Sensitive and Visual Detection of Brassica Yellows Virus Using Reverse Transcription Loop-Mediated Isothermal Amplification-Coupled CRISPR-Cas12 Assay.

Brassica yellows virus (BrYV) is an economically important virus on cruciferous species. In this study, a one-pot reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system was developed for the detection of BrYV. The limit of detection of this method reached 32.8 copies of the BrYV ORF5, which is 100-fold more sensitive than the RT-LAMP method. Moreover, there was no cross-reactivity with other rapeseed-infecting RNA viruses or poleroviruses. We dried the CRISPR/Cas12a reagent in a trehalose and pullulan mixture to retain its efficacy at the RT-LAMP temperature of 63°C in order to allow portable BrYV detection in a water bath. The entire process can be performed in about 1 h, and a positive result can be rapidly and conveniently detected using a handheld UV lamp. In the field, the RT-LAMP-CRISPR/Cas12a assay was accurate and had higher sensitivity than RT-LAMP and reverse transcription-polymerase chain reaction assays. The novel RT-LAMP-CRISPR/Cas12a assay allows convenient, portable, rapid, low-cost, highly sensitive, and specific detection of BrYV and has great potential for on-site monitoring of BrYV.

One-Step Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Detection of Canna Yellow Streak Virus.

Canna yellow streak virus (CaYSV) is a potyvirus that causes severe damage to the ornamental plant canna in the United Kingdom and Brazil. Here, we identified CaYSV in China by isolating total RNA from an infected plant, amplifying the virus genome segments, and cloning and sequencing the amplicons. After assembly, the full-length genome of the virus was obtained and uploaded to the NCBI database. Phylogenetic analysis results showed that the Guizhou isolate (OL546222) was most closely related to the KS isolate (MG545919.1). Virus detection is essential for virus disease control but the subclinical infection of CaYSV on canna in its early development increases the difficulty of CaYSV diagnosis. The goal of this study was to develop an efficient method for detection of CaYSV. We designed the primers, optimized the reaction conditions, and finally established a one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method. The product of RT-LAMP can be analyzed by both agarose gel electrophoresis and visible color change. The established one-step RT-LAMP assay showed high specificity and sensitivity in detecting CaYSV. This RT-LAMP method was also applied in analysis of 61 field samples collected from Guizhou and Jiangsu Provinces. The results showed that the infection rates of CaYSV on canna samples from these two provinces were very high (63 and 96% respectively).

Molecular Characterization of a Novel Polerovirus Infecting Soybean in China.

Poleroviruses are positive-sense, single-stranded viruses. In this study, we describe the identification of a novel polerovirus isolated from soybean displaying curled leaves. The complete viral genome sequence was identified using high-throughput sequencing and confirmed using rapid amplification of cDNA ends (RACE), RT-PCR and Sanger sequencing. Its genome organization is typical of the members of genus Polerovirus, containing seven putative open reading frames (ORFs). The full genome is composed of single-stranded RNA of 5822 nucleotides in length, with the highest nucleotide sequence identity (79.07% with 63% coverage) for cowpea polerovirus 2 (CPPV2). Amino acid sequence identities of the protein products between the virus and its relatives are below the threshold determined by the International Committee of Taxonomy of Viruses (ICTV) for species demarcation, and this strongly supports this virus' status as a novel species, for which the name soybean chlorotic leafroll virus (SbCLRV) is proposed. Recombination analysis identified a recombination event in the ORF5 of the 3' portion in the genome. Phylogenetic analyses of the genome and encoded protein sequences revealed that the new virus is closely related to phasey bean mild yellows virus, CPPV2 and siratro latent polerovirus. Subsequently, we demonstrated the infectivity of SbCLRV in Nicotiana benthamiana via infectious cDNA clone generation and agroinoculation.

Identification and genome analysis of a tomato zonate spot virus isolate from Bidens pilosa.

Bidens pilosa is a weed species that invades crop areas in tropical and subtropical regions. To date, only two potyviruses have been reported to infect B. pilosa. Here, we report the complete genome sequence of a tomato zonate spot tospovirus (TZSV) isolate from Bidens named TZSV-Bidens. The tripartite RNA of the TZSV-Bidens genome contains L, M, and S segments that are 8912, 4724, and 2997 nt in length, respectively. The genome contains five open reading frames (ORFs), with 92.23-95.01% amino acid sequence identity to the TZSV-YN isolate. Phylogenetic analysis based on amino acid sequences of members of the family Tospoviridae showed that TZSV-Bidens was grouped into a well-supported Eurasian cluster. The intergenic regions (IGRs) of the M and S RNAs are among the most variable regions and are far shorter than those of the TZSV-YN reference genome.

