Plant RNA Research Articles

Immunocapture-Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay for Detection of Plant RNA Viruses.

Loop-mediated isothermal amplification (LAMP) is a sensitive method that can rapidly amplify a specific nucleic acid target with high specificity. The LAMP reaction process has no denaturation step, instead DNA amplification occurs by strand displacement activity of the Bacillus stearothermophilus (Bst) DNA polymerase under isothermal conditions. It utilizes three sets of forward and reverse oligonucleotide primers specific to six distinct sequences on the target gene. These primers are used to generate amplification products that contain single-stranded loops, thereby allowing primers to bind to these sequences without the need for repeated cycles of thermal denaturation. For diagnosis of pathogens with RNA genome, LAMP has been merged with reverse transcription (RT) step to create RT-LAMP. To further reduce the cost of diagnosis and increase the throughput, immunocapture (IC) step was added to develop IC-RT-LAMP assay. Hence, this chapter focuses on utilizing IC-RT-LAMP assay to specifically identify severe strain of a plant virus from field samples.

Translation of Plant RNA Viruses.

Plant RNA viruses encode essential viral proteins that depend on the host translation machinery for their expression. However, genomic RNAs of most plant RNA viruses lack the classical characteristics of eukaryotic cellular mRNAs, such as mono-cistron, 5′ cap structure, and 3′ polyadenylation. To adapt and utilize the eukaryotic translation machinery, plant RNA viruses have evolved a variety of translation strategies such as cap-independent translation, translation recoding on initiation and termination sites, and post-translation processes. This review focuses on advances in cap-independent translation and translation recoding in plant viruses.

Metatranscriptomic Sequencing Suggests the Presence of Novel RNA Viruses in Rice Transmitted by Brown Planthopper.

Viral pathogens are a major threat to stable crop production. Using a backcross strategy, we find that integrating a dominant brown planthopper (BPH) resistance gene Bph3 into a high-yield and BPH-susceptible indica rice variety significantly enhances BPH resistance. However, when Bph3-carrying backcross lines are infested with BPH, these BPH-resistant lines exhibit sterile characteristics, displaying panicle enclosure and failure of seed production at their mature stage. As we suspected, BPH-mediated viral infections could cause the observed sterile symptoms, and we characterized rice-infecting viruses using deep metatranscriptomic sequencing. Our analyses revealed eight novel virus species and five known viruses, including a highly divergent virus clustered within a currently unclassified family. Additionally, we characterized rice plant antiviral responses using small RNA sequencing. The results revealed abundant virus-derived small interfering RNAs in sterile rice plants, providing evidence for Dicer-like and Argonaute-mediated immune responses in rice plants. Together, our results provide insights into the diversity of viruses in rice plants, and our findings suggest that multiple virus infections occur in rice plants.

An atypical class of non-coding small RNAs is produced in rice leaves upon bacterial infection

Non-coding small RNAs (sRNA) act as mediators of gene silencing and regulate plant growth, development and stress responses. Early insights into plant sRNAs established a role in antiviral defense and they are now extensively studied across plant–microbe interactions. Here, sRNA sequencing discovered a class of sRNA in rice (Oryza sativa) specifically associated with foliar diseases caused by Xanthomonas oryzae bacteria. Xanthomonas-induced small RNAs (xisRNAs) loci were distinctively upregulated in response to diverse virulent strains at an early stage of infection producing a single duplex of 20–22 nt sRNAs. xisRNAs production was dependent on the Type III secretion system, a major bacterial virulence factor for host colonization. xisRNA loci overlap with annotated transcripts sequences, with about half of them encoding protein kinase domain proteins. A number of the corresponding rice cis-genes have documented functions in immune signaling and xisRNA loci predominantly coincide with the coding sequence of a conserved kinase motif. xisRNAs exhibit features of small interfering RNAs and their biosynthesis depend on canonical components OsDCL1 and OsHEN1. xisRNA induction possibly mediates post-transcriptional gene silencing but they do not broadly suppress cis-genes expression on the basis of mRNA-seq data. Overall, our results identify a group of unusual sRNAs with a potential role in plant–microbe interactions.

