Plant Viroids Research Articles

Global Transcriptomic Analysis Reveals Insights into the Response of 'Etrog' Citron (Citrus medica L.) to Citrus Exocortis Viroid Infection.

Citrus exocortis viroid (CEVd) is the causal agent of citrus exocortis disease. We employed CEVd-infected ‘Etrog’ citron as a system to study the feedback regulation mechanism using transcriptome analysis in this study. Three months after CEVd infection, the transcriptome of fresh leaves was analyzed, and 1530 differentially expressed genes were detected. The replication of CEVd in citron induced upregulation of genes encoding key proteins that were involved in the RNA silencing pathway such as Dicer-like 2, RNA-dependent RNA polymerase 1, argonaute 2, argonaute 7, and silencing defective 3, as well as those genes encoding proteins that are related to basic defense responses. Many genes involved in secondary metabolite biosynthesis and chitinase activity were upregulated, whereas other genes related to cell wall and phytohormone signal transduction were downregulated. Moreover, genes encoding disease resistance proteins, pathogenicity-related proteins, and heat shock cognate 70 kDa proteins were also upregulated in response to CEVd infection. These results suggest that basic defense and RNA silencing mechanisms are activated by CEVd infection, and this information improves our understanding of the pathogenesis of viroids in woody plants.

Polyvalent detection of twelve viruses and four viroids affecting tomato by using a unique polyprobe

Non-radioactive molecular hybridization represents an attractive approach for the detection of multiple plant virus and/or viroids and a good alternative to the more extended serological and PCR-based detection methods. The use of polyprobes or riboprobes carrying partial sequences of different plant viruses or viroids fused in tandem, has permitted the detection of up to 10 different pathogens or the development of genus-specific probes. In the present article, the polyprobe technology has been adapted for the detection of the main viruses and viroids affecting tomato crops. To do this, three polyprobes have been developed covering four viroids (Poly4), twelve viruses (Poly12) or the four viroids plus the twelve viruses (poly16). The detection limit of the three polyprobes was comparable to the individual probes allowing the detection of up to 0,2 pg/μl of viral or viroidal RNA. A survey of 50 field samples revealed that all positive samples detected with the individual probes were also detected with the corresponding poly12 (98%) or poly16 (100%) probes. The analysis of tomato seeds revealed that both, single and polyprobes, were able to detect an infected seed in a pool of 250 healthy seeds. Finally, a ring-test analysis among six laboratories revealed a high reproducibility of the non-radioactive molecular hybridization procedure using the three polyprobes. The use of this technology in the routine analysis of tomato samples is discussed.

Combining a Simple Method for DNA/RNA/Protein Co-Purification and Arabidopsis Protoplast Assay to Facilitate Viroid Research.

Plant–viroid interactions represent a valuable model for delineating structure–function relationships of noncoding RNAs. For various functional studies, it is desirable to minimize sample variations by using DNA, RNA, and proteins co-purified from the same samples. Currently, most of the co-purification protocols rely on TRI Reagent (Trizol as a common representative) and require protein precipitation and dissolving steps, which render difficulties in experimental handling and high-throughput analyses. Here, we established a simple and robust method to minimize the precipitation steps and yield ready-to-use RNA and protein in solutions. This method can be applied to samples in small quantities, such as protoplasts. Given the ease and the robustness of this new method, it will have broad applications in virology and other disciplines in molecular biology.

Small RNA-Omics for Plant Virus Identification, Virome Reconstruction, and Antiviral Defense Characterization.

