The Nucleolar Fibrillarin Protein Is Required for Helper Virus-Independent Long-Distance Trafficking of a Subviral Satellite RNA in Plants.

RNA trafficking plays pivotal roles in regulating plant development, gene silencing, and adaptation to environmental stress. Satellite RNAs (satRNAs), parasites of viruses, depend on their helper viruses (HVs) for replication, encapsidation, and efficient spread. However, it remains largely unknown how satRNAs interact with viruses and the cellular machinery to undergo trafficking. Here, we show that the P20 protein of Bamboo mosaic potexvirus satRNA (satBaMV) can functionally complement in trans the systemic trafficking of P20-defective satBaMV in infected Nicotiana benthamiana The transgene-derived satBaMV, uncoupled from HV replication, was able to move autonomously across a graft union identified by RT-qPCR, RNA gel blot, and in situ RT-PCR analyses. Coimmunoprecipitation experiments revealed that the major nucleolar protein fibrillarin is coprecipitated in the P20 protein complex. Notably, silencing fibrillarin suppressed satBaMV-, but not HV-, phloem-based movement following grafting or coinoculation with HV Confocal microscopy revealed that the P20 protein colocalized with fibrillarin in the nucleoli and formed punctate structures associated with plasmodesmata. The mobile satBaMV RNA appears to exist as ribonucleoprotein (RNP) complex composed of P20 and fibrillarin, whereas BaMV movement proteins, capsid protein, and BaMV RNA are recruited with HV coinfection. Taken together, our findings provide insight into movement of satBaMV via the fibrillarin-satBaMV-P20 RNP complex in phloem-mediated systemic trafficking.

The stable association of virion with the triple-gene-block protein 3-based complex of Bamboo mosaic virus.

The triple-gene-block protein 3 (TGBp3) of Bamboo mosaic virus (BaMV) is an integral endoplasmic reticulum (ER) membrane protein which is assumed to form a membrane complex to deliver the virus intracellularly. However, the virus entity that is delivered to plasmodesmata (PD) and its association with TGBp3-based complexes are not known. Results from chemical extraction and partial proteolysis of TGBp3 in membrane vesicles revealed that TGBp3 has a right-side-out membrane topology; i.e., TGBp3 has its C-terminal tail exposed to the outer surface of ER. Analyses of the TGBp3-specific immunoprecipitate of Sarkosyl-extracted TGBp3-based complex revealed that TGBp1, TGBp2, TGBp3, capsid protein (CP), replicase and viral RNA are potential constituents of virus movement complex. Substantial co-fractionation of TGBp2, TGBp3 and CP, but not TGBp1, in the early eluted gel filtration fractions in which virions were detected after TGBp3-specific immunoprecipitation suggested that the TGBp2- and TGBp3-based complex is able to stably associate with the virion. This notion was confirmed by immunogold-labeling transmission electron microscopy (TEM) of the purified virions. In addition, mutational and confocal microscopy analyses revealed that TGBp3 plays a key role in virus cell-to-cell movement by enhancing the TGBp2- and TGBp3-dependent PD localization of TGBp1. Taken together, our results suggested that the cell-to-cell movement of potexvirus requires stable association of the virion cargo with the TGBp2- and TGBp3-based membrane complex and recruitment of TGBp1 to the PD by this complex.

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.

The reduction in σ-promoter recognition flexibility as induced by core RNAP is required for σ to discern the optimal promoter spacing.

Sigma (σ) factors are bacterial transcription initiation factors that direct transcription at cognate promoters. The promoters recognized by primary σ are composed of -10 and -35 consensus elements separated by a spacer of 17±1 bp for optimal activity. However, how the optimal promoter spacing is sensed by the primary σ remains unclear. In the present study, we examined this issue using a transcriptionally active Bacillus subtilis N-terminally truncated σA (SND100-σA). The results of the present study demonstrate that SND100-σA binds specifically to both the -10 and -35 elements of the trnS spacing variants, of which the spacer lengths range from 14 to 21 bp, indicating that simultaneous and specific recognition of promoter -10 and -35 elements is insufficient for primary σ to discern the optimal promoter spacing. Moreover, shortening in length of the flexible linker between the two promoter DNA-binding domains of σA also does not enable SND100-σA to sense the optimal promoter spacing. Efficient recognition of optimal promoter spacing by SND100-σA requires core RNAP (RNA polymerase) which reduces the flexibility of simultaneous and specific binding of SND100-σA to both promoter -10 and -35 elements. Thus the discrimination of optimal promoter spacing by σ is core-dependent.

The core-independent promoter-specific interaction of primary sigma factor.

