Zhenchao Wang

The Development and Application of a Dot-ELISA Assay for Diagnosis of Southern Rice Black-Streaked Dwarf Disease in the Field

Outbreaks of the southern rice black-streaked dwarf virus (SRBSDV) have caused significant crop losses in southern China in recent years, especially in 2010. There are no effective, quick and practicable methods for the diagnosis of rice dwarf disease that can be used in the field. Traditional reverse transcription-polymerase chain reaction (RT-PCR) methodology is accurate but requires expensive reagents and instruments, as well as complex procedures that limit its applicability for field tests. To develop a sensitive and reliable assay for routine laboratory diagnosis, a rapid dot enzyme-linked immunosorbent assay (dot-ELISA) method was developed for testing rice plants infected by SRBSDV. Based on anti-SRBSDV rabbit antiserum, this new dot-ELISA was highly reliable, sensitive and specific toward SRBSDV. The accuracy of two blotting media, polyvinylidene fluoride membrane (PVDF membrane) and nitrocellulose filter membrane (NC membrane), was compared. In order to facilitate the on-site diagnosis, three county laboratories were established in Shidian (Yunnan province), Jianghua (Hunan Province) and Libo (Guizhou province). Suspected rice cases from Shidian, Yuanjiang and Malipo in Yunnan province were tested and some determined to be positive for SRBSDV by the dot-ELISA and confirmed by the One Step RT-PCR method. To date, hundreds of suspected rice samples collected from 61 districts in southwestern China have been tested, among which 55 districts were found to have rice crops infected by SRBSDV. Furthermore, the test results in the county laboratories showed that Libo, Dehong (suspected samples were sent to Shidian) and Jianghua were experiencing a current SRBSDV outbreak.

Dufulin Activates HrBP1 to Produce Antiviral Responses in Tobacco

Background Dufulin is a new antiviral agent that is highly effective against plant viruses and acts by activating systemic acquired resistance (SAR) in plants. In recent years, it has been used widely to prevent and control tobacco and rice viral diseases in China. However, its targets and mechanism of action are still poorly understood. Methodology/Principal Findings Here, differential in-gel electrophoresis (DIGE) and classical two-dimensional electrophoresis (2-DE) techniques were combined with mass spectrometry (MS) to identify the target of Dufulin. More than 40 proteins were found to be differentially expressed (≥1.5 fold or ≤1.5 fold) upon Dufulin treatment in Nicotiana tabacum K326. Based on annotations in the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, these proteins were found to be related to disease resistance. Directed acyclic graph (DAG) analysis of the various pathways demonstrated harpin binding protein-1 (HrBP1) as the target of action of Dufulin. Additionally, western blotting, semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), and real time PCR analyses were also conducted to identify the specific mechanism of action of Dufulin. Our results show that activation of HrBP1 triggers the salicylic acid (SA) signaling pathway and thereby produces antiviral responses in the plant host. A protective assay based on lesion counting further confirmed the antiviral activity of Dufulin. Conclusion This study identified HrBP1 as a target protein of Dufulin and that Dufulin can activate the SA signaling pathway to induce host plants to generate antiviral responses.

Quantitative detection of relative expression levels of the whole genome of Southern rice black-streaked dwarf virus and its replication in different hosts

BackgroundIn recent years, a disease caused by Southern rice black-streaked dwarf virus (SRBSDV) has resulted in significant loss in rice production in Southern China and has spread quickly throughout East and Southeast Asia. This virus is transmitted by an insect vector, white-backed planthopper (WBPH) Sogatella furcifera (Hemiptera: Delphacidae), in a persistent propagative manner. Aside from rice, SRBSDV can also infect numerous Poaceae plants. However, the molecular mechanism of interaction between SRBSDV and its plant or insect vector remains unclear. In order to address this, we investigated the whole viral genome relative mRNA expression level in distinct hosts and monitored their expression level in real-time in rice plants.MethodsIn this study, a reliable, rapid, and sensitive method for detecting viral gene expression transcripts is reported. A SYBR Green I based real-time polymerase chain reaction (PCR) method was adopted for the quantitative detection of SRBSDV gene expression in different hosts and real-time changes in gene expression in rice.ResultsCompared to the relative mRNA expression level of the whole genome of SRBSDV, P3, P7-1, and P9-2 were dominantly expressed in rice and WBPH. Similarly, these genes also exhibited high expression levels in corn, suggesting that they have more important functions than other viral genes in the interaction between SRBSDV and hosts, and that they could be used as molecular detection target genes of SRBSDV. In contrast, the levels of P6 and P10 were relative low. Western blotting analysis partially was also verified our qPCR results at the level of protein expression. Analysis of the real-time changes in SRBSDV-infected rice plants revealed four distinct temporal expression patterns of the thirteen genes. Moreover, expression levels of P1 and other genes were significantly down-regulated on days 14 and 20, respectively.ConclusionSRBSDV genes showed similar expression patterns in distinct hosts (rice, corn, and WBPH), indicating that SRBSDV uses the same infection strategy in plant and insect hosts. P3, P7-1, and P9-2 were the dominantly expressed genes in the three tested hosts. Therefore, they are likely to be genes with the most crucial function and could be used as sensitive molecular detection targets for SRBSDV. Furthermore, real-time changes in SRBSDV genes provided a basis for understanding the mechanism of interaction between SRBSDV and its hosts.

