Beet Soil-Borne Virus Is a Helper Virus for the Novel Beta vulgaris Satellite Virus 1A.

Sugar beet (Beta vulgaris) is grown in temperate regions around the world as a source of sucrose used for natural sweetening. Sugar beet is susceptible to a number of viral diseases, but identification of the causal agent(s) under field conditions is often difficult due to mixtures of viruses that may be responsible for disease symptoms. In this study, the application of RNAseq to RNA extracted from diseased sugar beet roots obtained from the field and from greenhouse-reared plants grown in soil infested with the virus disease rhizomania (causal agent beet necrotic yellow vein virus; BNYVV) yielded genome-length sequences from BNYVV, as well as beet soil-borne virus (BSBV). The nucleotide identities of the derived consensus sequence of BSBV RNAs ranged from 99.4 to 96.7% (RNA1), 99.3 to 95.3% (RNA2), and 98.3 to 95.9% (RNA3) compared with published BSBV sequences. Based on the BSBV genome consensus sequence, clones of the genomic RNAs 1, 2, and 3 were obtained to produce RNA copies of the genome through in vitro transcription. Capped RNA produced from the clones was infectious when inoculated into leaves of Chenopodium quinoa and B. vulgaris, and extracts from transcript-infected C. quinoa leaves could infect sugar beet seedling roots through a vortex inoculation method. Subsequent exposure of these infected sugar beet seedling roots to aviruliferous Polymyxa betae, the protist vector of both BNYVV and BSBV, confirmed that BSBV derived from the infectious clones could be transmitted by the vector. Co-inoculation of BSBV synthetic transcripts with transcripts of a cloned putative satellite virus designated Beta vulgaris satellite virus 1A (BvSat1A) resulted in the production of lesions on leaves of C. quinoa similar to those produced by inoculation with BSBV alone. Nevertheless, accumulation of genomic RNA and the encoded protein of the satellite virus in co-inoculated leaves was readily detected on Northern and Western blots, respectively, whereas no accumulation of satellite virus products occurred when satellite virus RNA was inoculated alone. The predicted sequence of the detected protein encoded by BvSat1A bears hallmarks of coat proteins of other satellite viruses, and virions of a size consistent with a satellite virus were observed in samples testing positive for the virus. The results demonstrate that BSBV is a helper virus for the novel satellite virus BvSat1A.

First report of Pepper yellow dwarf strain of Beet curly top virus and Spinach curly top Arizona virus in red table beet in Idaho, United States.

