A transcriptional regulatory network of Rsv3-mediated extreme resistance against Soybean mosaic virus.

Resistance genes are an effective means for disease control in plants. They predominantly function by inducing a hypersensitive reaction, which results in localized cell death restricting pathogen spread. Some resistance genes elicit an atypical response, termed extreme resistance, where resistance is not associated with a hypersensitive reaction and its standard defense responses. Unlike hypersensitive reaction, the molecular regulatory mechanism(s) underlying extreme resistance is largely unexplored. One of the few known, naturally occurring, instances of extreme resistance is resistance derived from the soybean Rsv3 gene, which confers resistance against the most virulent Soybean mosaic virus strains. To discern the regulatory mechanism underlying Rsv3-mediated extreme resistance, we generated a gene regulatory network using transcriptomic data from time course comparisons of Soybean mosaic virus-G7-inoculated resistant (L29, Rsv3-genotype) and susceptible (Williams82, rsv3-genotype) soybean cultivars. Our results show Rsv3 begins mounting a defense by 6 hpi via a complex phytohormone network, where abscisic acid, cytokinin, jasmonic acid, and salicylic acid pathways are suppressed. We identified putative regulatory interactions between transcription factors and genes in phytohormone regulatory pathways, which is consistent with the demonstrated involvement of these pathways in Rsv3-mediated resistance. One such transcription factor identified as a putative transcriptional regulator was MYC2 encoded by Glyma.07G051500. Known as a master regulator of abscisic acid and jasmonic acid signaling, MYC2 specifically recognizes the G-box motif ("CACGTG"), which was significantly enriched in our data among differentially expressed genes implicated in abscisic acid- and jasmonic acid-related activities. This suggests an important role for Glyma.07G051500 in abscisic acid- and jasmonic acid-derived defense signaling in Rsv3. Resultantly, the findings from our network offer insights into genes and biological pathways underlying the molecular defense mechanism of Rsv3-mediated extreme resistance against Soybean mosaic virus. The computational pipeline used to reconstruct the gene regulatory network in this study is freely available at https://github.com/LiLabAtVT/rsv3-network.

Genetic and Phenotypic Analysis of Soybean mosaic virus Resistance in PI 88788 Soybean.

ABSTRACT Resistance to Soybean mosaic virus (SMV) was identified in PI 88788 soybean, a germ plasm accession from China that is used widely as a source of resistance to soybean cyst nematode. Strains SMV-G1 through -G7 infected the inoculated leaves of PI 88788 but were not detected in upper, noninoculated trifoliolate leaves. Inheritance of resistance was determined by inoculating progenies of crosses of PI 88788 with susceptible cvs. Essex and Lee 68 with SMV strains G1 and G7. Allelomorphic relationships with known genes for resistance to SMV were tested in crosses with the resistant genotypes PI 96983, L29, and V94-5152, possessing Rsv1, Rsv3, and Rsv4 genes, respectively. Data analyses showed that resistance in PI 88788 to SMV-G1 is controlled by a single, partially dominant gene; however, to SMV-G7, the same gene was completely dominant. The PI 88788 gene was independent of the Rsv1 and Rsv3 loci, but allelic to Rsv4 in V94-5152. Expression of the Rsv4 gene in PI 88788 resulted in a reduced number of infection sites and restricted short- and long-distance movement of virus, rather than hypersensitivity. A unique late susceptible phenotype was strongly associated with heterozygosity. This gene has potential value for use in gene pyramiding to achieve resistance to several SMV strains, as well as for rate-reducing resistance.

A plant-associated microbe genome initiative.

