The development and application of new crystallization method for tobacco mosaic virus coat protein.

BACKGROUND: Although tobacco mosaic virus (TMV) coat protein (CP) has been isolated from virus particles and its crystals have grown in ammonium sulfate buffers for many years, to date, no one has reported on the crystallization of recombinant TMV-CP connecting peptides expressed in E. coli. METHODS: In the present papers genetically engineered TMV-CP was expressed, into which hexahistidine (His) tags or glutathione-S-transferase (GST) tags were incorporated. Considering that GST-tags are long peptides and His-tags are short peptides, an attempt was made to grow crystals of TMV-CP cleaved GST-tags (WT-TMV-CP32) and TMV-CP incorporated His-tags (WT-His-TMV-CP12) simultaneously in ammonium sulfate buffers and commercial crystallization reagents. It was found that the 20S disk form of WT-TMV-CP32 and WT-His-TMV-CP12 did not form high resolution crystals by using various crystallization buffers and commercial crystallization reagents. Subsequently, a new experimental method was adopted in which a range of truncated TMV-CP was constructed by removing several amino acids from the N- or the C-terminal, and high resolution crystals were grown in ammonium sulfate buffers and commercial crystallization reagents. RESULTS: The new crystallization method was developed and 3.0 Å resolution macromolecular crystal was thereby obtained by removing four amino acids at the C-terminal of His-TMV-CP and connecting six His-tags at the N-terminal of His-TMV-CP (TR-His-TMV-CP19). The Four-layer aggregate disk structure of TR-His-TMV-CP19 was solved. This phenomenon showed that peptides at the C-terminus hindered the growth of high resolution crystals and the peptides interactions at the N-terminus were attributed to the quality of TMV-CP crystals. CONCLUSION: A 3.0 Å resolution macromolecular crystal of TR-His-TMV-CP19 was obtained and the corresponding structure was solved by removing four amino acids at the C-terminus of TMV-CP and connecting His-tags at the N-terminus of TMV-CP. It indicated that short peptides influenced the resolution of TMV-CP crystals.

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.

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.

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 CP(19)). Overall, N-His-TMV CP(19) 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 CP(19), 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 CP(19) 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.

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 K(326). 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.