Kian-Chung Lee

Salicylic acid-induced resistance to Cucumber mosaic virus in squash and Arabidopsis thaliana: Contrasting mechanisms of induction and antiviral action

Salicylic acid (SA)-induced resistance to Cucumber mosaic virus (CMV) in tobacco (Nicotiana tabacum) results from inhibition of systemic virus movement and is induced via a signal transduction pathway that also can be triggered by antimycin A, an inducer of the mitochondrial enzyme alternative oxidase (AOX). In Arabidopsis thaliana, inhibition of CMV systemic movement also is induced by SA and antimycin A. These results indicate that the mechanisms underlying induced resistance to CMV in tobacco and A. thaliana are very similar. In contrast to the situation in tobacco and A. thaliana, in squash (Cucurbita pepo), SA-induced resistance to CMV results from inhibited virus accumulation in directly inoculated tissue, most likely through inhibition of cell-to-cell movement. Furthermore, neither of the AOX inducers antimycin A or KCN induced resistance to CMV in squash. Additionally, AOX inhibitors that compromise SA-induced resistance to CMV in tobacco did not inhibit SA-induced resistance to the virus in squash. The results show that different host species may use significantly different approaches to resist infection by the same virus. These findings also imply that caution is required when attempting to apply findings on plant-virus interactions from model systems to a wider range of host species.

Cymbidium mosaic potexvirus RNA: complete nucleotide sequence and phylogenetic analysis

SummaryThe complete nucleotide sequence of the genomic RNA of cymbidium mosaic potexvirus (CymMV) was determined to be 6 227 nucleotides in length, excluding the poly (A) tail at the 3′ terminus. Similar to other potexviruses, its genome organisation is comprised of five major open reading frames (ORFs 1 to 5), encoding a Mr 160 KDa putative RNA-dependent RNA polymerase (RdRp); a Mr26KDa/13KDa/10KDa triple-gene-block (TGB) and a Mr 24 KDa coat protein. The CymMV encoded proteins shared a high degree of homology to their corresponding proteins of other members of the potexvirus group. The nucleotide sequence of the 5′ noncoding region (NCR) of CymMV and all other potexviruses initiates with GAAAA. CymMV possesses the shortest 5′ NCR among all potexviruses. Based on phylogenetic comparisons of RdRp and coat protein, CymMV shares a close relationship to PAMV, NMV, WClMV and SMYEaV. This is believed to be the first record of the complete nucleotide sequence of CymMV.

Breakage of resistance to Cucumber mosaic virus by co-infection with Zucchini yellow mosaic virus: enhancement of CMV accumulation independent of symptom expression.

Summary.Resistance to the cucumovirus Cucumber mosaic virus (CMV) in cucumber cv. Delila was manifested as a very low level of accumulation of viral RNA and capsid protein, and an absence of CMV-induced symptoms. In addition, resistance was observed at the single cell level, with a reduction in accumulation of CMV RNAs, compared to accumulation in cells of the susceptible cucumber cv. Bet Alpha. Resistance to CMV in cv. Delila was broken by co-infection with the potyvirus Zucchini yellow mosaic virus (ZYMV). Resistance breakage in cv. Delila plants was manifested by an increase in the accumulation of (+) and (−) CMV RNA as well as CMV capsid protein, with no increase in the level of accumulation of ZYMV. Resistance breakage in the resistant cultivar by ZYMV also occurred at the single cell level. Thus, synergistic interactions known to occur between a potyvirus and a cucumovirus led to resistance breakage during a double infection. However, resistance breakage was not accompanied by an increase in disease symptoms beyond those induced by ZYMV itself. On co-inoculation with an asymptomatic variant of ZYMV-AG an enhancement of CMV infection occurred without disease manifestation. Consequently, intensification of viral RNA and capsid protein accumulation can occur without a corresponding increase in disease development, suggesting that different host genes regulate viral accumulation and disease development in the CMV-resistant cucumber plants.

