A common challenge in pathogen discovery by deep sequencing approaches is to recognize viral or subviral pathogens in samples of diseased tissue that share no significant homology with a known pathogen. Here we report a homology-independent approach for discovering viroids, a distinct class of free circular RNA subviral pathogens that encode no protein and are known to infect plants only. Our approach involves analyzing the sequences of the total small RNAs of the infected plants obtained by deep sequencing with a unique computational algorithm, progressive filtering of overlapping small RNAs (PFOR). Viroid infection triggers production of viroid-derived overlapping siRNAs that cover the entire genome with high densities. PFOR retains viroid-specific siRNAs for genome assembly by progressively eliminating nonoverlapping small RNAs and those that overlap but cannot be assembled into a direct repeat RNA, which is synthesized from circular or multimeric repeated-sequence templates during viroid replication. We show that viroids from the two known families are readily identified and their fulllength sequences assembled by PFOR from small RNAs sequenced from infected plants. PFOR analysis of a grapevine library further identified a viroid-like circular RNA 375 nt long that shared no significant sequence homology with known molecules and encoded active hammerhead ribozymes in RNAs of both plus and minus polarities, which presumably self-cleave to release monomer from multimeric replicative intermediates. A potential application of the homology-independent approach for viroid discovery in plant and animal species where RNA replication triggers the biogenesis of siRNAs is discussed. T he term "viroid" was first introduced in 1971 to describe a novel class of free RNA nonviral pathogens found in plants (1-3). Viroids are single-stranded circular RNA molecules of 246-401 nt in length and do not encode any protein. Viroid RNAs display extensive intramolecular base pairing to give rod-like or quasirod-like conformations. Replication of viroids occurs via a rolling circle mechanism by host RNA polymerases to yield head-to-tail multiple-repeat replicative intermediates. Viroids identified to date infect only plants and belong to one of the two families. Viroids in the Pospiviroidae such as potato spindle tuber viroid (PSTVd) share a five-domain model including a central conserved region (CCR) and may all replicate in the nucleus. By contrast, viroids in the Avsunviroidae such as peach latent mosaic viroid (PLMVd) lack a CCR, encode a hammerhead ribozyme in both the positive and the negative strands that self-cleaves to release monomer from the multimeric intermediates, and may all replicate in the chloroplasts (1-3).Identification of new viroids requires purification and enrichment of the naked viroid RNA by 2D gel electrophoresis before cDNA synthesis and sequencing (4, 5). However, viroids generally occur at low concentrations in the infected host, making viroid discovery a challenging task for many plant pathology laborato...

Clinical isolates of coagulase-negative staphylococci often elaborate a biofilm involved in adherence to medical devices and resistance to host defenses. The biofilm contains the capsular polysaccharide/adhesin (PS/A), which mediates cell adherence to biomaterials, and another antigen, termed polysaccharide intercellular adhesin (PIA), which is thought to mediate bacterial accumulation into cellular aggregates. PIA is a polymer of β-1,6-linkedN-acetyl glucosamine residues with a molecular mass of <30,000 kDa. We found that recombinant Staphylococcus carnosus and Staphylococcus aureus carrying a plasmid with genes of the ica locus, which was reported to encode the biosynthetic proteins for production of PIA, were also able to synthesize PS/A. PS/A and a chemically and immunologically identical polysaccharide isolated from S. carnosus carrying theica genes on plasmid pCN27 were found to be high-molecular-mass (>250,000 kDa), acid-stable polymers of β-1,6-linked glucosamine substituted on the amino group primarily with succinate, although some preparations also contained acetate. Moreover, all recombinant staphylococcal strains with theica genes had the biologic properties previously attributed to PS/A. ica-positive strains readily formed an in vitro biofilm on plastic, adhered 3- to 10-fold more to catheters during a 30-min assay compared with control strains carrying only the cloning vector, adsorbed out antibodies to PS/A from immune serum, and elaborated a capsule visualized by immunoelectron microscopy with antisera to PS/A. These properties were also seen with PS/A-producing strains of Staphylococcus epidermidis, but not with transposon mutants lacking PS/A. An antiserum raised to PIA contained high-titer antibody to PS/A that was readily adsorbed out by PS/A-positive strains of S. epidermidis and recombinant strains of staphylococci carrying the ica genes. We conclude that the ica locus encodes production of PS/A and that the properties of S. epidermidis associated with initial bacterial adherence, biofilm formation, and intercellular adhesion can be correlated with elaboration of PS/A.

Plant viral diseases cause tremendous decreases in crop yield and quality. Natural products have always been a valuable source for lead discovery in medicinal and agricultural chemistry. A series of pimprinine alkaloids and their derivatives were prepared and identified by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS). The antiviral activities of these alkaloids against tobacco mosaic virus (TMV) were systematically investigated for the first time. Most of the compounds exhibited higher antiviral activities than ribavirin. Compounds 5l, 9h, and 10h, which had similar or higher antiviral activities than ningnanmycin (perhaps the most widely used antiviral agent at present), emerged as new antiviral pilot compounds. This systematic structure–activity-relationship research lays the foundation for simplifying the structure of these alkaloids. The ring-open products, acylhydrazones 9a–9u, were also found to possess good antiviral activities. Moreover, all the synthesized compounds displayed broad-spectrum fungicidal activities. This study provides important information for the research and development of pimprinine alkaloids as novel antiviral agents.

Summary Many plant viruses with monopartite or bipartite genomes have been developed as efficient expression vectors of foreign recombinant proteins. Nonetheless, due to lack of multiple insertion sites in these plant viruses, it is still a big challenge to simultaneously express multiple foreign proteins in single cells. The genome of Beet necrotic yellow vein virus ( BNYVV ) offers an attractive system for expression of multiple foreign proteins owning to a multipartite genome composed of five positive‐stranded RNA s. Here, we have established a BNYVV full‐length infectious cDNA clone under the control of the Cauliflower mosaic virus 35S promoter. We further developed a set of BNYVV ‐based vectors that permit efficient expression of four recombinant proteins, including some large proteins with lengths up to 880 amino acids in the model plant Nicotiana benthamiana and native host sugar beet plants. These vectors can be used to investigate the subcellular co‐localization of multiple proteins in leaf, root and stem tissues of systemically infected plants. Moreover, the BNYVV ‐based vectors were used to deliver Nb PDS guide RNA s for genome editing in transgenic plants expressing Cas9, which induced a photobleached phenotype in systemically infected leaves. Collectively, the BNYVV ‐based vectors will facilitate genomic research and expression of multiple proteins, in sugar beet and related crop plants.

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