Summary Conspicuous viral symptoms were seen on Tabernaemontana divaricata , a member of the family Apocynaceae, grown in a commercial nursery, in Israel. The symptoms varied widely and included chlorotic ringspots and banding, oak‐leaf patterns and mosaic. At the end of the winter, large yellow spots, which later became necrotic, appeared on fully expanded leaves. The necrotic zones later fell out, leaving “shot holes”. Preliminary analysis suggested that the disease was associated with a tobamovirus. Particles typical of a tobamovirus were observed by electron microscopy only in samples taken from symptomatic leaves. Apartial segment of the 5′‐terminus of the viral RNA, which comprised of 533 bp was cloned and sequenced. Comparison of the predicted amino acid sequence with those of other tobamoviruses revealed 94% identity with Tobacco mild green mosaic virus (TMGMV) genome. Primers specific to the coat protein (CP) gene of TMGMV used in a reverse transcription‐polymerase chain reaction assay (RT‐PCR) gave an expected amplification product of 455 bp. The amino acid composition of the cloned CP gene, which shows a complete identity to that of TMGMV, confirmed the identity of TMGMV infecting Tabernaemontana . Polyclonal antibodies prepared against the virus and used in double antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA) enabled specific detection of the virus in crude sap extracted from T. divaricata and mechanically inoculated indicator plants. This is the first report of TMGMV infection in Tabernaemontana and the first incidence of the virus in Israel.
A full-length cDNA clone of the RNA genome of the cucurbit potyvirus zucchini yellow mosaic virus (ZYMV) was constructed downstream from a bacteriophage T7 RNA polymerase promoter. A single extra guanosine residue not present in ZYMV RNA was added to the 5′ and 3′ ends. Capped (m7GpppG) ZYMV RNA transcripts were infectious in 10 of 91 Cucurbita pepo test plants; uncapped RNA transcripts were not infectious. The appearance of symptoms in plants inoculated with the infectious transcript was delayed for more than a week compared to plants inoculated with native viral RNA. The progeny virions recovered from infected plants had the same biological properties (aphid non-transmissibility and typical symptoms) as the parental virus. The progeny virions also reacted positively with ZYMV antiserum and ZYMV-specific probes by dot blot hybridization. The authenticity of the progeny virus was verified by identifying a specific molecular marker (C substituted for T in the 3′ non-coding region) using nucleotide sequence analysis.
Mutations in retinal primary cilia are responsible for human blindness but the mechanisms are not fully understood (Wheway et al., 2014). Characterizing the proteome of an organelle such as cilia, is a fruitful way to understand its function but methods often require considerable sample quantities. Here we develop a method to isolate the primary cilia of photoreceptor cells from bovine retinas. Through LC/MS/MS proteomics analysis we identify proteins enriched for cilia function including ciliopathy disease. This study shows our method can be used to isolate retinal primary cilia to obtain sufficient quantities of native protein samples.
