A High Throughput Soybean Gene Identification System Developed using Soybean Yellow Common Mosaic Virus (SYCMV)

Millet is a dangerous weed in crop fields. A lack of seed dormancy helps it to spread easily and be present in maize, wheat, and other crop fields. Our previous report revealed the possibility that millet can also play a role as a virus reservoir. In that study, we focused on visual symptoms and detected the presence of several viruses in millet using serological methods, which can only detect the presence of the investigated pathogen. In this current work, we used small RNA high-throughput sequencing as an unbiased virus diagnostic method to uncover presenting viruses in randomly sampled millet grown as a volunteer weed in two maize fields, showing stunting, chlorosis, and striped leaves. Our results confirmed the widespread presence of wheat streak mosaic virus at both locations. Moreover, barley yellow striate mosaic virus and barley virus G, neither of which had been previously described in Hungary, were also identified. As these viruses can cause severe diseases in wheat and other cereals, their presence in a weed implies a potential infection risk. Our study indicates that the presence of millet in fields requires special control to prevent the emergence of new viral diseases in crop fields.

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A cassava protoplast system for screening genes associated with the response to South African cassava mosaic virus

Virology Journal ◽

10.1186/s12985-020-01453-4 ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

Patience Chatukuta ◽

Marie Emma Christine Rey

Keyword(s):

Gene Expression ◽

Tissue Culture ◽

Mosaic Virus ◽

South African ◽

High Throughput ◽

E3 Ligase ◽

Turnaround Time ◽

African Cassava Mosaic Virus ◽

Likelihood Method ◽

Cassava Mosaic Virus

Abstract Background The study of transient gene expression in cassava plants during virus infection using existing protocols is laborious and may take approximately fifteen weeks due to cassava’s recalcitrance to transformation. The combination of a protoplast system with CRISPR-mediated gene editing promises to shorten the turnaround time from plant tissue culture to high-throughput gene expression screening for candidate genes. Here, we detail a protocol for screening genes associated with the response to South African cassava mosaic virus (SACMV) in cassava protoplasts, with reference to the ubiquitin E3 ligase gene, MeE3L. Methods Cassava protoplasts of model, and SACMV-susceptible and -tolerant genotypes, were transformed with SACMV infectious clones and/or a CRISPR-editing construct targeting the MeE3L using PEG4000-mediated transfection. DNA and RNA were extracted from transformed protoplasts at 24 h post-transfection. Relative SACMV DNA accumulation was determined via qPCR using DpnI-digested total DNA, MeE3L relative expression was determined via reverse transcriptase qPCR, and results were analysed using one-way ANOVA, Tukey’s HSD test and the 2−ΔΔCTstatistical method. The MeE3L exonic region was sequenced on the ABI 3500XL Genetic Analyzer platform; and sequences were analysed for mutations using MAFTT and MEGA-X software. Construction of a phylogenetic tree was done using the Maximum Likelihood method and Jones-Taylor-Thornton (JTT) matrix-based model. Results The differential expression of unedited and mutant MeE3L during SACMV infection of model, susceptible and tolerant cassava protoplasts was determined within 7 weeks after commencement of tissue culture. The study also revealed that SACMV DNA accumulation in cassava protoplasts is genotype-dependent and induces multiple mutations in the tolerant landrace MeE3L homolog. Notably, the susceptible cassava landrace encodes a RINGless MeE3Lwhich is silenced by SACMV-induced mutations. SACMV also induces mutations which silence the MeE3L RING domain in protoplasts from and tolerant cassava landraces. Conclusions This protocol presented here halves the turnaround time for high-throughput screening of genes associated with the host response to SACMV. It provides evidence that a cassava E3 ligase is associated with the response to SACMV and forms a basis for validation of these findings by in planta functional and interaction studies.

