scholarly journals Short interfering RNAs are Associated with Virus Resistance in a Woody Perennial Fruit Tree (Prunus domestica)

HortScience ◽  
2004 ◽  
Vol 39 (4) ◽  
pp. 862D-862
Author(s):  
Jean-Michel Hily ◽  
Ralph Scorza* ◽  
Michel Ravelonandro
Keyword(s):  
Virus Resistance ◽  
Rna Silencing ◽  
Transcriptional Gene Silencing ◽  
Marker Genes ◽  
Plum Pox Virus ◽  
Woody Perennials ◽  
Woody Perennial ◽  
Post Transcriptional Gene Silencing ◽  
High Level ◽  
Interfering Rna

We have shown that high-level resistance to plum pox virus (PPV) in transgenic plum clone C5 is based on post-transcriptional gene silencing (PTGS), otherwise termed RNA silencing (Scorza et al. Transgenic Res. 10:201-209, 2001). In order to more fully characterize RNA silencing in woody perennial crops, we investigated the production of short interfering RNA (siRNA) in transgenic plum clones C3 and C5, both of which harbor the capsid protein (CP) gene of PPV. We used as a control, plum PT-23, a clone only transformed with the two marker genes, NPTII and GUS. We show in the current report that C5 constitutively produces two classes of siRNA, the short (21-22 nucleotides) and long (≈27 nucleotides) species in the absence of PPV inoculation. Transgenic susceptible clone C3 and the control clone PT-23, when healthy, produce no siRNA. Upon infection, these clones produce only the short siRNA (21-22 nt). This siRNA production suggests that plum trees naturally respond to virus infection by initiating PTGS or PTGS-like mechanisms. This study also suggests that high-level virus resistance in woody perennials may require the production of both the short and long size classes of siRNA, as are produced by the resistant C5 plum clone.

scholarly journals Accumulation of the Long Clas of siRNA Is Associated with Resistance to Plum pox virus in a Transgenic Woody Perennial Plum Tree

2005 ◽  
Vol 18 (8) ◽  
pp. 794-799 ◽  
Author(s):  
Jean-Michel Hily ◽  
Ralph Scorza ◽  
Kevin Webb ◽  
Michel Ravelonandro
Keyword(s):  
Dna Methylation ◽  
Virus Resistance ◽  
Size Class ◽  
Plum Pox Virus ◽  
Prunus Species ◽  
Perennial Plant ◽  
Woody Perennial ◽  
High Level ◽  
Si Rna ◽  
Plum Tree

We investigated the hallmarks of posttranscription gene silencing (PTGS) in mature plants, embryos, and seedlings of the transgenic plum trees (Prunus sp.) that are resistant to Plum pox virus (PPV). We previously demonstrated that the transgene insert and resistance to PPV were mutually inherited in progeny of line C5. We show here that C5 constitutively produces a short (22 nt) and a long (25 to 26 nt) speciesshort interfering (si)RNA from embryo to mature plant in the absence of PPV inoculation. Unlike siRNA, methylation and transcription of the PPV-coat protein transgene were ‘o;re-set’ following seed germination. Uninoculated transgenic susceptible clones did not display DNA methylation, nor did they produce detectable levels of siRNA. Upon infection, susceptible clones, transgenic or untransformed, did produce siRNA but only the short 22-nt species. These findings show that plum trees respond to virus infection by initiating PTGS-like mechanisms that involve the production of siRNA. We further suggest that high-level virus resistance in transgenic Prunus species requires the production of the long-size class of siRNA. The research adds new insights into PTGS silencing in woody perennial plant species.

scholarly journals Cucumber mosaic virus Infection Transiently Breaks dsRNA-Induced Transgenic Immunity to Potato virus Y in Tobacco