In the fall 2021, red table beet plants (Beta vulgaris L. cv 'Eagle') exhibiting stunted growth with shorter petioles were observed at an incidence of 10 to 15 percent in a production field in Payette County, Idaho, United States. In addition to stunting, beet leaves displayed yellowing and mild curling and crumpling, and the roots exhibited hairy root symptoms (sFig.1). To identify potential causal viruses, total RNA was isolated from the leaf and root tissue using RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and subjected to high-throughput sequencing (HTS). Two libraries were prepared, one for the leaf sample and another for the root sample using a ribo-minus TruSeq Stranded Total RNA Library Prep kit (Illumina, San Diego, CA). HTS was performed with 150 bp paired-end sequencing on a NovaSeq 6000 (Novogene, Sacramento, CA). Following adapter trimming and removal of host transcripts, 5.9 and 16.2 million reads were obtained from the leaf and root samples, respectively. These reads were de novo assembled using the SPAdes assembler (Bankevitch et al., 2012; Prjibelski et al., 2020). The assembled leaf sample contigs were aligned to the NCBI non-redundant database to identify contigs matching known viruses. A single contig of 2845 nts that shared 96% coverage and 95.6% sequence identity to the pepper yellow dwarf strain of beet curly top virus (BCTV-PeYD, EU921828; Varsani et al., 2014), and 98% coverage and 98.39% identity with an isolate of BCTV-PeYD (KX529650) from Mexico, was identified in the leaf sample (GenBank Accession OP477336). To validate the HTS detection of BCTV-PeYD, total DNA was isolated from the leaf sample and a 454 bp fragment of the C1 gene (replication-associate protein) was PCR amplified and Sanger sequencing of the amplicon revealed 99.7% identity to the HTS assembled BCTV-PeYD sequence. In addition to the PeYD strain of BCTV, the Worland strain of BCTV (BCTV-Wor) was detected as a single 2930 nt contig with 100% coverage and 97.3% identity to the BCTV-Wor isolate CTS14-015 (KX867045) known to infect sugar beet in Idaho. Of note, there are 11 strains of BCTV and among those, the BCTV-Wor strain induces mild symptoms in sugar beet (Strausbaugh et al., 2017), whereas BCTV-PeYD was found only in pepper from New Mexico. Further, two contigs of 2201 nts and 523 nts were assembled generating a nearly complete genome of spinach curly top Arizona virus (SpCTAV) in the leaf sample with 99% coverage and 99.3% identity (GenBank Accession OQ703946) to the reference genome of SpCTAV (HQ443515; Hernandez-Zepeda et al., 2013). To validate the HTS results, total DNA was isolated from the leaf tissue and PCR amplified a 442 bp fragment that overlaps the V1, V2, and V3 ORFs and its sequence revealed 100% identity with the HTS assembled SpCTAV. The roots sample also showed HTS reads corresponding to BCTV-PeYD and SpCTAV. In addition, beet necrotic yellow vein virus (BNYVV) was detected in the root sample with 30% coverage, but no sequence reads matching to BNYVV was detected in the leaf sample. BNYVV is known to infect sugar beet causing rhizomania (Tamada et al., 1973; Schirmer et al., 2005). To further confirm the BNYVV HTS results, total RNA was extracted separately from the root and leaf tissue, and RT-PCR was performed with primers that were designed to amplify portions of BNYVV RNAs (Weiland et al., 2020). RT-PCR analysis generated the appropriate amplicons with expected sequences corresponding to the RNA-1, RNA-2, RNA-3, and RNA-4 of BNYVV as determined by Sanger sequencing implying BNYVV the causal agent of hairy root symptoms. Similar to observations seen for BNYVV infection in conventional sugar beet varieties, no amplification was detected for BNYVV in the RNA extracted from leaf tissue, indicating that the RT-PCR results are consistent with the HTS analysis. This is the first report of BCTV-PeYD and SpCTAV observed naturally infecting red table beet in Idaho suggesting the geographical expansion of these viruses. The co-existence of BCTV-PeYD and SpCTAV with limited host range needs to be investigated to determine the actual cause of the observed foliar symptoms. This report provides the basis for further research to understand the pathogenic nature of these viruses and their potential threat to red table beet and sugar beet production in Idaho.

First report of Tomato bushy stunt virus naturally infecting sugar beet in the United States.