ABSTRACT Plant-associated microorganisms are critical to agricultural and food security and are key components in maintaining the balance of our ecosystems. Some of these diverse microbes, which include viruses, bacteria, oomycetes, fungi, and nematodes, cause plant diseases, whereas others prevent diseases or enhance plant growth. Despite their importance, we know little about them on a genomic level. To intervene in disease and understand the basis of biological control or symbiotic relationships, a concerted and coordinated genomic analysis of these microbes is essential. Genome analysis, in this context, refers to the structural and functional analysis of the microbe DNA including the genes, the proteins encoded by those genes, as well as noncoding sequences involved in genome dynamics and function. The ultimate emphasis is on understanding genomic functions involved in plant associations. Members of The American Phytopathological Society (APS) developed a prioritized list of plant-associated microbes for genome analysis. With this list as a foundation for discussions, a Workshop on Genomic Analysis of Plant-Associated Microorganisms was held in Washington, D.C., on 9 to 11 April 2002. The workshop was organized by the Public Policy Board of APS, and was funded by the Department of Energy (DOE), the National Science Foundation (NSF), U.S. Department of Agriculture-Agricultural Research Service (USDA-ARS), and USDA-National Research Initiatives (USDA-NRI). The workshop included academic, industrial, and governmental experts from the genomics and microbial research communities and observers from the federal funding agencies. After reviewing current and near-term technologies, workshop participants proposed a comprehensive, international initiative to obtain the genomic information needed to understand these important microbes and their interactions with host plants and the environment. Specifically, the recommendations call for a 5-year, $500 million international public effort for genome analysis of plant-associated microbes. The goals are to (i) obtain genome sequence information for several representative groups of microbes; (ii) identify and determine function for the genes/proteins and other genomic elements involved in plant-microbe interactions; (iii) develop and implement standardized bioinformatic tools and a database system that is applicable across all microbes; and (iv) educate and train scientists with skills and knowledge of biological and computational sciences who will apply the information to the protection of our food sources and environment.

Diversity Among Isolates of Bean pod mottle virus.

ABSTRACT Isolates of Bean pod mottle virus (BPMV), a member of the genus Comovirus, collected from soybean fields in Kentucky, Virginia, Arkansas, and Iowa were classified into two distinct subgroups, I and II, based on nucleic acid hybridization analysis using cloned cDNA probes to RNA-1 from BPMV strains K-G7 and K-Ha1. Slot blot hybridization analysis using cloned cDNA probes to RNA-2 from the same two strains (K-G7 and K-Ha1), however, revealed that some of the isolates, initially classified as belonging to subgroup I after analysis with RNA-1 probes, are in fact natural reassortants between the two strain subgroups. This was corroborated by nucleotide sequence analysis of full-length cDNA clones of both RNA-1 and RNA-2 from a putative reassortant strain (K-Ho1). These results indicate that BPMV strain diversity is more complex than initially anticipated, and that the use of cloned probes to both genomic RNAs during nucleic acid hybridization analysis is required to unravel the extent of such diversity. In a field plot experiment, BPMV isolates that belong to distinct strain subgroups induced symptoms that varied in severity and in the level of yield losses. In this regard, the reassortant strain K-Ho1 caused the most serious damage compared with four other BPMV isolates tested. Furthermore, the soybean alleles Rsv(1) and Rsv(4), known to confer resistance against Soybean mosaic virus, a member of the genus Potyvirus, did not provide any protection against BPMV. Additionally, we developed a reverse transcription-polymerase chain reaction procedure based on the sequence of a highly conserved region in the capsid polyprotein coding sequence that provides efficient and highly sensitive detection of all BPMV isolates tested, regardless of their strain classification.

Tissue blot immunoassay and direct RT-PCR of cucumoviruses and potyviruses from the same NitroPure nitrocellulose membrane.

A method is described for using Nitropure nitrocellulose (NPN) membranes as an effective solid matrix for retrieval of template RNA of three potyviruses, Tobacco etch virus, Soybean mosaic virus and Turnip mosaic virus, and two cucumoviruses, Cucumber mosaic virus and Peanut stunt virus. These NPN membranes were also used for tissue blot immunosorbent assays (TBIAs) to identify and detect plant viruses. For RNA detection, discs from dried membranes blotted with infected tissue were minimally cleaned with Triton X-100 and placed directly into reverse transcription (RT) reactions to initiate cDNA synthesis. Aliquots of cDNA plus primers specific for coat protein produced PCR amplicons of expected sizes for each of the viruses. Intensity of PCR-amplified bands from cDNA transcribed from both non-processed and TBIA-processed NPN membranes was comparable to those using FTA Card protocols. Direct sequencing of PCR products yielded high quality runs enabling identification to species. NPN membranes retained immunologically detectable virus particles, as well as intact template viral RNA, for more than a year at room temperature. The quantity of amplification product decreased after several months of storage, but could be increased by increasing the number of PCR cycles.