Genomic analysis of the brassica pathogen turnip mosaic potyvirus reveals its spread along the former trade routes of the Silk Road.

Plant pathogens have agricultural impacts on a global scale and resolving the timing and route of their spread can aid crop protection and inform control strategies. However, the evolutionary and phylogeographic history of plant pathogens in Eurasia remains largely unknown because of the difficulties in sampling across such a large landmass. Here, we show that turnip mosaic potyvirus (TuMV), a significant pathogen of brassica crops, spread from west to east across Eurasia from about the 17th century CE. We used a Bayesian phylogenetic approach to analyze 579 whole genome sequences and up to 713 partial sequences of TuMV, including 122 previously unknown genome sequences from isolates that we collected over the past five decades. Our phylogeographic and molecular clock analyses showed that TuMV isolates of the Asian-Brassica/Raphanus (BR) and basal-BR groups and world-Brassica3 (B3) subgroup spread from the center of emergence to the rest of Eurasia in relation to the host plants grown in each country. The migration pathways of TuMV have retraced some of the major historical trade arteries in Eurasia, a network that formed the Silk Road, and the regional variation of the virus is partly characterized by different type patterns of recombinants. Our study presents a complex and detailed picture of the timescale and major transmission routes of an important plant pathogen.

[Viroids : infectious non coding RNAs]. / Les viroïdes : des ARN infectieux non codants.

Viroids are the smallest non-coding infectious RNAs (between 246 and 401 nucleotides) known to be highly structured and replicate autonomously in the host plants. Although they do not encode any peptides, viroids induce visible symptoms in susceptible host plants. This article provides an overview of their physical and biological properties, the diseases they cause and their significance for the plants. The mechanisms underlying the expression of symptoms in host plants, their detection and various strategies employed for diseases prevention are also developed.

Citrus exocortis viroid causes ribosomal stress in tomato plants.

Viroids are naked RNAs that do not code for any known protein and yet are able to infect plants causing severe diseases. Because of their RNA nature, many studies have focused on the involvement of viroids in RNA-mediated gene silencing as being their pathogenesis mechanism. Here, the alterations caused by the Citrus exocortis viroid (CEVd) on the tomato translation machinery were studied as a new aspect of viroid pathogenesis. The presence of viroids in the ribosomal fractions of infected tomato plants was detected. More precisely, CEVd and its derived viroid small RNAs were found to co-sediment with tomato ribosomes in vivo, and to provoke changes in the global polysome profiles, particularly in the 40S ribosomal subunit accumulation. Additionally, the viroid caused alterations in ribosome biogenesis in the infected tomato plants, affecting the 18S rRNA maturation process. A higher expression level of the ribosomal stress mediator NAC082 was also detected in the CEVd-infected tomato leaves. Both the alterations in the rRNA processing and the induction of NAC082 correlate with the degree of viroid symptomatology. Taken together, these results suggest that CEVd is responsible for defective ribosome biogenesis in tomato, thereby interfering with the translation machinery and, therefore, causing ribosomal stress.

First report of Grapevine yellow speckle viroid 1 infecting grapevine (Vitis vinifera L.) in Canada.

