ABSTRACT
Beet curly top virus (BCTV) limits sugarbeet production considerably. Previous studies have shown that infections are associated with the generation of defective DNAs (D-DNA) which may attenuate symptoms. Transgenic sugarbeet lines were established carrying a partial direct repeat construct of D-DNA in order to examine whether they are useful as a means of generating tolerance against BCTV. Thirty four independent transgenic lines were challenged. Viral full-length and D-DNAs were monitored by polymerase chain reaction (PCR) or rolling circle amplification (RCA) and restriction fragment length polymorphism (RFLP). The differential accumulation of both DNA species was compared with symptom severity during the course of infection. RCA/RFLP allowed the discrimination of two D-DNA classes which were either derived from the transgenic construct (D(0)) or had been generated de novo (D(n)). The statistical analysis of the results showed that the presence of D(0)-DNA correlated with increased symptom severity, whereas D(n)-DNAs correlated with attenuated symptoms.
Subject(s)
Beta vulgaris/immunology , DNA, Viral/biosynthesis , Defective Viruses/genetics , Defective Viruses/immunology , Geminiviridae/immunology , Geminiviridae/pathogenicity , Plant Diseases/prevention & control , Beta vulgaris/virology , DNA, Viral/genetics , DNA, Viral/isolation & purification , Defective Viruses/isolation & purification , Geminiviridae/isolation & purification , Genotype , Nucleic Acid Amplification Techniques , Plant Diseases/virology , Plants, Genetically Modified/immunology , Plants, Genetically Modified/virology , Polymorphism, Restriction Fragment LengthABSTRACT
Most whitefly-transmitted geminiviruses possess bipartite genomes comprising DNAs A and B. The production of viable pseudorecombinants by reassortment of infectious cloned components is generally limited to isolates/strains of a particular virus. Following exchange of cloned genomic components of Sida golden mosaic virus from Costa Rica (SiGMV/Co) and Sida golden mosaic virus from Honduras (SiGMV/Ho(yv)), the pseudorecombinant viruses were infectious in various plant species. Three DNA B components (B(1), B(2), B(3)), different in a few nucleotides, were isolated from Sida rhombifolia naturally infected with SiGMV/Ho(yv). Only SiGMV/Ho(yv) DNA B(2) was able to form a viable pseudorecombinant with SiGMV/Co DNA A. In protoplasts, as well as in inoculated leaves, SiGMV/Co DNA A trans-replicated the heterogenomic SiGMV/Ho(yv) DNA B(1) component, indicating that impaired movement is involved in the deficiency of SiGMV/Ho(yv) DNA B(1) to form a pseudorecombinant virus with SiGMV/Co DNA A. Even after extensive mutation analysis of SiGMV/Ho(yv) DNA B(1) and B(2), we were unable to pinpoint differences in SiGMV/Ho(yv) DNA B(2) that allowed the formation of a pseudorecombinant virus with SiGMV/Co DNA A. We observed a gradual increase of infectivity from noninfectious SiGMV/Co DNA A/SiGMV/Ho(yv) DNA B(1) and B(3) pseudorecombinant virus to pseudorecombinant viruses showing normal systemic spread of both genomic components associated with symptomatic plants.
Subject(s)
Geminiviridae/genetics , Reassortant Viruses/genetics , Adaptation, Physiological , Base Sequence , Central America , Costa Rica , DNA Replication , DNA, Viral , Geminiviridae/physiology , Honduras , Molecular Sequence Data , Mutagenesis , Plants, Toxic , Nicotiana/virology , Virus ReplicationABSTRACT
The nucleotide sequences of two Sida-infecting geminiviruses from Honduras were determined. The symptoms of both viruses are identical in Sida rhombifolia but different in Nicotiana benthamiana. An additional symptom of one virus was yellow vein clearing on infected N. benthamiana leaves. Both Sida golden mosaic viruses (SiGMV-Ho and SiGMV-Ho(yv)) have bipartite genomes (DNAs A and B). From the SiGMV-Ho(yv)-infected S. rhombifolia plant two different DNA B molecules were isolated and cloned. They differ in length by 24 nucleotides [SiGMV-Ho(yv) B1 (2593 nt) and B2 (2569 nt)] and at eight nucleotide positions. Both proteins encoded by DNA B (BV1 and BC1) are affected by these substitutions. Computer analysis shows that the bipartite genomes resemble those of other whitefly-transmitted geminiviruses. From homology analyses we conclude that both viruses are closely related but distinct. Comparison with a Sida-infecting virus from Costa Rica (SiGMV-Co) showed that the two viruses from Honduras are more similar to each other than either of them are to SiGMV-Co. Exchange of SiGMV-Ho and SiGMV-Ho(yv) genomic components resulted in viable pseudorecombinant viruses. SiGMV-Ho DNA A was able to produce a viable pseudorecombinant with SiGMV-Co DNA B while the reciprocal exchange was not infectious in N. benthamiana. SiGMV-Ho(yv) DNA A and SiGMV-Co DNA B produced a viable pseudorecombinant virus whereas only pseudorecombination of SiGMV-Co DNA A with SiGMV-Ho(yv) DNA B2, and not with DNA B1, was infectious in N. benthamiana.