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Glutamate decarboxylase of the parasitic arthropods Ctenocephalides felis and Rhipicephalus microplus: Gene identification, cloning, expression, assay development, identification of inhibitors by high throughput screening and comparison with the orthologs from Drosophila melanogaster and mouse

Insect Biochemistry and Molecular Biology ◽

10.1016/j.ibmb.2012.11.001 ◽

2013 ◽

Vol 43 (2) ◽

pp. 162-177 ◽

Cited By ~ 2

Author(s):

Thomas Ilg ◽

Michael Berger ◽

Sandra Noack ◽

Andreas Rohwer ◽

Michael Gaßel

Keyword(s):

Drosophila Melanogaster ◽

High Throughput ◽

Glutamate Decarboxylase ◽

High Throughput Screening ◽

Rhipicephalus Microplus ◽

Ctenocephalides Felis ◽

Assay Development ◽

Gene Identification

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Full Genome Evolutionary Studies of Wheat Streak Mosaic-Associated Viruses Using High-Throughput Sequencing

Frontiers in Microbiology ◽

10.3389/fmicb.2021.699078 ◽

2021 ◽

Vol 12 ◽

Author(s):

Carla Dizon Redila ◽

Savannah Phipps ◽

Shahideh Nouri

Keyword(s):

Mosaic Virus ◽

Rna Sequencing ◽

Great Plains ◽

High Throughput ◽

Phylogenetic Analyses ◽

Full Genome Sequence ◽

Mixed Infections ◽

Full Genome ◽

Genome Sequences ◽

Resistance Breaking

Wheat streak mosaic (WSM), a viral disease affecting cereals and grasses, causes substantial losses in crop yields. Wheat streak mosaic virus (WSMV) is the main causal agent of the complex, but mixed infections with Triticum mosaic virus (TriMV) and High plains wheat mosaic emaravirus (HPWMoV) were reported as well. Although resistant varieties are effective for the disease control, a WSMV resistance-breaking isolate and several potential resistance-breaking isolates have been reported, suggesting that viral populations are genetically diverse. Previous phylogenetic studies of WSMV were conducted by focusing only on the virus coat protein (CP) sequence, while there is no such study for either TriMV or HPWMoV. Here, we studied the genetic variation and evolutionary mechanisms of natural populations of WSM-associated viruses mainly in Kansas fields and fields in some other parts of the Great Plains using high-throughput RNA sequencing. In total, 28 historic and field samples were used for total RNA sequencing to obtain full genome sequences of WSM-associated viruses. Field survey results showed WSMV as the predominant virus followed by mixed infections of WSMV + TriMV. Phylogenetic analyses of the full genome sequences demonstrated that WSMV Kansas isolates are widely distributed in sub-clades. In contrast, phylogenetic analyses for TriMV isolates showed no significant diversity. Recombination was identified as the major evolutionary force of WSMV and TriMV variation in KS fields, and positive selection was detected in some encoding genomic regions in the genome of both viruses. Furthermore, the full genome sequence of a second Kansas HPWMoV isolate was reported. Here, we also identified previously unknown WSMV isolates in the Great Plains sharing clades and high nucleotide sequence similarities with Central Europe isolates. The findings of this study will provide more insights into the genetic structure of WSM-associated viruses and, in turn, help in improving strategies for disease management.

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Assessment of Common Soybean-Infecting Viruses in Ohio, USA, Through Multi-site Sampling and High-Throughput Sequencing

Plant Health Progress ◽

10.1094/php-rs-16-0018 ◽

2016 ◽

Vol 17 (2) ◽

pp. 133-140 ◽

Cited By ~ 1

Author(s):

Junping Han ◽

Leslie L. Domier ◽

Bryan J. Cassone ◽

Anne Dorrance ◽

Feng Qu

Keyword(s):

