Na-Sheng Lin

Roles of plant hormones in the regulation of host–virus interactions

Summary Hormones are tuners of plant responses to biotic and abiotic stresses. They are involved in various complicated networks, through which they modulate responses to different stimuli. Four hormones primarily regulate plant defence to pathogens: salicylic acid (SA), jasmonic acid (JA), ethylene (Et) and abscisic acid (ABA). In susceptible plants, viral infections result in hormonal disruption, which manifests as the simultaneous induction of several antagonistic hormones. However, these antagonistic hormones may exhibit some sequential accumulation in resistant lines. Virus propagation is usually restricted by the activation of the small interfering RNA (siRNA) antiviral machinery and/or SA signalling pathway. Several studies have investigated these two systems, using different model viruses. However, the roles of hormones other than SA, especially those with antagonistic properties, such as ABA, have been neglected. Increasing evidence indicates that hormones control components of the small RNA system, which regulates many processes (including the siRNA antiviral machinery and the microRNA system) at the transcriptional or post‐transcriptional level. Consequently, cross‐talk between the antagonistic SA and ABA pathways modulates plant responses at multiple levels. In this review, we summarize recent findings on the different roles of hormones in the regulation of plant–virus interactions, which are helping us to elucidate the fine tuning of viral and plant systems by hormones.

Satellite RNAs and Satellite Viruses of Plants

The view that satellite RNAs (satRNAs) and satellite viruses are purely molecular parasites of their cognate helper viruses has changed. The molecular mechanisms underlying the synergistic and/or antagonistic interactions among satRNAs/satellite viruses, helper viruses, and host plants are beginning to be comprehended. This review aims to summarize the recent achievements in basic and practical research, with special emphasis on the involvement of RNA silencing mechanisms in the pathogenicity, population dynamics, and, possibly, the origin(s) of these subviral agents. With further research following current trends, the comprehensive understanding of satRNAs and satellite viruses could lead to new insights into the trilateral interactions among host plants, viruses, and satellites.

Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes.

BackgroundPlant viruses can be employed as versatile vectors for the production of vaccines by expressing immunogenic epitopes on the surface of chimeric viral particles. Although several viruses, including tobacco mosaic virus, potato virus X and cowpea mosaic virus, have been developed as vectors, we aimed to develop a new viral vaccine delivery system, a bamboo mosaic virus (BaMV), that would carry larger transgene loads, and generate better immunity in the target animals with fewer adverse environmental effects.MethodsWe engineered the BaMV as a vaccine vector expressing the antigenic epitope(s) of the capsid protein VP1 of foot-and-mouth disease virus (FMDV). The recombinant BaMV plasmid (pBVP1) was constructed by replacing DNA encoding the 35 N-terminal amino acid residues of the BaMV coat protein with that encoding 37 amino acid residues (T128-N164) of FMDV VP1.ResultsThe pBVP1 was able to infect host plants and to generate a chimeric virion BVP1 expressing VP1 epitopes in its coat protein. Inoculation of swine with BVP1 virions resulted in the production of anti-FMDV neutralizing antibodies. Real-time PCR analysis of peripheral blood mononuclear cells from the BVP1-immunized swine revealed that they produced VP1-specific IFN-γ. Furthermore, all BVP1-immunized swine were protected against FMDV challenge.ConclusionChimeric BaMV virions that express partial sequence of FMDV VP1 can effectively induce not only humoral and cell-mediated immune responses but also full protection against FMDV in target animals. This BaMV-based vector technology may be applied to other vaccines that require correct expression of antigens on chimeric viral particles.

Complete nucleotide sequence of Dendrocalamus latiflorus and Bambusa oldhamii chloroplast genomes.

