Isolation and characterization of the 54-kDa and 22-kDa chitinase genes of Serratia marcescens KCTC2172

A DNA fragment (pCHI5422) containing two genes encoding a 54-kDa and a 22-kDa chitinase was isolated from a cosmid DNA library of Serratia marcescens KCTC2172. The complete nucleotide sequence of pCHI5422 consisting of 4581 bp was determined. The nucleotide sequence of the 22-kDa chitinase consists of 681 bp of open reading frame encoding 227 amino acids and is located 1422 bp downstream of the translation termination codon of the 54-kDa chitinase sequence. The 54-kDa chitinase gene consisted of 1497 bp in a single open reading frame encoding 499 amino acids. The genes encoding the 54-kDa and 22-kDa chitinase were separately subcloned in Escherichia coli and the individual chitinases were expressed and purified from the culture broth using chitin affinity chromatography. When chitohexaose was used as substrate, the major product of the enzymatic reaction of both the 54-kDa and 22-kDa chitinases was a (GlcNAc)₂ dimer with a minor amount of monomer. The specific activity of the 54-kDa and 22-kDa chitinases were 300 μM (min)⁻¹ mg⁻¹ and 17 μM (min)⁻¹ mg⁻¹ on the natural swollen chitin, respectively.     

对HCMV UL54 mRNA 片段特异性切割的M1GS构建

人巨细胞病毒是一种DNA病毒,在人群中一般呈亚临床感染和潜伏感染。为研究病毒基因沉默工具和抗病毒制剂,以人巨细胞病毒UL54基因mRNA序列设计互补的外部引导序列,共价结合到大肠杆菌来源RNaseP催化核心M1RNA上,从而构建成M1GS-T6核酶。通过对DNA聚合酶UL54基因亚克隆片段转录产物体外切割研究,证实该核酶具备对UL54mRNA片段的特异切割能力。     

The Tobacco mosaic virus 126-kDa protein associated with virus replication and movement suppresses RNA silencing

Systemic symptoms induced on Nicotiana tabacum cv. Xanthi by Tobacco mosaic virus (TMV) are modulated by one or both amino-coterminal viral 126- and 183-kDa proteins: proteins involved in virus replication and cell-to-cell movement. Here we compare the systemic accumulation and gene silencing characteristics of TMV strains and mutants that express altered 126- and 183-kDa proteins and induce varying intensities of systemic symptoms on N. tabacum. Through grafting experiments, it was determined that M(IC)1,3, a mutant of the masked strain of TMV that accumulated locally and induced no systemic symptoms, moved through vascular tissue but failed to accumulate to high levels in systemic leaves. The lack of M(IC)1,3 accumulation in systemic leaves was correlated with RNA silencing activity in this tissue through the appearance of virus-specific, approximately 25-nucleotide RNAs and the loss of fluorescence from leaves of transgenic plants expressing the 126-kDa protein fused with green fluorescent protein (GFP). The ability of TMV strains and mutants altered in the 126-kDa protein open reading frame to cause systemic symptoms was positively correlated with their ability to transiently extend expression of the 126-kDa protein:GFP fusion and transiently suppress the silencing of free GFP in transgenic N. tabacum and transgenic N. benthamiana, respectively. Suppression of GFP silencing in N. benthamiana occurred only where virus accumulated to high levels. Using agroinfiltration assays, it was determined that the 126-kDa protein alone could delay GFP silencing. Based on these results and the known synergies between TMV and other viruses, the mechanism of suppression by the 126-kDa protein is compared with those utilized by other originally characterized suppressors of RNA silencing.     

Evidence that the potyvirus P1 proteinase functions in trans as an accessory factor for genome amplification.