Chiral cinchona alkaloid-derived thiourea catalyst for enantioselective synthesis of novel β-amino esters by mannich reaction

A cinchona alkaloid-derived thiourea catalyst has been designed to access new asymmetric β-amino esters bearing benzothiazole moiety by utilizing a Mannich reaction between an imine and a malonate. A simultaneous activation of the two imine functionalities and malonate by the bifunctional chiral organocatalyst is proposed to account for the good yields (71-91%) and high enantiomeric excess (89.4-98.5%) under mild conditions.

Crystal Structure of a Four-Layer Aggregate of Engineered TMV CP Implies the Importance of Terminal Residues for Oligomer Assembly

Background Crystal structures of the tobacco mosaic virus (TMV) coat protein (CP) in its helical and disk conformations have previously been determined at the atomic level. For the helical structure, interactions of proteins and nucleic acids in the main chains were clearly observed; however, the conformation of residues at the C-terminus was flexible and disordered. For the four-layer aggregate disk structure, interactions of the main chain residues could only be observed through water–mediated hydrogen bonding with protein residues. In this study, the effects of the C-terminal peptides on the interactions of TMV CP were investigated by crystal structure determination. Methodology/Principal Findings The crystal structure of a genetically engineered TMV CP was resolved at 3.06 Å. For the genetically engineered TMV CP, a six-histidine (His) tag was introduced at the N-terminus, and the C-terminal residues 155 to 158 were truncated (N-His-TMV CP19). Overall, N-His-TMV CP19 protein self-assembled into the four-layer aggregate form. The conformations of residues Gln36, Thr59, Asp115 and Arg134 were carefully analyzed in the high radius and low radius regions of N-His-TMV CP19, which were found to be significantly different from those observed previously for the helical and four-layer aggregate forms. In addition, the aggregation of the N-His-TMV CP19 layers was found to primarily be mediated through direct hydrogen-bonding. Notably, this engineered protein also can package RNA effectively and assemble into an infectious virus particle. Conclusion The terminal sequence of amino acids influences the conformation and interactions of the four-layer aggregate. Direct protein–protein interactions are observed in the major overlap region when residues Gly155 to Thr158 at the C-terminus are truncated. This engineered TMV CP is reassembled by direct protein–protein interaction and maintains the normal function of the four-layer aggregate of TMV CP in the presence of RNA.

Methodology for antibody preparation and detection of southern rice black-streaked dwarf virus

Southern rice black-streaked dwarf virus (SRBSDV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in rice. Seven polypeptide fragments of the Putative uncharacterized protein (Pup) and the P10 proteins of SRBSDV were designed, synthesized, and used to immunize rabbits. Titers of polyclonal antibodies against the seven peptides were examined using indirect enzyme-linked immunosorbent assay (ELISA), and their specificities were investigated using western blotting. Indirect dot-immunobinding assay (DIBA) was also carried out at different dilutions against an antigen (rice extract). Antibody-1, which had the highest selectivity and titer, was then used to examine rice samples suspected of being infected with SRBSDV that were collected for over two years in different areas of China, using DIBA. Our results indicate that antibody-1 has the advantages of reliability, high sensitivity, and high specificity. Use of this antibody can help facilitate identification of the virus and its distribution in rice-growing areas where it causes significant problems.

