First Report of Mango Malformation Disease Caused by Fusarium mangiferae in Spain.

Mango (Mangifera indica L.) malformation disease (MMD) is one of the most important diseases affecting this crop worldwide, which causes severe economic losses because of the reduction of productivity. Symptoms of MMD in Spain were observed for the first time in April of 2006 in three mango orchards in the Axarquia Region (southern Spain). Symptoms included an abnormal development of vegetative shoots with shortened internodes and dwarfed leaves and hypertrophied short and thickened panicles. In the years of 2006, 2009, and 2010, isolates of Fusarium were obtained from vegetative shoots and floral tissue of symptomatic mango trees from 21 different orchards of cvs. Keitt, Kent, Osteen, Tommy Atkins, and a variety of minor commercial cultivars, all showing typical symptoms of MMD. Different Fusarium-like strains were isolated from infected tissues. Colonies from single-spored isolates possessed dark purple-to-salmon-colored mycelium when grown on potato dextrose agar medium. On fresh carnation leaf agar medium, mycelium contained aerial conidiophores possessing three- to five-celled macroconidia and abundant microconidia in false heads from mono- and polyphialides; while cream-orange-colored sporodochia were produced on the surface of the medium, typical for Fusarium mangiferae. The identification of 37 isolates was confirmed as F. mangiferae by species-specific PCR analysis with the primer pair 1-3 F/R that amplified a 608-bp DNA fragment from all Spanish isolates as well as a representative Israeli control strain, Fus 34, also designated as MRC7560 (2). Pathogenicity using four representative isolates, UMAF F02, UMAF F10, UMAF F17, and UMAF F38 of F. mangiferae from Spain as well as isolate MRC7560, was tested on 2-year-old healthy mango seedlings cv. Keitt by inoculating 15 buds from three different trees with a 20-µl conidial suspension (5 × 107 conidia per ml) per isolate (1). This experiment was conducted twice with two independent sets of plants and at different times (March and November 2010). Typical mango malformation symptoms were detected after bud break in March 2011, 5 and 12 months after inoculation. Symptoms were observed for 60% of the inoculated buds with the four F. mangiferae Spanish isolates and 75% with the MRC7560 control strain, but not with water-inoculated control plants. Recovered isolates from the infected floral and vegetative malformed buds were identical morphologically to those inoculated, and the specific 608-bp fragment described for F. mangiferae was amplified with specific-PCR, thus fulfilling Koch's postulates. To our knowledge, this is the first report of mango malformation disease caused by F. mangiferae in Spain and Europe. References: (1) S. Freeman et al. Phytopathology 89:456, 1999. (2) Q. I. Zheng and R. C. Ploetz. Plant Pathol. 51:208, 2002.

Sequence requirements for protein-primed initiation and elongation of phage O29 DNA replication.

The double-stranded linear DNA of Bacillus subtilis phage O29 is replicated by a mechanism in which a terminal protein (TP) acts as a primer. The second 3'-terminal nucleotide of the template directs the incorporation of the 5'-terminal nucleotide into the TP, giving rise to the initiation complex TP-dAMP. Elongation then proceeds by a sliding-back mechanism in which the dAMP covalently linked to the TP pairs to the 3'-terminal nucleotide of the template strand to recover full-length DNA. We have studied the sequence requirements for efficient initiation of replication using mutated TP-free double-stranded DNA fragments. Efficient initiation only requires the terminal repetition 5'-AA. The 3'-terminal T, although not used as template, increases the affinity of DNA polymerase for the initiator nucleotide; in addition, although to a minor extent, the third 3'-terminal position also directs the formation of the initiation complex and modulates the initiation rate at the second position. Efficient elongation requires a previous sliding-back, demanding again a repetition of two nucleotides at the 3' end; if the sliding-back is prevented, a residual elongation can proceed directly from the second position or after jumping back from the third to the first position.

Identification of residues within two regions involved in self-association of viral histone-like protein p6 from phage theta29.

Protein p6 of Bacillus subtilis phage theta29 is involved in the initiation of viral DNA replication and transcription by forming a multimeric nucleoprotein complex with the phage DNA. Based on this, together with its abundance and its capacity to bind to the whole viral genome, it has been proposed to be a viral histone-like protein. Protein p6 is in a monomer-dimer-oligomer equilibrium association. We have identified protein p6 mutants deficient in self-association by testing random mutants obtained by degenerated polymerase chain reaction in an in vivo assay for dimer formation. The mutations were mainly clustered in two regions located at the N terminus, and the central part of the protein. Site-directed single mutants, corresponding to those found in vivo, have been constructed and purified. Mutant p6A44V, located at the central part of the protein, showed an impaired dimer formation ability, and a reduced capacity to bind DNA and to activate the initiation of O29 DNA replication. Mutant p6I8T has at least 10-fold reduced self-association capacity, does not bind DNA nor activate O29 DNA initiation of replication. C-terminal deletion mutants showed an enhanced dimer formation capacity. The highly acidic tail, removed in these mutants, is proposed to modulate the protein p6 self-association.