Previous studies have led to a model in which the promoter-specific recognition of prokaryotic transcription initiation factor, sigma (σ), is core dependent. Most σ functions were studied on the basis of this tenet. Here, we provide in vitro evidence demonstrating that the intact Bacillus subtilis primary sigma, σ(A), by itself, is able to interact specifically with promoter deoxyribonucleic acid (DNA), albeit with low sequence selectivity. The core-independent promoter-specific interaction of the σ(A) is -10 specific. However, the promoter -10 specific interaction is unable to allow the σ(A) to discern the optimal promoter spacing. To fulfill this goal, the σ(A) requires assistance from core RNA polymerase (RNAP). The ability of σ, by itself, to interact specifically with promoter might introduce a critical new dimension of study in prokaryotic σ function.

Activation of the promoter of the fengycin synthetase operon by the UP element.

Bacillus subtilis F29-3 produces an antifungal peptidic antibiotic that is synthesized nonribosomally by fengycin synthetases. Our previous work established that the promoter of the fengycin synthetase operon is located 86 nucleotides upstream of the translational initiation codon of fenC. This investigation involved transcriptional fusions with a DNA fragment that contains the region between positions -105 and +80 and determined that deleting the region between positions -55 and -42 reduces the promoter activity by 64.5%. Transcriptional fusions in the B. subtilis DB2 chromosome also indicated that mutating the sequence markedly reduces the promoter activity. An in vitro transcription analysis confirmed that the transcription is inefficient when the sequence in this region is mutated. Electrophoretic mobility shift and footprinting analyses demonstrated that the C-terminal domain of the RNA polymerase alpha subunit binds to the region between positions -55 and -39. These results indicated that the sequence is an UP element. Finally, this UP element is critical for the production of fengycin, since mutating the UP sequence in the chromosome of B. subtilis F29-3 reduces the transcription of the fen operon by 85% and prevents the cells from producing enough fengycin to suppress the germination of Paecilomyces variotii spores on agar plates.

The two conserved cysteine residues of the triple gene block protein 2 are critical for both cell-to-cell and systemic movement of Bamboo mosaic virus.

The triple gene block protein 2 (TGBp2) of Bamboo mosaic virus (BaMV) is a transmembrane protein which is known to be required for the cell-to-cell movement of potexviruses. This protein has two conserved Cys residues, Cys-109 and Cys-112, at its C-terminal tail, which is supposed to be exposed on the outer surface of the endoplasmic reticulum (ER) membrane and ER-derived granular vesicles. In this study, we investigated the importance of these two Cys residues on the cell-to-cell and systemic movement of BaMV. Our results indicate that the Cys-to-Ala substitutions in TGBp2 make the cell-to-cell movement of BaMV relatively inefficient and the systemic movement of BaMV severely inhibited. Moreover, the defect in systemic movement is attributed to the inefficient transport of viral RNA in the phloem of petiole. Clearly, TGBp2 is critical not only for the cell-to-cell but also for the systemic movement of BaMV. In addition, the conserved Cys residues are important for the functioning of TGBp2.

Characterization of the RNA-binding properties of the triple-gene-block protein 2 of Bamboo mosaic virus.

The triple-gene-block protein 2 (TGBp2) of Bamboo mosaic virus (BaMV) is a transmembrane protein which was proposed to be involved in viral RNA binding during virus transport. Here, we report on the RNA-binding properties of TGBp2. Using tyrosine fluorescence spectroscopy and UV-crosslinking assays, the TGBp2 solubilized with Triton X-100 was found to interact with viral RNA in a non-specific manner. These results raise the possibility that TGBp2 facilitates intracellular delivery of viral RNA through non-specific protein-RNA interaction.

The cysteine residues at the C-terminal tail of Bamboo mosaic virus triple gene block protein 2 are critical for efficient plasmodesmata localization of protein 1 in the same block.

The movement of some plant viruses are accomplished by three proteins encoded by a triple gene block (TGB). The second protein (TGBp2) in the block is a transmembrane protein. This study was aimed to unravel the mechanism underlying the relatively inefficient cell-to-cell movement of Bamboo mosaic virus (BaMV) caused by amino acid substitutions for the three Cys residues, Cys-109, Cys-112 and Cys-119, at the C-terminal tail of TGBp2. Results from confocal microscopy revealed that substitutions of the three Cys residues of TGBp2, especially Cys-109 and Cys-112, would reduce the efficiency of TGBp2- and TGBp3-dependent PD localization of TGBp1. Moreover, there is an additive effect of the substitutions on reducing the efficiency of PD localization of TGBp1. These results indicate that the Cys residues in the C-terminal tail region of TGBp2 participate in the TGBp2- and TGBp3-dependent PD localization of TGBp1, and thus influence the cell-to-cell movement capability of BaMV.

