scholarly journals Mapping the RNA-binding domain on the Apple chlorotic leaf spot virus movement protein

2005 ◽  
Vol 86 (1) ◽  
pp. 225-229 ◽  
Author(s):  
Masamichi Isogai ◽  
Nobuyuki Yoshikawa

The RNA-binding properties of the cell-to-cell movement protein (MP) of Apple chlorotic leaf spot virus were analysed. MP was expressed in Escherichia coli and was used in UV-crosslinking analysis, using a digoxigenin–UTP-labelled RNA probe and gel-retardation analysis. The analyses demonstrated that MP bound cooperatively to single-stranded RNA (ssRNA). When analysed for NaCl dependence of the RNA-binding activity, the majority of the MP could bind ssRNA even in binding buffer with 1 M NaCl. Furthermore, competition binding experiments showed that the MP bound preferentially to ssRNA and single-stranded DNA without sequence specificity. MP deletion mutants were used to identify the RNA-binding domain by UV-crosslinking analysis. Amino acid residues 82–126 and 127–287 potentially contain two independently active, single-stranded nucleic acid-binding domains.

ChemBioChem ◽  
2005 ◽  
Vol 6 (8) ◽  
pp. 1391-1396 ◽  
Author(s):  
Marçal Vilar ◽  
Ana Saurí ◽  
Jose F. Marcos ◽  
Ismael Mingarro ◽  
Enrique Pérez-Payá

2007 ◽  
Vol 88 (1) ◽  
pp. 316-324 ◽  
Author(s):  
Hajime Yaegashi ◽  
Tsubasa Takahashi ◽  
Masamichi Isogai ◽  
Takashi Kobori ◽  
Satoshi Ohki ◽  
...  

Apple chlorotic leaf spot virus (ACLSV) is the type species of the genus Trichovirus and its single-stranded, plus-sense RNA genome encodes a 216 kDa protein (P216) involved in replication, a 50 kDa movement protein (P50) and a 21 kDa coat protein (CP). In this study, it was investigated whether these proteins might have RNA silencing-suppressor activities by Agrobacterium-mediated transient assay in the green fluorescent protein-expressing Nicotiana benthamiana line 16c. The results indicated that none of these proteins could suppress local silencing in infiltrated leaves. However, systemic silencing in upper leaves induced by both single- and double-stranded RNA could be suppressed by P50, but not by a frame-shift mutant of P50, P216 or CP. Moreover, when P50 was expressed separately from where silencing signals were generated in a leaf, systemic silencing in upper leaves was inhibited. Collectively, our data indicate that P50 acts as a suppressor of systemic silencing without interfering with local silencing, probably by inhibiting the movement of silencing signals.


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