Discovery of a Closterovirus Infecting Jujube Plants Grown at Aksu Area in Xinjiang of China.

Chinese jujube (Ziziphus jujuba Mill.) is a widely grown fruit crop at Aksu in Xinjiang Uygur Autonomous Region of China. Viral disease-like symptoms are common on jujube plants. Here, for the first time, we report a virus tentatively named persimmon ampelovirus jujube isolate (PAmpV-Ju) infecting jujube plants. The virus was identified using high-throughput sequencing from a jujube plant (ID: AKS15) and molecularly related to viruses in the family Closteroviridae. The genomic sequences of two PAmpV-Ju variants named AKS15-20 and AKS15-17 were determined by RT-PCR amplifications. The genome structure of PAmpV-Ju was identical to that of a recently reported persimmon ampelovirus (PAmpV) and consisted of seven open reading frames. The genomes of AKS15-20 and AKS15-17 shared 83.7% nt identity with each other, and the highest nt sequence identity of 79% with two variants of PAmpV. The incidence of PAmpV-Ju on Aksu jujube plants was evaluated by RT-PCR assays. The phylogenetic analysis of amplified partial sequences coding for polymerase, HSP70h, and CP revealed two phylogenetic clades represented by AKS15-20 and AKS15-17. Our study provides important evidence for understanding viruses infecting jujube plants and establishing efficient measures to prevent virus spread.

Complete genome sequence of a novel virus belonging to the genus Badnavirus in jujube (Ziziphus jujuba Mill.) in China.

The genome sequence of a novel circular DNA virus related to members of the genus Badnavirus was identified in diseased jujube trees by high-throughput sequencing and verified by conventional Sanger sequencing of cloned PCR products. The name "jujube badnavirus WS" (JuBWS) is proposed for this virus. Diseased jujube leaves showed yellow mosaic and malformation symptoms, with round chlorotic spots found on diseased fruit. The genome of this virus has a length of 6450 nt and has a typical badnavirus genomic structure with three open reading frames (ORFs). JuBWS was identified as a novel badnavirus based on nucleotide differences in the RNase (RT + RNase H) coding region of ORF3. The JuBWS sequence showed 70.48-76.41% nucleotide sequence identity to other known badnaviruses, thus meeting the taxonomic criterion for establishing a new species within the genus Badnavirus. This study suggested that the novel badnavirus might be a pathogen associated with jujube mosaic disease, and this will be investigated in the future.

A novel Actinidia cytorhabdovirus characterized using genomic and viral protein interaction features.

A novel cytorhabdovirus, tentatively named Actinidia virus D (AcVD), was identified from kiwifruit (Actinidia chinensis) in China using high-throughput sequencing technology. The genome of AcVD consists of 13,589 nucleotides and is organized into seven open reading frames (ORFs) in its antisense strand, coding for proteins in the order N-P-P3-M-G-P6-L. The ORFs were flanked by a 3' leader sequence and a 5' trailer sequence and are separated by conserved intergenic junctions. The genome sequence of AcVD was 44.6%-51.5% identical to those of reported cytorhabdoviruses. The proteins encoded by AcVD shared the highest sequence identities, ranging from 27.3% (P6) to 44.5% (L), with the respective proteins encoded by reported cytorhabdoviruses. Phylogenetic analysis revealed that AcVD clustered together with the cytorhabdovirus Wuhan insect virus 4. The subcellular locations of the viral proteins N, P, P3, M, G, and P6 in epidermal cells of Nicotiana benthamiana leaves were determined. The M protein of AcVD uniquely formed filament structures and was associated with microtubules. Bimolecular fluorescence complementation assays showed that three proteins, N, P, and M, self-interact, protein N plays a role in the formation of cytoplasm viroplasm, and protein M recruits N, P, P3, and G to microtubules. In addition, numerous paired proteins interact in the nucleus. This study presents the first evidence of a cytorhabdovirus infecting kiwifruit plants and full location and interaction maps to gain insight into viral protein functions.

Molecular Characteristics of Jujube Yellow Mottle-Associated Virus Infecting Jujube (Ziziphus jujuba Mill.) Grown at Aksu in Xinjiang of China.

Chinese jujube (Ziziphus jujuba Mill.) is a native fruit crop in China. Leaf mottle and dapple fruit disease is prevalent in cultivated jujube plants grown at Aksu in Xinjiang Uygur Autonomous Region of China. Jujube yellow mottle-associated virus (JYMaV), a tentative member in the genus Emaravirus, was recently identified from mottle-diseased jujube plants grown in Liaoning Province in China, but its incidence and genetic diversity in China is unknown. In this study, the genome sequences of three JYMaV isolates from two jujube cultivars and one jujube variant were determined by high-throughput sequencing (HTS) for small RNA and rRNA-depleted RNA coupled with RT-PCR assays. Comparison of these sequences together with sequences of the viral RNA segments derived by primer set 3C/5H-based RT-PCR revealed that genetic diversity was present in the virus populations and high sequence variation occurred at the non-translational regions of each of the viral genomic segments. Field investigation confirmed the close association of the virus with leaf mottle symptoms of jujube plants. Furthermore, this study revealed that P5 encoded in the viral RNA5 displayed a nuclear localization feature differing from the plasmodesma (PD) subcellular localization of the virus movement protein (P4), and the two proteins could interact with each other in the BiFC assays. Our study provides a snapshot of JYMaV genetic diversity in its natural hosts.

Cloning of a xylanase gene from Aspergillus usamii and its expression in Escherichia coli.

The complete gene xyn// that encodes endo-1,4-beta-xylanase secreted by Aspergillus usamii E001 was cloned and sequenced. The coding region of the gene is separated by only one intron. It encodes 184 amino acid residues of a protein with a calculated molecular weight of 19.8kDa plus a signal peptide of 27 amino acids. The amino acid sequence of the xyn// gene has higher similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The mature peptide encoding cDNA was subcloned into pET-28a(+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL, and xylanase activity was measured. The expressed fusion protein was analyzed by SDS-PAGE and a new specific band with molecular weight of about 20kDa was found when induced by IPTG. Enzyme activity assay verified the recombinant protein as a xylanase. A maximum activity of 49.6Umg(-1) was obtained from cellular extract of E. coli BL21-CodonPlus (DE3)-RIL harboring pET-28a-xyn//. The xylanase had optimal activity at pH 4.6 and 50 degrees C. This is the first report on the cloning of a xylanase gene from A. usamii.