Development of an indirect ELISA and immunocapture rt-PCR for Lily virus detection.
(10/18)
Multiple viruses such as Lily symptomless virus (LSV), Lily mottle virus (LMoV), and cucumber mosaic virus (CMV) are the most prevalent viruses infecting lilies in Korea. Leaf samples and bulbs showing characteristic symptoms of virus infection were collected from Gangwon, Chungnam, and Jeju provinces of Korea in 2008-2011. Coat protein (CP) genes of LSV and LMoV were amplified from collected samples by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into a pET21d(+) expression vector to generate recombinant CPs. The resulting carboxy-terminal His-tagged CPs were expressed in Escherichia coli strain BL21(DE3) by isopropyl-1-thio- beta-D-galactoside induction. The recombinant proteins were purified using Ni-NTA agarose beads, and the purified proteins were used as an immunogen to produce polyclonal antibodies in rabbits. The resulting polyclonal antisera recognized specifically LSV and LMoV from infected plant tissues in Western blotting assays. Indirect enzyme-linked immunosorbent assay and immunocapture RTPCR using these polyclonal antisera were developed for the sensitive, efficient, economic, and rapid detection of Lily viruses. These results suggest that large-scale bulb tests and economic detection of Lily viruses in epidemiological studies can be performed routinely using these polyclonal antisera. (+info)
First report of Cowpea mild mottle Carlavirus on yardlong bean (Vigna unguiculata subsp. sesquipedalis) in Venezuela.
(11/18)
An RNA virus-encoded zinc-finger protein acts as a plant transcription factor and induces a regulator of cell size and proliferation in two tobacco species.
(12/18)
Making sense of nuclear localization: a zinc-finger protein encoded by a cytoplasmically replicating plant RNA virus acts a transcription factor: a novel function for a member of large family of viral proteins.
(13/18)
Autocatalytic processing of the 223-kDa protein of blueberry scorch carlavirus by a papain-like proteinase.
(15/18)
The first open reading frame of the blueberry scorch carlavirus (BBScV) genome encodes a putative replication-associated protein of 223 kDa (p223). A pulse-chase analysis of viral RNA translated in vitro in rabbit reticulocyte lysate revealed that p223 was proteolytically processed. Using a full-length ORF 1 cDNA clone in a coupled in vitro transcription/translation reaction, we confirmed that the ORF 1 gene product of BBScV processes autocatalytically. From sequence alignments with phylogenetically related viruses, including tymoviruses, we predicted that p223 contained a papain-like proteinase domain with a putative catalytic cysteine994 and histidine1075. A second possible proteinase domain, which contained cysteine895 and histidine984 residues with similar spacing but was otherwise less similar to the viral papain-like proteinases, was identified immediately upstream of the predicted catalytic site. The cleavage site of the proteinase was predicted to be between the putative helicase and the polymerase domains, possibly between or close to glycine1472 and alanine1473. Supporting these predictions, deletion of the 2091 nucleotides encoding the C-terminal region of p223, which contained the putative RNA polymerase domain and the putative cleavage site of the polyprotein, abolished autoproteolysis. Deletion of the 2061 nucleotides encoding the N-terminal region, which contained the putative methyltransferase domain, did not affect autoproteolysis. Alteration of cysteine994, histidine1075, or glycine1472 abolished autoproteolysis in vitro, supporting the involvement of these residues at the catalytic site and cleavage site. Alteration of the upstream cysteine895 and histidine984 residues did not affect processing in vitro. Capped BBScV full-length transcripts containing mutations in the codons for either cysteine994 or histidine1075 were not infectious in the systemic host plants Chenopodium quinoa and C. amaranticolor, whereas alteration of glycine1472 signficantly decreased but did not abolish infectivity. Transcripts containing mutations in the codons for either cysteine895 or histidine984 also were infectious, but resulted in delayed symptom expression in plants. (+info)
Nucleotide sequences of apple stem pitting virus and of the coat protein gene of a similar virus from pear associated with vein yellows disease and their relationship with potex- and carlaviruses.
(16/18)
The nucleotide sequence (9306 nucleotides) of cDNA clones of apple stem pitting virus (ASPV) obtained from a double-stranded RNA template, extracted from diseased plant tissue, was determined. The genome is composed of five open reading frames (ORFs) encoding putative proteins with M(r)s of 247083, 25147, 12832, 7429 and 43712, and has a poly(A) tail. Using two oligonucleotides designed from the ASPV sequence information a 1598 bp fragment from near the 3' terminus of the viral RNA, containing the coat protein of M(r) 43,766, was amplified from vein yellows (VY)-infected pear plants by PCR. The sequence determined showed eight nucleotide changes resulting in five amino acid substitutions compared with the sequence of ASPV. When compared to potex-, carla-, clostero- and capilloviruses, the ASPV genome organization appeared to be most closely related to that of potexviruses, but with a larger coat protein of M(r) 44K (ORF5). The predicted coat protein size was confirmed by immunoblot analysis. The results show that ASPV does not fall into subgroup A of the closteroviruses but that it probably belongs in an as yet undefined group of viruses. They also suggest that the virus associated with VY is a strain of ASPV. (+info)