Quebec is the third largest wine grape producer in Canada in acreage, tonnage, and wine grape sales (Carisse et al. 2017; Ben Moussa et al. 2019). To evaluate the diversity of viruses infecting grapevine in Quebec, a total of 77 leaf tissue samples (cv. Vidal) were collected from July to October in 2020 in three different vineyards located in Frelighsburg, Hemmingford and Saint-Jacques-le-Mineur in Quebec, Canada. Double-stranded RNA was extracted from each sample and used for cDNA library preparation with the Nextera XT DNA Library Preparation Kit (Illumina) as described previously (Kesanakurti et al. 2016). High-throughput sequencing (HTS, 2x300 bp) was conducted on dual-indexed libraries in a v3 flow cell using the Illumina MiSeq platform (Adkar-Purushothama et al. 2020). The obtained raw FASTQ data was de-multiplexed into 154 separate sequence files, and the adapters and barcode sequences were trimmed. The quality of the sequences was verified using Trimmomatic V.0.32 and the "clean" sequences were analyzed using Virtool and VirFind virus detection pipelines described elsewhere (Ho and Tzanetakis 2014; Rott et al. 2017) to screen for all possible viruses in the databases. Over 100,000 reads per sample were obtained with a percentage of mapped viral reads ranging from 1.47 to 19.43% of total number of reads. Out of 77 samples, 16 revealed the sequence of grapevine yellow speckle viroid 1 (GYSVd-1), for which the length coverage ranged from 98.5 to 99.1%; the depth ranged from 2X to 856X. The GYSVd-1 positive sequence files were subjected to whole genome assembly on CLC genomics Workbench v20.0.4 with the isolate SY-BR from Brazil (KU880715) used as reference. Seven complete genomes of GYSVd-1 of 366-368 nucleotides (nt) in size were deposited (GenBank Acc. MW732682 to MW732688). BLASTN analysis of the sequences showed 98-100% nt identities with isolate SY-BR. Other viruses and viroids such as Grapevine fleck virus, Grapevine rupestris stem pitting-associated virus, Grapevine rupestris vein feathering virus and Hop stunt viroid were also detected. To confirm GYSVd-1 presence in Quebec vineyards, seven of the 16 HTS-positive grapevine leaf tissue samples were subjected to total RNA extraction, followed by RT-PCR assay as before (Adkar-Purushothama et al. 2015; Sahana et al. 2013); all were positive by RT-PCR. The PCR products were directly Sanger-sequenced, and they showed 100% nt identity to the HTS derived sequences. Three of the seven GYSVd-1 positive grapevines exhibited yellow leaf spots and flecks and tiny yellow leaves, but their mixed infection status makes definitive symptoms association difficult to determine. Previously, Hop stunt viroid was reported from grapevines in Canada (Xiao et al. 2019; Fall et al. 2020) but to the best of our knowledge, this is the first report of GYSVd-1 infecting grapevines in Canada, specifically in the province of Quebec. Further research is required to assess the GYSVd-1 related yield loss. Monitoring and testing for GYSVd-1 infection is necessary to prevent propagation of infected materials, spread, and potential negative impact for the Canadian grapevine industry.

Impact of Nucleic Acid Sequencing on Viroid Biology.

The early 1970s marked two breakthroughs in the field of biology: (i) The development of nucleotide sequencing technology; and, (ii) the discovery of the viroids. The first DNA sequences were obtained by two-dimensional chromatography which was later replaced by sequencing using electrophoresis technique. The subsequent development of fluorescence-based sequencing method which made DNA sequencing not only easier, but many orders of magnitude faster. The knowledge of DNA sequences has become an indispensable tool for both basic and applied research. It has shed light biology of viroids, the highly structured, circular, single-stranded non-coding RNA molecules that infect numerous economically important plants. Our understanding of viroid molecular biology and biochemistry has been intimately associated with the evolution of nucleic acid sequencing technologies. With the development of the next-generation sequence method, viroid research exponentially progressed, notably in the areas of the molecular mechanisms of viroids and viroid diseases, viroid pathogenesis, viroid quasi-species, viroid adaptability, and viroid-host interactions, to name a few examples. In this review, the progress in the understanding of viroid biology in conjunction with the improvements in nucleotide sequencing technology is summarized. The future of viroid research with respect to the use of third-generation sequencing technology is also briefly envisaged.

Identification and Molecular Mechanisms of Key Nucleotides Causing Attenuation in Pathogenicity of Dahlia Isolate of Potato Spindle Tuber Viroid.

While the potato spindle tuber viroid (PSTVd) variant, PSTVd-Dahlia (PSTVd-D or PSTVd-Dwt) induces very mild symptoms in tomato cultivar 'Rutgers', PSTVd-Intermediate (PSTVd-I or PSTVd-Iwt) induces severe symptoms. These two variants differ by nine nucleotides, of which six mutations are located in the terminal left (TL) to the pathogenicity (P) domains. To evaluate the importance of mutations located in the TL to the P domains, ten types of point mutants were created by swapping the nucleotides between the two viroid variants. Bioassay in tomato plants demonstrated that two mutants created on PSTVd-Iwt at positions 42 and 64 resulted in symptom attenuation. Phenotypic and RT-qPCR analysis revealed that mutation at position 42 of PSTVd-Iwt significantly reduced disease severity and accumulation of the viroid, whereas mutation at position 64 showed a significant reduction in stunting when compared to the PSTVd-Iwt infected plant. RT-qPCR analysis on pathogenesis-related protein 1b1 and chalcone synthase genes showed a direct correlation with symptom severity whereas the expansin genes were down-regulated irrespective of the symptom severity. These results indicate that the nucleotides at positions 42 and 64 are in concert with the ones at positions 43, 310, and 311/312, which determines the slower and stable accumulation of PSTVd-D without eliciting excessive host defense responses thus contributing in the attenuation of disease symptom.