Sugar beet (Beta vulgaris L.) is an important crop grown for its sucrose content used in sugar production around the world. Tomato bushy stunt virus (TBSV) is an RNA virus that belongs to the Tombusvirus genus of the family Tombusviridae (Hearne et al., 1990). The virus was first isolated from tomato, and it is known to infect a wide range of plants (Smith, 1935; Martelli et al., 1988; Hafez et al., 2010). In 1980, a natural infection of TBSV was reported in sugar beet leaves with chlorotic and necrotic ring spots and line pattern symptoms based on serological affinity to TBSV anti-sera in Czechoslovakia (Novak and Lanzova, 1980). In March 2021, sugarbeet plants showing stunted and bushy growth with yellowing and necrotic leaves were observed in a production field in the Imperial Valley of California. Harvested roots exhibited stunted and abnormal growth compared to roots from healthy plants (sFig. 1A). These symptoms prompted a screen for potential infection by TBSV. Root-tissue harvested from the symptomatic sugar beet was initially screened using a TBSV double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA; Agdia, Inc., Elkhart, IN), which reacted positive for TBSV. To obtain the full-length sequence of TBSV and potentially other viruses in the sample, total RNA isolated using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) from the root-tissue was subjected to high-throughput sequencing (HTS). Libraries were prepared using the TruSeq Stranded Total RNA Library Prep kit (Illumina, San Diego, CA) and sequenced using Illumina NovoSeq 6000 paired-end platform (Novogene, Sacramento, CA). A total of 52 million reads were obtained after removing the adapters and reads mapping to the host genome. These high-quality reads were de novo assembled into 75,891 contigs that are larger than 500 base pairs using the SPAdes assembler (Bankevitch et al., 2012; Prjibelski et al., 2020). The resulting contigs were searched for matching sequences to known viruses using the NCBI non-redundant database. A single contig of 4770 nts representing the full-length genome of TBSV was generated (Accession number OP477335), which showed 100% coverage to previously reported TBSV isolates 'statice' (AJ249740.1) and 'nipplefruit' (AY579432.1) with 92.19% and 91.25% nucleotide sequence identities, respectively, and thus confirming the presence of TBSV in sugar beet root-tissue. However, it showed 74% coverage with only 87% nucleotide identity to a previously reported Lettuce necrotic stunt virus (LNSV) from sugar beet, a tombusvirus that was re-classified as Moroccan pepper virus (MPV) due to high degree (>97%) of sequence identity (Obermeier et al., 2001; Wintermantel and Anchieta, 2012; Wintermantel and Hladky, 2013). The coat protein is conserved within species in tombusvirus, and it plays a significant role by providing serological relationships to tombusvirus taxonomy. The coat protein of TBSV-isolate of this study shared 98.45% and 96.91% identities at amino acid level with TBSV 'nipplefruit' (AY579432.1) and TBSV 'statice' (AJ249740.1) isolates, respectively. In contrast, it showed only 61.56% identity with the coat protein of MPV as shown in the phylogenetic tree indicating that the TBSV-isolate reported here is different from MPV (sFig. 2). To confirm the presence of TBSV, reverse-transcription (RT)-PCR was performed using the total RNA isolated from the root-tissue with primers (VR306: 5'-CGCTCACGAGCCCAGCATCCTTGA-3' and VR297: 5'-ACACCGCCACAGGAGCCATGATTG-3') designed based on the HTS data to amplify a portion of the TBSV genome. Sequencing of the RT-PCR product confirmed the presence of TBSV sequence with 99.1% identity to the TBSV-isolate identified in this study. Further, mechanical inoculation of total RNA isolated from the symptomatic sugar beet roots produced local lesions and systemic necrosis symptoms on the leaves of Chenopodium quinoa (sFig. 1B). Sequencing of the amplicon obtained using RT-PCR with primers VR306 and VR297 confirmed the presence of TBSV in C. quinoa. In addition to TBSV, several viral contigs representing Beet necrotic yellow vein virus were identified in the root-tissue indicating mixed infection in the field. To our knowledge, this is the first report that documents the occurrence of TBSV in sugar beet in the United States. Since TBSV is a soil-borne virus, our findings indicate the need for further studies focused on the frequency and coexistence of the TBSV with BNYVV in sugar beet production fields to understand the disease complexity resulting from potential mixed infections.

CRISPR-Based Isothermal Next-Generation Diagnostic Method for Virus Detection in Sugarbeet.

Rhizomania is a disease of sugarbeet caused by beet necrotic yellow vein virus (BNYVV) that significantly affects sugarbeet yield globally. Accurate and sensitive detection methods for BNYVV in plants and field soil are necessary for growers to make informed decisions on variety selection to manage this disease. A recently developed CRISPR-Cas-based detection method has proven highly sensitive and accurate in human virus diagnostics. Here, we report the development of a CRISPR-Cas12a-based method for detecting BNYVV in the roots of sugarbeet. A critical aspect of this technique is the identification of conditions for isothermal amplification of viral fragments. Toward this end, we have developed a reverse transcription (RT) recombinase polymerase amplification (RPA) for detecting BNYVV in sugarbeet roots. The RT-RPA product was visualized, and its sequence was confirmed. Subsequently, we designed and validated the cutting efficiency of guide RNA targeting BNYVV via in vitro activity assay in the presence of Cas12a. The sensitivity of CRISPR-Cas12a trans reporter-based detection for BNYVV was determined using a serially diluted synthetic BNYVV target sequence. Further, we have validated the developed CRISPR-Cas12a assay for detecting BNYVV in the root-tissue of sugarbeet bait plants reared in BNYVV-infested field soil. The results revealed that BNYVV detection is highly sensitive and specific to the infected roots relative to healthy control roots as measured quantitatively through the reporter signal. To our knowledge, this is the first report establishing isothermal RT-RPA- and CRISPR-based methods for virus diagnostic approaches for detecting BNYVV from rhizomania diseased sugarbeet roots.

Distinct and cooperative activities of HESO1 and URT1 nucleotidyl transferases in microRNA turnover in Arabidopsis.

