Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1991. / Vita. Abstract. Includes bibliographical references (leaves 19-30). Also available via the Internet.

Strain-specific resistance conditioned by a single dominant gene in soybean cv. York inoculated with SMV-G1, revealed no symptoms and no detectable viral replication (R). Unlike the hypersensitive response (HR), the R response did not result in localized virus and induction of a series of defense responses, but in inhibition of virus replication. However, this resistance was overcome by the resistance- breaking strain, SMV-G4, which induced lethal necrosis (N). Unlike HR, G4 strain was not restricted but spread ina restricted pattern along the vein, stem and into upper un-inoculated leaves where it induced necrosis. Like HR, PR proteins were found to accumulate in the N response and were named SPR (soybean pathogenesis-related) proteins. On the basis of major similarities in molecular weight characteristics and enzyme-substrate specificities, SPRs are proposed to be classed into four groups: SPR1; 1a, 1b, 1c, 1d; SPR2: 2a, 2b, 2c; SPR3: 3a, 3a’, 3b, 3c, 3d, 3e; SPR4. The functions of SPR1 and SPR4 groups have not yet been determined. The SPR2 group was identified as β-1,3-glucanases (GLN) and classed two subgroups. The SPR3 group was identified as chitinases (CHN) and classed three distinct subgroups. Like HR, the N response induced a series of defense responses with marked temporal increases, in a bimodal pattern, of phenylalanine ammonia-lyase (PAL) and peroxidase (POX) activities, bimodal pattern of PAL mRNA and chalcone synthase (CHS) mRNA induction, a single pattern of chitinase mRNA induction, and glyceollin and lignin accumulation. These marked increases occurred at or just before the time of initial appearance of necrotic lesions, or following necrosis development for localization of virus. Total RNA isolated from soybean inoculated with SMV-G4 were used for synthesis of cDNA by reverse transcription, and amplified using 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR)-specific primer sets by polymerase chain reaction. Amplified cDNAs were cloned into Bluescript vector (pKS⁻), transformed into E. coli, isolated pSOY HMGR and used as a probe for Northern analysis. We demonstrate that expression of HMGR mRNA is correlated with strain-specific resistance (R). SMV coat protein (CP) degraded in vitro by proteolysis during purification. The CP of SMV purified from infected leaves had an major size of 34 kD in SDS-PAGE with minor peptides of 32 and 31 kD. The minor peptides increased during the storage at 4°C or with trypsin treatment, and reacted with antiserum to intact virions. This heterogeneity of protein was not removed by alkaline phosphatase treatment for varying time intervals, and was not related with phosphorylation and dephosphorylation, or with virus maturation. These studies provided additional evidence of N- or C-terminal exposure on the particle’s surface. / Ph. D.

The genetic interactions among 9 soybean [<i>Glycine max</i> (L.) Merr.] cultivars and 6 strains of soybean mosaic virus (SMV) were investigated. The objectives were to identify genes and/or alleles conditioning resistant and necrotic reactions to SMV and to determine the genetic relationships among resistance genes from cultivars exhibiting differential responses to the SMV strains. Seven SMV-resistant (R) cultivars (‘PI 486355’, ‘Suweon 97’, ‘PI 96983’, ‘Ogden’, ‘York’, ‘Marshall’, and ‘Kwanggyo’) were crossed in all combinations among each other and with susceptible (S) cultivars ‘Essex’ and ‘Lee 68’. F₂ populations and F₂-derived F₃ lines were inoculated in field with the SMV type strain Gl and in the greenhouse with the virulent strains G4, G5, G6, G7, and G7A. All F₂ populations from R x S and necrotic (N) x S crosses having PI 96983, Ogden, York, Marshall, and Kwanggyo as either resistant or necrotic parents segregated 3R:1S and 3N:1S, respectively. F₂-derived F₃ progenies from R x S crosses exhibited an F₂ genotypic ratio of 1 homogeneous R : 2 segregating (3R:1S) : l homogeneous S. The results indicate that each of these five resistant parents has a single, dominant or partially dominant gene conditioning the resistant and necrotic reactions to SMV. No segregation for SMV reaction was evident in F₂ and F₃ generations from R x R, N x N, and S x S crosses among the five differential cultivars, indicating that the resistance genes in the five cultivars are alleles at a common locus. The alleles in PI 96983 and Ogden were previously labeled <i>Rsy</i> and <i>rsy^t</i>, respectively. Gene symbols, <i>Rsy^y</i>, <i>Rsy^m</i>, and <i>Rsy^k</i> are proposed for the resistance genes in York, Marshall, and Kwanggyo, respectively. It is also proposed that the gene symbol <i>rsy^t</i> be changed to <i>Rsy^t</i> to more accurately reflect its genetic relationship to the susceptible allele. The R x S crosses with PI 486355 and Suweon 97 as resistant parents segregated 15R:1S in the F₂ and 7 (all R) : 4 (3R:1S) : 4 (15R:1S) : 1 (all S) in the F₃, indicating that each has two independent genes for resistance to SMV. The F₂ plants of PI 486355 x Suweon 97 showed no segregation for SMV reaction, suggesting that they have at least one gene in common. The crosses among all 7 resistant parents produced no susceptible segregates when inoculated with strain G1. It is concluded that the 7 resistant cultivars each have a gene or allele at the <i>Rsy</i> locus. Data from the experiments furnished conclusive evidence that the necrotic reaction in segregating populations is highly associated with plants that are heterozygous for the resistance gene. / Ph. D.