RNA interference (RNAi)-based antiviral defense generates small interfering RNAs that represent the entire genome sequences of both RNA and DNA viruses as well as viroids and viral satellites. Therefore, deep sequencing and bioinformatics analysis of small RNA population (small RNA-ome) allows not only for universal virus detection and genome reconstruction but also for complete virome reconstruction in mixed infections. Viral infections (like other stress factors) can also perturb the RNAi and gene silencing pathways regulating endogenous gene expression and repressing transposons and host genome-integrated endogenous viral elements which can potentially be released from the genome and contribute to disease. This review describes the application of small RNA-omics for virus detection, virome reconstruction and antiviral defense characterization in cultivated and non-cultivated plants. Reviewing available evidence from a large and ever growing number of studies of naturally or experimentally infected hosts revealed that all families of land plant viruses, their satellites and viroids spawn characteristic small RNAs which can be assembled into contigs of sufficient length for virus, satellite or viroid identification and for exhaustive reconstruction of complex viromes. Moreover, the small RNA size, polarity and hotspot profiles reflect virome interactions with the plant RNAi machinery and allow to distinguish between silent endogenous viral elements and their replicating episomal counterparts. Models for the biogenesis and functions of small interfering RNAs derived from all types of RNA and DNA viruses, satellites and viroids as well as endogenous viral elements are presented and discussed.

Polyvalent Detection of Members of the Genus Potyvirus by Molecular Hybridization Using a Genus-Probe.

The use of a unique riboprobe named polyprobe, carrying partial sequences of different plant viruses or viroids fused in tandem, has permitted the polyvalent detection of up to 10 different pathogens by using a nonradioactive molecular hybridization procedure. In the present analysis, we have developed a unique polyprobe with the capacity to detect all members of the genus Potyvirus, which we have named genus-probe. To do this, we have exploited the capacity of the molecular hybridization assay to cross-hybridize with related sequences by reducing the hybridization temperature. We observed that sequences showing a percentage similarity of 68% or higher could be detected with the same probe by hybridizing at 50 to 55°C, with a detection limit of picograms of viral RNA comparable to the specific individual probes. According to this, we developed several polyvalent polyprobes, containing three, five, or seven different 500-nucleotide fragments of a conserved region of the NIb gene. The polyprobe carrying seven different conserved regions was able to detect all the 32 potyviruses assayed in the present work with no signal in the healthy tissue, indicating the potential capacity of the polyprobe to detect all described, and probably uncharacterized, potyviruses being then considered as a genus-probe. The use of this technology in routine diagnosis not only for Potyvirus but also to other viral genera is discussed.

Long-term preservation of potato leafroll virus, potato virus S, and potato spindle tuber viroid in cryopreserved shoot tips.

Availability of and easy access to diverse plant viruses and viroids is a prerequisite in applied and basic studies related to viruses and viroids. Long-term preservation of viruses and viroids is difficult. A protocol was described for long-term preservation of potato leafroll virus (PLRV), potato virus S (PVS), and potato spindle tuber viroid (PSTVd) in cryopreserved shoot tips of potato cv. Zihuabai. Shoot regrowth levels following cryopreservation were higher in 1.5mm-shoot tips (58-60%) than in 0.5-mm-ones (30-38%). All shoots recovered from 0.5-mm-shoot tips were PVS- and PSTVd-preserved, but none of them were PLRV-preserved. Cryopreservation of 1.5-mm-shoot tips resulted in 35% and 100% of PLRV- and PVS- and PSTVd-preserved shoots. Studies on cell survival patterns and virus localization provided explanations to the varying PLRV-preservation frequencies produced by cryopreservation of the two sizes of shoot tips. Although micropropagation efficiencies were low after 12weeks of subculture following cryopreservation, similar efficiencies were obtained after 16weeks of subculture in pathogen-preserved shoots recovered from cryopreservation, compared with the diseased in vitro stock shoots (the control). Pathogen concentrations in the three pathogens-preserved shoots analyzed by qRT-PCR were similar to those in micropropagated shoots. The three pathogens cryopreserved in shoot tips were readily transmitted by grafting and mechanical inoculation to potato plants. PLRV, PVS, and PSTVd represent a diverse range of plant viruses and viroid in terms of taxonomy and infectious ability. Therefore, shoot tip cryopreservation opens a new avenue for long-term preservation of the virus and viroid.

Recent Advances on the Multiplex Molecular Detection of Plant Viruses and Viroids.