Small RNA Derived from the Virulence Modulating Region of the Potato spindle tuber viroid Silences callose synthase Genes of Tomato Plants.

The tomato (Solanum lycopersicum) callose synthase genes CalS11-like and CalS12-like encode proteins that are essential for the formation of callose, a major component of pollen mother cell walls; these enzymes also function in callose formation during pathogen infection. This article describes the targeting of these callose synthase mRNAs by a small RNA derived from the virulence modulating region of two Potato spindle tuber viroid variants. More specifically, viroid infection of tomato plants resulted in the suppression of the target mRNAs up to 1.5-fold, depending on the viroid variant used and the gene targeted. The targeting of these mRNAs by RNA silencing was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Viroid mutants incapable of targeting callose synthase mRNAs failed to induce typical infection phenotypes, whereas a chimeric viroid obtained by swapping the virulence modulating regions of a mild and a severe variant of Potato spindle tuber viroid greatly affected the accumulation of viroids and the severity of disease symptoms. These data provide evidence of the silencing of multiple genes by a single small RNA derived from a viroid.

Alterations of the viroid regions that interact with the host defense genes attenuate viroid infection in host plant.

Understanding in intimate details how the viroid interaction with host's defense genes is a cornerstone for developing viroid resistant plants. In this present study, small RNAs (sRNA) derived from Potato spindle tuber viroid (PSTVd) were studied in silico in order to detect any interactions with the serine threonine kinase receptor, a transmembrane protein that plays a role in disease resistance in plants. Using molecular biology techniques, it was determined that PSTVd infection negatively affects at least three serine threonine kinase receptors as well as with three other genes that are known to be involved in the overall development of the tomato plants. The transient expression of these putative PSTVd-sRNAs, using the microRNA sequence as a backbone, in tomato plants induced phenotypes similar to viroid infection. Mutants created by altering the sequence of PSTVd in these regions failed to infect the tomato plant. The data presented here illustrates the importance of these regions in viroid survival, and suggests a possible avenue of exploration for the development of viroid resistant plants.

Detection, distribution, and genetic diversity of Australian grapevine viroid in grapevines in India.

Australian grapevine viroid (AGVd) is a viroid specific to grapevine with the least records in the world till date. Here, we report for the first time the presence of AGVd in grapevines in Indian sub-continent. The overall infection rate of AGVd in major grapevine producing areas in India was 9.3 %, which is conspicuously higher than the other regions of the world except for Tunisia and Iran. To understand the AGVd diversity in India, the genetic divergence was examined based on the disparity in the cultivars and the locations. Nucleotide sequence analysis revealed the existence of five major AGVd variants in India besides other 44 minor variants implying the "quasi-species" nature. Further, sequence alignment of all the Indian AGVd variants along with Australian type species underscored the presence of eleven mutation points which are archetypal for Indian AGVd, irrespective of the region, and cultivar of grapevines. Plotting of Indian AGVd sequence variants against Australian type species unveiled that all these eleven mutations are distributed on upper and lower left terminal and pathogenicity regions of the molecule. Phylogenetic analysis divulged all the major Indian AGVd variants formed two distinct clusters, suggesting the two separate evolutionary lineages of AGVd in Indian viticulture.

Hop Latent Viroid: A Hidden Threat to the Cannabis Industry.

Hop latent viroid (HLVd) is the biggest concern for cannabis and hop growers worldwide. Although most HLVd-infected plants remain asymptomatic, research on hops has demonstrated a decrease in both the α-bitter acid and terpene content of hop cones, which affects their economic value. The HLVd-associated "dudding" or "duds" disease of cannabis was first reported in 2019 in California. Since then, the disease has become widespread in cannabis-growing facilities across North America. Although severe yield loss associated with duds disease has been recorded, little scientific information is available to growers in order to contain HLVd. Consequently, this review aims to summarise all of the scientific information available on HLVd so as to be able to understand the effect of HLVd on yield loss, cannabinoid content, terpene profile, disease management and inform crop protection strategies.

