Genetic determinants for thrips transmission of tomato spotted wilt virus

by 1968- Sin, Sang-Hoon

SIN, SANG-HOON Genetic determinants for thrips transmission of Tomato spotted wilt virus. (Under the direction of Dr. James W. Moyer) Tomato spotted wilt virus (TSWV) is the type species of the genus Tospovirus in the family Bunyaviridae and is exclusively transmitted by thrips in nature. To determine the viral RNA segment(s) responsible for thrips transmissibility, a viral genetic system that included reassortment and segment-specific RFLP analysis was developed. A complete set of reassortants between TSWV-RG2, a thrips transmissible isolate, and TSWV-D, a thrips nontransmissible isolate was generated. Thrips transmission assays of the reassortants showed that reassortants containing the M RNA from TSWV-D, a thrips nontransmissible isolate, were not transmissible, whereas reassortants with the M RNA from TSWV-RG2, a thrips transmissible isolate, were transmissible, irrespective of the origin of the L and S RNA. This is compelling evidence that thrips transmissibility of TSWV resides on the M RNA. Initial attempts to delineate specific regions of the M RNA associated with thrips transmission and characterize genetic changes on the M RNA of the TSWV following repeated mechanical or thrips transmission were made by comparing the nucleotide sequence of the M RNAs of the 3 poorly-transmissible TSWV isolates and transmissibilityrestored isolates. After 24 transmission cycles by mechanical means or by thrips, whole M RNA sequences were determined by RT-PCR and sequencing. The mean mutation frequency of the NSm open reading frame (ORF) for the three isolates per passage was 9.2 X 10-5 nucleotide substitutions per base per mechanical or thrips passage. A similar pattern was observed in GN/GC ORF with 1.1 X 10-4 and 1.3 X 10-4 nucleotide substitutions per base per mechanical or thrips passage, respectively. Interestingly, two NS mutations and one synonymous S mutation in the GN/GC ORF were conserved in the three isolates after the series of mechanical transmission. Two NS mutations consist of a substitution of C86 to U (C86U) changing amino acid T29 to I (T29I) and U1373C (M458T), while one synonymous mutation was a substitution of G492A. In addition, two NS substitutions in the GN/GC ORF of thrips-nontransmissible TSWV-GT (TSWV-GTN) were found to be responsible for the loss of thrips transmissibility. Two NS substitutions were changes of U1976C (V659A) and A2150G (Y717C). An alternative strategy was employed to accomplish the fine mapping of determinants for thrips transmissibility on the M RNA. To increase the probability of identifying specific regions of the genome linked to transmissibility, thirty single lesion isolates (SLIs) were generated from TSWV-RG2P24, which was derived from TSWV-RG2 following 24 serial, mechanical passages in N. benthamiana. Three SLIs were transmissible, while 27 SLIs were nontransmissible by WFT. Sequence analysis of the M RNA, thrips transmission assays, Western blot analysis, and transmission electron microscopic studies revealed that a specific NS mutation (C1375A) in the GN/GC ORF of the M RNA resulted in the loss of thrips transmissibility without inhibition of virion assembly. This was in contrast to other nontransmissible SLIs which had frameshift and/or nonsense mutations in the GN/GC ORF but were defective in virion assembly. In addition, GC was only detectable in the transmissible isolates and C1375A mutants but not in the frameshift or nonsense mutants. This is the first report of a specific viral determinant associated with thrips transmission. These results also indicate that the glycoproteins may be dispensable for TSWV infection of plant hosts, but not for transmissibility by thrips.