Specific recognition of parental terminal protein by DNA polymerase for initiation of protein-primed DNA replication.

The linear genome of Bacillus subtilis phage phi29 has a protein covalently linked to the 5' ends, called parental terminal protein (TP), and is replicated using a free TP as primer. The initiation of phage phi29 DNA replication requires the formation of a DNA polymerase/TP complex that recognizes the replication origins located at the genome ends. The DNA polymerase catalyzes the formation of the initiation complex TP-dAMP, and elongation proceeds coupled to strand displacement. The same mechanism is used by the related phage Nf. However, DNA polymerase and TP from phi29 do not initiate the replication of Nf TP-DNA. To address the question of the specificity of origin recognition, we took advantage of the initiation reaction enhancement in the presence of Mn(2+), allowing us to detect initiation activity in heterologous systems in which DNA polymerase, TP, and template TP-DNA are not from the same phage. Initiation was selectively stimulated when DNA polymerase and TP-DNA were from the same phage, strongly suggesting that specific recognition of origins is brought through an interaction between DNA polymerase and parental TP.

Oligomeric structures of the phage phi29 histone-like protein p6.

Protein p6 of Bacillus subtilis phage phi29 has been described as a histone-like protein, playing a role in genome organization and compaction, on the basis of its high intracellular abundance, its pleiotropic effect, and its ability to bind and highly compact the whole phi29 DNA in vitro. Protein p6 forms large multimeric nucleoprotein complexes in which a right-handed superhelical DNA wraps toroidally around the protein core. Analytical ultracentrifugation analysis, at the concentration estimated in vivo (at least 1 mM), showed that protein p6 self-associates into elongated oligomers, suggesting that, in the absence of DNA, the protein could form a scaffold for DNA binding. In this work we have studied the structure of these oligomers by transmission electron microscopy and image processing. The results show that protein p6 aggregates into crooked-shaped oligomers, compatible with a helical structure. The oligomers could interact head-to-tail to form doughnut-shaped structures or they could grow into right-handed double-helical filaments by a nucleation-dependent polymerization process. The dimensions of the crooked-shaped structures are in agreement with that of the DNA in the nucleoprotein complex previously described. We propose that the crooked-shaped structures could act as a scaffold imposing the right-handed path followed by the DNA, and thus it could be considered a non-transient DNA chaperone.

Phage phi29 protein p6 is in a monomer-dimer equilibrium that shifts to higher association states at the millimolar concentrations found in vivo.

Protein p6 from Bacillus subtilis phage phi29 (Mr = 11 800) binds in vitro to DNA forming a large nucleoprotein complex in which the DNA wraps a multimeric protein core. The high intracellular abundance of protein p6 together with its ability to bind the whole phi29 DNA in vitro strongly suggests that it plays a role in viral genome organization. We have determined by sedimentation equilibrium analysis that protein p6 (1-100 microM range), in the absence of DNA, is in a monomer-dimer equilibrium, with an association constant (K2) of approximately 2 x 10(5) M-1. The intracellular concentration of protein p6 (approximately 1 mM) was estimated measuring the number of copies per cell (7 x 10(5)) and the cell volume (1 x 10(-15) L). At concentrations around 1 mM, protein p6 associates into oligomers. This self-association behavior is compatible with a dimer-hexamer model (K2,6 = 3.2 x 10(8) M-2) or with an isodesmic association of the dimer (K = 950 M-1), because the apparent weight-average molecular mass (Mw,a) does not reach saturation at the highest protein concentrations. The sedimentation coefficients of protein p6 monomer and dimer were 1.4 and 2.0, respectively, compatible with translational frictional ratios (f/fo) of 1.15 and 1.30, which slightly deviate from the hydrodynamics of a rigid globular protein. Taking together these results and considering the structure of the nucleoprotein complex, we speculate that the observed oligomers of protein p6 could mimic a scaffold on which DNA folds to form the nucleoprotein complex in vivo.

Activation of replication origins in phi29-related phages requires the recognition of initiation proteins to specific nucleoprotein complexes.

