Juan Antonio García

RNA-Dependent RNA Polymerase 1 from Nicotiana tabacum Suppresses RNA Silencing and Enhances Viral Infection in Nicotiana benthamiana

This work analyzes the surprising result that Nicotiana benthamiana transformed with RNA-dependent RNA polymerase 1 from Nicotiana tabacum (Nt-RDR1) is hypersusceptible to several viruses. It provides evidence supporting a dual role for RDR1 in contributing to salicylic acid–mediated antiviral defense at the same time as it suppresses RDR6-mediated antiviral RNA silencing. Endogenous eukaryotic RNA-dependent RNA polymerases (RDRs) produce double-stranded RNA intermediates in diverse processes of small RNA synthesis in RNA silencing pathways. RDR6 is required in plants for posttranscriptional gene silencing induced by sense transgenes (S-PTGS) and has an important role in amplification of antiviral silencing. Whereas RDR1 is also involved in antiviral defense in plants, this does not necessarily proceed through triggering silencing. In this study, we show that Nicotiana benthamiana transformed with RDR1 from Nicotiana tabacum (Nt-RDR1 plants) exhibits hypersusceptibility to Plum pox potyvirus and other viruses, resembling RDR6-silenced (RDR6i) N. benthamiana. Analysis of transient induction of RNA silencing in N. benthamiana Nt-RDR1 and RDR6i plants revealed that Nt-RDR1 possesses silencing suppression activity. We found that Nt-RDR1 does not interfere with RDR6-dependent siRNA accumulation but turns out to suppress RDR6-dependent S-PTGS. Our results, together with previously published data, suggest that RDR1 might have a dual role, contributing, on one hand, to salicylic acid–mediated antiviral defense, and suppressing, on the other hand, the RDR6-mediated antiviral RNA silencing. We propose a scenario in which the natural loss-of-function variant of RDR1 in N. benthamiana may be the outcome of selective pressure to maintain a high RDR6-dependent antiviral defense, which would be required to face the hypersensitivity of this plant to a large number of viruses.

Proteolytic activity of the plum pox potyvirus Nla-like protein in Escherichia coli

The nucleotide sequence of the small nuclear inclusion protein (NIa)-like cistron of plum pox potyvirus (PPV) has been determined. Viral proteolytic activity was expressed in Escherichia coli cells harboring plasmids with a PPV cDNA insert approximately 7000 nt long. Free PPV capsid protein was detected in these cells, but it was not produced when a mutation was introduced in the PPV cDNA insert which induced a Gln to Pro substitution at the large nuclear inclusion protein (NIb)-capsid protein junction. By mutational analysis, the NIa-like protein was determined to be responsible for the proteolytic activity. A Gln to Ser substitution at the presumed NIa-NIb junction, which inhibited proteolytic processing at the carboxyl end of the protease, had no effect on proteolytic cleavage at the NIb-capsid protein junction. In contrast with the high efficiency of proteolytic processing at the NIb-capsid protein cleavage site, processing at the ends of the PPV protease was not complete, suggesting that the PPV polyprotein, like that of other potyviruses, contains cleavage sites with different properties.

PROPERTIES OF THE ACTIVE PLUM POX POTYVIRUS RNA POLYMERASE COMPLEX IN DEFINED GLYCEROL GRADIENT FRACTIONS

As a first step in the study of the replication of plum pox virus (PPV) RNA, an in vitro virus-specific RNA polymerase activity was characterized in a crude membrane extract (Martin and Garcia, 1991). In this study, we report the fractionation of the crude membrane extract by centrifugation in glycerol gradients. The sedimentation properties after different treatments of the crude extract and its insensitivity to micrococcal nuclease treatment suggest that the RNA polymerase activity was localized in a defined and enclosed membranous structure. Subcellular membrane characterization of the different glycerol gradient fractions indicated that PPV-specific RNA synthesis occurred in fractions enriched in endoplasmic reticulum and tonoplast vesicles.