2003 ◽  
Vol 16 (10) ◽  
pp. 936-944 ◽  
Author(s):  
Neena Mitter ◽  
Emy Sulistyowati ◽  
Ralf G. Dietzgen
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Potato Virus ◽  
Potato Virus Y ◽  
Defense Mechanism ◽  
Transcriptional Gene Silencing ◽  
Mrna Levels ◽  
Post Transcriptional Gene Silencing ◽  
The Stability ◽  
High Level

Post-transcriptional gene silencing (PTGS), an intrinsic plant defense mechanism, can be efficiently triggered by double stranded (ds)RNA-producing transgenes and can provide high level virus resistance by specific targeting of cognate viral RNA. The discovery of virus-encoded suppressors of PTGS led to concerns about the stability of such resistance. Here, we show that Cucumber mosaic virus (CMV) is able to suppress dsRNA-induced PTGS and the associated Potato virus Y (PVY) immunity in tobacco. CMV suppression supported only a transient PVY accumulation and did not prevent recovery of the transgenic plants from PVY infection. CMV inoculation resulted in strongly increased transgene mRNA levels due to suppression of PTGS, but accumulation of PVY-specific small interfering (si)RNA was unaffected. However, PVY accumulation in previously immune plants resulted in increased PVY siRNA levels and transgene mRNA was no longer detected, despite the presence of CMV. Transgene mRNA returned to high levels once PVY was no longer detected in CMV-infected plants. Recovered and chronically CMV-infected tissues were immune to further PVY infection.

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

2007 ◽  
Vol 132 (6) ◽  
pp. 850-858 ◽  
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.

scholarly journals Virus and Viroid-Derived Small RNAs as Modulators of Host Gene Expression: Molecular Insights Into Pathogenesis

2021 ◽  
Vol 11 ◽  
Author(s):  
S. V. Ramesh ◽  
Sneha Yogindran ◽  
Prabu Gnanasekaran ◽  
Supriya Chakraborty ◽  
Stephan Winter ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Small Rnas ◽  
Rna Silencing ◽  
Transcriptional Gene Silencing ◽  
Genomic Rnas ◽  
Post Transcriptional Gene Silencing ◽  
Viroid Infection ◽  
Wide Range ◽  
Associated Symptoms ◽  
Sequence Complementarity

Virus-derived siRNAs (vsiRNAs) generated by the host RNA silencing mechanism are effectors of plant’s defense response and act by targeting the viral RNA and DNA in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) pathways, respectively. Contrarily, viral suppressors of RNA silencing (VSRs) compromise the host RNA silencing pathways and also cause disease-associated symptoms. In this backdrop, reports describing the modulation of plant gene(s) expression by vsiRNAs via sequence complementarity between viral small RNAs (sRNAs) and host mRNAs have emerged. In some cases, silencing of host mRNAs by vsiRNAs has been implicated to cause characteristic symptoms of the viral diseases. Similarly, viroid infection results in generation of sRNAs, originating from viroid genomic RNAs, that potentially target host mRNAs causing typical disease-associated symptoms. Pathogen-derived sRNAs have been demonstrated to have the propensity to target wide range of genes including host defense-related genes, genes involved in flowering and reproductive pathways. Recent evidence indicates that vsiRNAs inhibit host RNA silencing to promote viral infection by acting as decoy sRNAs. Nevertheless, it remains unclear if the silencing of host transcripts by viral genome-derived sRNAs are inadvertent effects due to fortuitous pairing between vsiRNA and host mRNA or the result of genuine counter-defense strategy employed by viruses to enhance its survival inside the plant cell. In this review, we analyze the instances of such cross reaction between pathogen-derived vsiRNAs and host mRNAs and discuss the molecular insights regarding the process of pathogenesis.