Protein p6 of Bacillus subtilis phage phi29 activates the initiation of viral DNA replication by forming a multimeric nucleoprotein complex at the origins of replication, located at both ends of the linear genome. This activation requires a precise positioning of the protein p6 array with respect to the initiation site. To investigate this activation mechanism, we have purified the phi29 protein p6 counterparts from the related phages Nf and GA-1 and analyzed the formation of complexes with DNA. In the homologous protein p6-DNA complexes the phi29 and Nf protein arrays showed an identical positioning, different than that of the GA-1 protein array. In contrast, in the heterologous complexes the protein showed a different arrangement except in the case of the Nf protein-phi29 DNA complex. We have also purified the proteins involved in the initiation of replication (terminal protein and DNA polymerase) from phages Nf and GA-1 and measured the ability of the different p6 proteins to activate homologous and heterologous replication origins. The results obtained indicate that the activation requires not only the formation of a specific nucleoprotein complex but also its specific recognition by the proteins involved in the initiation of DNA replication.

A genetic approach to the identification of functional amino acids in protein p6 of Bacillus subtilis phage phi 29.

Protein p6 of the Bacillus subtilis phage phi 29 is essential for in vivo viral DNA replication. This protein activates the initiation of phi 29 DNA replication in vitro by forming a multimeric nucleoprotein complex at the replication origins. The N-terminal region of protein p6 is involved in DNA binding, as shown by in vitro studies with p6 proteins altered by deletions or missense mutations. We report on the development of an in vivo functional assay for protein p6. This assay is based on the ability of protein p6-producing B. subtilis non-suppressor (su) cells to support growth of a phi 29 sus6 mutant phage. We have used this trans-complementation assay to investigate the effect on in vivo viral DNA synthesis of missense mutations introduced into the protein p6 N-terminal region. The alteration of lysine to alanine at position 2 resulted in a partially functional protein, whereas the replacement of arginine by alanine at position 6 gave rise to an inactive protein. These results indicate that arginine at position 6 is critical for the in vivo activity of protein p6. Our complementation system provides a useful genetic approach for the identification of functionally important amino acids in protein p6.

In vivo functional relationships among terminal proteins of Bacillus subtilis phi 29-related phages.

Gene 3 of the Bacillus subtilis phage phi 29 encodes the terminal protein (TP), which acts as a primer in the initiation of viral DNA replication. We have developed an in vivo functional assay for the phi 29 TP based on the ability of TP-producing B. subtilis non-suppressor (su-) cells to support DNA replication of a phi 29 sus3 mutant phage. This trans-complementation assay has been used to study in vivo functional relationships between the TP of phi 29 and related phages. Our results demonstrate that phi 29 TP functionally substitutes the TP of phage PZA, whereas replication of phage Nf DNA cannot take place in vivo using the phi 29 TP as a primer.

A new protein domain for binding to DNA through the minor groove.

Protein p6 of the Bacillus subtilis phage phi 29 binds with low sequence specificity to DNA through the minor groove, forming a multimeric nucleoprotein complex that activates the initiation of phi 29 DNA replication. Deletion analysis suggested that the N-terminal part of protein p6, predicted to form an amphipathic alpha-helix, is involved in DNA binding. We have constructed site-directed mutants at the polar side of the putative alpha-helix. DNA binding and activation of initiation of phi 29 DNA replication were impaired in most of the mutant proteins obtained. A 19 amino acid peptide comprising the N-terminus of protein p6 interacted with a DNA fragment containing high-affinity signals for protein p6 binding with approximately 50-fold higher affinity than the peptide corresponding to an inactive mutant. Both wild-type peptide and protein p6 recognized the same sequences in this DNA fragment. This result, together with distamycin competition experiments, suggested that the wild-type peptide also binds to DNA through the minor groove. In addition, CD spectra of the wild-type peptide showed an increase in the alpha-helical content when bound to DNA. All these results indicate that an alpha-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove.

DNA structure in the nucleoprotein complex that activates replication of phage phi 29.

Initiation of phage phi 29 DNA replication is activated by the viral protein p6 which forms a nucleoprotein complex at the replication origins, located at the linear genome ends. The complex consists of a DNA right-handed superhelix wrapped around a multimeric protein core. We have determined the superhelical path of the DNA in the complex, measuring the change in linking number induced by the protein, the surface-related helical repeat and the compaction of the DNA. One superhelical turn has approximately 63 bp (2.6 p6 dimers). Furthermore, we have determined that the DNA binding domain of protein p6 is located at the N-terminal region, predicted to form an amphipathic alpha-helix. We have obtained, by site-directed mutagenesis, protein p6 mutants in the polar side of the putative helix in which their DNA binding and replication activation properties were impaired or undetectable, in agreement with in vivo results.