Revisiting the Non-Coding Nature of Pospiviroids.

Viroids are small, circular, highly structured pathogens that infect a broad range of plants, causing economic losses. Since their discovery in the 1970s, they have been considered as non-coding pathogens. In the last few years, the discovery of other RNA entities, similar in terms of size and structure, that were shown to be translated (e.g., cirRNAs, precursors of miRNA, RNA satellites) as well as studies showing that some viroids are located in ribosomes, have reignited the idea that viroids may be translated. In this study, we used advanced bioinformatic analysis, in vitro experiments and LC-MS/MS to search for small viroid peptides of the PSTVd. Our results suggest that in our experimental conditions, even though the circular form of PSTVd is found in ribosomes, no produced peptides were identified. This indicates that the presence of PSTVd in ribosomes is most probably not related to peptide production but rather to another unknown function that requires further study.

A sensitive and reliable RT-nested PCR assay for detection of Citrus tristeza virus from naturally infected citrus plants.

A specific and sensitive reverse transcriptase-nested polymerase chain reaction assay (RT-nPCR) was developed for the detection of Citrus tristeza virus (CTV) from naturally infected citrus samples. Two sets of primer pairs were designed by alignment of nucleotide sequences available in GenBank database for different genotypes of CTV. RT-nPCR reaction components and thermal cycling parameters were optimized and reaction conditions were standardized. Sequencing of the PCR products from direct and nested-PCR reactions confirmed the specificity of both primer pairs. Presence of CTV specific amplicons in asymptomatic samples which were collected from diseased orchards indicated the sensitivity of the test. As RT-nPCR technique, developed in the present study, is specific and efficient in detecting CTV, this could be envisioned for diagnostic applications and surveillance.

sRNA Profiler: A User-Focused Interface for Small RNA Mapping and Profiling.

Viroids are circular, highly structured, single-stranded, non-coding RNA pathogens known to infect and cause disease in several plant species. They are known to trigger the host plant's RNA silencing machinery. The detection of viroid-derived small RNAs (vd-sRNA) in viroid-infected host plants opened a new avenue of study in host-viroid pathogenicity. Since then, several viroid research groups have studied the vd-sRNA retrieved from different host-viroid combinations. Such studies require the segregation of 21- to 24-nucleotide long small RNAs (sRNA) from a deep-sequencing databank, followed by separating the vd-sRNA from any sRNA within this group that showed sequence similarity with either the genomic or the antigenomic strands of the viroid. Such mapped vd-sRNAs are then profiled on both the viroid's genomic and antigenomic strands for visualization. Although several commercial interfaces are currently available for this purpose, they are all programmed for linear RNA molecules. Hence, viroid researchers must develop a computer program that accommodates the sRNAs derived from the circular viroid genome. This is a laborious process, and consequently, it often creates a bottleneck for biologists. In order to overcome this constraint, and to help the research community in general, in this study, a python-based pattern matching interface was developed so as to be able to both profile and map sRNAs on a circular genome. A "matching tolerance" feature has been included in the program, thus permitting the mapping of the sRNAs derived from the quasi-species. Additionally, the "topology" feature allows the researcher to profile sRNA derived from both linear and circular RNA molecules. The efficiency of the program was tested using previously reported deep-sequencing data obtained from two independent studies. Clearly, this novel software should be a key tool with which to both evaluate the production of sRNA and to profile them on their target RNA species, irrespective of the topology of the target RNA molecule.

Grapevine Virology in the Third-Generation Sequencing Era: From Virus Detection to Viral Epitranscriptomics.

Among all economically important plant species in the world, grapevine (Vitis vinifera L.) is the most cultivated fruit plant. It has a significant impact on the economies of many countries through wine and fresh and dried fruit production. In recent years, the grape and wine industry has been facing outbreaks of known and emerging viral diseases across the world. Although high-throughput sequencing (HTS) has been used extensively in grapevine virology, the application and potential of third-generation sequencing have not been explored in understanding grapevine viruses and their impact on the grapevine. Nanopore sequencing, a third-generation technology, can be used for the direct sequencing of both RNA and DNA with minimal infrastructure. Compared to other HTS methods, the MinION nanopore platform is faster and more cost-effective and allows for long-read sequencing. Due to the size of the MinION device, it can be easily carried for field viral disease surveillance. This review article discusses grapevine viruses, the principle of third-generation sequencing platforms, and the application of nanopore sequencing technology in grapevine virus detection, virus-plant interactions, as well as the characterization of viral RNA modifications.