3' uridylation is increasingly recognized as a conserved RNA modification process associated with RNA turnover in eukaryotes. 2'-O-methylation on the 3' terminal ribose protects micro(mi)RNAs from 3' truncation and 3' uridylation in Arabidopsis. Previously, we identified HESO1 as the nucleotidyl transferase that uridylates most unmethylated miRNAs in vivo, but substantial 3' tailing of miRNAs still remains in heso1 loss-of-function mutants. In this study, we found that among nine other potential nucleotidyl transferases, UTP:RNA uridylyltransferase 1 (URT1) is the single most predominant nucleotidyl transferase that tails miRNAs. URT1 and HESO1 prefer substrates with different 3' end nucleotides in vitro and act cooperatively to tail different forms of the same miRNAs in vivo. Moreover, both HESO1 and URT1 exhibit nucleotidyl transferase activity on AGO1-bound miRNAs. Although these enzymes are able to add long tails to AGO1-bound miRNAs, the tailed miRNAs remain associated with AGO1. Moreover, tailing of AGO1-bound miRNA165/6 drastically reduces the slicing activity of AGO1-miR165/6, suggesting that tailing reduces miRNA activity. However, monouridylation of miR171a by URT1 endows the miRNA the ability to trigger the biogenesis of secondary siRNAs. Therefore, 3' tailing could affect the activities of miRNAs in addition to leading to miRNA degradation.

The Arabidopsis nucleotidyl transferase HESO1 uridylates unmethylated small RNAs to trigger their degradation.

MicroRNAs (miRNAs), small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs) impact numerous biological processes in eukaryotes. In addition to biogenesis, turnover contributes to the steady-state levels of small RNAs. One major factor that stabilizes miRNAs and siRNAs in plants as well as siRNAs and piRNAs in animals is 2'-O-methylation on the 3' terminal ribose by the methyltransferase HUA ENHANCER1 (HEN1) [1-6]. Genetic studies with Arabidopsis, Drosophila, and zebrafish hen1 mutants show that 2'-O-methylation protects small RNAs from 3'-to-5' truncation and 3' uridylation, the addition of nontemplated nucleotides, predominantly uridine [2, 7, 8]. Uridylation is a widespread phenomenon that is not restricted to small RNAs in hen1 mutants and is often associated with their reduced accumulation ([7, 9, 10]; reviewed in [11]). The enzymes responsible for 3' uridylation of small RNAs when they lack methylation in plants or animals have remained elusive. Here, we identify the Arabidopsis HEN1 SUPPRESSOR1 (HESO1) gene as responsible for small RNA uridylation in hen1 mutants. HESO1 exhibits terminal nucleotidyl transferase activity, prefers uridine as the substrate nucleotide, and is completely inhibited by 2'-O-methylation. We show that uridylation leads to miRNA degradation, and the degradation is most likely through an enzyme that is distinct from that causing the 3' truncation in hen1 mutants.

Genome-wide analysis reveals rapid and dynamic changes in miRNA and siRNA sequence and expression during ovule and fiber development in allotetraploid cotton (Gossypium hirsutum L.).

BACKGROUND: Cotton fiber development undergoes rapid and dynamic changes in a single cell type, from fiber initiation, elongation, primary and secondary wall biosynthesis, to fiber maturation. Previous studies showed that cotton genes encoding putative MYB transcription factors and phytohormone responsive factors were induced during early stages of ovule and fiber development. Many of these factors are targets of microRNAs (miRNAs) that mediate target gene regulation by mRNA degradation or translational repression. RESULTS: Here we sequenced and analyzed over 4 million small RNAs derived from fiber and non-fiber tissues in cotton. The 24-nucleotide small interfering RNAs (siRNAs) were more abundant and highly enriched in ovules and fiber-bearing ovules relative to leaves. A total of 31 miRNA families, including 27 conserved, 4 novel miRNA families and a candidate-novel miRNA, were identified in at least one of the cotton tissues examined. Among 32 miRNA precursors representing 19 unique miRNA families identified, 7 were previously reported, and 25 new miRNA precursors were found in this study. Sequencing, miRNA microarray, and small RNA blot analyses showed a trend of repression of miRNAs, including novel miRNAs, during ovule and fiber development, which correlated with upregulation of several target genes tested. Moreover, 223 targets of cotton miRNAs were predicted from the expressed sequence tags derived from cotton tissues, including ovules and fibers. The cotton miRNAs examined triggered cleavage in the predicted sites of the putative cotton targets in ovules and fibers. CONCLUSIONS: Enrichment of siRNAs in ovules and fibers suggests active small RNA metabolism and chromatin modifications during fiber development, whereas general repression of miRNAs in fibers correlates with upregulation of a dozen validated miRNA targets encoding transcription and phytohormone response factors, including the genes found to be highly expressed in cotton fibers. Rapid and dynamic changes in siRNAs and miRNAs may contribute to ovule and fiber development in allotetraploid cotton.