Development of an RT-LAMP assay for the detection of maize yellow mosaic virus in maize

Maize is one of the most widely cultivated cereal crops worldwide. Maize yellow mosaic virus (MaYMV) (species Maize yellow mosaic virus, genus Polerovirus and family Luteoviridae) was first reported in maize from China. In this study, a one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting MaYMV. The optimal concentrations of betaine, Mg2+ and dNTPs for the assay were 0 M, 1.4 mM and 6 mM, respectively, and the optimal reaction time was 50 min. Using total plant RNA as the template, the detection limit of the RT-LAMP assay for MaYMV was 1 pg, while that of RT-PCR was 100 pg, indicating that the RT-LAMP assay developed was 100 times more sensitive than RT-PCR. Importantly, the RT-LAMP assay successfully detected MaYMV using rapidly extracted crude RNA from infected maize as a template. In conclusion, the RT-LAMP assay developed was a rapid, specific, sensitive and low-cost method for the detection of MaYMV in field samples of maize.

A Molecular switch for FLOWERING LOCUS C activation determines flowering time in Arabidopsis.

Plants have evolved sophisticated mechanisms to ensure flowering in favorable conditions for reproductive success. In the model plant Arabidopsis thaliana, FLOWERING LOCUS C (FLC) acts as a central repressor of flowering and the major determinant for winter cold requirement for flowering. FLC is activated in winter annuals by the FRIGIDA (FRI) activator complex containing FRI, FLC EXPRESSOR (FLX), and FLX-LIKE 4 (FLX4), among which FLX and FLX4 are also essential for establishing basal FLC expression in summer annuals. Here we show that a plant RNA polymerase II C-terminal domain phosphatase, C-TERMINAL DOMAIN PHOSPHATASE-LIKE 3 (CPL3), interacts with and dephosphorylates FLX4 through their scaffold protein FLX to inhibit flowering. CPL3-mediated dephosphorylation of FLX4 serves as a key molecular switch that enables binding of dephosphorylated FLX4 to the FLC locus to promote FLC expression, thus repressing flowering in both winter and summer annuals of Arabidopsis. Our findings reveal a molecular switch underlying the activation of FLC for flowering time control.

First report of dasheen mosaic virus infecting taro (Colocasia esculenta) in Louisiana.

Taro [Colocasia esculenta (L.) Schott], also called dasheen or malanga is an important staple crop in many tropical and subtropical countries (Chaïr et al. 2016). In October 2020, taro plants showing foliar symptoms consisting of mosaic, feathery mottle, and vein clearing patterns were observed in the Hilltop Arboretum, the Bluebonnet Swamp Nature Center, the Louisiana State University Agricultural Center Botanic Gardens, and the University Lake, in Baton Rouge, Louisiana. Unidentified aphids were also observed infesting the plants showing the described symptoms. From each location, two foliar samples from symptomatic and two from asymptomatic plants were collected and tested by ELISA using antiserum for general potyvirus group (Agdia, Elkhart, IN). Seven of eight symptomatic samples tested positive while the asymptomatic samples were negative. The seven positive samples were used to perform an additional ELISA test using antiserum specific for dasheen mosaic virus (DsMV) (Agdia). All seven samples tested positive for DsMV. To confirm the identity of the virus, total RNA was extracted from the seven samples using the PureLink® Plant RNA Reagent Kit (Invitrogen, Carlsbad, CA). After DNA digestion with PerfeCta® DNase I (Qiagen, Beverly, MA), the RNA was used to perform reverse transcription polymerase chain reaction (RT-PCR) with primer set DMV 5708-5731-F/DMV 6131-6154-R which is specific for DsMV (Wang et al. 2017). RT-PCR was performed using the AccessQuickTM RT-PCR System (Promega, Madison, WI) following the reaction conditions described by Wang et al. PCR products of the expected size (~447 bp) were obtained with all seven samples and were Sanger-sequenced. A consensus sequence (MW284936) was obtained with the two sequences from samples collected at the University Lake and aligned with other sequences available in the GenBank using BLASTn. Our isolate of DsMV showed 90.6% nt identity to an isolate of DsMV from Ethiopia (MG602229). Mechanical inoculations to healthy taro plants were conducted using leaf tissue of symptomatic plants as source of inoculum. Inoculated plants exhibited mosaic symptoms three weeks after inoculation and were ELISA-positive for DsMV. Symptomatology, serological tests, RT-PCR testing, and DNA sequencing of RT-PCR products support that the symptomatic taro plants were infected with DsMV. Taro is a crop in Hawaii, but in the contiguous United States, it is mostly grown as an ornamental and is considered an invasive species. Its distribution is restricted to the southern continental states and Hawaii (Cozad et al. 2018). CABI, EPPO (1998) lists the presence of DsMV in several states of the United States, including Louisiana; however, there is no record in the literature of the identification of this virus in Louisiana. The potential impact of DsMV in taro and related ornamental species in southern United States is unknown. To the best of our knowledge, this is the first report documenting DsMV infecting taro in Louisiana.

