Role of virus genes in seed and aphid transmission and development of a virus-induced gene silencing system to study seed development in soybean

Soybean mosaic virus (SMV) and Tobacco streak virus (TSV) are two important seed transmitted virus that infect soybean. SMV is a member of the family Potyviridae genus Potyvirus, and is transmitted by seed and aphids in soybean. The symptoms of SMV infection on soybean typically include mosaic and curling of leaves, reduced pod set and seed coat mottling. Although, the virus genes responsible for transmission by aphids have been studied extensively there has not been much research in understanding virus genes responsible for seed transmission. In this study I investigated the role of SMV genes in seed and aphid transmission, by designing recombinants between SMV isolates, SMV 413 (highly seed and aphid transmitted) and SMV G2 (lacking seed or aphid transmission). The SMV genes encoding the proteins P1, helper component protease (HC-Pro), and coat protein (CP) were the major determinants of seed transmission. The single amino acid mutation of G12 to D in the DAG motif of the CP and the amino acid Q264 to P in the C-terminus of the CP affected seed transmission of SMV. The aphid transmission study validated previous research and demonstrated that the amino acid motif DAG in the CP region and HC-Pro were important in aphid transmission. The severity of foliar symptoms was influenced by HC-Pro and seed coat mottling was determined by a single amino acid Q264 in the C-terminus of the CP. The seed transmitted virus TSV (genus Ilarvirus and family Bromoviridae) was utilized for construction of a DNA-based virus induced gene silencing (VIGS) vector for investigating genes expressed in the soybean seed. VIGS are reverse genetics tools comprised of virus vectors with a partial plant transcript insertion that can silence expression of the plant gene. TSV has a tripartite genome, and causes early symptoms such as leaf puckering and necrosis that are often followed by symptom recovery possibly induced by virus RNA silencing. Multicloning sites were inserted into all the three RNAs of TSV to facilitate the insertion of plant gene fragments for the development of a VIGS vector. TSV RNA2-based VIGS vector was the most stable and the integrity of plant gene inserts up to the size of 175 nucleotides were maintained in the vector. The small RNA (21-24 nucleotide size) deep sequencing of TSV-infected soybean plants with symptom recovery revealed that virus derived small interfering (vsi)RNA originated from hot spots within the TSV genome as indicated by the number of mapped sequences. The vsiRNA hot spots were not correlated to the presence of RNA secondary structure in the TSV genome and exhibited a bias for the sense strand. Most hot spots were located in the TSV RNA3 especially in the movement protein coding region. To further understand the effects of virus infection on micro (mi)RNA accumulation and function in plants showing symptom recovery, small RNAs and mRNAs were sequenced from three soybean cultivars either mock-inoculated or infected with TSV or Bean pod mottle virus (BPMV). BPMV (genus Comovirus and family Comoviridae) also causes severe symptoms initially followed by symptom recovery in soybean as observed in TSV. The soybean miRNA, gma-miRNA159a, that targets transcripts of a glutathione S-transferase (GST) and a MYB family transcription factor, and gma-miR166 that targets a HD-Zip transcription factor were down-regulated in virus-infected soybean exhibiting symptom recovery as indicated by the number of mapped sequences to the reported gma-miRNA transcript sequence. The GST, MYB factor and HD-Zip factor proteins may all be involved in programmed cell death that is associated with plant disease resistance. The mRNA-Seq analysis revealed that the accumulation of mRNAs encoding pathogenesis-related proteins, components of the salicylic acid mediated defense response and plant RNA virus infection associated proteins were up-regulated in virus-infected plants with symptom recovery. The transcripts for GST and lactoylglutathione lyase protein, involved in detoxification, were down-regulated. Changes in the accumulation of transcripts from other defense- and photosynthesis- related genes were cultivar dependent, as determined from the number of sequences that mapped to soybean transcripts