Controlled RISC loading efficiency of miR168 defined by miRNA duplex structure adjusts ARGONAUTE1 homeostasis.

Micro RNAs (miRNAs) are processed from precursor RNA molecules with precisely defined secondary stem-loop structures. ARGONAUTE1 (AGO1) is the main executor component of miRNA pathway and its expression is controlled via the auto-regulatory feedback loop activity of miR168 in plants. Previously we have shown that AGO1 loading of miR168 is strongly restricted leading to abundant cytoplasmic accumulation of AGO-unbound miR168. Here, we report, that intrinsic RNA secondary structure of MIR168a precursor not only defines the processing of miR168, but also precisely adjusts AGO1 loading efficiency determining the biologically active subset of miR168 pool. Our results show, that modification of miRNA duplex structure of MIR168a precursor fragment or expression from artificial precursors can alter the finely adjusted loading efficiency of miR168. In dcl1-9 mutant where, except for miR168, production of most miRNAs is severely reduced this mechanism ensures the elimination of unloaded AGO1 proteins via enhanced AGO1 loading of miR168. Based on this data, we propose a new competitive loading mechanism model for miR168 action: the miR168 surplus functions as a molecular buffer for controlled AGO1 loading continuously adjusting the amount of AGO1 protein in accordance with the changing size of the cellular miRNA pool.

Analysis of a novel RNA virus in a wild northern white-breasted hedgehog (Erinaceus roumanicus).

Tombusviruses are generally considered plant viruses. A novel tombus-/carmotetravirus-like RNA virus was identified in a faecal sample and blood and muscle tissues from a wild northern white-breasted hedgehog (Erinaceus roumanicus). The complete genome of the virus, called H14-hedgehog/2015/HUN (GenBank accession number MN044446), is 4,118 nucleotides in length with a readthrough stop codon of type/group 1 in ORF1 and lacks a poly(A) tract at the 3' end. The predicted ORF1-RT (RdRp) and the capsid proteins had low (31-33%) amino acid sequence identity to unclassified tombus-/noda-like viruses (Hubei tombus-like virus 12 and Beihai noda-like virus 10), respectively, discovered recently in invertebrate animals. An in vivo experimental plant inoculation study showed that an in vitro-transcribed H14-hedgehog/2015/HUN viral RNA did not replicate in Nicotiana benthamiana, Chenopodium quinoa, or Chenopodium murale, the most susceptible hosts for plant-origin tombusviruses.

Virus Detection by High-Throughput Sequencing of Small RNAs: Large-Scale Performance Testing of Sequence Analysis Strategies.

Recent developments in high-throughput sequencing (HTS), also called next-generation sequencing (NGS), technologies and bioinformatics have drastically changed research on viral pathogens and spurred growing interest in the field of virus diagnostics. However, the reliability of HTS-based virus detection protocols must be evaluated before adopting them for diagnostics. Many different bioinformatics algorithms aimed at detecting viruses in HTS data have been reported but little attention has been paid thus far to their sensitivity and reliability for diagnostic purposes. Therefore, we compared the ability of 21 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 12 plant viruses through a double-blind large-scale performance test using 10 datasets of 21- to 24-nucleotide small RNA (sRNA) sequences from three different infected plants. The sensitivity of virus detection ranged between 35 and 100% among participants, with a marked negative effect when sequence depth decreased. The false-positive detection rate was very low and mainly related to the identification of host genome-integrated viral sequences or misinterpretation of the results. Reproducibility was high (91.6%). This work revealed the key influence of bioinformatics strategies for the sensitive detection of viruses in HTS sRNA datasets and, more specifically (i) the difficulty in detecting viral agents when they are novel or their sRNA abundance is low, (ii) the influence of key parameters at both assembly and annotation steps, (iii) the importance of completeness of reference sequence databases, and (iv) the significant level of scientific expertise needed when interpreting pipeline results. Overall, this work underlines key parameters and proposes recommendations for reliable sRNA-based detection of known and unknown viruses.