Determination of polyprotein processing sites by amino terminal sequencing of nonstructural proteins encoded by plum pox potyvirus

Nonstructural proteins of plum pox potyvirus were partially purified following a procedure described for the isolation of tobacco etch virus nuclear inclusion proteins. Plum pox virus proteins with electrophoretic mobilities corresponding to 49, 59 and 68 kDa reacted with antibodies against the 49 kDa and 54 kDa components of the nuclear inclusions and the 70 kDa component of the cylindrical inclusions of tobacco etch virus, respectively. Further purification by size exclusion high performance liquid chromatography or SDS-polyacrylamide gel electrophoresis, and amino terminal amino acid sequencing permitted the location in the plum pox virus polyprotein of the cleavage sites from which the 49 kDa (NIa-type, protease), 59 kDa (NIb-type, putative RNA replicase), and 68 kDa (CI-type) proteins originate. A 110 kDa protein which copurified with the plum pox virus inclusion proteins reacted with both anti-NIa and anti-NIb sera and had the same amino terminus as the plum pox virus 49 kDa protein, indicating that it is a non-processed 49-59 kDa polypeptide.

Proteolytic activity of the plum pox potyvirus NIa-protein on excess of natural and artificial substrates in Escherichia coli.

The plum pox potyvirus (PPV) NIa protease expressed from a medium copy number plasmid was able to process an excess of substrate expressed from a high copy number plasmid, in a binary Escherichia coli expression system. The ΔB7 NIa protease mutant only partially processed the NIb‐CP junction but its efficiency was independent of the amount of substrate. The ΔB7 mutant essentially did not recognize an artificial cleavage site which was quite efficiently recognized by the wild‐type protease. No competitive inhibition of the proteolytic activity by the presence of excess of different protease mutants was observed.

A protein similar to Escherichia coli gro EL is present in Bacillus subtilis

Abstract We have found in Bacillus subtilis an oligomer that resembles closely the gro EL oligomer from Escherichia coli. These structures have similar morphology, dimensions and sedimentation coefficients, and both are based on a 65,000 Mr polypeptide. Furthermore, the oligomer from B. subtilis infected with phage φ29 copurifies with a viral protein (p10) that is involved in the early steps of phage head morphogenesis.

The orderly, in vitro emergence of DNA from bacteriophage φ29 particles

phi29 DNA-containing 12-13- particles (produced by infecting nonsuppressor hosts of Bacillus subtilis with phage containing suppressible mutations in cistrons 12 and 13) can be complemented with lysates containing proteins p12* and p13 to yield infectious phage. Complementation of these particles with lysates containing p12* but not p13 or complementation with purified p12* in the absence of p13 produces a structure (called complex) which has a markedly different organization. Electron microscopy and sedimentation analysis after digestion with DNase I or proteinase K indicate that complex is composed of an intact phage head with a genome-sized linear DNA molecule attached at the collar-tail region. EcoRI digestion establishes that the DNA molecule has a unique orientation. Gel electrophoresis indicates that p12*, the neck appendage protein, is transferred to the particles when complex is formed. Complex can also be produced by incubation of 12-13- particles at 42 degrees , by incubation at pH 6.0, or by incubation in the presence of 20 mM EDTA. Complex is also formed from DNA-containing 12- particles but to a lesser extent.

Proteolytic activity of plum pox virus—tobacco etch virus chimeric NIa proteases

Plasmids encoding chimeric NIa‐type proteases made of sequences from the polyviruses plum pox virus (PPV) and tobacco etch virus (TEV) have been constructed. Their proteolytic activity on the large nuclear inclusion protein (NIa)‐capsid protein (CP) junction of each virus was assayed in Escherichia coli cells. The amino half of the protease seemed to be involved neither in the enzymatic catalysis nor in substrate recognition. In spite of the large homology among the PPV and TEV NIa‐type proteases, the exchange of fragments from the carboxyl halves of the molecules usually caused a drastic decrease in the enzymatic activity. Inactive chimeric proteases did not interfere with cleavage by PPV wild type protease expressed from a second plasmid. The results suggest that the recognition and catalytic sites of the NIa proteases are closely interlinked and, although residues relevant for the correct interaction with the substrate could be present in other parts of the protein, a main determinant for substrate specificity should lie in a region situated, approximately, between positions 30 and 90 from the carboxyl end. This region includes the conserved His at position 360 of PPV or 355 of TEV, which has been postulated to interact with the Gin at position −1 of the cleavage sites.