Identification and Functional Analysis of Four RNA Silencing Suppressors in Begomovirus Croton Yellow Vein Mosaic Virus

Croton yellow vein mosaic virus (CYVMV), a species in the genus Begomovirus, is a prolific monopartite begomovirus in the Indian sub-continent. CYVMV infects multiple crop plants to cause leaf curl disease. Plants have developed host RNA silencing mechanisms to defend the threat of viruses, including CYVMV. We characterized four RNA silencing suppressors, namely, V2, C2, and C4 encoded by CYVMV and betasatellite-encoded C1 protein (βC1) encoded by the cognate betasatellite, croton yellow vein betasatellite (CroYVMB). Their silencing suppressor functions were verified by the ability of restoring the β-glucuronidase (GUS) activity suppressed by RNA silencing. We showed here for the first time that V2 was capable of self-interacting, as well as interacting with the V1 protein, and could be translocalized to the plasmodesmata in the presence of CYVMV. The knockout of either V2 or V1 impaired the intercellular mobility of CYVMV, indicating their novel coordinated roles in the cell-to-cell movement of the virus. As pathogenicity determinants, each of V2, C2, and C4 could induce typical leaf curl symptoms in Nicotiana benthamiana plants even under transient expression. Interestingly, the transcripts and proteins of all four suppressors could be detected in the systemically infected leaves with no correlation to symptom induction. Overall, our work identifies four silencing suppressors encoded by CYVMV and its cognate betasatellite and reveals their subcellular localizations, interaction behavior, and roles in symptom induction and intercellular virus movement.

The Titer of Citrus Yellow Vein Clearing Virus is Positively Associated with the Severity of Symptoms in Infected Citrus Seedlings

Citrus yellow vein clearing virus is a new member of the genus Mandarivirus in the family Alphaflexiviridae. Citrus yellow vein clearing virus (CYVCV) is the causal agent of citrus yellow vein clearing disease and is widely distributed in Pakistan, India, Turkey, and China. CYVCV is transmitted from citrus to citrus by Dialeurodes citri, grafting, and contaminated knife blades, threatening citrus production. In this study, four infectious full-length cDNA clones of CYVCV (namely AY112, AY132, AY212, and AY221) derived from CYVCV isolate AY were obtained through yeast homologous recombination and inoculated to ‘Eureka’ lemon (Citrus limon Burm. f.) by Agrobacterium-mediated vacuum infiltration. Pathogenicity analysis indicated that the clones AY212 and AY221 caused more severe symptoms than AY112 and AY132. Northern blot and quantitative reverse transcription PCR (qRT-PCR) analyses showed that the titers of virulent clones (AY212 and AY221) were significantly higher than those of attenuated clones (AY112 and AY132) in the infected ‘Eureka’ lemon (Citrus limon Burm. f.) seedlings. Subsequent comparative studies of viral infectivity, accumulation, and symptoms induced by AY221 in nine citrus cultivars indicated that (i) the infectivity of AY221 varied from 25% to 100% among different cultivars; (ii) ‘Oota’ ponkan (C. reticulata L.) showed the lowest infection rate with mild symptoms, which might be a useful resource for CYVCY-resistance genes; (iii) CYVCV titer was positively associated with the symptom development in infected citrus seedlings. In general, this report revealed the biological properties of CYVCV, thus laying a foundation for further investigation of pathogenic mechanisms in this virus.

Decision Strategies for Absorbance Readings from an Enzyme-Linked Immunosorbent Assay—A Case Study about Testing Genotypes of Sugar Beet (Beta vulgaris L.) for Resistance against Beet Necrotic Yellow Vein Virus (BNYVV)

The Beet necrotic yellow vein virus (BNYVV) causes rhizomania in sugar beet (Beta vulgaris L.), which is one of the most destructive diseases in sugar beet worldwide. In breeding projects towards resistance against BNYVV, the enzyme-linked immunosorbent assay (ELISA) is used to determine the virus concentration in plant roots and, thus, the resistance levels of genotypes. Here, we present a simulation study to generate 10,000 small samples from the estimated density functions of ELISA values from susceptible and resistant sugar beet genotypes. We apply receiver operating characteristic (ROC) analysis to these samples to optimise the cutoff values for sample sizes from two to eight and determine the false positive rates (FPR), true positive rates (TPR), and area under the curve (AUC). We present, furthermore, an alternative approach based upon Bayes factors to improve the decision procedure. The Bayesian approach has proven to be superior to the simple cutoff approach. The presented results could help evaluate or improve existing breeding programs and help design future selection procedures based upon ELISA. An R-script for the classification of sample data based upon Bayes factors is provided.