Non-targeted effects of virus-induced gene silencing vectors on host endogenous gene expression.

Virus-induced gene silencing (VIGS) uses recombinant viruses to study gene function; however, the effect of the virus vector itself on the gene expression of the host is not always considered. In our work, we investigated non-targeted gene expression changes of the host in order to see how often these changes appear. Effects of various VIGS vector infections were analysed by monitoring gene expression levels of housekeeping genes by Northern blot analysis in four different hosts. We found that non-targeted changes happens very often. More importantly, these non-targeted effects can cause drastic changes in the gene-expression pattern of host genes that are usually used as references in these studies. We have also found that in a tobacco rattle virus (TRV)-based VIGS, the presence of foreign sequences in the cloning site of the vector can also have a non-targeted effect, and even the use of an internal control can lead to unpredicted changes. Our results show that although VIGS is a very powerful technique, the VIGS vector, as a pathogen of the host, can cause unwanted changes in its gene-expression pattern, highlighting the importance of careful selection of both the genes to be tested and those to be used as references in the planned experiments.

Independent parallel functions of p19 plant viral suppressor of RNA silencing required for effective suppressor activity.

Plant viruses ubiquitously mediate the induction of miR168 trough the activities of viral suppressors of RNA silencing (VSRs) controlling the accumulation of ARGONAUTE1 (AGO1), one of the main components of RNA silencing based host defence system. Here we used a mutant Tombusvirus p19 VSR (p19-3M) disabled in its main suppressor function, small interfering RNA (siRNA) binding, to investigate the biological role of VSR-mediated miR168 induction. Infection with the mutant virus carrying p19-3M VSR resulted in suppressed recovery phenotype despite the presence of free virus specific siRNAs. Analysis of the infected plants revealed that the mutant p19-3M VSR is able to induce miR168 level controlling the accumulation of the antiviral AGO1, and this activity is associated with the enhanced accumulation of viral RNAs. Moreover, saturation of the siRNA-binding capacity of p19 VSR mediated by defective interfering RNAs did not influence the miR168-inducing activity. Our data indicate that p19 VSR possesses two independent silencing suppressor functions, viral siRNA binding and the miR168-mediated AGO1 control, both of which are required to efficiently cope with the RNA-silencing based host defence. This finding suggests that p19 VSR protein evolved independent parallel capacities to block the host defence at multiple levels.

Identification of Nicotiana benthamiana microRNAs and their targets using high throughput sequencing and degradome analysis.

BACKGROUND: Nicotiana benthamiana is a widely used model plant species for research on plant-pathogen interactions as well as other areas of plant science. It can be easily transformed or agroinfiltrated, therefore it is commonly used in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification. To discover and characterize the miRNAs and their cleaved target mRNAs in N. benthamiana, we sequenced small RNA transcriptomes and degradomes of two N. benthamiana accessions and validated them by Northern blots. RESULTS: We used a comprehensive molecular approach to detect and to experimentally validate N. benthamiana miRNAs and their target mRNAs from various tissues. We identified 40 conserved miRNA families and 18 novel microRNA candidates and validated their target mRNAs with a genomic scale approach. The accumulation of thirteen novel miRNAs was confirmed by Northern blot analysis. The conserved and novel miRNA targets were found to be involved in various biological processes including transcription, RNA binding, DNA modification, signal transduction, stress response and metabolic process. Among the novel miRNA targets we found the mRNA of REPRESSOR OF SILENCING (ROS1). Regulation of ROS1 by a miRNA provides a new regulatory layer to reinforce transcriptional gene silencing by a post-transcriptional repression of ROS1 activity. CONCLUSIONS: The identified conserved and novel miRNAs along with their target mRNAs also provides a tissue specific atlas of known and new miRNA expression and their cleaved target mRNAs of N. benthamiana. Thus this study will serve as a valuable resource to the plant research community that will be beneficial well into the future.

