Virus induced gene silencing confirms oligogenic inheritance of brown stem rot resistance in soybean

Authors: MCCABE, CHANTAL; Lincoln, Lori; O`Rourke, Jamie; Graham, Michelle

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/11/2023
Publication Date: 1/8/2024
Citation: McCabe, C.E., Lincoln, L.M., O'Rourke, J.A., Graham, M.A. 2024. Virus induced gene silencing confirms oligogenic inheritance of brown stem rot resistance in soybean. Frontiers in Plant Science. 14. https://doi.org/10.3389/fpls.2023.1292605.
DOI: https://doi.org/10.3389/fpls.2023.1292605

Interpretive Summary: Brown stem rot (BSR), caused by the soil borne fungal pathogen Phialophora gregata, is one of the top ten yield reducing pathogens for soybeans grown in the Northern United States. BSR symptoms are often confused with other soybean diseases or nutrient stress, making it difficult to characterize and deploy resistance in soybean breeding programs. Different genetic studies have suggested BSR resistance could be controlled by a single gene or as many as three tightly linked genes, all located on the same interval of chromosome 16. This interval contains 107 receptor-like proteins (RLPs), similar to disease resistance genes characterized from other species. The RLPs are arranged in distinct clusters, each with different sequence signature. To gain insight into BSR resistance mechanisms, we took advantage of virus induced gene silencing, which allowed us to turn off different RLP clusters or cluster combinations. If a cluster was required for resistance, turning it off would result in resistant plants that became susceptible to BSR. Using this approach, we identified two clusters that are required for resistance to BSR in a line carrying the Rbs1 resistance gene. Using whole genome expression analyses, we developed a network of genes regulated by the two clusters of RLPs to confer resistance. Improved understanding of BSR resistance mechanisms will enable faster identification of novel resistant germplasm and easier integration of resistance into elite soybean germplasm.

Technical Abstract: 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 GWA studies 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 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 resistance gene 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 phenotypes were 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 in the Rbs locus confer resistance to P. gregata. Failure of B1a/B2 to silence resistance in PI 437833 and PI 437970 suggest 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.