Viral elements and host cellular proteins in intercellular movement of Bamboo mosaic virus.

As a member of the genus Potexvirus, Bamboo mosaic virus (BaMV) also belongs to the plant viruses that encode triple gene block proteins (TGBps) for intercellular movement within the host plants. Recent studies of the movement mechanisms of BaMV have revealed similarities and differences between BaMV and other potexviruses. This review focuses on the general aspects of viral and host elements involved in BaMV movement, the interactions among these elements, and the possible pathways for intra- and intercellular trafficking of BaMV. Major features of BaMV trafficking that have not been demonstrated in other potexviruses include: (i) the involvement of replicase, (ii) fine regulation by coat protein phosphorylation, (iii) the key roles played by TGBp3, (iv) the use of virions as the major transported form, and (v) the involvement of specific host factors, such as Ser/Thr kinase-like protein of Nicotiana benthamiana. We also highlight areas for future study that will provide a more comprehensive understanding of the detailed interactions among viral movement proteins and host factors, as well as the regulatory mechanisms of virus movement. Finally, a model based on the current knowledge is proposed to depict the diverse abilities of BaMV to utilize a wide range of mechanisms for efficient intercellular movement.

The core-independent promoter-specific binding of Bacillus subtilis σB.

Bacillus subtilis σ(D) is an alternative σ factor that possesses a core-independent promoter -10 element binding specificity despite the lack of a distinct footprint on its cognate promoter. We wished to determine whether this property is common to alternative σ factors. To this end, we over-expressed B. subtilis σ(B) in Escherichia coli and analyzed its DNA binding ability by electrophoretic mobility shift assay and DNase I footprinting. The major complex formed by σ(B) and its cognate promoter DNA is heparin-sensitive. However, in contrast to the -10 element binding specificity observed for B. subtilis σ(D) , the promoter binding of σ(B) is specific for the -35 element. These and other results clearly demonstrate that alternative σ factors possess different promoter-binding characteristics, and make core-independent contributions to recognition of their cognate promoters.

The importance of region 2.1 in sustaining the functional structure of the Bacillus subtilis sigma(A) factor.

Region 2.1 of the sigma factor is once proposed to be involved in core binding, and certain bulky hydrophobic amino acids in region 2.1 are thought to make contact with the conserved isoleucine residues in the promoter -10 binding region on the same protein. To examine the roles of the contact between these two regions in sigma(A) structure and function, sigma(A )factor with L145A, I149A, or Y153A was created, and the effects of each substitution on the growth of Bacillus subtilis and on the structural and functional properties of sigma(A) were analyzed. Our data revealed that the growth potential of B. subtilis was significantly affected by each of the substitutions of sigma(A) at elevated temperature. The growth defect was most pronounced with the strain containing L145A-sigma(A); it possessed a low growth potential even at 37 degrees C. In parallel, changes in the structural stability and core-binding activity of sigma(A) and in the promoter-binding and transcription activities of sigma(A)-RNA polymerase were observed for each of the substitutions, with the most drastic effects exerted by L145A. Clearly, region 2.1 of sigma(A) has extra functions, such as the binding of RNA polymerase to promoter DNA, other than the known core-binding ability. Moreover, the multiple effects of each of the substitutions on sigma(A) demonstrate that the contacts between the hydrophobic amino acids in region 2.1 and those in the promoter -10 binding region are critical to the maintenance of the functional sigma(A) structure and that L145 in region 2.1 plays an important role in this respect.

Topological properties of the triple gene block protein 2 of Bamboo mosaic virus.

The triple gene block protein 2 (TGBp2) of Bamboo mosaic virus (BaMV) has been proposed to be a transmembrane protein; however, its features remain unclear. Here, we used biochemical approaches to determine its topological properties. Our data reveal that TGBp2 is mainly associated with the endoplasmic reticulum membrane. The resistance of TGBp2 in proteoliposomes, prepared from both the BaMV-infected tissues and in vitro reconstitution system, to both chemical extraction and trypsin digestion confirmed that it is indeed an integral membrane protein. On the basis of the minor change in the size of the major stable TGBp2-derived tryptic fragment from the monomeric TGBp2, as well as the sensitivity of the cysteine residues at the C-terminal tail of TGBp2 to maleimide modification, we suggest that TGBp2 adopts a topology with both its short N- and C-terminal tails exposed to the outer surface of the endoplasmic reticulum. Moreover, TGBp2 is able to self-assemble as revealed by the significant increase in multimeric TGBp2 when the TGBp2-containing proteoliposomes were treated with chemical crosslinker or oxidation agent.

