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.Work in RF laboratory has been supported by the Spanish Ministerio de Economía y Competitividad (grant BFU2014-56812-P), and in MB laboratory by Consiglio per la Ricerca in Agricoltura e l’analisi dell’Economia Agraria (CREA).Peer reviewedPeer Reviewe

Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs: Apple chlorotic leaf spot virus as a case study

Background Approaches to simplify and streamline the construction of full-length infectious cDNA clones (FL-cDNAs) are needed. Among desirable improvements are the ability to use total nucleic acids (TNA) extracts from infected hosts (to bypass viral purification limitations) for the direct one-step amplification of large FL-cDNAs, the possibility to inoculate plants with uncloned FL-cDNAs and the simplified cloning of these large molecules. Results Using the 7.55 kb genome of Apple chlorotic leaf spot trichovirus (ACLSV) approaches allowing the rapid generation from TNA extracts of FL-cDNAs under the control of the T7 promoter and the successful inoculation of plants using in vitro transcripts obtained from these uncloned amplification products have been developed. We also show that the yeast homologous recombination system permits efficient cloning of FL-cDNAs and the simultaneous one-step tailoring of a ternary Yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector allowing efficient inoculation of both herbaceous and woody host plants by agroinfiltration. Conclusions The fast and efficient strategies described here should have broad applications, in particular for the study "difficult" plant viruses, such as those infecting woody hosts, and potentially for other, non plant-infecting viral agents

Rancang Bangun Sistem Pengukuran Posisi Target dengan Kamera Stereo untuk Pengarah Senjata Otomatis

Salah satu hal yang penting dalam mengarahkan senjata secara otomatis ke target adalah informasi posisi dari target terhadap senjata. Terdapat banyak metode untuk mengetahui posisi target. Salah satunya adalah dengan metode pengukuran triangulasi. Metode ini membutuhkan minimal dua citra untuk medapatkan informasi jarak target terhadap kamera. Kemudian, informasi jarak tersebut bisa diolah untuk mengetahui posisi target terhadap senjata. Di dalam sistem ini, stereo visual digunakan untuk mendukung proses pengukuran triangulasi. Stereo visual menggunakan dua kamera untuk menghasilkan dua citra. Dalam sistem ini, salah satu kamera bertindak sebagai pemilih target. Citra yang ditangkap dua kamera tersebut akan diproses oleh processing unit untuk mendapatkan informasi posisi target terhadap senjata. Informasi ini digunakan untuk menggerakkan motor pada platform senjata agar senjata mengarah ke target. Hasil pengujian yang dilakukan pada sistem ini adalah sistem dapat menentukan posisi target yang dipilih oleh operator dan juga dapat mengarahkan senjata ke arah target tersebut. Akurasi tertinggi dalam penentuan posisi target dicapai ketika jarak antar dua kamera sekitar 30 cm

Guidelines for the reliable use of high throughput sequencing technologies to detect plant pathogens and pests.

High-throughput sequencing (HTS) technologies have the potential to become one of the most signi cant advances in molecular diagnostics. Their use by researchers to detect and characterize plant pathogens and pests has been growing steadily for more than a decade and they are now envisioned as a routine diagnostic test to be deployed by plant pest diagnostics laboratories. Nevertheless, HTS technologies and downstream bioinformatics analysis of the generated datasets represent a complex process including many steps whose reliability must be ensured. The aim of the present guidelines is to provide recommendations for researchers and diagnosticians aiming to reliably use HTS technologies to detect plant pathogens and pests. These guidelines are generic and do not depend on the sequencing technology or platform. They cover all the adoption processes of HTS technologies from test selection to test validation as well as their routine implementation. A special emphasis is given to key elements to be considered: undertaking a risk analysis, designing sample panels for validation, using proper controls, evaluating performance criteria, con rming and interpreting results. These guidelines cover any HTS test used for the detection and identi cation of any plant pest (viroid, virus, bacteria, phytoplasma, fungi and fungus-like protists, nematodes, arthropods, plants) from any type of matrix. Overall, their adoption by diagnosticians and researchers should greatly improve the reliability of pathogens and pest diagnostics and foster the use of HTS technologies in plant health

