Molecular Hybridization Techniques for Detecting and Studying Viroids

Pallás, Vicente; Sánchez‐Navarro, J. A.; Kinard, Gary; Serio, Francesco Di

doi:10.1016/b978-0-12-801498-1.00035-8

Cited by 10 publications

(6 citation statements)

References 49 publications

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“…Digoxigenin (DIG)-labeled riboprobes 1 (binding to positions from 1588 to 1934 nt of dsRNA1), 2 and 3 (116–355 and 177–485 nt of dsRNA3, respectively) were synthesized in vitro transcription based on the corresponding cDNAs inserted into the pGEM-T easy vector with T7 RNA polymerase (Takara, Beijing, China) as previously described [ 25 ]. Nucleic acids were spotted (for dot blotting) or electro-transferred (for Northern blot) to positively-charged nylon membranes (Roche Diagnostics), and hybridized with DIG-labeled riboprobes as previously described [ 25 ].…”

Section: Methodsmentioning

confidence: 99%

A special satellite-like RNA of a novel hypovirus from Pestalotiopsis fici broadens the definition of fungal satellite

et al. 2023

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Satellites associated with plant or animal viruses have been largely detected and characterized, while those from mycoviruses together with their roles remain far less determined. Three dsRNA segments (dsRNA 1 to 3 termed according to their decreasing sizes) were identified in a strain of phytopathogenic fungus Pestalotiopsis fici AH1-1 isolated from a tea leaf. The complete sequences of dsRNAs 1 to 3, with the sizes of 10316, 5511, and 631 bp, were determined by random cloning together with a RACE protocol. Sequence analyses support that dsRNA1 is a genome of a novel hypovirus belonging to genus Alphahypovirus of the family Hypoviridae, tentatively named Pestalotiopsis fici hypovirus 1 (PfHV1); dsRNA2 is a defective RNA (D-RNA) generating from dsRNA1 with septal deletions; and dsRNA3 is the satellite component of PfHV1 since it could be co-precipitated with other dsRNA components in the same sucrose fraction by ultra-centrifuge, suggesting that it is encapsulated together with PfHV1 genomic dsRNAs. Moreover, dsRNA3 shares an identical stretch (170 bp) with dsRNAs 1 and 2 at their 5′ termini and the remaining are heterogenous, which is distinct from a typical satellite that generally has very little or no sequence similarity with helper viruses. More importantly, dsRNA3 lacks a substantial open reading frame (ORF) and a poly (A) tail, which is unlike the known satellite RNAs of hypoviruses, as well as unlike those in association with Totiviridae and Partitiviridae since the latters are encapsidated in coat proteins. As up-regulated expression of RNA3, dsRNA1 was significantly down-regulated, suggesting that dsRNA3 negatively regulates the expression of dsRNA1, whereas dsRNAs 1 to 3 have no obvious impact on the biological traits of the host fungus including morphologies and virulence. This study indicates that PfHV1 dsRNA3 is a special type of satellite-like nucleic acid that has substantial sequence homology with the host viral genome without encapsidation in a coat protein, which broadens the definition of fungal satellite.

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Section: Methodsmentioning

confidence: 99%

A special satellite-like RNA of a novel hypovirus from Pestalotiopsis fici broadens the definition of fungal satellite

et al. 2023

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“…However, even though two‐dimensional PAGE and sequential PAGE could confirm the presence of a viroid‐like agent, further sequencing is required to identify HSVd, making it extremely laborious to detect the pathogen unequivocally. That problem was solved with the development of hybridization‐based methods (Li et al, 1995; Owens & Diener, 1981; Pallas et al, 2017). The use of HSVd‐specific probes was crucial for the diagnosis of this pathogen, especially in fruit trees (Astruc et al, 1996).…”

Section: Survey and Diagnostic Methodsmentioning

confidence: 99%

Hop stunt viroid: A polyphagous pathogenic RNA that has shed light on viroid–host interactions

Marquez‐Molins

Gómez

Pallás

2020

Molecular Plant Pathology

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Taxonomy Hop stunt viroid (HSVd) is the type species of the genus Hostuviroid (family Pospiviroidae). The other species of this genus is Dahlia latent viroid, which presents an identical central conserved region (CCR) but lacks other structural hallmarks present in Hop stunt viroid. HSVd replication occurs in the nucleus through an asymmetric rolling‐circle model as in the other members of the family Pospiviroidae, which also includes the genera Pospiviroid, Cocadviroid, Apscaviroid, and Coleoviroid. Physical properties Hop stunt viroid consists of a single‐stranded, circular RNA of 295–303 nucleotides depending on isolates and sequence variants. The most stable secondary structure is a rod‐like or quasi‐rod‐like conformation with two characteristic domains: a CCR and a terminal conserved hairpin similar to that of cocadviroids. HSVd lacks a terminal conserved region. Hosts and symptoms HSVd infects a very broad range of natural hosts and has been reported to be the causal agent of five different diseases (citrus cachexia, cucumber pale fruit, peach and plum apple apricot distortion, and hop stunt). It is distributed worldwide. Transmission HSVd is transmitted mechanically and by seed.