Plant viruses are still one of the main contributors to economic losses in agriculture. It has been estimated that plant viruses can cause as much as 50 billion euros loss worldwide, per year. This situation may be worsened by recent climate change events and the associated changes in disease epidemiology. Reliable and early detection methods are still one of the main and most effective actions to develop control strategies for plant viral diseases. During the last years, considerable progress has been made to develop tools with high specificity and low detection limits for use in the detection of these plant pathogens. Time and cost reductions have been some of the main objectives pursued during the last few years as these increase their feasibility for routine use. Among other strategies, these objectives can be achieved by the simultaneous detection and (or) identification of several viruses in a single assay. Nucleic acid-based detection techniques are especially suitable for this purpose. Polyvalent detection has allowed the detection of multiple plant viruses at the genus level. Multiplexing RT polymerase chain reaction (PCR) has been optimized for the simultaneous detection of more than 10 plant viruses/viroids. In this short review, we provide an update on the progress made during the last decade on techniques such as multiplex PCR, polyvalent PCR, non-isotopic molecular hybridization techniques, real-time PCR, and array technologies to allow simultaneous detection of multiple plant viruses. Also, the potential and benefits of the powerful new technique of deep sequencing/next-generation sequencing are described.

Viruses infecting main ornamental plants: an overview

Ornamental plants are cultivated for decorative and utility purposes in urban landscapes throughout the world. They are much valued for their aesthetic properties and constitute an important part of the global horticulture industry. Plant viruses and viroids of various taxonomic groups have a significant negative impact on ornamentals provoking a wide range of symptoms, reducing both decorative value and quality of propagated material and causing large economic damage. A significant growth of the ornamental plants market in recent years promotes the spread of viral diseases. Therefore, systematization of data on virus and viroid phytopathogens diversity in these cultures is an urgent research task. Among the most popular ornamentals are chrysanthemum, rose, clematis, canna, and lavender. More than fifty viruses and viroids from 17 different families (including two viroid families) have been identified in these crops to date. In the presented review, we described the variety of these pathogens and their effect on the above-mentioned ornamentals

Circular RNA shifts the miRNA binding panel and takes a role in hepatocellular carcinoma progression

Circular RNAs (circRNAs) are a novel type of RNA molecule that covalently joint its 5' and 3' end together and form a closed single strand annular. CircRNAs were first reported by Sanger et al . as a plant viroid RNA in 1976 (1). In decades, circRNAs were rarely investigated mainly because they are usually expressed at a relative low level.

Circular RNAs: a novel tool in cardiovascular biomarker development?

As we move towards cardiovascular personalised medicine, the development of novel biomarkers to assist in the decision-making process is a hot topic in translational research. In recent years, the non-coding transcriptome has emerged as a novel source of indicators with clinical applications. A vast number of publications have highlighted the potential of different members of this family as biomarkers, especially microRNAs and long non-coding RNAs. Recent advances in RNA sequencing and bioinformatic analysis have drawn attention to another promising class of non-coding RNAs: circular RNAs (circRNAs). CircRNAs are single-stranded and covalently closed RNA molecules that lack free caps or poly(A) tails. Initially discovered in plant viroids as early as the 1970s, circRNAs were originally considered splicing by-products or background noise. Nonetheless, transcriptome-wide analyses have identified and characterised thousands of circRNAs in diverse human cells, with key functions in the regulation of cellular processes. Indeed, various authors have proposed a causative role in a number of pathological conditions, including cardiovascular disease (CVD). Furthermore, circRNAs have the optimal chemical and biological properties to serve as interesting biomarkers. CircRNAs are free of exonuclease-mediated degradation and have a longer half-life than most linear RNAs, and their expression is cell- and developmental stage- and disease-specific. The presence of circRNAs has been demonstrated in clinical specimens, including whole blood, plasma and serum. Here, we review publications that have evaluated the potential of circRNAs as biomarkers for the diagnosis and prediction of cardiovascular conditions, including atherosclerosis, hypertension, aneurysm and the risk of adverse events. In addition, we discuss current methodological limitations and proposed future steps for their application as clinical indicators.

