Brome mosaic virus as a model for engineered resistance to plant viral infections

Abstract

The ability to engineer resistance to plant viral infection to food crops represents a milestone in plant molecular biology. Successful approaches in transgenic plants have expressed a portion of the pathogen's own genome to ameliorate viral infections. Called pathogen derived resistance (PDR), the mechanism(s) through which interference has been mediated remain largely obscure. Using brome mosaic virus (BMV) as a model system for studying PDR, I have concentrated on cis and trans acting sequences within the genomic BMV RNAs that direct viral replication and transcription. I report that the presence or absence of wt or mutant transcripts of BMV RNA-3 during transfection of barley protoplasts can result in profoundly different (+):(-) strand molar ratios for progeny RNAs. My findings implicate the subgenomic promoter of RNA-3 as the primary determinant of asymmetric replication. I examined the effect of adding various (-) sense RNAs corresponding to this region in cotransfections with wt BMV RNAs. Accumulation of progeny RNAs-1 and -2 was reduced by >90% in the presence of (-) sense intercistronic sequences. This trans interference was concentration dependent, not an antisense effect, and was mediated by previously identified regulatory sequences within this region. Evidence is also shown for the activity of a host-encoded RNA-dependent RNA-polymerase in barley protoplasts. In another study, transcripts corresponding to the 200 nucleotide (-) strand promoter or its antisense complement were tested for their ability to interfere with viral replication. Both approaches dramatically reduced progeny accumulation in a concentration dependent manner. The appearance of complementary (-) strands indicated that sense transcripts could serve as templates for (-) strand synthesis. Additional studies transformed rice with constructs designed to express the (-) strand promoter or coat protein cistron. Both reduced BMV replication in rice protoplasts. The lack of detectable coat protein indicated that protection was mediated through viral RNA sequences. These studies demonstrate that BMV can serve as a model system for engineered resistance to plant viral infections.