Mechanism of regulation of prokaryotic tubulin-like GTPase FtsZ by membrane protein EzrA.

At initiation of cell division, FtsZ, a tubulin-like GTPase, assembles into a so-called Z-ring structure at the site of division. The formation of Z ring is negatively regulated by EzrA, which ensures only one ring at the midcell per cell cycle. The mechanism leading to the negative regulation of Z-ring formation by EzrA has been analyzed. Our data reveal that the interaction between EzrA and FtsZ not only reduces the GTP-binding ability of FtsZ but also accelerates the rate of GTP hydrolysis, both of which are unfavorable for the polymerization of FtsZ. Moreover, the acceleration in rate of GTP hydrolysis by EzrA is attributed to stabilization of the transition state for GTP hydrolysis and reduction in the affinity of GDP for FtsZ. Clearly, EzrA is able to modify the GTP hydrolysis cycle of FtsZ. On the basis of these results, a model for how EzrA acts to negatively regulate Z-ring formation is proposed.

Role of the sigma factor in transcription initiation in the absence of core RNA polymerase.

Sigma factors (sigmas) are bacterial transcription factors that bind core RNA polymerase (RNAP) and direct transcription initiation at cognate promoter sites. However, most of their functions have been investigated in the context of RNAP. This has made the exact function of sigma, and the importance of core RNAP in modulating sigma function, ambiguous. Here we identify a Bacillus subtilis mutant sigma(A) that is independently capable of specific binding and melting of the promoter DNA. Interestingly, specific and independent promoter binding of sigma is sufficient for the temperature- and Mg(2+)-independent melting of promoter DNA around the transcription start site, in contrast to the temperature- and Mg(2+)-dependent melting by RNAP around the promoter -10 element. Thus core RNAP is able to negatively modulate the sigma-initiated melting of the transcription start site and, by sensing the changes in temperature and Mg(2+) concentration, to regulate the efficiency of promoter -10 melting.

Movement of potexviruses requires species-specific interactions among the cognate triple gene block proteins, as revealed by a trans-complementation assay based on the bamboo mosaic virus satellite RNA-mediated expression system.

The intra- and intercellular transport of potexviruses require interactions among viral RNA, coat protein and elements of the triple gene block proteins (TGBps). In this study, the requirement of bamboo mosaic virus (BaMV) TGBps for movement functions and the compatibilities with those of two potexviruses, Potato virus X (PVX) and Foxtail mosaic virus (FoMV), were examined using a satellite RNA-mediated trans-complementation assay system. Single or multiple TGBps of BaMV, PVX and FoMV were expressed from BaMV satellite RNA (satBaMV RNA) vectors to complement the functions of green fluorescent protein-tagged, movement-defective BaMV with mutation(s) in the matching gene(s). It was found that individual BaMV TGBps expressed from the satellite vector could function normally in trans, whereas bi-gene BaMV TGBp constructs in which the expression of TGBp3 might be impaired and individual TGBp genes from PVX or FoMV could not complement the movement functions of the defective helper viruses. Furthermore, alterations of the ratio among TGBps by ectopic expression of individual components of TGBps from satBaMV RNA vectors did not affect the cell-to-cell movement capabilities of wild-type BaMV significantly. The results indicate that species-specific interactions among movement proteins are obligatory for the cell-to-cell movement of BaMV and possibly other potexviruses.

Anchorage of cyclodextrin glucanotransferase on the outer membrane of Escherichia coli.

The gene encoding cyclodextrin glucanotransferase (CGTase) was successfully cloned from B. macerans by PCR. A recombinant plasmid pCS005 with a gene encoding the Lpp-OmpA-CGTase trifusion protein was constructed and transformed into E. coli for the surface display of CGTase. Results of immunoblotting analysis and protease accessibility on the fractionated cell membranes confirmed that the Lpp-OmpA-CGTase trifusion protein was successfully anchored on the outer membrane of E. coli. However, only 50% of the membrane-anchored trifusion proteins were displayed on the outer surface of E. coli with the remaining 50% un-translocated. The low efficiency of surface display is attributed to the large size of CGTase. Only a trace amount of CGTase activity was detected for both the whole cells and the cell debris fractions. Because the results of the protease accessibility study suggested that the trypsin-resistant conformation of CGTase was preserved in the membrane-anchored CGTase, we believe that the lack of enzyme activity is mainly due to the inaccessibility of the CGTase active site, near the N-terminus, for substrate molecules. It can be estimated that the critical size for surface display of protein in E. coli is approximately 70 kDa.