The Use of High-Throughput Sequencing for the Study and Diagnosis of Plant Viruses and Viroids in Pollen.

This protocol details the wet lab preparation, extraction of fruit pollen samples, and analysis of the sequencing data following Illumina NextSeq small and total RNA sequencing. The protocol was developed for virus and viroid detection using NGS sequencing and was based on the results of a comparison between different extraction methods followed by yield, RNA purity, and integrity assessment. Moreover, the advantage of an additional ribosomal (r)RNA depletion step to the total RNA extraction protocol was evaluated. The smallRNA procedure is the preferred method of choice. If the total RNA protocol is chosen, the use of the mirVana kit followed by an rRNA depletion step is the best option. The library preparation and sequencing steps were outsourced. As a final step in the data analysis, the VirusDetect software was used to detect the viruses and viroids in the pollen samples.

Next Generation Sequencing for Detection and Discovery of Plant Viruses and Viroids: Comparison of Two Approaches.

Next generation sequencing (NGS) technologies are becoming routinely employed in different fields of virus research. Different sequencing platforms and sample preparation approaches, in the laboratories worldwide, contributed to a revolution in detection and discovery of plant viruses and viroids. In this work, we are presenting the comparison of two RNA sequence inputs (small RNAs vs. ribosomal RNA depleted total RNA) for the detection of plant viruses by Illumina sequencing. This comparison includes several viruses, which differ in genome organization and viroids from both known families. The results demonstrate the ability for detection and identification of a wide array of known plant viruses/viroids in the tested samples by both approaches. In general, yield of viral sequences was dependent on viral genome organization and the amount of viral reads in the data. A putative novel Cytorhabdovirus, discovered in this study, was only detected by analysing the data generated from ribosomal RNA depleted total RNA and not from the small RNA dataset, due to the low number of short reads in the latter. On the other hand, for the viruses/viroids under study, the results showed higher yields of viral sequences in small RNA pool for viroids and viruses with no RNA replicative intermediates (single stranded DNA viruses).

The occurrence of viruses and viroids in ornamental citrus mother plants in Tuscany (Central Italy)

Citrus tristeza closterovirus (CTV) has been found several times in the last decades in Italy, and plant protection services are involved in monitoring and surveillance. Although orchards linked to the citrus industry are well monitored, there is an underestimated risk of viruses or virus-like diseases in ornamental nurseries. Our aim was to modify a CTV monitoring program to include other viruses (Citrus variegation virus, CVV; Citrus psorosis virus, CPsV) and viroids (Citrus exocortis viroid, CEVd; Hop stunt viroid, HSVd; Citrus bent leaf viroid, CBLVd; Citrus dwarfing viroid, CDVd; Citrus bark cracking viroid, CBCVd). Ornamental mother plants were monitored for four years in 15 nurseries in two locations in central Italy using inexpensive multiplex RT-PCR protocols. CTV incidence was 1.6–13.5%, with an average distribution of 11.9%. The average incidence of CVV and CPsV was 6.3% and 2.7%, respectively. Higher CTV, CVV and CPsV incidences were observed in C. x paradisi, C. grandis and C. x clementina. The most widespread viroid identified was CEVd (32.9%), frequently observed in C. x limonia and C. limon. HSVd (10.5%), and CDVd (7.1%) were mostly found in C. x limonia. Lower infection rates were observed for CBLVd (2.0%) and CBCVd (1.4%). However, the nurseries’ response to the virus alert by the protection services was only partially effective. Although the CTV incidence was lower in nurseries re-checked after the initial detection, it was not eradicated from two nurseries out of three, and the occurrence of viroids was reduced in just one nursery. Given that dangerous viruses along with the concomitant spread of viroids have unfortunately become a fact of every day life, multiplex RT-PCR diagnoses are likely to play an increasing role in warning nursery managers of possible infections.

