ◽

Cited By ~ 3

Author(s):

Yoshitsugu Takabatake ◽

Yoshitaka Isaka ◽

Enyu Imai

Keyword(s):

Small Interfering Rna ◽

Hairpin Rna ◽

Small Hairpin Rna ◽

Rna Targeting ◽

Interfering Rna

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Large-Scale, High-Throughput Validation of Short Hairpin RNA Sequences for RNA Interference

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057105284342 ◽

2006 ◽

Vol 11 (3) ◽

pp. 236-246 ◽

Cited By ~ 6

Author(s):

Laurence H. Lamarcq ◽

Bradley J. Scherer ◽

Michael L. Phelan ◽

Nikolai N. Kalnine ◽

Yen H. Nguyen ◽

...

Keyword(s):

High Throughput ◽

Large Scale ◽

Strong Support ◽

Gc Content ◽

Rapid Identification ◽

Hairpin Rna ◽

Rna Sequences ◽

Short Hairpin ◽

Short Hairpin Rnas ◽

Interfering Rna

A method for high-throughput cloning and analysis of short hairpin RNAs (shRNAs) is described. Using this approach, 464 shRNAs against 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAs against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for positionspecific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNAinterference (RNAi).

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Viral Vectors Applied for RNAi-Based Antiviral Therapy

Viruses ◽

10.3390/v12090924 ◽

2020 ◽

Vol 12 (9) ◽

pp. 924 ◽

Cited By ~ 2

Author(s):

Kenneth Lundstrom

Keyword(s):

Antiviral Therapy ◽

Viral Vectors ◽

Rna Virus ◽

Micro Rna ◽

Attractive Alternative ◽

Rna Modifications ◽

Hairpin Rna ◽

Pathogenic Viruses ◽

Short Hairpin ◽

Interfering Rna

RNA interference (RNAi) provides the means for alternative antiviral therapy. Delivery of RNAi in the form of short interfering RNA (siRNA), short hairpin RNA (shRNA) and micro-RNA (miRNA) have demonstrated efficacy in gene silencing for therapeutic applications against viral diseases. Bioinformatics has played an important role in the design of efficient RNAi sequences targeting various pathogenic viruses. However, stability and delivery of RNAi molecules have presented serious obstacles for reaching therapeutic efficacy. For this reason, RNA modifications and formulation of nanoparticles have proven useful for non-viral delivery of RNAi molecules. On the other hand, utilization of viral vectors and particularly self-replicating RNA virus vectors can be considered as an attractive alternative. In this review, examples of antiviral therapy applying RNAi-based approaches in various animal models will be described. Due to the current coronavirus pandemic, a special emphasis will be dedicated to targeting Coronavirus Disease-19 (COVID-19).

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Characterization of Sour Cherry Isolates of Plum pox virus from the Volga Basin in Russia Reveals a New Cherry Strain of the Virus

Phytopathology ◽

10.1094/phyto-11-12-0285-r ◽

2013 ◽

Vol 103 (9) ◽

pp. 972-979 ◽

Cited By ~ 27

Author(s):

Miroslav Glasa ◽

Yuri Prikhodko ◽

Lukáš Predajňa ◽

Alžbeta Nagyová ◽

Yuri Shneyder ◽

...

Keyword(s):

Virus Disease ◽

Phylogenetic Analyses ◽

Natural Infection ◽

Sour Cherry ◽

Fruit Trees ◽

Plum Pox Virus ◽

Volga River ◽

Distinct Cluster ◽

Genomic Characterization ◽

Cherry Trees

Plum pox virus (PPV) is the causal agent of sharka, the most detrimental virus disease of stone fruit trees worldwide. PPV isolates have been assigned into seven distinct strains, of which PPV-C regroups the genetically distinct isolates detected in several European countries on cherry hosts. Here, three complete and several partial genomic sequences of PPV isolates from sour cherry trees in the Volga River basin of Russia have been determined. The comparison of complete genome sequences has shown that the nucleotide identity values with other PPV isolates reached only 77.5 to 83.5%. Phylogenetic analyses clearly assigned the RU-17sc, RU-18sc, and RU-30sc isolates from cherry to a distinct cluster, most closely related to PPV-C and, to a lesser extent, PPV-W. Based on their natural infection of sour cherry trees and genomic characterization, the PPV isolates reported here represent a new strain of PPV, for which the name PPV-CR (Cherry Russia) is proposed. The unique amino acids conserved among PPV-CR and PPV-C cherry-infecting isolates (75 in total) are mostly distributed within the central part of P1, NIa, and the N terminus of the coat protein (CP), making them potential candidates for genetic determinants of the ability to infect cherry species or of adaptation to these hosts. The variability observed within 14 PPV-CR isolates analyzed in this study (0 to 2.6% nucleotide divergence in partial CP sequences) and the identification of these isolates in different localities and cultivation conditions suggest the efficient establishment and competitiveness of the PPV-CR in the environment. A specific primer pair has been developed, allowing the specific reverse-transcription polymerase chain reaction detection of PPV-CR isolates.

