Chunhong Wei

The Rice Dwarf Virus P2 Protein Interacts with ent-Kaurene Oxidases in Vivo, Leading to Reduced Biosynthesis of Gibberellins and Rice Dwarf Symptoms

The mechanisms of viral diseases are a major focus of biology. Despite intensive investigations, how a plant virus interacts with host factors to cause diseases remains poorly understood. The Rice dwarf virus (RDV), a member of the genus Phytoreovirus, causes dwarfed growth phenotypes in infected rice (Oryza sativa) plants. The outer capsid protein P2 is essential during RDV infection of insects and thus influences transmission of RDV by the insect vector. However, its role during RDV infection within the rice host is unknown. By yeast two-hybrid and coimmunoprecipitation assays, we report that P2 of RDV interacts with ent-kaurene oxidases, which play a key role in the biosynthesis of plant growth hormones gibberellins, in infected plants. Furthermore, the expression of ent-kaurene oxidases was reduced in the infected plants. The level of endogenous GA1 (a major active gibberellin in rice vegetative tissues) in the RDV-infected plants was lower than that in healthy plants. Exogenous application of GA3 to RDV-infected rice plants restored the normal growth phenotypes. These results provide evidence that the P2 protein of RDV interferes with the function of a cellular factor, through direct physical interactions, that is important for the biosynthesis of a growth hormone leading to symptom expression. In addition, the interaction between P2 and rice ent-kaurene oxidase-like proteins may decrease phytoalexin biosynthesis and make plants more competent for virus replication. Moreover, P2 may provide a novel tool to investigate the regulation of GA metabolism for plant growth and development.

Arabidopsis Putative Deacetylase AtSRT2 Regulates Basal Defense by Suppressing PAD4, EDS5 and SID2 Expression

The silent information regulator protein (Sir2) and its homologs are NAD+-dependent deacetylase enzymes that play important roles in a variety of physiological processes. However, the functions of the Sir2 family in plants are poorly understood. Here, we report that Arabidopsis AtSRT2, a homolog of yeast Sir2, negatively regulates plant basal defense against the pathogen Pseudomonas syringae pv. tomato DC3000 (PstDC3000). In response to PstDC3000 infection, the expression of AtSRT2 was down-regulated in a salicylic acid (SA)-independent manner. In addition, knock-out of AtSRT2 (srt2) enhanced resistance against PstDC3000 and increased expression of pathogenesis-related gene 1 (PR1). Conversely, overexpression of AtSRT2 resulted in hypersusceptibility to PstDC3000 and impaired PR1 induction. Consistent with this phenotype, expression of PAD4, EDS5 and SID2, three essential genes in the SA biosynthesis pathway, were increased in the srt2 mutant and decreased in AtSRT2-overexpressing plants. Taken together, these results demonstrate that AtSRT2 is a negative regulator of basal defense, possibly by suppressing SA biosynthesis.

Viral suppression of RNA silencing.

Gene silencing (RNA silencing) plays a fundamental role in antiviral defense in plants, fungi and invertebrates. Viruses encode proteins that suppress gene silencing to counter host defense. Viral suppressors of RNA silencing (VSRs) have been identified from almost all plant virus genera and some viruses of insects and mammals. Recent studies have revealed that VSRs counter host defense and interfere with host gene regulation by interacting with RNA or important components of the RNA silencing pathway. Here, we review the current understanding of the complex mechanisms of VSRs that have been revealed by recent studies.

RNA-dependent RNA polymerase 6 of rice (Oryza sativa) plays role in host defense against negative-strand RNA virus, Rice stripe virus.

RNA-dependent RNA polymerases (RDRs) from fungi, plants and some invertebrate animals play fundamental roles in antiviral defense. Here, we investigated the role of RDR6 in the defense of economically important rice plants against a negative-strand RNA virus (Rice stripe virus, RSV) that causes enormous crop damage. In three independent transgenic lines (OsRDR6AS line A, B and C) in which OsRDR6 transcription levels were reduced by 70-80% through antisense silencing, the infection and disease symptoms of RSV were shown to be significantly enhanced. The hypersusceptibilities of the OsRDR6AS plants were attributed not to enhanced insect infestation but to enhanced virus infection. The rise in symptoms was associated with the increased accumulation of RSV genomic RNA in the OsRDR6AS plants. The deep sequencing data showed reduced RSV-derived siRNA accumulation in the OsRDR6AS plants compared with the wild type plants. This is the first report of the antiviral role of a RDR in a monocot crop plant in the defense against a negative-strand RNA virus and significantly expands upon the current knowledge of the antiviral roles of RDRs in the defense against different types of viral genomes in numerous groups of plants.