Assembly of phage phi 29 genome with viral protein p6 into a compact complex.

The formation of a multimeric nucleoprotein complex by the phage phi 29 dsDNA binding protein p6 at the phi 29 DNA replication origins, leads to activation of viral DNA replication. In the present study, we have analysed protein p6-DNA complexes formed in vitro along the 19.3 kb phi 29 genome by electron microscopy and micrococcal nuclease digestion, and estimated binding parameters. Under conditions that greatly favour protein-DNA interaction, the saturated phi 29 DNA-protein p6 complex appears as a rigid, rod-like, homogeneous structure. Complex formation was analysed also by a psoralen crosslinking procedure that did not disrupt complexes. The whole phi 29 genome appears, under saturating conditions, as an irregularly spaced array of complexes approximately 200-300 bp long; however, the size of these complexes varies from approximately 2 kb to 130 bp. The minimal size of the complexes, confirmed by micrococcal nuclease digestion, probably reflects a structural requirement for stability. The values obtained for the affinity constant (K(eff) approximately 10(5) M-1) and the cooperativity parameter (omega approximately 100) indicate that the complex is highly dynamic. These results, together with the high abundance of protein p6 in infected cells, lead us to propose that protein p6-DNA complexes could have, at least at some stages, during infection, a structural role in the organization of the phi 29 genome into a nucleoid-type, compact nucleoprotein complex.

Phage phi 29 protein p6: a viral histone-like protein.

Phage phi 29 protein p6 is one of the most abundant viral proteins in phi 29-infected B subtilis cells, constituting about 4% of the total cellular proteins (about 3 x 10(6) copies/cell) at late infection. Electron microscopic studies showed that, in vitro, protein p6 forms heterogeneously-sized complexes all along phi 29 DNA, suggesting that protein p6 may have a role in genome packaging and organization. The low stability of the protein p6-phi 29 DNA complexes observed in vitro could reflect the dynamic nature of these complexes, to allow replication, transcription, and encapsidation of the genome. The protein p6-DNA complex consists of a DNA right-handed superhelix wrapped around a multimeric protein core. The DNA in this complex is strongly distorted and compacted. Protein p6 recognition signals have been mapped near the ends of the linear phi 29 DNA and act as nucleation sites for complex formation. Protein p6 does not recognize a specific sequence, but sequences with specific bendable properties that would favor the formation of the complex. Protein p6 represses transcription from the phi 29 C2 early promoter, and activates initiation of phi 29 DNA replication that occurs from both DNA ends. The formation of nucleoprotein complexes at the origins of replication, as well as the specific positioning of protein p6 with respect to the DNA ends are required for the activation of replication. This suggests that the proteins involved in the initiation step of phi 29 DNA replication, either directly interact with protein p6, or recognize a conformational change at a specific location in the DNA. The mechanism of activation could be the local and transient unpairing of DNA at specific sites, facilitated by the strong distortion of DNA conformation in the nucleoprotein complex.

Multimeric complexes formed by DNA-binding proteins of low sequence specificity.

Some proteins bind to double-stranded DNA with low sequence specificity, forming regular multimeric complexes that extend over large regions of DNA, strongly distorting its conformation. Formation of these complexes at particular DNA sites usually depends on the structural ability of the DNA to follow the path imposed by the protein array. These complexes are found in both prokaryotic and eukaryotic organisms and participate in processes such as DNA replication, transcription and packaging.

Superhelical path of the DNA in the nucleoprotein complex that activates the initiation of phage phi 29 DNA replication.

Initiation of bacteriophage phi 29 DNA replication is activated by protein p6, a viral double-stranded DNA-binding protein that forms a nucleoprotein complex at the viral replication origins. This complex consists of a DNA right-handed superhelix wrapped around a multimeric protein p6 core with protein p6 dimers regularly bound every 24 base-pairs (bp). In this paper, we have constructed a concatemer formed by direct repeats of a 24 bp sequence previously proposed to act as a signal for protein p6 binding at a phi 29 replication origin. DNase I footprinting shows that protein p6 binds to the concatemer in a similar way to the phi 29 DNA replication origins but with higher affinity, indicating that the 24 bp sequence is a recognition signal for protein p6. Furthermore, the concatemer was cloned in a plasmid and, by electron microscopy, it was shown to be the highest-affinity protein p6 binding region present in the plasmid. Based on these observations, the linking number change restrained by protein p6 has been measured in a series of plasmids containing concatemers with different numbers of 24 bp repeats; from the values obtained the linking number change restrained by a single protein p6 dimer has been estimated (delta Lkd = 0.1). In addition, when protein p6-DNA complexes fixed with glutaraldehyde were analysed by electron microscopy, it was observed that protein p6 compacts 4.2-fold the length of naked DNA. These data, together with the previously known value of the surface-related DNA helical repeat in the complex (12 bp), completely define the superhelical path of the DNA in the complex: one superhelical turn approximately involves 63 bp and 2.6 protein p6 dimers, and the DNA superhelix has a diameter of 6.6 nm and a slope of 14 degrees. The data obtained also indicate that the DNA in the protein p6-DNA complex is undertwisted (11.5 bp/turn) and strongly bent (66 degrees/12 bp). These DNA conformational changes might contribute to the activation of phi 29 DNA initiation of replication by protein p6.