The tobacco etch potyvirus (TEV) polyprotein is proteolytically processed by three viral proteinases (NIa, HC-Pro, and P1). While the NIa and HC-Pro proteinases each provide multiple functions essential for viral infectivity, the role of the P1 proteinase beyond its autoproteolytic activity is understood poorly. To determine if P1 is necessary for genome amplification and/or virus movement from cell to cell, a mutant lacking the entire P1 coding region (delta P1 mutant) was produced with a modified TEV strain (TEV-GUS) expressing beta-glucuronidase (GUS) as a reporter, and its replication and movement phenotypes were assayed in tobacco protoplasts and plants. The delta P1 mutant accumulated in protoplasts to approximately 2 to 3% the level of parental TEV-GUS, indicating that the P1 protein may contribute to but is not strictly required for viral RNA amplification. The delta P1 mutant was capable of cell-to-cell and systemic (leaf-to-leaf) movement in plants but at reduced rates compared with parental virus. This is in contrast to the S256A mutant, which encodes a processing-defective P1 proteinase and which was nonviable in plants. Both delta P1 and S256A mutants were complemented by P1 proteinase expressed in a transgenic host. In transgenic protoplasts, genome amplification of the delta P1 mutant relative to parental virus was stimulated five- to sixfold. In transgenic plants, the level of accumulation of the delta P1 mutant was stimulated, although the rate of cell-to-cell movement was the same as in nontransgenic plants. Also, the S256A mutant was capable of replication and systemic infection in P1-expressing transgenic plants. These data suggest that, in addition to providing essential processing activity, the P1 proteinase functions in trans to stimulate genome amplification.     

Extreme resistance to potato virus X infection in plants expressing a modified component of the putative viral replicase.

Three types of mutation were introduced into the sequence encoding the GDD motif of the putative replicase component of potato virus X (PVX). All three mutations rendered the viral genome completely noninfectious when inoculated into Nicotiana clevelandii or into protoplasts of Nicotiana tabacum (cv. Samsun NN). In order to test whether these negative mutations could inactivate the viral genome in trans, the mutant genes were expressed in transformed N.tabacum (cv. Samsun NN) under control of the 35S RNA promoter of cauliflower mosaic virus and the transformed lines were inoculated with PVX. In 10 lines tested in which the GDD motif was expressed as GAD or GED there was no effect on susceptibility to PVX. In two of four lines transformed to express the ADD form of the conserved motif, the F1 and F2 progeny plants were highly resistant to infection by PVX, although only to strains closely related to the source of the transgene. The resistance was associated with suppression of PVX accumulation in the inoculated and systemic leaves and in protoplasts of the transformed plants, although some low level viral RNA production was observed in the inoculated but not the systemic leaves when the inoculum was as high as 100 or 250 micrograms/ml PVX RNA. These results suggest for a plant virus, as reported previously for Q beta phage, that virus resistance may be engineered by expression of dominant negative mutant forms of viral genes in transformed cells.     

Analysis of the role of brome mosaic virus 1a protein domains in RNA replication, using linker insertion mutagenesis

Brome mosaic virus (BMV) belongs to a "superfamily" of plant and animal positive-strand RNA viruses that share, among other features, three large domains of conserved sequence in nonstructural proteins involved in RNA replication. Two of these domains reside in the 109-kDa BMV 1a protein. To examine the role of 1a, we used biologically active cDNA clones of BMV RNA1 to construct a series of linker insertion mutants bearing two-codon insertions dispersed throughout the 1a gene. The majority of these mutations blocked BMV RNA replication in protoplasts, indicating that both intervirally conserved domains function in RNA replication. Coinoculation tests with a large number of mutant combinations failed to reveal detectable complementation between mutations in the N- and C-terminal conserved domains, implying that these two domains either function in some directly interdependent fashion or must be present in the same protein. Four widely spaced mutations with temperature-sensitive (ts) defects in RNA replication were identified, including a strongly ts insertion near the nucleotide-binding consensus of the helicaselike C-terminal domain. Temperature shift experiments with this mutant show that 1a protein is required for continued accumulation of all classes of viral RNA (positive strand, negative strand, and subgenomic) and is required for at least the first 10 h of infection. ts mutations were also identified in the 3' noncoding region of RNA1, 5' to conserved sequences previously implicated in cis for replication. Under nonpermissive conditions, the cis-acting partial inhibition of RNA1 accumulation caused by these noncoding mutations was also associated with reduced levels of the other BMV genomic RNAs. Comparison with previous BMV mutant results suggests that RNA replication is more sensitive to reductions in expression of 1a than of 2a, the other BMV-encoded protein involved in replication.     

