1.11.74
GENOMIC REASSORTMENT OF TOMATO SPOTTED WILT TOSPOVIRUS AS A MECHANISM OF RESISTANCE BREAKING: INVOLVEMENT AND ANALYSIS OF THE INTERGENIC REGION

JW MOYER, K HOFFMANN and WP QIU

Dept. of Plant Pathology, North Carolina State University, Raleigh, NC 276951 7616, USA

Background and objectives
Tomato spotted wilt virus (TSWV) has become of increasing importance in many agronomic and ornamental crops, causing significant losses worldwide. Recently, TSWV-derived resistance has been developed as a potential strategy of managing the disease. It has long been recognized that TSWV is capable of overcoming virus resistance conferred by host genes. However, the impact of TSWV-derived resistance upon the viral population remains unknown. To better understand the resistance-breaking mechanisms, we have chosen TSWV-derived resistance as a model to study how tospoviruses defeat resistance genes.

Materials and methods
A genetic system of mapping TSWV phenotypes to specific genome segments was developed, using genetic markers on the L, M and S RNA to track the parental origin of the genome segments in the progeny isolates. A mixed viral population, derived from two suppressed isolates, TSWV-D and TSWV-10, was subjected to selection pressure from the constitutive expression of a TSWV-derived transgene in the host plant. The genomic composition of the resulting viral population was determined and compared to progeny isolates developed on non-transgenic control plants.

Results and conclusions
We determined that TSWV isolates readily exchange or re-assort genome segments in a non-random fashion. The genotype analysis of viral genomic segments demonstrated that the mixed viral population was driven to form a specific re-assortant, LI0M10SD, in the presence of the TSWV-derived selection pressure, but remained as a heterogenous mixture in the absence of the selection pressure. A genotype assay of 120 local lesion isolates from the first, fourth and seventh transfers confirmed the shift of the genomic composition. The result also suggested that the individual segments of the LI0M10SD re-assortant were each specifically selected in response to the TSWV-derived resistance rather than the overall genome. The S RNA following the seventh transfer on the resistant plants remained essentially identical to TSWV-D S RNA, indicating that no molecular recombination occurred between S RNA of TSWV-10, D or the transgene (derived from TSWV-D S RNA). The competitiveness of the TSYN-D S RNA relative to the TSWV-10 S RNA was associated with the intergenic region (IGR). The less competitive S RNA possessed 62 additional nucleotides including a 33 nt repeat. This is the first evidence that supports the hypothesis that the IGR of the S RNA of TSWV serves a regulatory function in RNA synthesis in vivo. Preliminary analysis of the M RNA has also revealed duplicate sequences and deletions in the IGR. In addition, our observations indicated that TSWV adapts to a new host genotype through genome segment reassortment.