Plant virus-mediated induction of miR168 is associated with repression of ARGONAUTE1 accumulation.

Virus infections induce the expression of ARGONAUTE1 (AGO1) mRNA and in parallel enhance the accumulation of miR168 (regulator of AGO1 mRNA). Here, we show that in virus-infected plants the enhanced expression of AGO1 mRNA is not accompanied by increased AGO1 protein accumulation. We also show that the induction of AGO1 mRNA level is a part of the host defence reaction, whereas the induction of miR168, which overlaps spatially with virus-occupied sectors, is mediated mainly by the Tombusvirus p19 RNA-silencing suppressor. The absence of p19 results in the elimination of miR168 induction and accompanied with the enhanced accumulation of AGO1 protein. In transient expression study, p19 mediates the induction of miR168 and the down-regulation of endogenous AGO1 level. P19 is not able to efficiently bind miR168 in virus-infected plants, indicating that this activity is uncoupled from the small RNA-binding capacity of p19. Our results imply that plant viruses can inhibit the translational capacity of AGO1 mRNA by modulating the endogenous miR168 level to alleviate the anti-viral function of AGO1 protein.

Viromes of Plants Determined by High-Throughput Sequencing of Virus-Derived siRNAs.

Plants growing in open airfields can be infected by several viruses even as a multiple infection. Virus infection in crops can lead to a serious damage to the harvest. In addition, virus presence in grapevine, fruit trees, and tuberous vegetables, propagated vegetatively affects the phytosanitary status of the propagation material (both the rootstock and the variety) having profound effect on the lifetime and health of the new plantations. The fast evolution of sequencing techniques provides a new opportunity for metagenomics-based viral diagnostics. Small interfering (si) RNAs produced by the RNA silencing-based host immune system during viral infection can be sequenced by high-throughput techniques and analyzed for the presence of viruses, revealing the presence of all known viral pathogens in the sample and therefore opening new avenues in virus diagnostics. This method is based on Illumina sequencing and bioinformatics analysis of virus-derived siRNAs in the host. Here we describe a protocol for this challenging technique step by step with notes, to ensure success for every user.

Viromes of Monocotyledonous Weeds Growing in Crop Fields Reveal Infection by Several Viruses Suggesting Their Virus Reservoir Role.

In 2019, random samples of Panicum miliaceum growing as a weed were surveyed to uncover their virus infections at two locations in Hungary. This pilot study revealed infection with three viruses, two appearing for the first time in the country. As follow-up research, in the summer of 2021, we collected symptomatic leaves of several monocotyledonous plants in the same locations and determined their viromes using small RNA high-throughput sequencing (HTS). As a result, we have identified the presence of wheat streak mosaic virus (WSMV), barley yellow striate mosaic virus (BYSMV), barley virus G (BVG), and two additional viruses, namely Aphis glycines virus 1 (ApGlV1) and Ljubljana dicistrovirus 1 (LDV1), which are described for the first time in Hungary. New hosts of the viruses were identified: Cynodon dactylon is a new host of BYSMV and LDV1, Echinocloa crus-galli is a new host of BVG, ApGlV1 and LDV1, Sorghum halepense is a new host of ApGlV1, and Panicum miliaceum is a new host of LDV1. At the same time, Zea mays is a new host of ApGlV1 and LDV1. Small RNA HTS diagnosed acute infections but failed to detect persistent ones, which could be revealed using RT-PCR. The infection rates at the different locations and plant species were different. The phylogenetic analyses of the sequenced virus variants suggest that the tested monocotyledonous weeds can host different viruses and play a virus reservoir role. Viral spread from the reservoir species relies on the activity of insect vectors, which is why their management requires an active role in plant protection strategies, which need careful planning in the changing environment.

The First Virome of a Russian Vineyard.