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“…RT-PCR detection, RNA blot analysis, and co-precipitate analysis RT-PCR amplification was performed using a specific primer pair derived from the dsRNA 1 sequence (PfHV1-1F: 5′-TTCGATTTCAACGCCAGGTC-3′; PfHV1-1R: 5′-GCCGGGTCTATCGTCTTTTC-3′) (Table S1), generating a 307-bp fragment with an annealing temperature of 56°C in a PCR Thermal Cycler (Model PTC-100, MJ Research, USA). Digoxigenin (DIG)-labeled riboprobes 1 (binding to positions from 1588 to 1934 nt of dsRNA1), 2 and 3 (116-355 and 177-485 nt of dsRNA3, respectively) were synthesized in vitro transcription based on the corresponding cDNAs inserted into the pGEM-T easy vector with T7 RNA polymerase (Takara, Beijing, China) as previously described (22). Nucleic acids were spotted (for dot blotting) or electro-transferred (for Northern blot) to positively-charged nylon membranes (Roche Diagnostics), and hybridized with DIG-labeled riboprobes as previously described (22).…”

Section: Cloning Sequencing and Sequence Analysismentioning

confidence: 99%

“…Digoxigenin (DIG)-labeled riboprobes 1 (binding to positions from 1588 to 1934 nt of dsRNA1), 2 and 3 (116-355 and 177-485 nt of dsRNA3, respectively) were synthesized in vitro transcription based on the corresponding cDNAs inserted into the pGEM-T easy vector with T7 RNA polymerase (Takara, Beijing, China) as previously described (22). Nucleic acids were spotted (for dot blotting) or electro-transferred (for Northern blot) to positively-charged nylon membranes (Roche Diagnostics), and hybridized with DIG-labeled riboprobes as previously described (22).…”

Section: Cloning Sequencing and Sequence Analysismentioning

confidence: 99%

A special satellite-like dsRNA of a novel hypovirus from Pestalotiopsis fici broadens the definition of satellite

Han

Liu

Kong

et al. 2022

Preprint

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Satellites associated with plant or animal viruses have been largely detected and characterized, while those from mycoviruses together with their roles remain far less determined. Three dsRNA segments (dsRNA 1 to 3 termed according to their decreasing sizes) were identified in a strain of phytopathogenic fungus Pestalotiopsis fici AH1-1 isolated from a tea leaf. The complete sequences of dsRNAs 1 to 3, with the sizes of 10316, 5511, and 631 bp, were determined by random cloning together with a RACE protocol. Sequence analyses support that dsRNA1 is a genome of a novel hypovirus belonging to a newly proposed genus "Alphahypovirus" of the family Hypoviridae, tentatively named Pestalotiopsis fici hypovirus 1 (PfHV1); dsRNA2 is a defective RNA (D-RNA) generating from dsRNA1 with septal deletions; and dsRNA3 is the satellite component of PfHV1 since it could be co-precipitated with other dsRNA components in the same sucrose fraction by ultra-centrifuge, suggesting that it is encapsulated together with PfHV1 genomic dsRNAs. Moreover, dsRNA3 shares an identical stretch (170 bp) with dsRNAs 1 and 2 at their 5′ termini and the remaining is heterogenous, which is distinct from a typical satellite that generally has very little or no sequence similarity with helper viruses. More importantly, dsRNA3 lacks a substantial open reading frame (ORF) and a poly (A) tail, which is unlike the known satellite dsRNAs of hypoviruses, as well as unlike those in association with Totiviridae and Partitiviridae since the latters are encapsidated in coat proteins. As up-regulated expression of dsRNA3, dsRNA1 was significantly down-regulated, suggesting that dsRNA3 negatively regulates the expression of dsRNA1, whereas dsRNAs 1 to 3 have no obvious impact on the biological traits of the host fungus including morphologies and virulence. This study indicates that PfHV1 dsRNA3 is a special type of satellite-like nucleic acid that has substantial sequence homology with the host viral genome without encapsidation in a coat protein, which broadens the definition of satellite.

show abstract

Molecular Hybridization Techniques for Detecting and Studying Viroids

Cited by 10 publications

References 49 publications

A special satellite-like RNA of a novel hypovirus from Pestalotiopsis fici broadens the definition of fungal satellite

A special satellite-like RNA of a novel hypovirus from Pestalotiopsis fici broadens the definition of fungal satellite

Hop stunt viroid: A polyphagous pathogenic RNA that has shed light on viroid–host interactions

A special satellite-like dsRNA of a novel hypovirus from Pestalotiopsis fici broadens the definition of satellite

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