In Situ Localization of Barley Yellow Dwarf Virus-PAV 17-kDa Protein and Nucleic Acids in Oats

Barley yellow dwarf virus strain PAV (BYDV-PAV) RNA and the 17-kDa protein were localized in BYDV-PAV-infected oat cells using in situ hybridization and in situ immunolocalization assays, respectively. The in situ hybridization assay showed labeling of filamentous material in the nucleus, cytoplasm, and virus-induced vesicles with both sense and antisense nucleic acid probes, suggesting that the filamentous material found in BYDV-PAV-infected cells contains viral RNA. BYDV-PAV negative-strand RNA was detected before virus particles were observed, which indicates that RNA replication is initiated before synthesis of viral coat protein in the cytoplasm. The 17-kDa protein was associated with filamentous material in the cytoplasm, nucleus, and virus-induced vesicles. The labeling densities observed using antibodies against the 17-kDa protein were similar in the nucleus and cytoplasm. No labeling of the 17-kDa protein was observed in plasmodesmata, but filaments in the nuclear pores occasionally were labeled. Since BYDV-PAV RNA and 17-kDa protein colocalized within infected cells, it is possible that single-stranded viral RNA is always associated with the 17-kDa protein in vivo. The 17-kDa protein may be required for viral nucleic acid filaments to traverse the nuclear membrane or other membrane systems.

Download Full-text

Molecular cytogenetic analysis of Agropyron chromatin specifying resistance to barley yellow dwarf virus in wheat

Genome ◽

10.1139/g96-044 ◽

1996 ◽

Vol 39 (2) ◽

pp. 336-347 ◽

Cited By ~ 55

Author(s):

Uwe Hohmann ◽

Winfried Busch ◽

Katia Badaeva ◽

Bernd Friebe ◽

Bikram S. Gill

Keyword(s):

In Situ Hybridization ◽

Barley Yellow Dwarf Virus ◽

Genomic In Situ Hybridization ◽

Yellow Dwarf ◽

Deletion Mapping ◽

Addition Line ◽

C Banding ◽

Barley Yellow Dwarf ◽

Yellow Dwarf Virus

Nine families of bread wheat (TC5, TC6, TC7, TC8, TC9, TC10, TC14, 5395-(243AA), and 5395) with resistance to barley yellow dwarf virus and containing putative translocations between wheat and a group 7 chromosome of Agropyron intermedium (L1 disomic addition line, 7Ai#1 chromosome) induced by homoeologous pairing or tissue culture were analyzed. C-banding, genomic in situ hybridization (GISH), and restriction fragment length polymorphism (RFLP) in combination with repetitive Agropyron-specific sequences and deletion mapping in wheat were used to determine the relative locations of the translocation breakpoints and the size of the transferred alien chromatin segments in hexaploid wheat–Agropyron translocation lines. All homoeologous compensating lines had complete 7Ai#1 or translocated 7Ai#1–7D chromosomes that substitute for chromosome 7D. Two complete 7Ai#1 (7D) substitution lines (5395-(243AA) and 5395), one T1BS–7Ai#1S∙7Ai#1L addition line (TC7), and two different translocation types, T7DS–7Ai#1S∙7Ai#1L (TC5, TC6, TC8, TC9, and TC10) and T7DS∙7DL–7Ai#1L (TC14), substituting for chromosome 7D were identified. The substitution line 5395-(243AA) had a reciprocal T1BS∙1BL–4BS/T1BL–4BS∙4BL translocation. TC14 has a 6G (6B) substitution. The RFLP data from deletion mapping studies in wheat using 37 group 7 clones provided 10 molecular tagged chromosome regions for homoeologous and syntenic group 7 wheat or Agropyron chromosomes. Together with GISH we identified three different sizes of the transferred Agropyron chromosome segments with approximate breakpoints at fraction length (FL) 0.33 in the short arm of chromosome T7DS–7Ai#1S∙7Ai#1L (TC5, TC6, TC8, TC9, and TC10) and another at FL 0.37 of the nonhomoeologous translocated chromosome T1BS–7Ai#1S∙7Ai#1L (TC7). One breakpoint was identified in the long arm of chromosome T7DS∙7DL–7Ai#1L (TC14) at FL 0.56. We detected some nonreciprocal translocations for the most proximal region of the chromosome arm of 7DL, which resulted in small duplications. Key words : C-banding, genomic in situ hybridization (GISH), physical mapping, translocation mapping, RFLP analysis.

Download Full-text

Comparison of Techniques for Detection of Barley Yellow Dwarf Virus-PAV-IL

Plant Disease ◽

10.1094/pdis.1997.81.11.1236 ◽

1997 ◽

Vol 81 (11) ◽

pp. 1236-1240 ◽

Cited By ~ 17

Author(s):

Antonia R. Figueira ◽

Leslie L. Domier ◽

Cleora J. D'Arcy

Keyword(s):

Nucleic Acid ◽

Barley Yellow Dwarf Virus ◽

Infected Tissue ◽

Yellow Dwarf ◽

Nucleic Acid Hybridization ◽

Dwarf Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Hybridization Technique ◽

Barley Yellow Dwarf ◽

Yellow Dwarf Virus

Detection of barley yellow dwarf virus (BYDV)-PAV-IL by an improved nucleic acid hybridization technique, using a nonradioactive probe with chromogenic and chemiluminescent substrates, was compared with detection by polymerase chain reaction (PCR), double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with polyclonal antibodies, and triple antibody sandwich ELISA with polyclonal and monoclonal antibodies. Each method was used to detect purified virus and virus in sap extracts from infected oat leaves. The detection limits for both ELISA procedures were 1 ng of purified BYDV-PAV-IL and the equivalent of 78 ng of infected tissue. Nucleic acid hybridization with either chemiluminescent or chromogenic substrates also detected as little as 1 ng of purified BYDV-PAV-IL, but it was slightly more sensitive at detecting virus in tissue extracts (25 ng of infected tissue). The most sensitive detection technique was PCR amplification, which could detect as little as 0.1 pg of RNA extracted from purified virus and detected viral RNA in the equivalent of 0.5 pg of infected leaf tissue.

Download Full-text