Although bamboo is one of the most important woody crops in Asia, information on its genome is still very limited. To investigate the relationship among Poaceae members and to understand the mechanism of albino mutant generation in vitro, the complete chloroplast genome of two economically important bamboo species, Dendrocalamus latiflorus Munro and Bambusa oldhamii Munro, was determined employing a strategy that involved polymerase chain reaction (PCR) amplification using 443 novel primers designed to amplify the chloroplast genome of these two species. The lengths of the B. oldhamii and D. latiflorus chloroplast genomes are 139,350 and 139,365 bp, respectively. The organization structure and the gene order of these two bamboos are identical to other members of Poaceae. Highly conserved chloroplast genomes of Poaceae facilitated sequencing by the PCR method. Phylogenetic analysis using both chloroplast genomes confirmed the results obtained from studies on chromosome number and reproductive organ morphology. There are 23 gaps, insertions/deletions > 100 bp, in the chloroplast genomes of 10 genera of Poaceae compared in this study. The phylogenetic distribution of these gaps corresponds to their taxonomic placement. The sequences of these two chloroplast genomes provide useful information for studying bamboo evolution, ecology and biotechnology.

Hsp90 interacts specifically with viral RNA and differentially regulates replication initiation of Bamboo mosaic virus and associated satellite RNA.

Host factors play crucial roles in the replication of plus-strand RNA viruses. In this report, a heat shock protein 90 homologue of Nicotiana benthamiana, NbHsp90, was identified in association with partially purified replicase complexes from BaMV-infected tissue, and shown to specifically interact with the 3′ untranslated region (3′ UTR) of BaMV genomic RNA, but not with the 3′ UTR of BaMV-associated satellite RNA (satBaMV RNA) or that of genomic RNA of other viruses, such as Potato virus X (PVX) or Cucumber mosaic virus (CMV). Mutational analyses revealed that the interaction occurs between the middle domain of NbHsp90 and domain E of the BaMV 3′ UTR. The knockdown or inhibition of NbHsp90 suppressed BaMV infectivity, but not that of satBaMV RNA, PVX, or CMV in N. benthamiana. Time-course analysis further revealed that the inhibitory effect of 17-AAG is significant only during the immediate early stages of BaMV replication. Moreover, yeast two-hybrid and GST pull-down assays demonstrated the existence of an interaction between NbHsp90 and the BaMV RNA-dependent RNA polymerase. These results reveal a novel role for NbHsp90 in the selective enhancement of BaMV replication, most likely through direct interaction with the 3′ UTR of BaMV RNA during the initiation of BaMV RNA replication.

Identification of the RNA-binding sites of the triple gene block protein 1 of bamboo mosaic potexvirus

The triple gene block protein 1 (TGBp1) encoded by open reading frame 2 of bamboo mosaic potexvirus (BaMV) was overexpressed in Escherichia coli and purified in order to test its RNA-binding activity. UV crosslinking assays revealed that the RNA-binding activity was present mainly in the soluble fraction of the refolded TGBp1. The binding activity was nonspecific and salt concentration-dependent: activity was present at 0-50 mM NaCl but was almost abolished at 200 mM. The RNA-binding domain was located by deletion mutagenesis to the N-terminal 3-24 amino acids of TGBp1. Sequence alignment analysis of the N-terminal 25 amino acids of the TGBp1 homologues of potexviruses identified three arginine residues. Arg-to-Ala substitution at any one of the three arginines eliminated most of the RNA-binding activity, indicating that they were all critical to the RNA-binding activity of the TGBp1 of BaMV.

Glyceraldehyde 3-Phosphate Dehydrogenase Negatively Regulates the Replication of Bamboo Mosaic Virus and Its Associated Satellite RNA