Soybean mosaic virus (SMV), a potyvirus detected worldwide, can cause serious diseases in soybean (Glycine max L. Merr.). Host resistance to SMV conferred by a single dominant gene, Rsvl, was studied as a model to gain insights of plant virus resistance genes, and to facilitate the breeding of resistant cultivars. DNA restriction fragment length polymorphisms (RFLPs) and microsatellites (or simple sequence repeats, SSRs) were used as genetic markers to identify the chromosomal location of Rsvl1 in a cross between PI 96983 (resistant) and a susceptible cultivar. Twenty five RFLP and three SSR loci polymorphic between the parental lines were analyzed in 107 F, individuals. Genotypes of Rsv1 were determined by inoculating F2.3 progeny with SMV-G1. Genetic analysis revealed that one SSR (HSP176L) and two RFLP (pA186 and pK644a) markers are closely linked to Rsv1, with a distance of 0.5, 1.5, and 2.1 cM, respectively. The tight linkages of the three markers to Rsv1 were confirmed by SSR and RFLP analysis of three near isogenic lines (NILs) of Rsv1 derived from PI 96983 or Marshall. The three Rsv1-linked markers were then used to screen 67 diverse soybean types. These marker loci showed a remarkably high level of polymorphism, indicating a possible association between disease resistance and rapid sequence divergence. At each Rsv1-linked marker locus, one SSR allele or RFLP haplotype is highly correlated with SMV resistance. These resistance markers, especially the SSR allele at HSP176L which can be detected by the polymerase chain reaction (PCR), may be useful for germplasm screening. The grouping of the 67 accessions according to their Rsv1-linked multilocus marker haplotypes agrees with available pedigree information. A set of differential cultivars known to contain Rsv1 clustered into putative Rsv1- carrying groups. Based on molecular marker analysis and previous inheritance studies, 37 of the 45 resistance accessions probably derive their resistance from Rsv1. The remaining eight accessions include Columbia (Rsv3), and the other potentially diverse resistance sources. A heat shock protein (HSP) multigene family, HSP176L included, was analyzed for its positional proximity to the Rsv1 gene cluster. A technique termed amplified sequence length polymorphism (ASLP) was developed to convert known DNA sequences to PCR-based genetic markers. Among six pairs of HSP primers used, two (HSP175E and 185C) detected ASLPs between the parents, and segregated in the F₂ population with a size of 174. HSP175E was found to be closely-linked (0.7 cM) to HSP176L, both of which are Class I small HSP genes. HSP185C, however, was mapped to a different linkage group, suggesting that it may belong to another family. ADR11, a member of auxin down-regulated (ADR) multigene family, is known to be linked to HSP173B, also a Class I gene but not mappable in this population. ASLP analysis of ADR11 in a set of Rsv1 NILs indicates that it is linked to Rsv1, and ADR11 co-segregates with HSP175E in the F, population. Thus, the Class I small HSP multigene family including HSP176L, 175E, and 173B, and possibly a family of ADR genes, is located near the Rsvi resistance gene cluster. / Ph. D.