Recent origination of circular RNAs in plants

Circular RNA (circRNA) is a kind of new regulatory RNA with diverse biological functions. Numerous circRNAs have been identified in many plant species; however, evolution of plant circRNAs remains largely unknown. In this study, we assembled full-length sequences of 6519 rice (Oryza sativa) circRNAs and analyzed their conservation in another 46 plant species based on comparison of sequences and expression patterns. We found that, at the genomic level, 8.7% of the 6519 circRNAs were conserved in dicotyledonous plants and 49.1% in Oryza genus. Meanwhile, 57.8% of parental protein-coding genes of the rice circRNAs originated recently after divergence of monocotyledonous plants, implying recent origin of the majority of rice circRNAs, a conclusion further supported by the results based on analysis of 4663 full-length circRNAs in Arabidopsis thaliana. Accordingly, we proposed three models to address the origination of different types of circRNAs. Taken together, the results obtained in this study provide new insights for the evolutionary dynamics of plant circRNAs and candidate circRNAs for further functional exploration.

Plant and animal small RNA communications between cells and organisms.

Since the discovery of eukaryotic small RNAs as the main effectors of RNA interference in the late 1990s, diverse types of endogenous small RNAs have been characterized, most notably microRNAs, small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs). These small RNAs associate with Argonaute proteins and, through sequence-specific gene regulation, affect almost every major biological process. Intriguing features of small RNAs, such as their mechanisms of amplification, rapid evolution and non-cell-autonomous function, bestow upon them the capacity to function as agents of intercellular communications in development, reproduction and immunity, and even in transgenerational inheritance. Although there are many types of extracellular small RNAs, and despite decades of research, the capacity of these molecules to transmit signals between cells and between organisms is still highly controversial. In this Review, we discuss evidence from different plants and animals that small RNAs can act in a non-cell-autonomous manner and even exchange information between species. We also discuss mechanistic insights into small RNA communications, such as the nature of the mobile agents, small RNA signal amplification during transit, signal perception and small RNA activity at the destination.

Genome-Wide Identification and Analysis of Small Nucleolar RNAs and Their Roles in Regulating Latex Regeneration in the Rubber Tree (Hevea brasiliensis).

Small nucleolar RNAs (snoRNAs) are a class of conserved nuclear RNAs that play important roles in the modification of ribosomal RNAs (rRNAs) in plants. In rubber trees, rRNAs are run off with latex flow during tapping and need to be regenerated for maintaining the functions of the laticifer cells. SnoRNAs are expected to play essential roles in the regeneration of rRNAs. However, snoRNAs in the rubber tree have not been sufficiently characterized thus far. In this study, we performed nuclear RNA sequencing (RNA-seq) to identify snoRNAs globally and investigate their roles in latex regeneration. We identified a total of 3,626 snoRNAs by computational prediction with nuclear RNA-seq data. Among these snoRNAs, 50 were highly expressed in latex; furthermore, the results of reverse transcription polymerase chain reaction (RT-PCR) showed the abundant expression of 31 of these snoRNAs in latex. The correlation between snoRNA expression and adjusted total solid content (TSC/C) identified 13 positively yield-correlated snoRNAs. To improve the understanding of latex regeneration in rubber trees, we developed a novel insulated tapping system (ITS), which only measures the latex regenerated in specific laticifers. Using this system, a laticifer-abundant snoRNA, HbsnoR28, was found to be highly correlated with latex regeneration. To the best of our knowledge, this is the first report to globally identify snoRNAs that might be involved in latex regeneration regulation and provide new clues for unraveling the mechanisms underlying the regulation of latex regeneration.

Tissue-specific gene expression shows a cynipid wasp repurposes oak host gene networks to create a complex and novel parasite-specific organ.