Interaction Research on the Antiviral Molecule Dufulin Targeting on Southern Rice Black Streaked Dwarf Virus P9-1 Nonstructural Protein

Southern rice black streaked dwarf virus (SRBSDV) causes severe harm to rice production. Unfortunately, studies on effective antiviral drugs against SRBSDV and interaction mechanism of antiviral molecule targeting on SRBSDV have not been reported. This study found dufulin (DFL), an ideal anti-SRBSDV molecule, and investigated the interactions of DFL targeting on the nonstructural protein P9-1. The biological sequence information and bonding characterization of DFL to four kinds of P9-1 protein were described with fluorescence titration (FT) and microscale thermophoresis (MST) assays. The sequence analysis indicated that P9-1 had highly-conserved C- and N-terminal amino acid residues and a hypervariable region that differed from 131 aa to 160 aa. Consequently, wild-type (WT-His-P9-1), 23 C-terminal residues truncated (TR-ΔC23-His-P9-1), 6 N-terminal residues truncated (TR-ΔN6-His-P9-1), and Ser138 site-directed (MU-138-His-P9-1) mutant proteins were expressed. The FT and MST assay results indicated that DFL bounded to WT-His-P9-1 with micromole affinity and the 23 C-terminal amino acids were the potential targeting site. This system, which combines a complete sequence analysis, mutant protein expression, and binding action evaluating system, could further advance the understanding of the interaction abilities between antiviral drugs and their targets.

Development of proteomic technology of shotgun and label free combined with multiple reaction monitoring to simultaneously detect southern rice black-streaked dwarf virus and rice ragged stunt virus

The co-infection of rice caused by southern rice black-streaked dwarf virus (SRBSDV) and rice ragged stunt virus (RRSV) was widely found at many regions, such as Yunnan Province, China, and North and Central Vietnam. These rice viral diseases lead to seriously yield loss of rice. In this study, the proteomics technology of shotgun and label free combined with multiple reaction monitoring (MRM) was developed to detect rice sample of a single or/and co-infection. The shotgun assay indicated that some proteins coded by SRBSDV and RRSV were detected via the mode of in-gel digestion, except for P5-2, P7-2 and P9-2 of SRBSDV and P4b, P5, P6, P8a and P8b of RRSV. The technology of label free combined with MRM indicated that P2, P5-1, P4, P8, P7-1, P6, P9-1 and P10 of SRBSDV and P1, P3 and P9 of RRSV were higher abundance in rice plant, and P5-2, P7-2 and P9-2 of SRBSDV and P4b and P5 of RRSV were lower abundance in viruliferous-rice plant. So SRBSDV P9-1 and RRSV P3 was selected as marker molecule to be used in detection technology, and the label free combined with MRM technology was established to detect two kinds of rice virus.

Screening anti-southern rice black-streaked dwarf virus drugs based on S7-1 gene expression in rice suspension cells.

Southern rice black-streaked dwarf virus (SRBSDV) is a rice pathogen that had an outbreak in southern China in 2010 and caused significant crop losses. Therefore, screening for effective antiviral drugs against SRBSDV is very important. This study used rice suspension cells infected with SRBSDV by polyethylene glycol-mediated uptake for screening antiviral drugs. SRBSDV P7-1, which is coded by the S7-1 gene, has an intrinsic ability to self-interact to form tubules that play an important role in viral infection. Therefore, relative expression level of the SRBSDV S7-1 gene in infected rice suspension cells was assayed by real-time quantitative polymerase chain reaction to evaluate the antiviral activities of various drugs. Dufulin displayed the highest inhibitory activity against SRBSDV S7-1 expression. In addition, changes in peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL) activities were determined in inoculated and noninoculated cells. The results showed that both POD and PPO activities increased upon dufulin treatment. Furthermore, the validity of this approach was confirmed in an in vivo experiment in which dufulin was found to effectively inhibit SRBSDV.

Dot immunobinding assay method with chlorophyll removal for the detection of southern rice black-streaked dwarf virus.

Southern rice black-streaked dwarf virus (SRBSDV), a new virus from Fiji, has seriously damaged rice crops in southern China and northern Vietnam in recent years. This virus is difficult to diagnose in the early stages of infection, and is very destructive at the late stage. In the present study, a dot immunobinding assay (DIBA) that has a high sensitivity for diagnosing SRBSDV was developed. Two kinds of treatment for the DIBA were evaluated to determine the most effective one for removing chlorophyll interferences via rice extraction. The first included several reagents to remove chlorophyll, namely, the alkaline reagents like magnesium oxide and alumina oxide, the adsorbent reagents like activated carbon and bentonite, as well as the extraction agent acetone. The second and third treatments, which were used to remove chlorophyll in blot membrane-nitrocellulose and polyvinylidene fluoride (PVDF), included several organic solvents containing methanol, ethanol, acetone, ethyl acetate, and diethyl ether. The results showed that activated carbon and methanol yielded the best contrasting purple color for the infected samples by decreasing the chlorophyll content.