The Antifungal Effects of Citral on Magnaporthe oryzae Occur via Modulation of Chitin Content as Revealed by RNA-Seq Analysis.

The natural product citral has previously been demonstrated to possess antifungal activity against Magnaporthe oryzae. The purpose of this study was to screen and annotate genes that were differentially expressed (DEGs) in M. oryzae after treatment with citral using RNA sequencing (RNA-seq). Thereafter, samples were reprepared for quantitative real-time PCR (RT-qPCR) analysis verification of RNA-seq data. The results showed that 649 DEGs in M. oryzae were significantly affected after treatment with citral (100 µg/mL) for 24 h. Kyoto Encyclopedia of Genes and Genomes (KEGG) and a gene ontology (GO) analysis showed that DEGs were mainly enriched in amino sugar and nucleotide sugar metabolic pathways, including the chitin synthesis pathway and UDP sugar synthesis pathway. The results of the RT-qPCR analysis also showed that the chitin present in M. oryzae might be degraded to chitosan, chitobiose, N-acetyl-D-glucosamine, and ß-D-fructose-6-phosphate following treatment with citral. Chitin degradation was indicated by damaged cell-wall integrity. Moreover, the UDP glucose synthesis pathway was involved in glycolysis and gluconeogenesis, providing precursors for the synthesis of polysaccharides. Galactose-1-phosphate uridylyltransferase, which is involved in the regulation of UDP-α-D-galactose and α-D-galactose-1-phosphate, was downregulated. This would result in the inhibition of UDP glucose (UDP-Glc) synthesis, a reduction in cell-wall glucan content, and the destruction of cell-wall integrity.

Characterization of maize translational responses to sugarcane mosaic virus infection.

Sugarcane mosaic virus (SCMV) frequently causes dramatic losses in maize production as the main pathogen of maize dwarf mosaic disease. It is important to understand the translational responses in maize to SCMV infection since viruses have to recruit host translation apparatus to express their proteins. However, due to technical limitations, research on virus translation lags far behind that on transcription. Here, we studied the relationship between systemic symptom expression and virus accumulation and found that both SCMV RNA and proteins accumulated rapidly during the systemic infection process in which varying degrees of chlorosis to mosaic symptoms developed on non-inoculated leaves. In addition, we applied ribosome profiling, which couples polysomal mRNA isolation with high-throughput sequencing, on the symptomatic leaves infected with SCMV to unravel the translational responses of maize to viral infection on a genome-wide scale. The results showed that only the genomic positive-stranded RNA of SCMV was involved in translation, and SCMV only occupied a small amount of translational resources of host plant at the early stage of infection. Further analyses on a global gene expression and gene ontology (GO) enrichment revealed that photosynthesis and metabolism were dramatically repressed at both transcriptional and translational levels. Altogether, our results laid a foundation for dissecting the molecular mechanism of plant translational responses to viral infection.

Virus-Derived Small Interfering RNAs Affect the Accumulations of Viral and Host Transcripts in Maize.

RNA silencing is a conserved surveillance mechanism against invading viruses in plants, which involves the production of virus-derived small interfering RNAs (vsiRNAs) that play essential roles in the silencing of viral RNAs and/or specific host transcripts. However, how vsiRNAs function to target viral and/or host transcripts is poorly studied, especially in maize (Zea mays L.). In this study, a degradome library constructed from Sugarcane mosaic virus (SCMV)-inoculated maize plants was analyzed to identify the cleavage sites in viral and host transcripts mainly produced by vsiRNAs. The results showed that 42 maize transcripts were possibly cleaved by vsiRNAs, among which several were involved in chloroplast functions and in biotic and abiotic stresses. In addition, more than 3000 cleavage sites possibly produced by vsiRNAs were identified in positive-strand RNAs of SCMV, while there were only four cleavage sites in the negative-strand RNAs. To determine the roles of vsiRNAs in targeting viral RNAs, six vsiRNAs were expressed in maize protoplast based on artificial microRNAs (amiRNAs), of which four could efficiently inhibit the accumulations of SCMV RNAs. These results provide new insights into the genetic manipulation of maize with resistance against virus infection by using amiRNA as a more predictable and useful approach.