Every organism on Earth depends on interactions with other organisms to survive. In each of these interactions, an organism must utilize the limited toolbox of genes and proteins it possesses to successfully manipulate or cooperate with another species, but it can also co-opt the genome machinery of its partner to expand its available tools. Insect-induced plant galls are an extreme example of this, wherein an insect hijacks the plant's genome to direct the initiation and development of galls consisting of plant tissue. However, previous transcriptomic studies have not evaluated individual tissues within a gall to determine the full extent to which a galling insect manipulates its host plant. Here we demonstrate that the cynipid wasp Dryocosmus quercuspalustris creates a complex parasite-specific organ from red oak tissue via massive changes in host gene expression. Our results show that the gall wasp is not merely modifying oak leaf tissue but creating extensive changes in gene expression between galled and ungalled tissue (differential expression in 28% of genes) and distinct gall tissue types (20% of genes). The outer gall tissue shows increases in various plant defence systems, which is consistent with its predicted functional role of protecting the wasp larva. The inner larval capsule shows suppression of large parts of the plant innate immune system and evidence for the wasp utilizing the plant's RNA interference mechanisms, which may be a potential mechanism for the wasp's control on gall growth.

Characterization of small interfering RNAs derived from pepper mild mottle virus in infected pepper plants by high-throughput sequencing

Pepper mild mottle virus (PMMoV) infects pepper plants and induces severe yield losses in China. However, the molecular interaction between PMMoV and pepper plants is largely unknown. RNA silencing is a eukaryotically conserved mechanism against viruses mediated by virus-derived small interfering RNAs (vsiRNAs) in plants. In this study, the profiles of vsiRNAs from PMMoV in infected pepper plants were obtained by high-throughput sequencing. The results showed that vsiRNAs were predominantly 21 and 22 nucleotides (nts) in length, and had a U bias at the 5′-terminal. The single-nucleotide resolution maps revealed that vsiRNAs were heterogeneously distributed throughout PMMoV genomic RNAs and hotspots of sense and antisense strands were mainly located in the RdRp and CP coding regions. The host transcripts targeted by vsiRNAs were predicted and they are mainly involved in physiological pathways related to stress response, cell regulation, and metabolism process. In addition, PMMoV infection induced significant up-regulation of CaAGO1a/1b/2, CaDCL2 and CaRDR1 gene transcripts in pepper plants, which are important components involved in antiviral RNA silencing pathway. Taken together, our results suggest the possible roles of vsiRNAs in PMMoV-pepper interactions.

The Intriguing Conundrum of a Nonconserved Multifunctional Protein of Citrus Tristeza Virus That Interacts with a Viral Long Non-Coding RNA.

Citrus tristeza virus (CTV), the largest non-segmented plant RNA virus, has several peculiar features, among which is the production of a 5′-terminal long non-coding RNA (lncRNA) termed low-molecular-weight tristeza 1 (LMT1). In this study, we found that p33, a unique viral protein that performs multiple functions in the virus infection cycle, specifically binds LMT1, both in vivo and in vitro. These results were obtained through the expression of p33 under the context of the wild type virus infection or along with a mutant CTV variant that does not produce LMT1 as well as via ectopic co-expression of p33 with LMT1 in Nicotiana benthamiana leaves followed by RNA immunoprecipitation and rapid amplification of cDNA ends assays. Further experiments in which a recombinant p33 protein and an in vitro transcribed full-length LMT1 RNA or its truncated fragments were subjected to an electrophoretic mobility shift assay demonstrated that p33 binds to at least two distinct regions within LMT1. To the best of our knowledge, this is the first report of a plant virus protein binding to a lncRNA produced by the same virus. The biological significance of the interaction between these two viral factors is discussed.

