The Soybean Rpp3 Gene Encodes a TIR-NBS-LRR Protein that Confers Resistance to Phakopsora pachyrhizi.

Soybean rust is an economically significant disease caused by the fungus Phakopsora pachyrhizi that negatively impacts soybean (Glycine max [L.] Merr.) production throughout the world. Susceptible plants infected by P. pachyrhizi develop tan-colored lesions on the leaf surface that give rise to funnel-shaped uredinia as the disease progresses. While most soybean germplasm is susceptible, seven genetic loci (Rpp1 to Rpp7) that provide race-specific resistance to P. pachyrhizi (Rpp) have been identified. Rpp3 was first discovered and characterized in the soybean accession PI 462312 (Ankur), and it was also determined to be one of two Rpp genes present in PI 506764 (Hyuuga). Genetic crosses with PI 506764 were later used to fine-map the Rpp3 locus to a 371-kb region on chromosome 6. The corresponding region in the susceptible Williams 82 (Wm82) reference genome contains several homologous nucleotide binding site-leucine rich repeat (NBS-LRR) genes. To identify Rpp3, we designed oligonucleotide primers to amplify Rpp3 candidate (Rpp3C) NBS-LRR genes at this locus from PI 462312, PI 506764, and Wm82 using polymerase chain reaction (PCR). Five Rpp3C genes were identified in both Rpp3-resistant soybean lines, and co-silencing these genes compromised resistance to P. pachyrhizi. Gene expression analysis and sequence comparisons of the Rpp3C genes in PI 462312 and PI 506764 suggest that a single candidate gene, Rpp3C3, is responsible for Rpp3-mediated resistance. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Rpp1 Encodes a ULP1-NBS-LRR Protein That Controls Immunity to Phakopsora pachyrhizi in Soybean.

Phakopsora pachyrhizi is the causal agent of Asian soybean rust. Susceptible soybean plants infected by virulent isolates of P. pachyrhizi are characterized by tan-colored lesions and erumpent uredinia on the leaf surface. Germplasm screening and genetic analyses have led to the identification of seven loci, Rpp1 to Rpp7, that provide varying degrees of resistance to P. pachyrhizi (Rpp). Two genes, Rpp1 and Rpp1b, map to the same region on soybean chromosome 18. Rpp1 is unique among the Rpp genes in that it confers an immune response (IR) to avirulent P. pachyrhizi isolates. The IR is characterized by a lack of visible symptoms, whereas resistance provided by Rpp1b to Rpp7 results in red-brown foliar lesions. Rpp1 maps to a region spanning approximately 150 kb on chromosome 18 between markers Sct_187 and Sat_064 in L85-2378 (Rpp1), an isoline developed from Williams 82 and PI 200492 (Rpp1). To identify Rpp1, we constructed a bacterial artificial chromosome library from soybean accession PI 200492. Sequencing of the Rpp1 locus identified three homologous nucleotide binding site-leucine rich repeat (NBS-LRR) candidate resistance genes between Sct_187 and Sat_064. Each candidate gene is also predicted to encode an N-terminal ubiquitin-like protease 1 (ULP1) domain. Cosilencing of the Rpp1 candidates abrogated the immune response in the Rpp1 resistant soybean accession PI 200492, indicating that Rpp1 is a ULP1-NBS-LRR protein and plays a key role in the IR.

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

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.

Genotypic Characterization of the U.S. Peanut Core Collection.

Cultivated peanut (Arachis hypogaea) is an important oil, food, and feed crop worldwide. The USDA peanut germplasm collection currently contains 8,982 accessions. In the 1990s, 812 accessions were selected as a core collection on the basis of phenotype and country of origin. The present study reports genotyping results for the entire available core collection. Each accession was genotyped with the Arachis_Axiom2 SNP array, yielding 14,430 high-quality, informative SNPs across the collection. Additionally, a subset of 253 accessions was replicated, using between two and five seeds per accession, to assess heterogeneity within these accessions. The genotypic diversity of the core is mostly captured in five genotypic clusters, which have some correspondence with botanical variety and market type. There is little genetic clustering by country of origin, reflecting peanut's rapid global dispersion in the 18th and 19th centuries. A genetic cluster associated with the hypogaea/aequatoriana/peruviana varieties, with accessions coming primarily from Bolivia, Peru, and Ecuador, is consistent with these having been the earliest landraces. The genetics, phenotypic characteristics, and biogeography are all consistent with previous reports of tetraploid peanut originating in Southeast Bolivia. Analysis of the genotype data indicates an early genetic radiation, followed by regional distribution of major genetic classes through South America, and then a global dissemination that retains much of the early genetic diversity in peanut. Comparison of the genotypic data relative to alleles from the diploid progenitors also indicates that subgenome exchanges, both large and small, have been major contributors to the genetic diversity in peanut.

Virus-Induced Gene Silencing and Transient Gene Expression in Soybean (Glycine max) Using Bean Pod Mottle Virus Infectious Clones.

Virus-induced gene silencing (VIGS) is a powerful and rapid approach for determining the functions of plant genes. The basis of VIGS is that a viral genome is engineered so that it can carry fragments of plant genes, typically in the 200 to 300 base pair size range. The recombinant viruses are used to infect experimental plants, and wherever the virus invades, the target gene or genes will be silenced. VIGS is thus transient, and in the span of a few weeks, it is possible to design VIGS constructs and then generate loss-of-function phenotypes through RNA silencing of the target genes. In soybean (Glycine max), the Bean pod mottle virus (BPMV) has been engineered to be valuable tool for silencing genes with diverse functions and also for over-expression of foreign genes. This protocol describes a method for designing BPMV constructs and using them to silence or transiently express genes in soybean. © 2016 by John Wiley & Sons, Inc.