Microbial Diversity in the Phyllosphere and Rhizosphere of an Apple Orchard Managed under Prolonged "Natural Farming" Practices.

Microbial diversity in an apple orchard cultivated with natural farming practices for over 30 years was compared with conventionally farmed orchards to analyze differences in disease suppression. In this long-term naturally farmed orchard, major apple diseases were more severe than in conventional orchards but milder than in a short-term natural farming orchard. Among major fungal species in the phyllosphere, we found that Aureobasidium pullulans and Cryptococcus victoriae were significantly less abundant in long-term natural farming, while Cladosporium tenuissimum predominated. However, diversity of fungal species in the phyllosphere was not necessarily the main determinant in the disease suppression observed in natural farming; instead, the maintenance of a balanced, constant selection of fungal species under a suitable predominant species such as C. tenuissimum seemed to be the important factors. Analysis of bacteria in the phyllosphere revealed Pseudomonas graminis, a potential inducer of plant defenses, predominated in long-term natural farming in August. Rhizosphere metagenome analysis showed that Cordyceps and Arthrobotrys, fungal genera are known to include insect- or nematode-infecting species, were found only in long-term natural farming. Among soil bacteria, the genus Nitrospira was most abundant, and its level in long-term natural farming was more than double that in the conventionally farmed orchard.

Current overview on viroid-host interactions.

Viroids are one of the most enigmatic highly structured, circular, single-stranded RNA phytopathogens. Although they are not known to code for any peptide, viroids induce visible symptoms in susceptible host plants that resemble those associated with many plant viruses. It is known that viroids induce disease symptoms by direct interaction with host factors; however, the precise mechanism by which this occurs remains poorly understood. Studies on the host's responses to viroid infection, host susceptibility and nonhost resistance have been underway for several years, but much remains to be done in order to fully understand the complex nature of viroid-host interactions. Recent progress using molecular biology techniques combined with computational algorithms, in particular evidence of the role of viroid-derived small RNAs in the RNA silencing pathways of a disease network, has widened the knowledge of viroid pathogenicity. The complexity of viroid-host interactions has been revealed in the past decades to include, but not be limited to, the involvement of host factors, viroid structural complexity, and viroid-induced ribosomal stress, which is further boosted by the discovery of long noncoding RNAs (lncRNAs). In this review, the current understanding of the viroid-host interaction has been summarized with the goal of simplifying the complexity of viroid biology for future research. This article is categorized under: RNA in Disease and Development > RNA in Disease.

Insights Into Potato Spindle Tuber Viroid Quasi-Species From Infection to Disease.

Viroids are non-coding RNA plant pathogens that are characterized by their possession of a high mutation level. Although the sequence heterogeneity in viroid infected plants is well understood, shifts in viroid population dynamics due to mutations over the course of infection remain poorly understood. In this study, the ten most abundant sequence variants of potato spindle tuber viroid RG1 (PSTVd) expressed at different time intervals in PSTVd infected tomato plants were identified by high-throughput sequencing. The sequence variants, forming a quasi-species, were subjected to both the identification of the regions favoring mutations and the effect of the mutations on viroid secondary structure and viroid derived small RNAs (vd-sRNA). At week 1 of PSTVd infection, 25% of the sequence variants were similar to the "master" sequence (i.e., the sequence used for inoculation). The frequency of the master sequence within the population increased to 70% at week 2 after PSTVd infection, and then stabilized for the rest of the disease cycle (i.e., weeks 3 and 4). While some sequence variants were abundant at week 1 after PSTVd infection, they tended to decrease in frequency over time. For example, the variants with insertions at positions 253 or 254, positions that could affect the Loop E as well as the metastable hairpin I structure that has been shown important during replication and viroid infectivity, resulted in decreased frequency. Data obtained by in silico analysis of the viroid derived small RNAs (vd-sRNA) was also analyzed. A few mutants had the potential of positively affecting the viroid's accumulation by inducing the RNA silencing of the host's defense related genes. Variants with mutations that could negatively affect viroid abundance were also identified because their derived vd-sRNA were no longer capable of targeting any host mRNA or of changing its target sequence from a host defense gene to some other non-important host gene. Together, these findings open avenues into understanding the biological role of sequence variants, this viroid's interaction with host components, stable and metastable structures generated by mutants during the course of infection, and the influence of sequence variants on stabilizing viroid population dynamics.