The P2 capsid protein of the nonenveloped rice dwarf phytoreovirus induces membrane fusion in insect host cells

Insect transmission is an essential process of infection for numerous plant and animal viruses. How an insect-transmissible plant virus enters an insect cell to initiate the infection cycle is poorly understood, especially for nonenveloped plant and animal viruses. The capsid protein P2 of rice dwarf virus (RDV), which is nonenveloped, is necessary for insect transmission. Here, we present evidence that P2 shares structural features with membrane-fusogenic proteins encoded by enveloped animal viruses. When RDV P2 was ectopically expressed and displayed on the surface of insect Spodoptera frugiperda cells, it induced membrane fusion characterized by syncytium formation at low pH. Mutational analyses identified the N-terminal and a heptad repeat as being critical for the membrane fusion-inducing activity. These results are corroborated with results from RDV-infected cells of the insect vector leafhopper. We propose that the RDV P2-induced membrane fusion plays a critical role in viral entry into insect cells. Our report that a plant viral protein can induce membrane fusion has broad significance in studying the mechanisms of virus entry into insect cells and insect transmission of nonenveloped plant and animal viruses.

Identification of a Naturally Occurring Recombinant Isolate of Sugarcane Mosaic Virus Causing Maize Dwarf Mosaic Disease

The complete nucleotide sequence of a potyvirus causing severe maize dwarf mosaic disease in Shaanxi province, northwestern China was determined (GenBank accession No. AY569692). The full genome is 9596 nucleotides in length excluding the 3 ′-terminal poly (A) sequence. It contains a large open reading frame (ORF) flanked by a 149 nt 5′-untranslated region (UTR) and a 255 nt 3′-UTR. The putative polyprotein encoded by this large ORF comprises of 3063 amino acid residues. Sequence comparisons and phylogenetic analyses showed that this potyvirus is an isolate of Sugarcane mosaic virus (SCMV). The entire sequences shared identities of 89.6–97.6 % and 79.3–93.3% with 9 sequenced SCMV isolates at the nucleotide and deduced amino acid levels, respectively. But it showed much lower identities with Maize dwarf mosaic virus (MDMV), Sorghum mosaic virus (SrMV) and Johnsongrass mosaic virus (JGMV) isolates. The putative coat protein sequence is identical to that of a Chinese maize isolate SCMV-HZ. However, partition comparisons and phylogenetic profile analyses of the viral nucleotide sequences indicated that it is a recombinant isolate of SCMV. The recombination sites are located within the 6K1 and CI coding regions.

Rice black‐streaked dwarf virus minor core protein P8 is a nuclear dimeric protein and represses transcription in tobacco protoplasts

Virus‐encoding nuclear transcriptional regulators play important roles in the viral life cycle. Most of these proteins exhibit intrinsic transcriptional activation or repression activity, and are involved in the regulation of the expression of virus genome itself or important cellular genes to facilitate viral replication and inhibit antiviral responses. Here, we report that the minor core protein P8 of Rice black‐streaked dwarf virus, a dsRNA virus infecting host plants and insects, is targeted to the nucleus of insect and plant cells via its N‐terminal 1–40 amino acids and possesses potent active transcriptional repression activity in Bright Yellow‐2 tobacco suspension cells. Moreover, P8, like many transcriptional regulatory proteins, is capable of forming homo‐dimers within insect cells and in vitro. All these data suggest that P8 is likely to enter the nucleus of host cell and play an important role as a negative transcriptional regulator of host gene expression during the process of virus–host interaction.