Modelling temperature-dependent dynamics of single and mixed infections in a plant virus

Multiple viral infection is an important issue in health and agriculture with strong impacts on society and the economy. Several investigations have dealt with the population dynamics of viruses with different dynamic properties, focusing on strain competition during multiple infections and the effects on viruses’ hosts. Recent interest has been on how multiple infections respond to abiotic factors such as temperature (T). This is especially important in the case of plant pathogens, whose dynamics could be affected significantly by global warming. However, few mathematical models incorporate the effect of T on parasite fitness, especially in mixed infections. Here, we investigate simple mathematical models incorporating thermal reaction norms (TRNs), which allow for quantitative analysis. A logistic model is considered for single infections, which is extended to a Lotka-Volterra competition model for mixed infections. The dynamics of these two models are investigated, focusing on the roles of T-dependent replication and competitive interactions in both transient and asymptotic dynamics. We determine the scenarios of co-existence and competitive exclusion, which are separated by a transcritical bifurcation. To illustrate the applicability of these models, we ran single- and mixed-infection experiments in plants growing at 20 ∘C and 30 ∘C using two strains of the plant RNA virus Pepino mosaic virus. Using a macroevolutionary algorithm, we fitted the models to the data by estimating the TRNs for both strains in single infections. Then, we used these TRNs to feed the mixed-infection model estimating the strength of competition. We found an asymmetrical pattern in which each strain dominated at different T values due to differences in their TRNs. We also identified that T can modify competition interference greatly for both isolates. The models proposed here can be useful for investigating the outcomes of multiple-infection dynamics under abiotic changes and have implications for the understanding of viral responses to global warming.

First report of Sweet potato virus G in sweet potato in Italy

Sweet potato (Ipomoea batatas) is the sixth most important food crop in terms of production globally. In Italy, sweet potato is mainly cultivated in Veneto province (northeast Italy), and to a lesser extent in Lazio and Apulia provinces, on a total area of about 400 hectares. Among the sweet potato pests, viruses are the second most important constraint next to sweet potato weevil (Barkessa, 2018). In 2020, tubers of six different genotypes, with uncertain geographical origins, were sent by a grower from Veneto province to IPSP-CNR, with the aim of ascertaining their phytosanitary status. Two tubers of each genotype were grown in an insect-proof greenhouse (26/22°C, day/night) under natural light conditions and transplanted in 25 cm diameter pots containing peat bog-moss as a substrate. After about one month, leaves of only one genotype (identified as DP/578) showed light diffuse chlorotic symptoms (Figures 1, 2). Sap extracts from both plants of each of the six genotypes were tested with ELISA using broad-spectrum potyvirus antibodies (Bioreba, Switzerland). Only the symptomatic genotype DP/578 tested positive to potyvirus antibodies. Total RNA was extracted from the leaves of the two plants of DP/578 and from a healthy plant, with the Plant RNA Kit (Omega Bio-Tek, USA), and subjected to RT-PCR using the universal Potyviridae forward primer (5'-GGBAAYAAYAGYGGDCARCC-3'; Gibbs & Mackenzie, 1997) and the reverse primer SPFMV-Rv (5'-TTGCACACCCCTCATTCCYAAG-3'; Parrella et al., 2006). An amplification product of approximately 1700 bp was produced only from samples with symptoms but not from the healthy control. Sequences obtained from the two symptomatic plants were identical (GenBank Accession No. MW456564) and showed highest identity (98.2%) with the CH2-32-1 isolate of Sweet potato virus G (SPVG) from China (MK778832). SPVG alone is not considered a real threat to sweet potato production since infected plants rarely display symptoms (Rännäli et al., 2008). Significant yield losses have been recorded only when SPVG co-infects sweet potato with other potyviruses such as Sweet potato feathery mottle virus (SPFMV) (Clark & Hoy, 2006). Previously, outbreaks of SPFMV were reported in sweet potato cultivation in central Italy (Parrella et al., 2006). In the current study, attempts to amplify other sweet potato closely related potyviruses were unsuccessful using RT-PCR developed by Li et al. (2012). Based on our observation and results (symptoms recorded and serological detection of SPVG), the yield effects of SPVG may have been overlooked in sweet potato. Considering the number of existing SPVG strains and of sweet potato landraces/varieties, further investigation would be desirable to understand the real incidence of SPVG in individual sweet potato crops and its effects on production.

Multiplex Assay of Viruses Integrating Recombinase Polymerase Amplification, Barcode-Anti-Barcode Pairs, Blocking Anti-Primers, and Lateral Flow Assay.