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New Insight on the Role of Plasminogen Receptor in Cancer Progression

Cancer Growth and Metastasis ◽

10.4137/cgm.s27335 ◽

2015 ◽

Vol 8 ◽

pp. CGM.S27335 ◽

Cited By ~ 18

Author(s):

Seema Kumari ◽

Ramarao Malla

Keyword(s):

Cancer Progression ◽

Receptor Gene ◽

Present Review ◽

Annexin Ii ◽

Invasion And Metastasis ◽

Hairpin Rna ◽

Diagnostic Strategies ◽

Surface Receptors ◽

Interfering Rna

Objective Plasminogen system plays a crucial role in physiological and pathological events related to tissue regeneration, wound healing, immune response, angiogenesis, invasion and metastasis. It gets activated when plasminogen associates with its cell surface receptors. Latest information on some of the well-explored plasminogen receptors such as annexin II-S100A10, cytokeratin 8, α-enolase, plasminogen receptor (KT) (Plg-R(KT)) and histone H2B has been discussed in the present review. These receptors can pave the way for effective new therapeutic and diagnostic strategies to counteract malignant diseases. Conclusion The present review concludes the key role of plasminogen receptors in extracellular matrix degradation, infiltration into surrounding tissues, neovascularization, invasion, metastasis and drug resistance. This review also discusses the possible effect of blocking these plasminogen receptors with monoclonal antibodies and DNA-based vaccination or silencing plasminogen receptor gene using small interfering RNA or short hairpin RNA to counteract cancer invasion and metastasis.

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Generation of Transgenic Rootstock Plum ((Prunus pumila L. × P. salicina Lindl.) × (P. cerasifera Ehrh.)) Using Hairpin-RNA Construct for Resistance to the Plum pox virus

Agronomy ◽

10.3390/agronomy8010002 ◽

2017 ◽

Vol 8 (1) ◽

pp. 2 ◽

Cited By ~ 6

Author(s):

Tatiana Sidorova ◽

Alexander Pushin ◽

Dmitry Miroshnichenko ◽

Sergey Dolgov

Keyword(s):

Plum Pox Virus ◽

Hairpin Rna

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Rapid Construction of Small Interfering RNA-Expressing Adenoviral Vectors on the Basis of Direct Cloning of Short Hairpin RNA-Coding DNAs

Human Gene Therapy ◽

10.1089/hum.2006.129 ◽

2007 ◽

Vol 18 (1) ◽

pp. 74-80 ◽

Cited By ~ 10

Author(s):

Hiroyuki Mizuguchi ◽

Naoko Funakoshi ◽

Tetsuji Hosono ◽

Fuminori Sakurai ◽

Kenji Kawabata ◽

...

Keyword(s):

Small Interfering Rna ◽

Adenoviral Vectors ◽

Short Hairpin Rna ◽

Hairpin Rna ◽

Direct Cloning ◽

Rapid Construction ◽

Short Hairpin ◽

Interfering Rna

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The Kinesin KIF1C and Microtubule Plus Ends Regulate Podosome Dynamics in Macrophages

Molecular Biology of the Cell ◽

10.1091/mbc.e05-11-1010 ◽

2006 ◽

Vol 17 (6) ◽

pp. 2811-2823 ◽

Cited By ~ 80

Author(s):

Petra Kopp ◽

Reiner Lammers ◽

Martin Aepfelbacher ◽

Günther Woehlke ◽

Thomas Rudel ◽

...

Keyword(s):

Molecular Basis ◽

Small Interfering Rna ◽

Migration And Invasion ◽

Nonmuscle Myosin ◽

Subcellular Targeting ◽

Hairpin Rna ◽

Monocytic Cells ◽

Human Macrophages ◽

Short Hairpin ◽

Interfering Rna

Microtubules are important for the turnover of podosomes, dynamic, actin-rich adhesions implicated in migration and invasion of monocytic cells. The molecular basis for this functional dependency, however, remained unclear. Here, we show that contact by microtubule plus ends critically influences the cellular fate of podosomes in primary human macrophages. In particular, we identify the kinesin KIF1C, a member of the Kinesin-3 family, as a plus-end–enriched motor that targets regions of podosome turnover. Expression of mutation constructs or small interfering RNA-/short hairpin RNA-based depletion of KIF1C resulted in decreased podosome dynamics and ultimately in podosome deficiency. Importantly, protein interaction studies showed that KIF1C binds to nonmuscle myosin IIA via its PTPD-binding domain, thus providing an interface between the actin and tubulin cytoskeletons, which may facilitate the subcellular targeting of podosomes by microtubules. This is the first report to implicate a kinesin in podosome regulation and also the first to describe a function for KIF1C in human cells.

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