Molecular characterization of horse nettle virus A, a new member of subgroup B of the genus Nepovirus.

A new positive-strand RNA virus was discovered in a horse nettle plant, using high-throughput sequencing (HTS), and its complete genome, consisting of RNA1 and RNA2, which are 7522 and 4710 nucleotides in length, respectively, was characterized. Each genome segment contains a single open reading frame flanked by 5' and 3' untranslated regions (UTRs), followed by a poly(A) tail at the 3' end. The encoded proteins have the highest amino acid sequence identity (55% and 45%) to the polyprotein encoded by RNA1 of tomato black ring virus (TBRV) and RNA2 of potato virus B (PVB), respectively. Its genome organization and phylogenetic relationship to other nepoviruses suggested that this virus is a novel member of subgroup B, and recombination analysis revealed its evolutionary history within the subgroup. These results suggest the new virus, provisionally named "horse nettle virus A", represents a new species within the genus Nepovirus.

High-throughput detection of a large set of viruses and viroids of pome and stone fruit trees by multiplex PCR-based amplicon sequencing.

A comprehensive diagnostic method of known plant viruses and viroids is necessary to provide an accurate phytosanitary status of fruit trees. However, most widely used detection methods have a small limit on either the number of targeted viruses/viroids or the number of samples to be evaluated at a time, hampering the ability to rapidly scale up the test capacity. Here we report that by combining the power of high multiplexing PCR (499 primer pairs) of small amplicons (120-135bp), targeting 27 viruses and 7 viroids of fruit trees, followed by a single high-throughput sequencing (HTS) run, we accurately diagnosed the viruses and viroids on as many as 123 pome and stone fruit tree samples. We compared the accuracy, sensitivity, and reproducibility of this approach and contrast it with other detection methods including HTS of total RNA (RNA-Seq) and individual RT-qPCR for every fruit tree virus or viroid under the study. We argue that this robust and high-throughput cost-effective diagnostic tool will enhance the viral/viroid knowledge of fruit trees while increasing the capacity for large scale diagnostics. This approach can also be adopted for the detection of multiple viruses and viroids in other crops.

Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level.

Small proteins are a new and expanding area of research. Many characterized small proteins are composed of a single hydrophobic α-helix, and the functional requirements of their limited amino acid sequence are not well understood. One hydrophobic small protein, CydX, has been shown to be a component of the cytochrome bd oxidase complex in Escherichia coli, and is required for enzyme function. To investigate small protein sequence specificity, an alanine scanning mutagenesis on the small protein CydX was conducted using mutant alleles expressed from the E. coli chromosome at the wild-type locus. The resulting mutant strains were assayed for CydX function. No single amino acid was required to maintain wild-type resistance to ß-mercaptoethanol. However, substitutions of 10-amino acid blocks indicated that the N-terminus of the protein was required for wild-type CydX activity. A series of double mutants showed that multiple mutations at the N-terminus led to ß-mercaptoethanol sensitivity in vivo. Triple mutants showed both in vivo and in vitro phenotypes. Together, these data provide evidence suggesting a high level of functional plasticity in CydX, in which multiple amino acids may work cooperatively to facilitate CydX function.