First report of clover yellow vein virus on orchid (Dendrobium sp.) in South Korea

Orchid is one of the most popular and commercially important cultivated flowers in the world. Among many orchid species, Dendrobium species are popular cut flowers and potted plants in South Korea. In March 2019, 10 Dendrobium orchid plants in a greenhouse in Daegu, South Korea showed large chlorotic blotches, mosaic and mottle symptoms. One leaf each from the 10 symptomatic orchid plants by leaf dip-preparations and transmission electron microscopy (JEM-1400; JEOL Inc., Tokyo, Japan) after leaf dip-preparations (Brenner and Horne 1959; Richert-Pöggeler et al. 2019). Typical potyvirus-like particles of flexuous and filamentous shape and ∼ 760 × 15 nm length/width were observed in all tested samples. The presence of potyvirus was confirmed by serological detection with a commercially available ImmunoStrip® for potyvirus group (Agdia, Elkhart, USA). In contrast, a negative result was obtained for a virus-free Dendrobium plant by the serological test. The two most common viruses in orchids, namely odontoglossum ringspot virus (ORSV) and cymbidium mosaic virus (CymMV) in all Dendrobium samples were not detected in any samples by an ImmunoStrip® for ORSV and CymMV (Agdia, Elkhart, USA). To determine the species of the virus, total RNA was extracted from all 10 ImmunoStrip®-positive samples using the RNeasy plant mini kit (Qiagen, Hilden, Germany). Subsequently, reverse transcription-PCR (RT-PCR) products (~1,625 bp) were amplified using potyvirus- specific primer pair (Gibbs and Mackenzie, 1997) and sequenced by the Sanger method at Macrogen (Seoul, South Korea). Sequencing results showed 100% nucleotide identity among 10 samples. Thus, one sequence was chosen for identification of virus species using sequence comparison. BLASTn analysis showed that the nucleotide sequence and its deduced amino acid sequence of the amplicon shared 95.4-98.7% and 96.2-99.6% identity to multiple clover yellow vein virus (ClYVV) sequences (e.g., accession no. AB011819) in GenBank. To further confirm the presence of ClYVV and determine if other viral agents were present in the samples, total RNA from three of the 10 symptomatic plants was depleted of ribosomal RNAs and subjected to high-throughput sequencing (HTS) analysis on a HiSeq 4000 platform (Macrogen Inc., Seoul, South Korea). A total of 3,764,432, 4,203,881, and 4,139,775 of 150-bp paired-end clean reads were obtained for the three samples. After de novo assembly of the reads with Trinity (Haas et al. 2013), 5, 6 and 7 contigs were obtained and searched with BLASTn against NCBI viral refseq database. Eighteen contigs from all three samples sized at 2,176-9,432 nt exhibited 94.0-97.9% nucleotide identity with the complete genome sequences of other ClYVV isolates (e.g., accession no. AB011819) deposited in Genbank; no other viruses were identified by HTS. The complete genome sequence (9,585 nucleotides in length) of ClYVV Dendrobium isolate (ClYVV-Den) was determined using ClYVV-specific primers (Takahashi et al., 1997) and the sequence of CIYVV-Den was deposited to GenBank (Accession no. LC506604). Together, these results support that symptomatic Dendrobium orchids were infected with ClYVV-Den in this study. ClYVV has been previously reported affecting Calanthe orchids in Japan (Inouye et al., 1988; Ikegami et al., 1995). Our results suggest that ClYVV may be detrimental to the production of Dendrobium orchids or commercial ornamental crops in South Korea. To our knowledge, this is the first report of ClYVV in Dendrobium sp. in South Korea.

Molecular evolution and genetic analysis of Mesta yellow vein mosaic virus and associated betasatellites

Mesta yellow vein mosaic disease (MYVMD), one of the major diseases circulating mesta growing regions of Indian sub-continent, is responsible for serious yield loss in mesta crops. A complex of monopartite begomovirus, Mesta yellow vein mosaic virus (MYVMV) and associated betasatellite, is reported in several studies as the causal agent of MYVMD. However, all-inclusive molecular evolutionary analysis of so far available MYVMVs and associated betasatellites disseminating in this region is still lacking. In this study, by estimating and analyzing various indexes of population genetics and evolutionary parameters, we discussed the sources of genetic variations, population dynamics and different forces acting on the evolution of MYVMVs and associated betasatellites. The study finds recombination as a vital force in the evolution and diversification of begomovirus complexes in different geographic locations however, betasatellites were found to be exposed to more diverse recombination events compared to MYVMVs. Indian isolates are reported to have high frequency of polymorphism in this study which suggests a balancing selection or expansion occurring in Indian populations of begomoviruses. Higher degree of genetic differentiation and lower rate of gene flow calculated between the viral populations of Bangladesh and Pakistan is justified by the relatively far geographical distance between these two countries. Although the study detects overall purifying selection, the degrees of constraints acting on individual gene tested are found different. Coat protein (AV1) is estimated with very high nucleotide substitution rate which is very likely to result from the strongest purifying selection pressure (dN/dS = 0.131) calculated in this study on coat protein. The findings of this study on different evolutionary forces that shape the emergence and diversification of MYVMVs and associated betasatellites may provide directions towards future evolutionary trend analysis and development of comprehensive disease control strategies for begomoviruses.