Suppression of RNA-dependent RNA polymerase 6 in tomatoes allows potato spindle tuber viroid to invade basal part but not apical part including pluripotent stem cells of shoot apical meristem.

RNA-dependent RNA polymerase 6 (RDR6) is one of the key factors in plant defense responses and suppresses virus or viroid invasion into shoot apical meristem (SAM) in Nicotiana benthamiana. To evaluate the role of Solanum lycopersicum (Sl) RDR6 upon viroid infection, SlRDR6-suppressed (SlRDR6i) 'Moneymaker' tomatoes were generated by RNA interference and inoculated with intermediate or lethal strain of potato spindle tuber viroid (PSTVd). Suppression of SlRDR6 did not change disease symptoms of both PSTVd strains in 'Moneymaker' tomatoes. Analysis of PSTVd distribution in shoot apices by in situ hybridization revealed that both PSTVd strains similarly invade the basal part but not apical part including pluripotent stem cells of SAM in SlRDR6i plants at a low rate unlike a previous report in N. benthamiana. In addition, unexpectedly, amount of PSTVd accumulation was apparently lower in SlRDR6i plants than in control tomatoes transformed with empty cassette in early infection especially in the lethal strain. Meanwhile, SlRDR6 suppression did not affect the seed transmission rates of PSTVd. These results indicate that RDR6 generally suppresses PSTVd invasion into SAM in plants, while suppression of RDR6 does not necessarily elevate amount of PSTVd accumulation. Additionally, our results suggest that host factors such as RDR1 other than RDR6 may also be involved in the protection of SAM including pluripotent stem cells from PSTVd invasion and effective RNA silencing causing the decrease of PSTVd accumulation during early infection in tomato plants.

Suppression of RNA-Dependent RNA Polymerase6 Favors the Accumulation of Potato Spindle Tuber Viroid in Nicotiana Benthamiana.

To date, two plant genes encoding RNA-dependent RNA polymerases (RdRs) that play major roles in the defense against RNA viruses have been identified: (i) RdR1, which is responsible for the viral small RNAs (vsRNAs) found in virus-infected plants, and, (ii) RdR6, which acts as a surrogate in the absence of RdR1. In this study, the role of RdR6 in the defense against viroid infection was examined by knock-down of RdR6 followed by potato spindle tuber viroid (PSTVd) infection. The suppression of RdR6 expression increased the plant's growth, as was illustrated by the plant's increased height. PSTVd infection of RdR6 compromised plants resulted in an approximately three-fold increase in the accumulation of viroid RNA as compared to that seen in control plants. Additionally, RNA gel blot assay revealed an increase in the number of viroids derived small RNAs in RdR6 suppressed plants as compared to control plants. These data provide a direct correlation between RdR6 and viroid accumulation and indicate the role of RDR6 in the plant's susceptibility to viroid infection.

Viroid-derived small RNA induces early flowering in tomato plants by RNA silencing.

Viroid infection often leads to early flowering in the host plant. This report describes the targeting of the FRIGIDA-like protein 3 (FRL3) mRNA in tomato plants by a small RNA derived from the conserved left terminal region of the potato spindle tuber viroid (PSTVd). This targeting leads to the silencing of the FRL3 mRNA. Viroid infection assays using a severe variant of PSTVd induced early flowering in tomato plants by the down-regulation of greater amounts of the target than did a mild PSTVd variant. The targeting of the FRL3 mRNA by RNA silencing was validated by both an artificial microRNA experiment transiently expressing viroid-derived small RNAs in tomato plants, and by 5' RNA ligase-mediated rapid amplification of cDNA ends (RACE). These data unambiguously demonstrated the role of small RNAs in the early flowering seen in viroid-infected plants.