A multiplex assay based on recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a desirable tool for many areas. This multiplex assay could be efficiently realized using single-stranded (ss) DNAs located in separate zones on the test strip and bound complementary ssDNA tags of double-stranded (ds) DNA amplicons. Here, we investigate how to enrich multiplex assay capabilities using ssDNAs. Bifunctional oligonucleotide probes integrating (1) a forward primer for RPA, (2) a C9 spacer to stop polymerase, and (3) a ssDNA tag for binding at test strip are developed. The amplicons have a unique individual ssDNA tag at one end and a universal label of fluorescein introducing through a reverse primer at the other end. A conjugate of gold nanoparticles (GNP) with antibodies to fluorescein is used to detect all amplicons. The remainder of primers after RPA interacting with GNP conjugate was found to be a limiting factor for sensitive and specific multiplex assay. The addition of anti-RPA-primers before the use of test strips was proposed to simply and effectively eliminate remaining primers. This approach was successfully applied for the detection of three priority plant RNA viruses: potato virus Y (PVY), -S (PVS) and potato leafroll virus (PLRV). The total time of the assay is 30 min. The multiplex RPA-LFT detected at least 4 ng of PVY per g of plant leaves, 0.04 ng/g for PVS, and 0.04 ng/g for PLRV. The testing of healthy and infected potato samples showed concordance between the developed assay and reverse transcription-polymerase chain reaction. Thus, the capabilities of the proposed universal modules (ssDNA anchors, bifunctional probes, and blocking anti-primers) for multiplex detection of RNA analytes with high specificity and sensitivity were demonstrated.

Historical Perspective on the Discovery of the Quasispecies Concept.

Viral quasispecies are dynamic distributions of nonidentical but closely related mutant and recombinant viral genomes subjected to a continuous process of genetic variation, competition, and selection that may act as a unit of selection. The quasispecies concept owes its theoretical origins to a model for the origin of life as a collection of mutant RNA replicators. Independently, experimental evidence for the quasispecies concept was obtained from sampling of bacteriophage clones, which revealed that the viral populations consisted of many mutant genomes whose frequency varied with time of replication. Similar findings were made in animal and plant RNA viruses. Quasispecies became a theoretical framework to understand viral population dynamics and adaptability. The evidence came at a time when mutations were considered rare events in genetics, a perception that was to change dramatically in subsequent decades. Indeed, viral quasispecies was the conceptual forefront of a remarkable degree of biological diversity, now evident for cell populations and organisms, not only for viruses. Quasispecies dynamics unveiled complexities in the behavior of viral populations,with consequences for disease mechanisms and control strategies. This review addresses the origin of the quasispecies concept, its major implications on both viral evolution and antiviral strategies, and current and future prospects.

A vast pool of lineage-specific microproteins encoded by long non-coding RNAs in plants.

Pervasive transcription of eukaryotic genomes results in expression of long non-coding RNAs (lncRNAs) most of which are poorly conserved in evolution and appear to be non-functional. However, some lncRNAs have been shown to perform specific functions, in particular, transcription regulation. Thousands of small open reading frames (smORFs, <100 codons) located on lncRNAs potentially might be translated into peptides or microproteins. We report a comprehensive analysis of the conservation and evolutionary trajectories of lncRNAs-smORFs from the moss Physcomitrium patens across transcriptomes of 479 plant species. Although thousands of smORFs are subject to substantial purifying selection, the majority of the smORFs appear to be evolutionary young and could represent a major pool for functional innovation. Using nanopore RNA sequencing, we show that, on average, the transcriptional level of conserved smORFs is higher than that of non-conserved smORFs. Proteomic analysis confirmed translation of 82 novel species-specific smORFs. Numerous conserved smORFs containing low complexity regions (LCRs) or transmembrane domains were identified, the biological functions of a selected LCR-smORF were demonstrated experimentally. Thus, microproteins encoded by smORFs are a major, functionally diverse component of the plant proteome.

Viruses Without Borders: Global Analysis of the Population Structure, Haplotype Distribution, and Evolutionary Pattern of Iris Yellow Spot Orthotospovirus (Family Tospoviridae, Genus Orthotospovirus).