First Report of Grapevine geminivirus A from Grapevines in China

HomePlant DiseaseVol. 101, No. 7First Report of Grapevine geminivirus A from Grapevines in China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Grapevine geminivirus A from Grapevines in ChinaX. D. Fan, Z. P. Zhang, F. Ren, G. J. Hu, Z. N. Li, and Y. F. DongX. D. FanSearch for more papers by this author, Z. P. ZhangSearch for more papers by this author, F. RenSearch for more papers by this author, G. J. HuSearch for more papers by this author, Z. N. LiSearch for more papers by this author, and Y. F. DongSearch for more papers by this authorAffiliationsAuthors and Affiliations X. D. Fan Z. P. Zhang F. Ren G. J. Hu Z. N. Li Y. F. Dong , National Center for Eliminating Viruses from Deciduous Fruit Tree, Research Institute of Pomology, Chinese Academy of Agriculture Sciences, Xingcheng, China. Published Online:9 May 2017https://doi.org/10.1094/PDIS-01-17-0106-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat The viruses belonging to the family Geminiviridae have circular, single-stranded DNA genomes, and some geminiviruses can cause great damage to agriculture. Recently, a new geminivirus, tentatively named Grapevine geminivirus A (GGVA), was reported in grapevines (Al Rwahnih et al. 2017). In the present study, bark scrapings from an ‘Ayaneskeal’ grapevine with foliar ringspots were used for the extraction of small RNAs and sequencing in 2012. The analysis generated approximately 29,319,407 Illumina reads of 16 to 28 nt in length and more than 463,611 reads were mapped to the GGVA genome. BLAST analysis showed also the presence of Grapevine leafroll-associated virus-3, Grapevine berry inner necrosis virus, Grapevine yellow speckle viroid 1, Grapevine yellow speckle viroid 2, and Hop stunt viroid. The full GGVA genome was amplified using two overlapping primers designed based on the sequences of the assembled contig [(23-1F/2R (5′-TGTCGGCCATGTCAGGGTGTT-3′) and (5′-GGCATAGATGATACAATTGCA-3′)]. Sequencing showed that the GGVA genome of isolate LN_ANSJ (GenBank accession no. KX950822) was 2,905 nt in length, and was 98% identical with the previously reported GGVA isolate NP (KX570610). The genome of LN_ANSJ had the same six open reading frames (ORFs) as the NP isolate, and the same nonanucleotide stem-loop sequence “TAATATT↓AC” in the intergenic region. Furthermore, two primers, C1-1F/1R (5′-CAGTATCTTCTGAGGCACGA-3′/5′-GCTAACGACGCTTATGCTGA-3′) and V1-1F/1R (5′-AAGAGGAAATCTTTCGCACC-3′/5′-AATTGACTCGATTACGACCAG-3′), were designed based on the sequences of C1 and V1 genes to amplify a 703-bp and 568-bp fragment in the Rep and CP genes of GGVA, respectively. In a survey of 69 grapevines of 20 different grapevine cultivars collected from four different provinces (Liaoning, Shandong, Ningxia, and Sichuan) in China, PCR testing using the primers C1-1F/1R and V1-1F/1R showed that 55% (38/69) of the samples tested positive for GGVA, and the incidence was high in the common cultivars and rootstocks such as Summer Black (100%, 6/6), Ruby Seedless (100%, 6/6), Cabernet Franc (71.4%, 5/7), and Beta grapevines (42.9%, 6/14). GGVA was not found in the native Amur grapevine ‘Beibinghong’ (0/8). Some PCR products were cloned and sequenced and sequencing confirmed them to be virus-specific. In the tested grapevines, most samples did not exhibit any symptoms, except for three grapevines which had foliar ringspots. The results suggested that GGVA may be widely distributed in China and may not cause obvious symptoms in many grapevine cultivars. To the best of our knowledge, this is the first report of GGVA in China.Reference:Al Rwahnih, M., et al. 2017. Phytopathology 107:240. https://doi.org/10.1094/PHYTO-07-16-0282-R Link, ISI, Google ScholarThis work was supported by the China Agricultural Research System (no. CARS-30-bc-3).DetailsFiguresLiterature CitedRelated Vol. 101, No. 7 July 2017SubscribeISSN:0191-2917e-ISSN:1943-7692 Metrics Article History Issue Date: 20 Jun 2017Published: 9 May 2017First Look: 27 Mar 2017Accepted: 17 Mar 2017 Pages: 1333-1333 Information© 2017 The American Phytopathological SocietyCited byDevelopment and application of crude sap-based recombinase polymerase amplification assay for the detection and occurrence of grapevine geminivirus A in Indian grapevine cultivars9 March 2023 | Frontiers in Plant Science, Vol. 14A thermal cycler-based, homogenization-free plant total nucleic acid extraction method for plant viruses and viroids assayJournal of Virological Methods, Vol. 313Grapevine berry inner necrosis virusCABI Compendium, Vol. CABI CompendiumGrapevine geminivirus ACABI Compendium, Vol. CABI CompendiumGrapevine yellow speckle viroid 2CABI Compendium, Vol. CABI CompendiumDevelopment of a Full-Length Infectious cDNA Clone of the Grapevine Berry Inner Necrosis Virus11 October 2020 | Plants, Vol. 9, No. 10Molecular Characterization and Genomic Function of Grapevine Geminivirus A2 September 2020 | Frontiers in Microbiology, Vol. 11First Report of Grapevine Geminivirus A in Vitis in New ZealandS. Veerakone, A. G. Blouin, R. A. Barrero, K. R. Napier, R. M. MacDiarmid, and L. I. Ward19 November 2019 | Plant Disease, Vol. 104, No. 2Pest categorisation of non‐EU viruses and viroids of Vitis L.EFSA Journal, Vol. 17, No. 9First Report of Grapevine geminivirus A in Diverse Vitis Species in KoreaY. Jo, H. Choi, M. K. Song, J. S. Park, J. W. Lee, and W. K. Cho26 October 2017 | Plant Disease, Vol. 102, No. 1