Characterization of a DNA binding protein of bacteriophage PRD1 involved in DNA replication.

Escherichia coli phage PRD1 protein P12, involved in PRD1 DNA replication in vivo, has been highly purified from E. coli cells harbouring a gene XII-containing plasmid. Protein P12 binds to single-stranded DNA as shown by gel retardation assays and nuclease protection experiments. Binding of protein P12 to single-stranded DNA increases about 14% the contour length of the DNA as revealed by electron microscopy. Binding to single-stranded DNA seems to be cooperative, and it is not sequence specific. Protein P12 also binds to double-stranded DNA although with an affinity 10 times lower than to single-stranded DNA. Using the in vitro phage phi 29 DNA replication system, it is shown that protein P12 stimulates the overall phi 29 DNA replication.

A novel nucleoprotein complex at a replication origin.

The viral protein p6, required for the protein-primed initiation of replication of Bacillus subtilis phage phi 29, forms a nucleoprotein complex at the viral replication origins that shows novel features. Deoxyribonuclease I and hydroxyl radical footprinting data, as well as the induction of positive supercoiling, support a model in which a DNA right-handed superhelix tightly wraps around a multimeric p6 core. The interaction occurs through the DNA minor groove. The activity of p6 not only requires the formation of the complex but also its correct positioning, indicating that the other proteins involved in the initiation of replication recognize, at a precise position, either the p6 core or the DNA conformational change induced by p6.

Functional domain for priming activity in the phage phi 29 terminal protein.

By site-directed mutagenesis we have changed into Cys the Ser232 of the phi 29 terminal protein (TP) involved in the covalent linkage to dAMP for the initiation of replication. The mutant TP, highly purified, had about 0.7% of the priming activity of the wild-type (wt) protein p3. The linkage between the mutant protein p3 and dAMP was more labile to piperidine treatment than the serine-dAMP linkage in the wt protein p3, suggesting the presence of a different kind of linkage, Cys-dAMP. In the other three mutant TPs, residues Leu220, Ser223 and Ser226 were independently changed into Pro; the purified TP mutants had about 3%, 140% and 1% of the priming activity of the wt p3, respectively. All the mutant TP were able to interact with the phi 29 DNA polymerase and with DNA, suggesting that Leu220 and Ser226, in addition to Ser232, form part of a functional domain involved in the process of initiation of DNA replication.

Signals at the bacteriophage phi 29 DNA replication origins required for protein p6 binding and activity.

Protein p6 of Bacillus subtilis phage phi 29 binds specifically to the ends of the viral DNA that contain the replication origins, giving rise to a nucleoprotein structure. DNA regions recognized by protein p6 have been mapped by deletion analysis and DNase I footprinting. Main protein p6-recognition signals have been located between nucleotides 62 and 125 at the right phi 29 DNA end and between nucleotides 46 and 68 at the left end. In addition, recognition signals are also present at other sites within 200-300 bp at each phi 29 DNA end. Protein p6 does not seem to recognize a specific sequence in the DNA, but rather a structural feature, which could be bendability. The formation of the protein p6-DNA nucleoprotein complex is likely to be the structural basis for the protein p6 activity in the initiation of replication.

Site-directed mutagenesis in the DNA linking site of bacteriophage phi 29 terminal protein: isolation and characterization of a Ser232----Thr mutant.

By site-directed mutagenesis we have changed the serine residue 232 of the phi 29 terminal protein, involved in the covalent linkage to dAMP for the initiation of replication, into a threonine residue. The mutant terminal protein has been purified to homogeneity and shown to be inactive in the formation of the initiation complex; nevertheless, the mutant protein retains its ability to interact with the phi 29 DNA polymerase and with the DNA. The results obtained indicate a high specificity in the linking site of the terminal protein.