Iris yellow spot, caused by Iris yellow spot orthotospovirus (IYSV) (Genus: Orthotospovirus, Family: Tospoviridae), is an important disease of Allium spp. The complete N gene sequences of 142 IYSV isolates of curated sequence data from GenBank were used to determine the genetic diversity and evolutionary pattern. In silico restriction fragment length polymorphism (RFLP) analysis, codon-based maximum likelihood studies, genetic differentiation and gene flow within the populations of IYSV genotypes were investigated. Bayesian phylogenetic analysis was carried out to estimate the evolutionary rate. In silico RFLP analysis of N gene sequences categorized IYSV isolates into two major genotypes viz., IYSV Netherlands (IYSVNL; 55.63%), IYSV Brazil (IYSVBR; 38.73%) and the rest fell in neither group [IYSV other (IYSVother; 5.63%)]. Phylogenetic tree largely corroborated the results of RFLP analysis and the IYSV genotypes clustered into IYSVNL and IYSVBR genotypes. Genetic diversity test revealed IYSVother to be more diverse than IYSVNL and IYSVBR. IYSVNL and IYSVBR genotypes are under purifying selection and population expansion, whereas IYSVother showed decreasing population size and hence appear to be under balancing selection. IYSVBR is least differentiated from IYSVother compared to IYSVNL genotype based on nucleotide diversity. Three putative recombinant events were found in the N gene of IYSV isolates based on RDP analysis, however, RAT substantiated two among them. The marginal likelihood mean substitution rate was 5.08 × 10–5 subs/site/year and 95% highest posterior density (HPD) substitution rate between 5.11 × 10–5 and 5.06 × 10–5. Findings suggest that IYSV continues to evolve using population expansion strategies. The substitution rates identified are similar to other plant RNA viruses.

First report of Rose leaf rosette-associated virus infecting rose (Rosa spp.) in California, USA.

Rose leaf rosette-associated virus (RLRaV) is a member of genus Closterovirus, family Closteroviridae. The virus was first discovered in China in 2015 from a mixed infected wild rose (Rosa multiflora Thunb.) showing small leaf rosettes on branches, dieback and severe decline symptoms (He et al. 2015). In 2013, a rose plant (cv. Roses Are Red) was introduced to Foundation Plant Services (FPS, UC-Davis) rose collection. The plant was originated from a private rose breeder collection located in California. In 2019, total nucleic acids (TNA) were isolated from leaf tissues of one asymptomatic plant (Roses Are Red plant) using MagMax Plant RNA Isolation Kit (Thermo Fisher Scientific, USA). Extracted TNA were screened by reverse-transcription quantitative PCR (RT-qPCR) for six common viruses infecting roses, including prunus necrotic ringspot virus (PNRSV), apple mosaic virus (ApMV), rose spring dwarf associated virus (RSDaV), rose yellow vein virus (RYVV), rose rosette virus (RRV), and blackberry chlorotic ringspot virus (BCRV); however, the results were negative. Therefore, the sample was subjected to high throughput sequencing (HTS). Briefly, TNA was depleted of rRNA and advanced for cDNA library preparation using TruSeq Stranded Total RNA kit (Illumina, USA). HTS was performed on Illumina NextSeq 500 platform. The raw reads were trimmed, de novo assembled, and subsequently were annotated using tBLASTx algorithm (Al Rwahnih et al. 2018). HTS generated 23.6 million 75 nucleotide (nt) single-end raw data reads. De novo assembly generated a contig (16,528 nts) resembling RLRaV reference sequence (KJ748003) with 74% identity at the nucleotide level. Putative coat protein and heat shock protein 70-like protein were identified based on >90% identity with RLRaV genes. To confirm HTS results, RT-PCR was performed using two primer sets, 1) Clo-F4916 (5'-GGTGTTCCAACGCTATCGTG-3') and Clo-R5215 (5'- TGTCCTCAAACCGCCTACAT-3') targeting nucleotide sequences of putative polyprotein 1a, and 2) Clo-F10006 (5'-GATTCCGCGGACGAATTAAT-3') and Clo-R10311 (5'-GGTAACCGAAAGGTAAAGTATTC-3') targeting nucleotide sequences of putative protein p25. The RLRaV amplicons with expected size of 300 nt were confirmed using bidirectional Sanger sequencing. The near-complete sequence of the new RLRaV isolate was deposited in GenBank under accession number MW056181. In addition, HTS analysis showed that RLRaV was in mixed infection with two mycoviruses (rose cryptic virus with 8,267 mapped reads and rose partitivirus with 7,283 mapped readss). To our knowledge, this is the first report of RLRaV affecting roses in California. Further research is needed to determine the prevalence of RLRaV in California as well as evaluation of RLRaV effect on rose performance.