Graduate Thesis Or Dissertation
 

Interactions between pea seed-borne mosaic virus pathotype 1 and Pisum sativum resistance gene sbm-1

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  • Pea seed-borne mosaic potyvirus pathotype 1 (PSbMV-P1) has the ability to infect most genotypes of Pisum sativum. The exception are those genotypes that are homozygous recessive for the sbm-1 gene. The life cycle of PSbMV pathotype 4 (PSbMV-P4) is unaffected by the sbm-1/sbm-1 genotype. Infectious clones of Pl-P4 recombinants were used to define the genomic segment in P1 that is inhibited by the sbm-1 gene. Transcripts generated in vitro from these clones were initially tested for infectivity by mechanical inoculation onto the susceptible genotype Early Freezer 680 (EF680). Those recombinants that proved to be infectious were then tested for pathogenicity to PI269818, a sbm-1 /sbm-1 genotype. The P4 genomic substitution, which enabled P1 to infect PI269818, was made progressively smaller until one PSbMV-P1 coding region was established as the determinant for infectivity in PI269818. This study demonstrated that the VPg coding region is responsible for the inability of PSbMV-P1 to infect PI269818. Whether P1 life cycle disruption occurred at the nucleotide or amino acid levels is unknown. In conjunction with the definition of the P1 coding region inhibited by sbm-1, the point of virus life-cycle disruption was investigated. PSbMV-P1 resistant plants were inoculated with P1 purified virus or RNA. A time line of infection was established for inoculated and noninoculated leaves. Protoplasts generated from P1 susceptible- and resistant-plant leaves were transfected with P1 RNA, followed by ELISA testing for presence of P1 coat protein. Whereas P1 RNA was fully infectious to EF680 protoplasts, P1 coat protein was undetectable in PI269818 protoplasts transfected with P1 RNA. RT-PCR of RNA extracted from PI269818 P1 inoculated leaves revealed no P1 RNA amplification. When P1 inoculated PI269818 leaves were used to inoculate EF680, no viral infection was detected. These results suggest that a complete lack of P1 replication is occurring in sbm-1 /sbm-1 peas. The potyviral VPg protein has been implicated in viral replication. This study demonstrates that the VPg is the determinant of P1 pathogenicity in PI269818 and consistent with the proposed role of VPg in viral replication, PSbMV-P1 viral life-cycle disruption in the sbm-1/sbm-1 genotype occurs at an early time in viral replication.
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