Small RNAs serve as a genetic buffer against genomic shock in Arabidopsis interspecific hybrids and allopolyploids.

Small RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs), and trans-acting siRNAs (tasiRNAs), control gene expression and epigenetic regulation. Although the roles of miRNAs and siRNAs have been extensively studied, their expression diversity and evolution in closely related species and interspecific hybrids are poorly understood. Here, we show comprehensive analyses of miRNA expression and siRNA distributions in two closely related species Arabidopsis thaliana and Arabidopsis arenosa, a natural allotetraploid Arabidopsis suecica, and two resynthesized allotetraploid lines (F(1) and F(7)) derived from A. thaliana and A. arenosa. We found that repeat- and transposon-associated siRNAs were highly divergent between A. thaliana and A. arenosa. A. thaliana siRNA populations underwent rapid changes in F(1) but were stably maintained in F(7) and A. suecica. The correlation between siRNAs and nonadditive gene expression in allopolyploids is insignificant. In contrast, miRNA and tasiRNA sequences were conserved between species, but their expression patterns were highly variable between the allotetraploids and their progenitors. Many miRNAs tested were nonadditively expressed (deviating from the mid-parent value, MPV) in the allotetraploids and triggered unequal degradation of A. thaliana or A. arenosa targets. The data suggest that small RNAs produced during interspecific hybridization or polyploidization serve as a buffer against the genomic shock in interspecific hybrids and allopolyploids: Stable inheritance of repeat-associated siRNAs maintains chromatin and genome stability, whereas expression variation of miRNAs leads to changes in gene expression, growth vigor, and adaptation.

Degradation of microRNAs by a family of exoribonucleases in Arabidopsis.

microRNAs (miRNAs) play crucial roles in numerous developmental and metabolic processes in plants and animals. The steady-state levels of miRNAs need to be properly controlled to ensure normal development. Whereas the framework of miRNA biogenesis is established, factors involved in miRNA degradation remain unknown. Here, we show that a family of exoribonucleases encoded by the SMALL RNA DEGRADING NUCLEASE (SDN) genes degrades mature miRNAs in Arabidopsis. SDN1 acts specifically on single-stranded miRNAs in vitro and is sensitive to the 2'-O-methyl modification on the 3' terminal ribose of miRNAs. Simultaneous knockdown of three SDN genes in vivo results in elevated miRNA levels and pleiotropic developmental defects. Therefore, we have uncovered the enzymes that degrade miRNAs and demonstrated that miRNA turnover is crucial for plant development.

The FHA domain proteins DAWDLE in Arabidopsis and SNIP1 in humans act in small RNA biogenesis.

Proteins containing the forkhead-associated domain (FHA) are known to act in biological processes such as DNA damage repair, protein degradation, and signal transduction. Here we report that DAWDLE (DDL), an FHA domain-containing protein in Arabidopsis, acts in the biogenesis of miRNAs and endogenous siRNAs. Unlike mutants of genes known to participate in the processing of miRNA precursors, such as dcl1, hyponastic leaves1, and serrate, ddl mutants show reduced levels of pri-miRNAs as well as mature miRNAs. Promoter activity of MIR genes, however, is not affected by ddl mutations. DDL is an RNA binding protein and is able to interact with DCL1. In addition, we found that SNIP1, the human homolog of DDL, is involved in miRNA biogenesis and interacts with Drosha. Therefore, we uncovered an evolutionarily conserved factor in miRNA biogenesis. We propose that DDL participates in miRNA biogenesis by facilitating DCL1 to access or recognize pri-miRNAs.

Small RNA metabolism in Arabidopsis.

The Arabidopsis genome encodes two major classes of 20-24-nucleotide riboregulators: microRNAs and small interfering RNAs. These small RNAs act as sequence-specific repressors of target gene expression, either at the transcriptional level through DNA and/or histone methylation or at the post-transcriptional level through transcript cleavage or translational inhibition. Small RNAs are processed from precursor RNAs by one or more of the four DICER-LIKE RNase III enzymes, modified by HUA ENHANCER 1, a small RNA methyltransferase, and loaded onto an argonaute protein-containing RNA-induced silencing complex. Here, we review the biogenesis of small RNAs, and we discuss the major outstanding questions in small RNA metabolism and function.