Phylogenetic Analysis of Triple Gene Block Viruses Based on the TGB 1 Homolog Gene Indicates a Convergent Evolution

The complete nucleotide sequence of the triple gene block one (TGB 1) of cymbidium mosaic potexvirus (CymMV) was compared to those from other potex-, carla-, furo- and hordeiviruses. Seven conserved motifs in the TGB 1, including the ATP-GTP binding domain (P-Loop) consensus GXXGKTSTS, were found in all four virus genera. We propose that all TGBV can be classified into phylogenetic clusters based on their TGB 1 homolog genes. These clusters can be further delineated to form subgroups. The first cluster comprises the potexviruses which are further subdivided into three subgroups; BaMV, FMV, PlaMV and PapMV (subgroup Ia); CymMV, PAMV, NMV, SMYEaV and WClMV (subgroup Ib) and PVX (subgroup Ic). The second cluster comprises carlaviruses with a dual subgrouping; CVB, LSV, PVM, PMV and ASPV (subgroup IIa) and LVX (subgroup IIb). The third cluster carries the most diverse of TGBV comprising furoviruses PCV, PMTV and BSBV (subgroup IIIa) and hordeiviruses PSLV, BSMV and LRSV (subgroup IIIb). The phylogenetic relationships of triple gene block viruses (TGBV) based on the TGB 1 homolog gene indicates a convergent evolution.

Cucurbit protoplast isolation for the study of plant virus replication.

A cucurbit protoplast isolation protocol was established for the study of plant virus replication in vivo. This protocol is applicable to both cucumber and squash leaf tissue with significant increases in yields of viable protoplasts suitable for electroporation, compared to other published methods. A combination of Cellulase RS, Macerozyme R10 and mannitol was used as digestion enzymes and osmoticum. An average of 1.7x10(7) protoplasts per gram of fresh leaf tissue were obtained from cucumber cultivar Bet-alpha. Both cucumber cultivar Shimson and squash cultivar First Taste produced an average yield of 6.0x10(6) protoplasts per g of fresh leaf tissue. Electroporation of 10 microg of Zucchini yellow mosaic potyvirus (ZYMV-S) RNA into the protoplasts resulted in virus replication and synthesis of coat protein (CP). SDS-PAGE and immunoblotting were used to detect the CP 48 h post-electroporation. This protocol is highly reproducible and will assist researchers who require cucurbit protoplasts to study virus replication.

Sequence and Phylogenetic Analysis of the Cytoplasmic Inclusion Protein Gene of Zucchini Yellow Mosaic Potyvirus: Its Role in Classification of the Potyviridae

The cytoplasmic inclusion (CI) gene of a Singapore isolate of zucchini yellow mosaic virus (ZYMV-S) was sequenced and compared with CI of 14 other potyviruses. In addition to the consensus sequence GAVGSGKST of nucleotide binding motif (NTBM) which is implicated as a membrane-binding component of the RNA helicase complex, five other conserved motifs were found. Phylogenetic trees were constructed from sequence data for the CI and the coat protein. Similar branching patterns obtained from both CI and coat protein analyses suggests that phylogenetic relationship among potyviruses can be determined using the CI. We propose that phylogenetic analysis of CI gene may be used as an alternative approach for the study of evolution within the family Potyviridae.

Purification, crystallization and X-ray analysis of Hibiscus chlorotic ringspot virus

Hibiscus chlorotic ringspot virus (HCRSV), a Carmovirus, occurs worldwide and induces chlorotic ringspots on leaves, stunting and flower distortion in Hibiscus species, including kenaf. The HCRSV capsid has T = 3 icosahedral symmetry and contains 180 copies of the coat protein. A virus yield of 48-70 mg per 100 g of infected kenaf leaves was achieved with an improved purification scheme involving sucrose-cushion and sucrose density-gradient centrifugation. The virus was crystallized using PEG 8000 and 2,3-butanediol as co-precipitants. The crystals belonged to the cubic space group P23, with unit-cell parameter a = 392 A, and diffracted X-rays to at least 4.5 A resolution.

Microfluidic chips for viral RNA extraction & detection

Sensing biomolecules at minute quantities demands laborious and skill-laden laboratory protocols for sample preparation and amplification in order to improve signal-to-noise ratio. Nucleic-acid-based detection of viral particles in whole blood requires separation of viral particles from blood cells followed by extraction, amplification, and detection of viral nucleic acids. Here, three microfluidic chips have been independently shown to be capable of performing each critical step. Separation of viral particles involves a flow-through, shear-type microfilter chip that can handle large volume of blood. The remaining chips, although developed for genomic DNA, have been adopted for extraction and amplification of viral RNA. In the extraction chip, protein coatings around viral particles are chemically broken to liberate viral RNA which can reversibly bind to the chip surface under high-salt conditions. Viral RNA can be eluted out with a low-salt buffer after removal of unwanted debris. In the amplification chip, viral RNA is first transcribed into cDNA and then multiplied exponentially in copies by a continuous, isothermal, enzyme-based technique known as Nucleic Acid Sequence-Based Amplification (NASBA). The amplicons are detected on the same chip using DNA probes conjugated with horse radish peroxide (HRP) for colorimetry