Complete sequence and genomic annotation of carrot torradovirus 1.

Carrot torradovirus 1 (CaTV1) is a new member of the genus Torradovirus within the family Secoviridae. CaTV1 genome sequences were obtained from a previous next-generation sequencing (NGS) study and were compared to other members and tentative new members of the genus. The virus has a bipartite genome, and RACE was used to amplify and sequence each end of RNA1 and RNA2. As a result, RNA1 and RNA2 are estimated to contain 6944 and 4995 nucleotides, respectively, with RNA1 encoding the proteins involved in virus replication, and RNA2 encoding the encapsidation and movement proteins. Sequence comparisons showed that CaTV1 clustered within the non-tomato-infecting torradoviruses and is most similar to motherwort yellow mottle virus (MYMoV). The nucleotide sequence identities of the Pro-Pol and coat protein regions were below the criteria established by the ICTV for demarcating species, confirming that CaTV1 should be classified as a member of a new species within the genus Torradovirus.

Transcriptome sequencing identifies novel persistent viruses in herbicide resistant wild-grasses.

Herbicide resistance in wild grasses is widespread in the UK, with non-target site resistance (NTSR) to multiple chemistries being particularly problematic in weed control. As a complex trait, NTSR is driven by complex evolutionary pressures and the growing awareness of the role of the phytobiome in plant abiotic stress tolerance, led us to sequence the transcriptomes of herbicide resistant and susceptible populations of black-grass and annual rye-grass for the presence of endophytes. Black-grass (Alopecurus myosuroides; Am) populations, displaying no overt disease symptoms, contained three previously undescribed viruses belonging to the Partititiviridae (AMPV1 and AMPV2) and Rhabdoviridae (AMVV1) families. These infections were widespread in UK black-grass populations and evidence was obtained for similar viruses being present in annual rye grass (Lolium rigidum), perennial rye-grass (Lolium perenne) and meadow fescue (Festuca pratensis). In black-grass, while no direct causative link was established linking viral infection to herbicide resistance, transcriptome sequencing showed a high incidence of infection in the NTSR Peldon population. The widespread infection of these weeds by little characterised and persistent viruses and their potential evolutionary role in enhancing plant stress tolerance mechanisms including NTSR warrants further investigation.

Detection and transmission of Carrot torrado virus, a novel putative member of the Torradovirus genus.

A new Torradovirus tentatively named Carrot torrado virus (CaTV) was an incidental finding following a next generation sequencing study investigating internal vascular necrosis in carrot. The closest related viruses are Lettuce necrotic leaf curl virus (LNLCV) found in the Netherlands in 2011 and Motherwort yellow mottle virus (MYMoV) found in Korea in 2014. Primers for reverse transcriptase-PCR (RT-PCR) and RT-qPCR were designed with the aim of testing for the presence of virus in plant samples collected from the field. Both methods successfully amplified the target from infected samples but not from healthy control samples. The specificity of the CaTV assay was also checked against other known carrot viruses and no cross-reaction was seen. A comparative study between methods showed RT-qPCR was the most reliable method, giving positive results in samples where RT-PCR fails. Evaluation of the Ct values following RT-qPCR and a direct comparison demonstrated this was due to improved sensitivity. The previous published Torradovirus genus specific RT-PCR primers were tested and shown to detect CaTV. Also, virus transmission experiments carried out suggest that unlike other species of the same genus, Carrot torrado virus could be aphid-transmitted.

Low-density lipoprotein receptor is a calcium/magnesium sensor - role of LR4 and LR5 ion interaction kinetics in low-density lipoprotein release in the endosome.

The low-density lipoprotein receptor (LDLR) captures circulating lipoproteins and delivers them in the endosome for degradation. Its function is essential for cholesterol homeostasis, and mutations in the LDLR are the major cause of familiar hypercholesterolemia. The release of LDL is usually attributed to endosome acidification. As the pH drops, the affinity of the LDLR/LDL complex is reduced, whereas the strength of a self-complex formed between two domains of the receptor (i.e. the LDL binding domain and the ß-propeller domain) increases. However, an alternative model states that, as a consequence of a drop in both pH and Ca(2+) concentration, the LDLR binding domain is destabilized in the endosome, which weakens the LDLR/LDL complex, thus liberating the LDL particles. In the present study, we test a key underlying assumption of the second model, namely that the lipoprotein binding repeats of the receptor (specifically repeats 4 and 5, LR4 and LR5) rapidly sense endosomal changes in Ca(2+) concentration. Our kinetic and thermodynamic analysis of Ca(2+) and Mg(2+) binding to LR4 and LR5, as well as to the tandem of the two (LR4-5), shows that both repeats spontaneously release Ca(2+) in a time scale much shorter than endosomal delivery of LDL, thus acting as Ca(2+) sensors that become unfolded under endosomal conditions. Our analysis additionally explains the lower Ca(2+) affinity of repeat LR4, compared to LR5, as arising from a very slow Ca(2+) binding reaction in the former, most likely related to the lower conformational stability of apolipoprotein LR4, compared to apolipoprotein LR5, as determined from thermal unfolding experiments and molecular dynamics simulations.