Among other pathogens, more than 80 viruses infect grapevine. The aim of this work was to study the virome diversity of grapevine viruses and mycoviruses of a vineyard using high-throughput sequencing technologies. The grapevine virome was studied in symptomatic vines of the Rkatsiteli cultivar (V. vinifera) collected at the vineyards of the Krasnodar Krai in Russia. Ribosomal-depleted total RNA and isolated small RNAs were used for library preparation and high-throughput sequencing. Six grapevine-infecting viruses and two viroids were validated by RT-PCR and analyzed phylogenetically. We identified the presence of grapevine leafroll-associated virus 3, grapevine Pinot gris virus, grapevine virus T, grapevine rupestris stem-pitting-associated virus, grapevine fleck virus, and grapevine rupestris vein feathering virus, as well as two viroids, grapevine yellow speckle viroid 1 and hop stunt viroid. We also studied the mycovirome of the vineyard and identified nine viruses with single-stranded positive-sense RNA genomes: alternaria arborescens mitovirus 1, botrytis cinerea mitovirus 1, botrytis cinerea mitovirus 2, botrytis cinerea mitovirus 3, botrytis cinerea mitovirus 4, sclerotinia sclerotiorum mitovirus 3, botrytis cinerea hypovirus 1, grapevine-associated narnavirus 1, and botrytis virus F. In addition, we identified botrytis cinerea hypovirus 1 satellite-like RNA and two single-stranded negative-sense RNA viruses. This is the first study of grapevine mycoviruses in Russia. The obtained result will contribute to the development of biocontrol strategies in the future.

Analysis of Virus-Derived siRNAs in Strawberry Plants Co-Infected with Multiple Viruses and Their Genotypes.

Plants can be infected with multiple viruses. High-throughput sequencing tools have enabled numerous discoveries of multi-strain infections, when more than one viral strain or divergent genomic variant infects a single plant. Here, we investigated small interfering RNAs (siRNAs) in a single strawberry plant co-infected with several strains of strawberry mottle virus (SMoV), strawberry crinkle virus (SCV) and strawberry virus 1 (StrV-1). A range of plants infected with subsets of the initial viral species and strains that were obtained by aphid-mediated transmission were also evaluated. Using high-throughput sequencing, we characterized the small RNA fractions associated with different genotypes of these three viruses and determined small RNA hotspot regions in viral genomes. A comparison of virus-specific siRNA (vsiRNA) abundance with relative viral concentrations did not reveal any consistent agreement. Strawberry mottle virus strains exhibiting considerable variations in concentrations were found to be associated with comparable quantities of vsiRNAs. Additionally, by estimating the specificity of siRNAs to different viral strains, we observed that a substantial pool of vsiRNAs could target all SMoV strains, while strain-specific vsiRNAs predominantly targeted rhabdoviruses, SCV and StrV-1. This highlights the intricate nature and potential interference of the antiviral response within a single infected plant when multiple viruses are present.

Small RNA Profiling of Aster Yellows Phytoplasma-Infected Catharanthus roseus Plants Showing Different Symptoms.

Micropropagated Catharantus roseus plants infected with 'Candidatus Phytoplasma asteris' showed virescence symptoms, witches' broom symptoms, or became asymptomatic after their planting in pots. Nine plants were grouped into three categories according to these symptoms, which were then employed for investigation. The phytoplasma concentration, as determined by qPCR, correlated well with the severity of symptoms. To reveal the changes in the small RNA profiles in these plants, small RNA high-throughput sequencing (HTS) was carried out. The bioinformatics comparison of the micro (mi) RNA and small interfering (si) RNA profiles of the symptomatic and asymptomatic plants showed changes, which could be correlated to some of the observed symptoms. These results complement previous studies on phytoplasmas and serve as a starting point for small RNA-omic studies in phytoplasma research.

Clematis vitalba Is a Natural Host of the Novel Ilarvirus, Prunus Virus I.