Peanut Stripe Potyvirus Resistance in Peanut (Arachis Hypogaea L.) Plants Carrying Viral Coat Protein Gene Sequences

Peanut (Arachis hypogaea L.) lines exhibiting high levels of resistance to peanut stripe virus (PStV) were obtained following microprojectile bombardment of embryogenic callus derived from mature seeds. Fertile plants of the commercial cultivars Gajah and NC7 were regenerated following co-bombardment with the hygromycin resistance gene and one of two forms of the PStV coat protein (CP) gene, an untranslatable, full length sequence (CP2) or a translatable gene encoding a CP with an N-terminal truncation (CP4). High level resistance to PStV was observed for both transgenes when plants were challenged with the homologous virus isolate. The mechanism of resistance appears to be RNA-mediated, since plants carrying either the untranslatable CP2 or CP4 had no detectable protein expression, but were resistant or immune (no virus replication). Furthermore, highly resistant, but not susceptible CP2 T0 plants contained transgene-specific small RNAs. These plants now provide important germplasm for peanut breeding, particularly in countries where PStV is endemic and poses a major constraint to peanut production.     

Replication of tobacco mosaic virus RNA

The replication of tobacco mosaic virus (TMV) RNA involves synthesis of a negative-strand RNA using the genomic positive-strand RNA as a template, followed by the synthesis of positive-strand RNA on the negative-strand RNA templates. Intermediates of replication isolated from infected cells include completely double-stranded RNA (replicative form) and partly double-stranded and partly single-stranded RNA (replicative intermediate), but it is not known whether these structures are double-stranded or largely single-stranded in vivo. The synthesis of negative strands ceases before that of positive strands, and positive and negative strands may be synthesized by two different polymerases. The genomic-length negative strand also serves as a template for the synthesis of subgenomic mRNAs for the virus movement and coat proteins. Both the virus-encoded 126-kDa protein, which has amino-acid sequence motifs typical of methyltransferases and helicases, and the 183-kDa protein, which has additional motifs characteristic of RNA-dependent RNA polymerases, are required for efficient TMV RNA replication. Purified TMV RNA polymerase also contains a host protein serologically related to the RNA-binding subunit of the yeast translational initiation factor, eIF3. Study of Arabidopsis mutants defective in RNA replication indicates that at least two host proteins are needed for TMV RNA replication. The tomato resistance gene Tm-1 may also encode a mutant form of a host protein component of the TMV replicase. TMV replicase complexes are located on the endoplasmic reticulum in close association with the cytoskeleton in cytoplasmic bodies called viroplasms, which mature to produce 'X bodies'. Viroplasms are sites of both RNA replication and protein synthesis, and may provide compartments in which the various stages of the virus mutiplication cycle (protein synthesis, RNA replication, virus movement, encapsidation) are localized and coordinated. Membranes may also be important for the configuration of the replicase with respect to initiation of RNA synthesis, and synthesis and release of progeny single-stranded RNA.     

Transgenic cucumbers harboring the 54-kDa putative gene of Cucumber fruit mottle mosaic tobamovirus are highly resistant to viral infection and protect non-transgenic scions from soil infection

Cucumber fruit mottle mosaic tobamovirus (CFMMV) causes severe mosaic symptoms and yellow mottling on leaves and fruits and, occasionally, severe wilting of cucumber (Cucumis sativus L.) plants. No genetic source of resistance against this virus has been identified in cucumber. The gene coding for the putative 54-kDa replicase gene of CFMMV was cloned into an Agrobacterium tumefaciens binary vector, and transformation was performed on cotyledon explants of a parthenocarpic cucumber cultivar. R1 seedlings were screened for resistance to CFMMV by symptom expression, back inoculation on an alternative host and ELISA. From a total of 14 replicase-containing R1 lines, eight resistant lines were identified. Line I44 – homozygous for the putative 54-kDa replicase gene – was immune to CFMMV infection by mechanical and graft inoculation, and to root infection following planting in CFMMV-infested soil. A substantial delay of symptom appearance was observed following infection by three additional cucurbit-infecting tobamoviruses. When used as a rootstock, line I44 protected susceptible cucumber scions from soil infection by CFMMV. This paper is the first report on protection of a susceptible cultivar against a soil-borne viral pathogen, by grafting onto a transgenic rootstock.     