Transcription regulation of ezrA and its effect on cell division of Bacillus subtilis.

The EzrA protein of Bacillus subtilis is a negative regulator for FtsZ (Z)-ring formation. It is able to modulate the frequency and position of Z-ring formation during cell division. The loss of this protein results in cells with multiple Z rings located at polar as well as medial sites; it also lowers the critical concentration of FtsZ required for ring formation (P. A. Levin, I. G. Kurster, and A. D. Grossman, Proc. Natl. Acad. Sci. USA 96:9642-9647, 1999). We have studied the regulation of ezrA expression during the growth of B. subtilis and its effects on the intracellular level of EzrA as well as the cell length of B. subtilis. With the aid of promoter probing, primer extension, in vitro transcription, and Western blotting analyses, two overlapping sigmaA-type promoters, P1 and P2, located about 100 bp upstream of the initiation codon of ezrA, have been identified. P1, supposed to be an extended -10 promoter, was responsible for most of the ezrA expression during the growth of B. subtilis. Disruption of this promoter reduced the intracellular level of EzrA very significantly compared with disruption of P2. Moreover, deletion of both promoters completely abolished EzrA in B. subtilis. More importantly, the cell length and percentage of filamentous cells of B. subtilis were significantly increased by disruption of the promoter(s). Thus, EzrA is required for efficient cell division during the growth of B. subtilis, despite serving as a negative regulator for Z-ring formation.

The temperature sensitivity of Bacillus subtilis DB1005 is due to insufficient activity, rather than insufficient concentration, of the mutant delta A factor.

The delta A factor of Bacillus subtilis DB1005 contains two amino acid substitutions (I198A and I202A) in the promoter-10 binding region. It has been confirmed that this delta factor is responsible for the temperature sensitivity of B. subtilis DB1005. An investigation was conducted into how the mutant delta A could cause temperature-sensitive (Ts) cell growth by analysing its structural stability, cellular concentration and transcriptional activity. The mutant delta A was unstable even at the permissive temperature of 37 degrees C (t1/2 59 min), whereas the wild-type counterpart was fairly stable under the same conditions (t1/2 > 600 min). However, neither wild-type delta A nor mutant delta A was stable at 49 degrees C (t1/2 34 min and 23 min, respectively). Analyses of the rates of delta A synthesis revealed that B. subtilis DB1005 was able to compensate for unstable delta A by elevating the level of delta A at 37 degrees C but not at 49 degrees C. Moreover, overexpression of the mutant delta A at 49 degrees C could not suppress the Ts phenotype of B. subtilis DB1005. This indicates that the temperature sensitivity of B. subtilis DB1005 is not due to insufficient delta A concentration in the cell. The greater decline of an already reduced activity of the mutant delta A at 49 degrees C suggests that the temperature sensitivity of B. subtilis DB1005 is instead the result of a very low activity of delta A; probably below a critical level necessary for cell growth.

Properties of Bacillus subtilis sigma A factors with region 1.1 and the conserved Arg-103 at the N terminus of region 1.2 deleted.

sigma factors in the sigma(70) family can be classified into the primary and alternative sigma factors according to their physiological functions and amino acid sequence similarities. The primary sigma factors are composed of four conserved regions, with the conserved region 1 being divided into two subregions. Region 1.1, which is absent from the alternative sigma factor, is poor in conservation; however, region 1.2 is well conserved. We investigated the importance of these two subregions to the function of Bacillus subtilis sigma(A), which belongs to a subgroup of the primary sigma factor lacking a 254-amino-acid spacer between regions 1 and 2. We found that deletion of not more than 100 amino acid residues from the N terminus of sigma(A), which removed part or all region 1.1, did not affect the overall transcription activity of the truncated sigma(A)-RNA polymerase in vitro, indicating that region 1.1 is not required for the functioning of sigma(A) in RNA polymerase holoenzyme. This finding is consistent with the complementation data obtained in vivo. However, region 1.1 is able to negatively modulate the promoter DNA-binding activity of the sigma(A)-RNA polymerase. Further deletion of the conserved Arg-103 at the N terminus of region 1.2 increased the content of stable secondary structures of the truncated sigma(A) and greatly reduced the transcription activity of the truncated sigma(A)-RNA polymerase by lowering the efficiency of transcription initiation after core binding of sigma(A). More importantly, the conserved Arg-103 was also demonstrated to be critical for the functioning of the full-length sigma(A) in RNA polymerase.