Natural Infection of Pomegranate (Punica Granatum) by Apple Dimple Fruit Viroid

The analysis by high throughput sequencing (HTS) and RT-PCR of Spanish pomegranate fruits showing yellow rings revealed the presence of viroid isolates closely related to fig isolates of apple dimple fruit viroid (ADFVd). The analysis of pomegranate public RNASeq data (Sequence Reads Archives, SRAs) from Israel provided evidence for the presence of similar ADFVd isolates in pomegranate trees in this country. In addition, reads or contigs of plum viroid I (PVd-I) isolates were also identified in two of the analyzed SRA datasets from Israel, suggesting the presence of this second viroid in pomegranate. Full length ADFVd genomic sequences have been recovered, increasing knowledge on the diversity of this viroid and on the pomegranate virome in which only four viruses and one viroid had previously been reported

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.Peer reviewe

Scientific Opinion on the pest categorisation of Eotetranychus lewisi

The Panel on Plant Health performed a pest categorisation of the Lewis spider mite, Eotetranychus lewisi, for the European Union (EU). The Lewis spider mite is a well-defined and distinguishable pest species that has been reported from a wide range of hosts, including cultivated species. Its distribution in the EU territory is restricted to (i) Madeira in Portugal; and to (ii) Poland where few occurrences were reported in glasshouses only. The pest is listed in Annex IIAI of Council Directive 2000/29/EC. A potential pathway of introduction and spread is plants traded from outside Europe and between Member States. The Lewis spider mite has the potential to establish in most part of the EU territory based on climate similarities with the distribution area outside the EU and the widespread availability of hosts present both in open fields and in protected cultivations. With regards to the potential consequences, one study is providing quantitative data on impact showing that the pest can reduce yield and affect quality of peaches and poinsettias, and only few studies describe the general impact of the pest on cultivated hosts. Although chemical treatments are reported to be effective in controlling the Lewis spider mite, it is mentioned as a growing concern for peaches, strawberries, raspberries and vines in the Americas. Overall, Eotetranychus lewisi meets the pest categorisation criteria defined in the International Standards for Phytosanitary Measures No 11 for a quarantine pest and in No 21 for a regulated non-quarantine pest

Scientific Opinion on the pest categorisation of Strawberry vein banding virus

The Panel on Plant Health performed a pest categorisation of Strawberry vein banding virus (SVBV) for the European Union (EU) territory. SVBV is a well-defined virus species of the genus Caulimovirus for which the entire genome sequence is known and molecular detection assays are available. SVBV is transmitted by vegetative multiplication of infected hosts and through the activity of aphid vectors, the most efficient being Chaetosiphon spp. The virus is reported from all continents and is present in three EU Member States: the Czech Republic, Italy and Slovakia. The host range of SVBV is restricted to cultivated and wild strawberries. It is listed in Annex IAI of Directive 2000/29/EC. SVBV is not expected to be affected by ecoclimatic conditions wherever its hosts are present and has the potential to establish in large parts of the EU territory, and to subsequently spread through the action of its Chaetosiphon fragaefolii vector, which is present in many Member States. SVBV does not cause severe symptoms, and modern cultivars are mostly symptomless if infected with SVBV alone. SVBV can, however, contribute to more severe symptoms when it occurs in mixed infections with other strawberry viruses. Despite this, SVBV is considered a minor problem in strawberry production as a consequence of modern practices including the systematic use of certified planting materials and the use of short crop cycles, which have greatly reduced the impact of strawberry viruses. Overall, SVBV does not have the potential to be a quarantine pest as, given current agricultural practices, it does not fulfil the pest categorisation criteria defined in the International Standards for Phytosanitary Measures No 11 of having a severe impact. However, SVBV has the potential to be a regulated non-quarantine pest because it fulfils all pest categorisation criteria defined in the International Standards for Phytosanitary Measures No 21