Clematis vitalba L. is a climbing shrub and a pioneer plant in abandoned orchards or vineyards that are widespread in temperate climate zones. In past years, several viruses infecting the Clematis species have been identified, including different ilarviruses. Prunus virus I (PrVI) is a recently described ilarvirus, which has been shown to infect sweet cherries and peaches in Greece. Moreover, its presence has been detected in ornamental Clematis in Russia. In the present work, we analyzed the virome of wildly growing C. vitalba plants from Hungary, Slovakia and Croatia showing different kinds of symptoms using high-throughput sequencing (HTS) of small RNAs or ribodepleted RNAs. Applying HTS enabled us to identify the presence of PrVI in C. vitalba, and the bioinformatic analyses were further validated with RT-PCR using PrVI-specific primers and Sanger dideoxy sequencing. Nearly full genome sequences of all three viral RNAs of one Hungarian, two Slovak and one Croatian isolate were determined. Their phylogenetic analysis showed high similarity to each other and to other PrVI isolates described from Central Europe. As the sampled plants were co-infected with other viruses, it is not possible to determine a direct correlation between the infection with PrVI and the observed symptoms. Analyses of different Prunus species in stock collection showed infection of several peach and sweet cherry varieties in Hungary. Our results expand the knowledge on the natural host range of PrVI and highlight the necessity to evaluate alternative plant hosts (even non-Prunus) of PrVI and the role of the virus in the etiology of the potential diseases.

Virus-induced gene silencing of Mlo genes induces powdery mildew resistance in Triticum aestivum.

Powdery mildew is one of the most important cereal diseases worldwide. Genetic analysis has revealed that mutant alleles of the Mlo gene cause broad-spectrum resistance against this pathogen in barley. In this study, the possibility of inducing broad-spectrum powdery mildew resistance against this pathogen by RNAi of the barley Mlo ortholog in wheat was examined using virus-induced gene silencing (VIGS). A clear correlation was found between resistance and accumulation of Mlo-specific siRNAs, raising the possibility of designing powdery mildew resistance in wheat by RNA silencing using both transgenic and non-transgenic approaches.

Grapevine Pinot Gris Virus Is Present in Different Non-Vitis Hosts.

Grapevine Pinot gris virus (GPGV) was described in Italy using a metagenomic approach: next-generation sequencing of the virus-derived small RNAs. Since that time, it has been reported all over the world. The presence of GPGV is associated with grapevine disease, but most of the time, the disease is asymptomatic. Although the host range of this virus has not been investigated, it has been found in the non-Vitis hosts, Silene latifolia and Chenopodium album. We investigated the presence of GPGV in grapevine and other plant species growing as weeds in the vineyard. Using RT-PCR, we identified GPGV in seven non-Vitis hosts: Ailanthus, Asclepias, Crataegus, Fraxinus, Rosa, Rubus, and Sambucus. In the case of Rosa and Rubus, this finding was supported by Northern blot detection of the virus. GPGV strains in non-Vitis hosts belong to the asymptomatic clade, and are clustered according to their original geographic locations. The presence of GPGV in species other than grapevine shows that besides well-known vector and propagating material-based infections, other possible entry sites for the virus can exist, which have to be taken into consideration when developing reliable regulation strategies.

Viromes of Hungarian Peach Trees Identified by High-Throughput Sequencing of Small RNAs.

Peach trees can be infected with viruses and viroids. As we do not have efficient plant protection methods against these pathogens, the prevention of infection is crucial. Fruit trees are maintained by vegetative propagation. Planting material such as certified mother trees and rootstocks should be free from viruses and viroids, and this status has to be regularly checked to prevent infections. We surveyed certified peach trees for the presence of viruses and viroids using small RNA high-throughput sequencing (HTS), an unbiased virus diagnostic method. The results of the bioinformatic analysis of HTS were validated by other molecular methods including RT-PCR, Northern blot hybridization and loop-mediated isothermal amplification (LAMP). We found the presence of plum pox virus and peach latent mosaic viroid (PLMVd) in the vector-free isolator houses, whose presence should be regularly tested. Moreover, we detected frequent infection with recently described viruses such as nectarine stem pitting-associated virus and peach-associated luteovirus (PaLV). During the survey, PLMVd and PaLV were detected for the first time in Hungary. The analysis of the presenting virus variants and possible sources of infection suggests that the source of the viral infection could be the infected propagating material. Our study emphasizes the importance of using sensitive and trustworthy diagnostic techniques to be able to detect viral infections and successfully prevent their spread by propagation material.