Identification of a ribonucleoprotein intermediate of tomato mosaic virus RNA replication complex formation

The replication of eukaryotic positive-strand RNA virus genomes occurs in the membrane-bound RNA replication complexes. Previously, we found that the extract of evacuolated tobacco BY-2 protoplasts (BYL) is capable of supporting the translation and subsequent replication of the genomic RNAs of plant positive-strand RNA viruses, including Tomato mosaic virus (ToMV). Here, to dissect the process that precedes the formation of ToMV RNA replication complexes, we prepared membrane-depleted BYL (mdBYL), in which the membranes were removed by centrifugation. In mdBYL, ToMV RNA was translated to produce the 130-kDa and 180-kDa replication proteins, but the synthesis of any ToMV-related RNAs did not occur. When BYL membranes were added back to the ToMV RNA-translated mdBYL after the termination of translation with puromycin, ToMV RNA was replicated. Using a replication-competent ToMV derivative that encodes the FLAG-tagged 180-kDa replication protein, it was shown by affinity purification that a complex that contained the 130-kDa and 180-kDa proteins and ToMV genomic RNA was formed after translation in mdBYL. When the complex was mixed with BYL membranes, ToMV RNA was replicated, which suggests that this ribonucleoprotein complex is an intermediate of ToMV RNA replication complex formation. We have named this ribonucleoprotein complex the "pre-membrane-targeting complex." Our data suggest that the formation of the pre-membrane-targeting complex is coupled with the translation of ToMV RNA, while posttranslationally added exogenous 180-kDa protein and replication templates can contribute to replication and can be replicated, respectively. Based on these results, we discuss the mechanisms of ToMV RNA replication complex formation.     

A 43-kDa protein of Treponema denticola is essential for dentilisin activity

A protease of Treponema denticola, dentilisin, is thought to be part of a complex with 43- and 38-kDa proteins. A sequence encoding a 43-kDa protein was located in the 3' region of the prcA gene upstream of the dentilisin gene (prtP). The 43-kDa protein was apparently generated from digestion of PrcA. To clarify the function of the protein, we constructed a mutant of the 43-kDa protein following homologous recombination. The mutant lacked detectable dentilisin activity. Immunoblot analysis demonstrated that the dentilisin protein was degraded in the mutant. The results of real-time polymerase chain reaction suggested that prtP mRNA expression in the mutant was somewhat decreased compared with the wild-type strain. These data suggest that the 43-kDa protein is involved in the stabilization of the dentilisin protein.     

Plant viral synergism: the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses.

Synergistic viral diseases of higher plants are caused by the interaction of two independent viruses in the same host and are characterized by dramatic increases in symptoms and in accumulation of one of the coinfecting viruses. In potato virus X (PVX)/potyviral synergism, increased pathogenicity and accumulation of PVX are mediated by the expression of potyviral 5' proximal sequences encoding P1, the helper component proteinase (HC-Pro), and a fraction of P3. Here, we report that the same potyviral sequence (termed P1/HC-Pro) enhances the pathogenicity and accumulation of two other heterologous viruses: cucumber mosaic virus and tobacco mosaic virus. In the case of PVX-potyviral synergism, we show that the expression of the HC-Pro gene product, but not the RNA sequence itself, is sufficient to induce the increase in PVX pathogenicity and that both P1 and P3 coding sequences are dispensable for this aspect of the synergistic interaction. In protoplasts, expression of the potyviral P1/HC-Pro region prolongs the accumulation of PVX (-) strand RNA and transactivates expression of a reporter gene from a PVX subgenomic promoter. Unlike the synergistic enhancement of PVX pathogenicity, which requires only expression of HC-Pro, the enhancement of PVX (-) strand RNA accumulation in protoplasts is significantly greater when the entire P1/HC-Pro sequence is expressed. These results indicate that the potyviral P1/HC-Pro region affects a step in disease development that is common to a broad range of virus infections and suggest a mechanism involving transactivation of viral replication.     

Transgenic tobacco plants expressing the bacterial mc gene resist virus infection

A bacterial rnc gene coding for a double-stranded RNA-dependent RNase III endoribonuclease and a mutant, rnc70, were expressed in tobacco plants. The RNase III protein produced in the transgenic plants was the same size as the bacterial protein. Expression of the wild-type gene could cause stunting in some plant lines, but not in others. Expression of the mutant protein did not affect normal growth and development of the transgenic plants. Transgenic plants of the R1 and R2 generations, expressing the wild type, as well as a mutant protein, were resistant to infection by three disparate RNA plant viruses with a divided genome but not against two viruses with a single-stranded RNA genome. Introduction of the rnc gene in crop plants may provide resistance to economically important virus diseases.