Diagnostic Parameters of Northern Blot Hybridization Technique for Detection of Citrus Viroids in Field-grown Plants

Diagnostic Parameters of Northern Blot Hybridization Technique for Detection of Citrus Viroids in Field-grown Plants

Euphresco project VirusCollect – fulfilling the need for a common collection of plant viruses and viroids for reference

The availability and accessibility of suitably characterized plant virus and viroid isolates for reference is vital for research and diagnostic laboratories. To ensure the long‐term availability of isolates and reference materials, there is a need for international collaboration. The Euphresco (European Plant Health Research Coordination) project VirusCollect aimed to establish a platform to link collections of viruses and viroids maintained by individual institutions via Q‐bank (http://www.q-bank.eu/), a database on plant pests and diseases. Within the VirusCollect project, standard operating procedures were developed and implemented by the participating laboratories to guarantee the quality of isolates and data. In addition, over 135 virus and viroid isolates of phytosanitary and/or economic importance were added to Q‐bank, which now provides links to over 500 virus isolates of almost 100 species, in addition to basic information on many more plant viruses. VirusCollect has enabled the first step in collaboration between curators and standardization of maintenance of virus collections. The project established the basis for improving the quality of individual collections and the layout of Q‐bank as a platform to share data and information. The follow‐up project, VirusCollect II, enables further international collaboration to ensure future access to reliable collections of plant viruses and viroids.

A Framework for the Evaluation of Biosecurity, Commercial, Regulatory, and Scientific Impacts of Plant Viruses and Viroids Identified by NGS Technologies.