The diversity of post-transcriptional gene silencing mediated by small silencing RNAs in plants

2020 ◽  
Vol 64 (6) ◽  
pp. 919-930 ◽  
Author(s):  
Huijuan Tan ◽  
Bosheng Li ◽  
Hongwei Guo
Keyword(s):  
Gene Silencing ◽  
Genome Stability ◽  
Transcriptional Gene Silencing ◽  
Rnase Iii ◽  
Post Transcriptional Gene Silencing ◽  
Endogenous Genes ◽  
Foreign Genes ◽  
Interfering Rna ◽  
Distinct Features ◽  
Initiating Factors

Abstract In plants, post-transcriptional gene silencing (PTGS) tightly regulates development, maintains genome stability and protects plant against foreign genes. PTGS can be triggered by virus infection, transgene, and endogenous transcript, thus commonly serves as an RNA-based immune mechanism. Accordingly, based on the initiating factors, PTGS can be divided into viral-PTGS, transgene-PTGS, and endo-gene-PTGS. Unlike the intensely expressed invading transgenes and viral genes that frequently undergo PTGS, most endogenous genes do not trigger PTGS, except for a few that can produce endogenous small RNAs (sRNAs), including microRNA (miRNA) and small interfering RNA (siRNA). Different lengths of miRNA and siRNA, mainly 21-, 22- or 24-nucleotides (nt) exert diverse functions, ranging from target mRNA degradation, translational inhibition, or DNA methylation and chromatin modifications. The abundant 21-nt miRNA or siRNA, processed by RNase-III enzyme DICER-LIKE 1 (DCL1) and DCL4, respectively, have been well studied in the PTGS pathways. By contrast, the scarceness of endogenous 22-nt sRNAs that are primarily processed by DCL2 limits their research, although a few encouraging studies have been reported recently. Therefore, we review here our current understanding of diverse PTGS pathways triggered by a variety of sRNAs and summarize the distinct features of the 22-nt sRNA mediated PTGS.

scholarly journals Homology-Dependent Virus Resistance in Transgenic Plants with the Coat Protein Gene of Sweet Potato Feathery Mottle Potyvirus: Target Specificity and Transgene Methylation

1999 ◽  
Vol 89 (5) ◽  
pp. 385-391 ◽  
Author(s):  
S. Sonoda ◽  
M. Mori ◽  
M. Nishiguchi
Keyword(s):  
Coat Protein ◽  
Sweet Potato ◽  
Virus Resistance ◽  
Potato Virus X ◽  
Transcriptional Gene Silencing ◽  
Target Specificity ◽  
Post Transcriptional Gene Silencing ◽  
Resistant Lines ◽  
Nontranslated Region ◽  
Transgenic Lines

Nicotiana benthamiana plants were transformed with the coat protein (CP) coding sequence and the 3′ nontranslated region (NTR) of the severe strain of sweet potato feathery mottle potyvirus (SPFMV-S). Regenerated lines were screened for virus resistance using recombinant potato virus X (PVX) engineered to contain the sequence homologous to the transgene. Out of 19 transgenic lines, 7 showed virus resistance after inoculation by the recombinant PVX. In most of the resistant lines, relatively low steady-state accumulation of the CP gene mRNA and little or no protein products were observed, suggesting that the resistance was manifested by a post-transcriptional gene-silencing mechanism. The resistant lines could be divided into two groups according to the target specificity of the silencing mechanism; one group recognizing the 3′ part of the transgene mRNA and the other not only the 3′ part, but also the 5′ and the central part of the transgene mRNA. Particular regions of the transgene corresponding to the RNA target in the resistant lines were differentially methylated compared with the transgene sequence in a susceptible line.

scholarly journals Hibiscus chlorotic ringspot virus coat protein inhibits trans-acting small interfering RNA biogenesis in Arabidopsis

2008 ◽  
Vol 89 (9) ◽  
pp. 2349-2358 ◽  
Author(s):  
Chunying Meng ◽  
Jun Chen ◽  
Shou-wei Ding ◽  
Jinrong Peng ◽  
Sek-Man Wong
Keyword(s):  
Coat Protein ◽  
Gene Silencing ◽  
Rna Silencing ◽  
Small Interfering Rna ◽  
Fluorescent Protein ◽  
Cauliflower Mosaic Virus ◽  
Ringspot Virus ◽  
Pcr Analysis ◽  
Transcriptional Gene Silencing ◽  
Interfering Rna