Mosaic Virus ◽

High Throughput ◽

High Throughput Sequencing ◽

Virus Disease ◽

Soybean Mosaic Virus ◽

Alfalfa Mosaic Virus ◽

Plant Viruses ◽

Yellow Mosaic Virus ◽

Tobacco Streak Virus ◽

Streak Virus

Multi-site sampling was conducted during 2011 and 2012 to assess the scope of virus disease problems of soybean in Ohio, USA. A total of 259 samples were collected from 80 soybean fields distributed in 42 Ohio counties, accounting for more than 90% of major soybean-growing counties in Ohio. A high-throughput RNA-Seq approach was adopted to identify all viruses in the samples that share sufficient sequence similarities with known plant viruses. To minimize sequencing costs, total RNA extracted from up to 20 samples were first pooled to make up regional pools, resulting in eight regional pools per year in both 2011 and 2012. These regional pools were further pooled into two yearly master pools of RNA, and sequenced using the Illumina's HiSeq2000 platform. Bioinformatic analyses of sequence reads led to the identification of signature sequences of nine different viruses. The originating locations of these viruses were then mapped with PCR or RT-PCR. This study confirmed the widespread distribution of Bean pod mottle virus, Soybean vein necrosis virus, Tobacco ringspot virus, and Tobacco streak virus in Ohio. It additionally revealed occasional association of Alfalfa mosaic virus, Bean yellow mosaic virus, Clover yellow vein virus, Soybean mosaic virus, and Soybean Putnam virus with Ohio soybean. This is the first statewide survey of soybean viruses in Ohio, and provides the much-needed baseline information for management of virus diseases of soybean. Accepted for publication 20 May 2016. Published 10 June 2016.

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Double-Stranded RNA High-Throughput Sequencing Reveals a New Cytorhabdovirus in a Bean Golden Mosaic Virus-Resistant Common Bean Transgenic Line

Viruses ◽

10.3390/v11010090 ◽

2019 ◽

Vol 11 (1) ◽

pp. 90 ◽

Cited By ~ 8

Author(s):

Dione M. T. Alves-Freitas ◽

Bruna Pinheiro-Lima ◽

Josias C. Faria ◽

Cristiano Lacorte ◽

Simone G. Ribeiro ◽

...

Keyword(s):

Phaseolus Vulgaris ◽

Common Bean ◽

Mosaic Virus ◽

High Throughput ◽

Transgenic Line ◽

High Throughput Sequencing ◽

Open Reading Frames ◽

Virus Species ◽

Homologous Proteins ◽

Bean Golden Mosaic Virus

Using double-strand RNA (dsRNA) high-throughput sequencing, we identified five RNA viruses in a bean golden mosaic virus (BGMV)-resistant common bean transgenic line with symptoms of viral infection. Four of the identified viruses had already been described as infecting common bean (cowpea mild mottle virus, bean rugose mosaic virus, Phaseolus vulgaris alphaendornavirus 1, and Phaseolus vulgaris alphaendornavirus 2) and one is a putative new plant rhabdovirus (genus Cytorhabdovirus), tentatively named bean-associated cytorhabdovirus (BaCV). The BaCV genome presented all five open reading frames (ORFs) found in most rhabdoviruses: nucleoprotein (N) (ORF1) (451 amino acids, aa), phosphoprotein (P) (ORF2) (445 aa), matrix (M) (ORF4) (287 aa), glycoprotein (G) (ORF5) (520 aa), and an RNA-dependent RNA polymerase (L) (ORF6) (114 aa), as well as a putative movement protein (P3) (ORF3) (189 aa) and the hypothetical small protein P4. The predicted BaCV proteins were compared to homologous proteins from the closest cytorhabdoviruses, and a low level of sequence identity (15–39%) was observed. The phylogenetic analysis shows that BaCV clustered with yerba mate chlorosis-associated virus (YmCaV) and rice stripe mosaic virus (RSMV). Overall, our results provide strong evidence that BaCV is indeed a new virus species in the genus Cytorhabdovirus (family Rhabdoviridae), the first rhabdovirus to be identified infecting common bean.

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