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Mosaic symptoms were seen on the ornamental plant Bupleurum falcatum (Apiaceae) grown in a commercial nursery in Israel. Examination by electron microscopy (EM) of samples from diseased plants revealed elongated filamentous virus particles. Here we provide evidence that B. falcatum is an alternate host for Lettuce mosaic potyvirus (LMV). Several lines of evidence indicated that Bupleurum was infected by LMV, including double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), immunosorbent electron microscopy, and sap and aphid transmission of LMV to lettuce and other host plants. Additionally, general primers derived from a conserved region of the 3’-terminus of the potyvirus genome were used in a reverse transcription-polymerase chain reaction (RT-PCR) assay and gave an expected amplification product of 672 bp. A similar approach was carried out to amplify the corresponding fragment from LMV-infected lettuce. The PCR product was cloned and sequenced; it encompassed an open reading frame encoding 153 amino acids and a 3’ untranslated region (UTR) of 273 nucleotides, very similar to the coat protein (CP) and the 3‵UTR nucleotide sequence, respectively, of other LMV isolates. Analysis of the amino acid sequences of the putative CP of the Bupleurum isolate showed complete identity to the lettuce isolate of LMV. The Bupleurum isolate had no new nucleotide changes which have not been established in other published LMV strain sequences. It is concluded that LMV is the causal agent of the disease in B. falcatum in Israel. [ABSTRACT FROM AUTHOR]

The biological, serological, and molecular characteristics of a newly isolated L4 resistance-breaking isolate of Pepper mild mottle virus (PMMoV) were studied. The new pathotype of PMMoV is closely related to the Israeli pathotypes P1,2 and P1,2,3 of the virus; however, the mosaic symptoms caused by this new pathotype on pepper plants with an L4 genotype were more severe than the mild mosaic symptoms caused by other common pathotypes of the virus in susceptible plants. The predicted amino acid sequence of the putative coat protein (CP) of the newly described pathotype has two amino acid mismatches when compared with the CP of pathotype P1,2, leucine to glutamine at position 47, and alanine to glycine at position 87. The CP of the new pathotype has one amino acid mismatch when compared with P1,2,3, having alanine instead of glycine at position 87. Based on its biological characteristics, the new pathotype was designated P1,2,3,4 of PMMoV-Is. A method is described for the differentiation among the three PMMoV pathotypes using restriction cleavage analysis of reverse-transcription polymerase chain reaction products made from virus-infected plants. An additional unique MnlI site in the CP gene of the newly isolated P1,2,3,4 allows its distinction from the other two isolates, while BglI cleaved only products of the P1,2 pathotype. [ABSTRACT FROM AUTHOR]

Citrus tristeza virus (CTV) is probably the most destructive viral pathogen of citrus. It causes chronic losses to commercial citrus production in all citrus-growing areas. The complete sequences of at least 42 genomes of different CTV strains have been obtained using different technologies including sequencing of multiple overlapped RT-PCR-amplified fragments with sizes of less than 4 kb, or from small viral RNA (svRNA), through next-generation high-throughput sequencing (NGS) technologies. The large size of CTV genome (>19.2 kb) makes it impractical to obtain and amplify full-length cDNA in a single step. The strategy of ligation of multiple cDNA fragments to assemble a full-length cDNA clone involves several serial cloning steps and sometimes subcloning phases using enzymatic digestion with restriction nucleases and ligation reactions. In this protocol, we describe a strategy to clone the entire genome of CTV obtained from two RT-PCR amplified products. These 5'- and 3'-genomic halves, which were designed to be overlapped in 15 nt in their 3'- and 5'-ends, respectively, were used as templates for further overlapped PCR to amplify the entire ~20 kb CTV genome. The resultant full cDNA PCR product was then inserted into pCAMBIA-binary vector.

The biological, serological, and molecular characteristics of a newly isolated L 4 resistance-breaking isolate of Pepper mild mottle virus (PMMoV) were studied. The new pathotype of PMMoV is closely related to the Israeli pathotypes P 1,2 and P 1,2,3 of the virus; however, the mosaic symptoms caused by this new pathotype on pepper plants with an L 4 genotype were more severe than the mild mosaic symptoms caused by other common pathotypes of the virus in susceptible plants. The predicted amino acid sequence of the putative coat protein (CP) of the newly described pathotype has two amino acid mismatches when compared with the CP of pathotype P 1,2 , leucine to glutamine at position 47, and alanine to glycine at position 87. The CP of the new pathotype has one amino acid mismatch when compared with P 1,2,3 , having alanine instead of glycine at position 87. Based on its biological characteristics, the new pathotype was designated P 1,2,3,4 of PMMoV-Is. A method is described for the differentiation among the three PMMoV pathotypes using restriction cleavage analysis of reverse-transcription polymerase chain reaction products made from virus-infected plants. An additional unique MnlI site in the CP gene of the newly isolated P 1,2,3,4 allows its distinction from the other two isolates, while BglI cleaved only products of the P 1,2 pathotype.

The gene encoding the wild type Integrase protein of coliphage HK022 was integrated chromosomally and expressed in Arabidopsis thaliana plants. Double-transgenic plants cloned with the int gene as well as with a T-DNA fragment carrying the proper att sites in a tandem orientation showed that Int catalyzed a site-specific integration reaction (attP x attB) as well as a site-specific excision reaction (attL x attR). The reactions took place without the need to provide any of the accessory proteins that are required by Int in the bacterial host. When expressed in tobacco plants a GFP-Int fusion exhibits a predominant nuclear localization.

Binding('tagging') of a virus-specific oligonucleotide 'sticker' to RNA transcripts copied from PCR products caused retardation of transcript mobility in gel. This enables detection of specific sequences within the RNA transcripts, and a virus strain (PVY(NTN)) could thus be positively identified. We have demonstrated further that oligonucleotides that contained virus sequences originated from different genomic locations varied in their inhibitory effect on the rate of transcript migration in gel; thus, the most effective oligonucleotide could be chosen. Combinations of different strain-specific oligonucleotides had additive retarding effects on transcript migration. The conditions for annealing oligonucleotides to the RNA transcripts were studied, including concentrations of oligonucleotides and salt. A higher electrophoresis temperature (up to 45 degrees C) reduced the gel retardation phenomena, which indicated a conformation mechanism. The applicability of 'tagging' of RNA transcripts with a strain-specific oligonucleotide for virus strain differentiation is discussed.