ABSTRACT The identification of cellular proteins associated with virus replicase complexes is crucial to our understanding of virus-host interactions, influencing the host range, replication, and virulence of viruses. A previous in vitro study has demonstrated that partially purified Bamboo mosaic virus (BaMV) replicase complexes can be employed for the replication of both BaMV genomic and satellite BaMV (satBaMV) RNAs. In this study, we investigated the BaMV and satBaMV 3′ untranslated region (UTR) binding proteins associated with these replicase complexes. Two cellular proteins with molecular masses of ∼35 and ∼55 kDa were specifically cross-linked with RNA elements, whereupon the ∼35-kDa protein was identified as the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Gel mobility shift assays confirmed the direct interaction of GAPDH with the 3′ UTR sequences, and competition gel shift analysis revealed that GAPDH binds preferentially to the positive-strand BaMV and satBaMV RNAs over the negative-strand RNAs. It was observed that the GAPDH protein binds to the pseudoknot poly(A) tail of BaMV and stem-loop-C poly(A) tail of satBaMV 3′ UTR RNAs. It is important to note that knockdown of GAPDH in Nicotiana benthamiana enhances the accumulation of BaMV and satBaMV RNA; conversely, transient overexpression of GAPDH reduces the accumulation of BaMV and satBaMV RNA. The recombinant GAPDH principally inhibits the synthesis of negative-strand RNA in exogenous RdRp assays. These observations support the contention that cytosolic GAPDH participates in the negative regulation of BaMV and satBaMV RNA replication.

The abscisic acid pathway has multifaceted effects on the accumulation of Bamboo mosaic virus.

Accepted 29 October 2013. Abscisic acid (ABA) plays a key role in modulating plant responses to different biotic and abiotic stresses. However, the effect of ABA on virus infection is not fully understood. Here, we describe the effects of the ABA pathway on the accumulation of Bamboo mosaic virus (BaMV) and Cucumber mosaic virus (CMV) in two different hosts: Arabidopsis thaliana and Nicotiana benthamiana. We report that ABA2 plays a critical role in the accumulation of BaMV and CMV. Mutants downstream of ABA2 (aao3, abi1-1, abi3-1, and abi4-1) were susceptible to BaMV, indicating that the ABA pathway downstream of ABA2 is essential for BaMV resistance. The aba2-1 mutant decreased the accumulation of BaMV (+)RNA, (-)RNA, and coat protein, with the most dramatic effect being observed for (-)RNA. These findings were further validated by the use of virus-induced gene silencing and enzyme-linked immunosorbent assay in N. benthamiana. In addition, infecting N. benthamiana with BaMV or CMV increased ABA contents and activated the SA and ABA pathways, thereby disrupting the antagonism between these two cascades. Our findings uncover a novel role for ABA2 in supporting BaMV and CMV accumulation, distinct from the opposing role of its downstream genes.

Incidence of bamboo mosaic potexvirus in Taiwan

Bamboo mosaic virus (BaMV) was immunologically detected by direct tissue blotting in 11 bamboo species, including three varieties and three cultivars, in Taiwan. Viral RNA was detected in one additional bamboo species, Melocanna baccifera, by hybridization with a specific riboprobe for BaMV RNA. Most infected bamboo species have pachymorph rhizomes and belong to the genera Bambusa and Dendrocalamus. Species with other types of rhizomes were only rarely infected

Transgenic resistance to Cymbidium mosaic virus in Dendrobium expressing the viral capsid protein gene.

A Taiwan isolate of Cymbidium mosaic virus (CymMV-CS) was isolated from infected Cymbidium sinesis Willd. The cDNA of the capsid protein (CP) gene was synthesized and sequenced. Alignment of this CP gene with other reported CPs revealed homologies of 92–98% at the nucleotide level and 98–99% at the amino acid level. To generate virus-resistant varieties, the CymMV-CS CP gene was transformed into Dendrobium protocorms through particle bombardment. Transformants were selected on medium supplemented with 20 mg/L hygromycin and the presence of the transgene was confirmed by polymerase chain reaction, Southern, Northern and Western blot analyses. Transgenic Dendrobium harboring the CymMV CP gene expressed a very low level of virus accumulation four months post-inoculation with CymMV, as detected by ELISA. The transgenic plants exhibited much milder symptoms than the non-transgenic plants upon challenge with CymMV virions