Comparative analysis of TMV-Cg and TMV-U1 detection methods in infected Arabidopsis thaliana.
The common strain of the tobacco mosaic virus (TMV-U1), and the crucifer-infecting tobacco mosaic virus (TMV-Cg), both members of Tobamovirus genus, infect efficiently the solanaceous plants such as tomato and tobacco. The crucifer-infecting tobacco mosaic virus (TMV-Cg) also infects Arabidopsis thaliana plant, spreading systemically without causing severe symptoms. In contrast, Arabidopsis is a poor host for TMV-U1 infection. Within the past 10 years, Arabidopsis has developed into a powerful model system for studying plant-pathogen interaction. However, a detailed analysis comparing the accuracy of various viral detection methods has not been reported previously. Four detection methods were evaluated in A. thaliana (ecotype Po-1), infected with TMV-U1 or TMV-Cg. Western blots, enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ RNA hybridization methods were used to determine viral spread at various days post inoculation (dpi) in inoculated and apical non-inoculated leaves. The detection of viral spread of TMV-U1 and TMV-Cg in Arabidopsis, using these four detection methods, supports previous studies, which demonstrate that the systemic spreads of these two viruses differ in Arabidopsis. Western blotting and ELISA detected TMV-Cg at 5dpi, and TMV-U1 at 12 dpi in systemic tissues. Viral spread was detected earlier when using RNA detection methods. Reverse transcriptase-polymerase chain reaction (RT-PCR) was very sensitive for detecting TMV-Cg in A. thaliana, but less sensitive for TMV-U1 detection. In situ RNA hybridization showed differential distribution of TMV-Cg and TMV-U1 in the inoculated leaf and systemic tissues.
Pereda
S
S
Department of Molecular Genetics and Microbiology, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago, Chile.
Ehrenfeld
N
N
Medina
C
C
Delgado
J
J
Arce-Johnson
P
P
D017360:Arabidopsis / Q000821:virology*; D015153:Blotting, Western; D019607:Brassicaceae / Q000821:virology; D004797:Enzyme-Linked Immunosorbent Assay; D009693:Nucleic Acid Hybridization; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013997:Time Factors; D014027:Tobacco Mosaic Virus / Q000302:isolation & purification*; D014773:Virology / Q000379:methods*