Many plant and animal viruses have evolved suppressor proteins to block host RNA silencing at various stages of the RNA silencing pathways. Hibiscus chlorotic ringspot virus (HCRSV) coat protein (CP) is capable of suppressing the transiently expressed sense-RNA-induced post-transcriptional gene silencing (PTGS) in Nicotiana benthamiana. Here, constitutively expressed HCRSV CP from transgenic Arabidopsis was found to be able to rescue expression of the silenced GUS transgene. The HCRSV CP-transgenic Arabidopsis (line CP6) displayed several developmental abnormalities: elongated, downwardly curled leaves and a lack of coordination between stamen and carpel, resulting in reduced seed set. These abnormalities are similar to those observed in mutations of the genes of Arabidopsis RNA-dependent polymerase 6 (rdr6), suppressor of gene silencing 3 (sgs3), ZIPPY (zip) and dicer-like 4 (dcl4). The accumulation of microRNA (miRNA) miR173 remained stable; however, the downstream trans-acting small interfering RNA (ta-siRNA) siR255 was greatly reduced. Real-time PCR analysis showed that expression of the ta-siRNA-targeted At4g29770, At5g18040, PPR and ARF3 genes increased significantly, especially in the inflorescences. Genetic crossing of CP6 with an amplicon-silenced line (containing a potato virus X–green fluorescent protein transgene under the control of the 35S cauliflower mosaic virus promoter) suggested that HCRSV CP probably interfered with gene silencing at a step after RDR6. The reduced accumulation of ta-siRNA might result from the interference of HCRSV CP with Dicer-like protein(s), responsible for the generation of dsRNA in ta-siRNA biogenesis.

scholarly journals Long-Term Efficacy and Safety of RNAi-Mediated Virus Resistance in ‘HoneySweet’ Plum

2021 ◽  
Vol 12 ◽  
Author(s):  
Khushwant Singh ◽  
Ann M. Callahan ◽  
Brenda J. Smith ◽  
Tadeusz Malinowski ◽  
Ralph Scorza ◽  
...  
Keyword(s):  
Health Risks ◽  
Virus Resistance ◽  
The United States ◽  
Genetically Engineered ◽  
Plum Pox Virus ◽  
Potential Health ◽  
Rna Profiling ◽  
Term Efficacy ◽  
Interfering Rna

Interfering RNA technology has been established as an effective strategy to protect plants against viral infection. Despite this success, interfering RNA (RNAi) has rarely been applied due to the regulatory barriers that confront genetically engineered plants and concerns over possible environmental and health risks posed by non-endogenous small RNAs. ‘HoneySweet’ was developed as a virus-resistant plum variety that is protected by an RNAi-mediated process against Sharka disease caused by the plum pox virus. ‘HoneySweet’ has been approved for cultivation in the United States but not in countries where the plum pox virus is endemic. In this study, we evaluated the long-term efficacy of virus resistance in ‘HoneySweet,’ the nature and stability of its sRNA profile, and the potential health risks of consuming ‘HoneySweet’ plums. Graft-challenged ‘HoneySweet’ trees carrying large non-transgenic infected limbs remained virus-free after more than 10 years in the field, and the viral sequences from the non-transgenic infected limbs showed no evidence of adaptation to the RNAi-based resistance. Small RNA profiling revealed that transgene-derived sRNA levels were stable across different environments and, on average, were more than 10 times lower than those present in symptom-less fruits from virus-infected trees. Comprehensive 90-day mouse feeding studies showed no adverse health impacts in mice, and there was no evidence for potential siRNA off-target pathologies predicted by comparisons of the most abundant transgene-derived sRNAs to the mouse genome. Collectively, the data confirmed that RNAi provides a highly effective, stable, and safe strategy to combat virus diseases in crop plants.

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