Detection of Apple Hammerhead Viroid, Apple Luteovirus 1 and Citrus Concave Gum-Associated Virus in Apple Propagation Materials and Orchards in the Czech Republic and Hungary.

Grafting cultivars onto rootstocks is a widely used practice by the apple industry predominantly aimed at faster fruit bearing. Using high-throughput sequencing, we revealed the presence of recently described viral agents, namely apple hammerhead viroid (AHVd), apple luteovirus 1 (ALV-1), and citrus concave gum-associated virus (CCGaV), in germplasm collections and production orchards in the Czech Republic and Hungary. The HTS results were validated with RT-(q)PCR, and Northern blotting techniques. To obtain further insight about the presence of these agents, RT-PCR based surveys were carried out and showed their widespread presence alone or in mixed infections. The pathogens were present both in production areas and in feral samples. In addition, rootstock-to-scion transmission of ALV-1 and CCGaV was confirmed using commercial rootstock materials. Phylogenetic relationships based on partial sequences of distinct variants were also investigated. Furthermore, the rosy apple aphid was found to be ALV-1-positive, suggesting that it might be a potential vector of the virus.

High-Throughput Sequencing of Small RNAs for Diagnostics of Grapevine Viruses and Viroids in Russia.

The use of high-throughput sequencing (HTS) technology has led to significant progress in the identification of many viruses and their genetic variants. In this study, we used the HTS platform to sequence small RNAs (sRNAs) of grapevine to study the virome. Isolation of RNA was performed using symptomatic grapevines collected from commercial vineyards in Krasnodar Krai in 2017-2018. To determine the viromes of vineyards, we used an integrated approach that included a bioinformatic analysis of the results of sRNA HTS and the molecular method RT-PCR, which made it possible to identify 13 viruses and 4 viroids. Grapevine leafroll-associated virus 4 (GLRaV-4), Grapevine Syrah Virus-1 (GSyV-1), Raspberry bushy dwarf virus (RBDV), Australian grapevine viroid (AGVd), and Grapevine yellow speckle viroid 2 (GYSVd-2) were identified for the first time in Russia. Out of 38 samples analyzed, 37 had mixed infections with 4-11 viruses, indicating a high viral load. Analysis of the obtained sequences of fragments of virus genomes made it possible to identify recombination events in GLRaV-1, GLRaV-2, GLRaV-3, GLRaV-4, GVT, GPGV, GRSPaV, GVA, and GFLV. The obtained results indicate a wide spread of the viruses and a high genetic diversity in the vineyards of Krasnodar Krai and emphasize the urgent need to develop and implement long-term strategies for the control of viral grapevine diseases.

Variable Populations of Grapevine Virus T Are Present in Vineyards of Hungary.

Grapevine virus T (GVT) is a recently described foveavirus, which was identified from a transcriptome of a Teroldego grapevine cultivar in 2017. Recently, we surveyed vineyards and rootstock plantations in Hungary using small RNA (sRNA) high-throughput sequencing (HTS), at a time when GVT had not yet been described. A re-analysis of our sRNA HTS datasets and a survey of grapevines by RT-PCR revealed the presence of GVT in most of the vineyards tested, while at rootstock fields its presence was very rare. The presence and high variability of the virus in the country was confirmed by sequence analysis of strains originating from different vineyards. In this study, we demonstrate the presence of GVT in Hungary and show its high diversity, suggesting that GVT presence may not seriously affect grapevine health and that it could have been present in European vineyards for a long time as a latent infection.