Insulin Receptor Substrate 1/2 (IRS1/2) Regulates Wnt/β-Catenin Signaling through Blocking Autophagic Degradation of Dishevelled2

Background: IRS1/2 is a critical component of insulin signaling, but it remains unclear whether IRS1/2 functions on Wnt signaling. Results: IRS1/2 interacts with and stabilizes Dvl2 by suppressing its autophagic degradation, leading to promotion of Wnt-mediated EMT and cell proliferation. Conclusion: IRS1/2 positively regulates Wnt/β-catenin signaling through Dvl2. Significance: The IRS1/2-Dvl2 node might be implicated in tumorigenesis and metastasis. Wnt signaling plays a pivotal role in cell proliferation, tissue homeostasis, and tumorigenesis. Dishevelled (Dvl) is a central node of Wnt signaling. Insulin receptor substrates (IRSs), as a critical component of insulin signaling, are involved in cell proliferation, metabolism, and cancer development. In this study, we report that IRS1/2 promotes Wnt/β-catenin signaling by stabilizing Dvl2. We found that IRS1/2 interacts with Dvl2. Overexpression of IRS1/2 increased the protein level of Dvl2 and promoted canonical Wnt signaling, as evidenced by the increased T cell-specific factor 4 transcriptional activity and the up-regulation of expression of CYCLIN D1 and c-MYC, two Wnt target genes critical for cell growth, whereas depletion of IRS1/2 reduced the level of Dvl2 and attenuated Wnt/β-catenin signaling. Biochemical analyses revealed that IRS1/2 decreased Lys-63-linked ubiquitination of Dvl2 and stabilized Dvl2 protein via suppressing its autophagy-mediated degradation. We further revealed that IRS1/2 blocks autophagy-induced formation of the Dvl2-p62/SQSTM1 complex, resulting in disabled association of Dvl2 to autophagosomes. We demonstrated that IRS1/2 promoted the induction of epithelial-mesenchymal transition (EMT) and cell proliferation in response to Wnt stimulation, whereas depletion of Dvl2 impaired the IRS1/2-mediated EMT and cell growth. Our findings revealed that IRS1/2 promotes EMT and cell proliferation through stabilizing Dvl2.

Biochemical and structural properties of cyanases from Arabidopsis thaliana and Oryza sativa.

Cyanate is toxic to all organisms. Cyanase converts cyanate to CO2 and NH3 in a bicarbonate-dependent reaction. The biophysical functions and biochemical characteristics of plant cyanases are poorly studied, although it has been investigated in a variety of proteobacteria, cyanobacteria and fungi. In this study, we characterised plant cyanases from Arabidopsis thaliana and Oryza sativa (AtCYN and OsCYN). Prokaryotic-expressed AtCYN and OsCYN both showed cyanase activity in vitro. Temperature had a similar influence on the activity of both cyanases, but pH had a differential impact on AtCYN and OsCYN activity. Homology modelling provided models of monomers of AtCYN and OsCYN, and a coimmunoprecipitation assay and gel filtration indicated that AtCYN and OsCYN formed homodecamers. The analysis of single-residue mutants of AtCYN indicated that the conserved catalytic residues also contributed to the stability of the homodecamer. KCNO treatment inhibited Arabidopsis germination and early seedling growth. Plants containing AtCYN or OsCYN exhibited resistance to KCNO stress, which demonstrated that one role of cyanases in plants is detoxification. Transcription level of AtCYN was higher in the flower than in other organs of Arabidopsis. AtCYN transcription was not significantly affected by KCNO treatment in Arabidopsis, but was induced by salt stress. This research broadens our knowledge on plant detoxification of cyanate via cyanase.

OsRDR6 plays role in host defense against double-stranded RNA virus, Rice Dwarf Phytoreovirus

RNAi is a major antiviral defense response in plant and animal model systems. RNA-dependent RNA polymerase 6 (RDR6) is an essential component of RNAi, which plays an important role in the resistance against viruses in the model plants. We found previously that rice RDR6 (OsRDR6) functioned in the defense against Rice stripe virus (RSV), and Rice Dwarf Phytoreovirus (RDV) infection resulted in down-regulation of expression of RDR6. Here we report our new findings on the function of OsRDR6 against RDV. Our result showed that down-regulation of OsRDR6 through the antisense (OsRDR6AS) strategy increased rice susceptibility to RDV infection while over-expression of OsRDR6 had no effect on RDV infection. The accumulation of RDV vsiRNAs was reduced in the OsRDR6AS plants. In the OsRDR6 over-expressed plants, the levels of OsRDR6 RNA transcript and protein were much higher than that in the control plants. Interestingly, the accumulation level of OsRDR6 protein became undetectable after RDV infection. This finding indicated that the translation and/or stability of OsRDR6 protein were negatively impacted upon RDV infection. This new finding provides a new light on the function of RDR6 in plant defense response and the cross-talking between factors encoded by host plant and double-stranded RNA viruses.