Recent advances in high-throughput sequencing technologies and bioinformatics have generated huge new opportunities for discovering and diagnosing plant viruses and viroids. Plant virology has undoubtedly benefited from these new methodologies, but at the same time, faces now substantial bottlenecks, namely the biological characterization of the newly discovered viruses and the analysis of their impact at the biosecurity, commercial, regulatory, and scientific levels. This paper proposes a scaled and progressive scientific framework for efficient biological characterization and risk assessment when a previously known or a new plant virus is detected by next generation sequencing (NGS) technologies. Four case studies are also presented to illustrate the need for such a framework, and to discuss the scenarios.

Functional analysis of a viroid RNA motif mediating cell-to-cell movement in Nicotiana benthamiana.

Cell-to-cell trafficking through different cellular layers is a key process for various RNAs including those of plant viruses and viroids, but the regulatory mechanisms involved are still not fully elucidated and good model systems are important. Here, we analyse the function of a simple RNA motif (termed 'loop19') in potato spindle tuber viroid (PSTVd) which is required for trafficking in Nicotiana benthamiana leaves. Northern blotting, reverse transcriptase PCR (RT-PCR) and in situ hybridization analyses demonstrated that unlike wild-type PSTVd, which was present in the nuclei in all cell types, the trafficking-defective loop19 mutants were visible only in the nuclei of upper epidermal and palisade mesophyll cells, which shows that PSTVd loop19 plays a role in mediating RNA trafficking from palisade to spongy mesophyll cells in N.benthamiana leaves. Our findings and approaches have broad implications for studying the RNA motifs mediating trafficking of RNAs across specific cellular boundaries in other biological systems.

Next-Generation Sequencing and Genome Editing in Plant Virology.

Next-generation sequencing (NGS) has been applied to plant virology since 2009. NGS provides highly efficient, rapid, low cost DNA, or RNA high-throughput sequencing of the genomes of plant viruses and viroids and of the specific small RNAs generated during the infection process. These small RNAs, which cover frequently the whole genome of the infectious agent, are 21–24 nt long and are known as vsRNAs for viruses and vd-sRNAs for viroids. NGS has been used in a number of studies in plant virology including, but not limited to, discovery of novel viruses and viroids as well as detection and identification of those pathogens already known, analysis of genome diversity and evolution, and study of pathogen epidemiology. The genome engineering editing method, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been successfully used recently to engineer resistance to DNA geminiviruses (family, Geminiviridae) by targeting different viral genome sequences in infected Nicotiana benthamiana or Arabidopsis plants. The DNA viruses targeted include tomato yellow leaf curl virus and merremia mosaic virus (begomovirus); beet curly top virus and beet severe curly top virus (curtovirus); and bean yellow dwarf virus (mastrevirus). The technique has also been used against the RNA viruses zucchini yellow mosaic virus, papaya ringspot virus and turnip mosaic virus (potyvirus) and cucumber vein yellowing virus (ipomovirus, family, Potyviridae) by targeting the translation initiation genes eIF4E in cucumber or Arabidopsis plants. From these recent advances of major importance, it is expected that NGS and CRISPR-Cas technologies will play a significant role in the very near future in advancing the field of plant virology and connecting it with other related fields of biology.

Application of a simple and affordable protocol for isolating plant total nucleic acids for RNA and DNA virus detection

Standard molecular methods for plant virus diagnosis require the purification of RNA or DNA extracts from a large number of samples, with sufficient concentration and quality for their use in PCR, RT-PCR, or qPCR analysis. Most methods are laborious and use either hazardous and/or costly chemicals. A previously published protocol for RNA isolation from several plant species yields high amounts of good quality RNA-DNA mixture in a simple, safe and inexpensive manner. In the present work, this method was tested to obtain RNA-DNA extracts from leaves of tomato, potato and three species of citrus, and was compared with two commercial kits. The results demonstrated that this protocol offers at least comparable nucleic acid quality, quantity and purity to those provided by commercial phenol-based or spin column systems and that are suitable to be used in PCR, RT-PCR and qPCR for virus and viroid detection. Because of its easy implementation and the use of safe and inexpensive reagents, it can be easily implemented to work in plant virus and viroid detection in different plant species.