Dataset Information

Temporal and spatial Bean pod mottle virus-induced gene silencing in soybean.

ABSTRACT: Virus-induced gene silencing (VIGS) is a powerful reverse genetics tool in plant science. In this study, we investigated the temporal and spatial silencing patterns achieved by Bean pod mottle virus (BPMV)-based VIGS in soybean using virus constructs targeting green fluorescence protein (GFP). Silencing GFP enabled an in-depth analysis of silencing in soybean tissues over time in a transgenic line constitutively expressing GFP. We discovered evidence for variable GFP silencing based on insert orientation and targeted region in the coding sequence. A 3' sequence in reverse orientation produced the strongest silencing phenotypes. Furthermore, we documented that BPMV VIGS can achieve widespread silencing in a broad range of tissues, including leaves, stems, flowers and roots. Near-complete silencing was attained in leaves and flowers. Although weaker than in shoots, the observed gene silencing in soybean roots will also allow reverse genetics studies in this tissue. When GFP fluorescence was assayed in cross-sections of stems and leaf petioles, near-complete and uniform silencing was observed in all cell types. Silencing was observed from as early as 2 weeks post-virus inoculation in leaves to 7 weeks post-virus inoculation in flowers, suggesting that this system can induce and maintain silencing for significant durations.

SUBMITTER: Juvale PS

PROVIDER: S-EPMC6638800 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Temporal and spatial Bean pod mottle virus-induced gene silencing in soybean.

Juvale Parijat S PS Hewezi Tarek T Zhang Chunquan C Kandoth Pramod Kaitheri PK Mitchum Melissa G MG Hill John H JH Whitham Steven A SA Baum Thomas J TJ

Molecular plant pathology 20120627 9

Virus-induced gene silencing (VIGS) is a powerful reverse genetics tool in plant science. In this study, we investigated the temporal and spatial silencing patterns achieved by Bean pod mottle virus (BPMV)-based VIGS in soybean using virus constructs targeting green fluorescence protein (GFP). Silencing GFP enabled an in-depth analysis of silencing in soybean tissues over time in a transgenic line constitutively expressing GFP. We discovered evidence for variable GFP silencing based on insert or ...[more]

PMID: 22738403

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Project description:Brown Stem Rot (BSR), caused by the soil borne fungal pathogen Phialophora gregata, can reduce soybean yields by as much as 38%. Previous allelism studies identified three Resistant to brown stem Rot genes (Rbs1, Rbs2, and Rbs3), all mapping to large, overlapping regions on soybean chromosome 16. However, recent fine-mapping and genome wide association studies (GWAS) suggest Rbs1, Rbs2, and Rbs3 are alleles of a single Rbs locus. To address this conflict, we characterized the Rbs locus using the Williams82 reference genome (Wm82.a4.v1). We identified 120 Receptor-Like Proteins (RLPs), with hallmarks of disease resistance receptor-like proteins (RLPs), which formed five distinct clusters. We developed virus induced gene silencing (VIGS) constructs to target each of the clusters, hypothesizing that silencing the correct RLP cluster would result in a loss of resistance phenotype. The VIGS constructs were tested against P. gregata resistant genotypes L78-4094 (Rbs1), PI 437833 (Rbs2), or PI 437970 (Rbs3), infected with P. gregata or mock infected. No loss of resistance phenotype was observed. We then developed VIGS constructs targeting two RLP clusters with a single construct. Construct B1a/B2 silenced P. gregata resistance in L78-4094, confirming at least two genes confer Rbs1-mediated resistance to P. gregata. Failure of B1a/B2 to silence resistance in PI 437833 and PI 437970 suggests additional genes confer BSR resistance in these lines. To identify differentially expressed genes (DEGs) responding to silencing, we conducted RNA-seq of leaf, stem and root samples from B1a/B2 and empty vector control plants infected with P. gregata or mock infected. B1a/B2 silencing induced DEGs associated with cell wall biogenesis, lipid oxidation, the unfolded protein response and iron homeostasis and repressed numerous DEGs involved in defense and defense signaling. These findings will improve integration of Rbs resistance into elite germplasm and provide novel insights into fungal disease resistance.

Dataset Information

Temporal and spatial Bean pod mottle virus-induced gene silencing in soybean.

Publications

Temporal and spatial Bean pod mottle virus-induced gene silencing in soybean.

Similar Datasets

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