Cucurbit aphid-borne yellows virus Is Prevalent in Field-Grown Cucurbit Crops of Southeastern Spain.

Despite the importance of field-grown cucurbits in Spain, only limited information is available about the impact of disease on their production. During the 2003 and 2004 growing seasons, systematic surveys were carried out in open field melon (Cucumis melo) and squash (Cucurbita pepo) crops of Murcia Province (Spain). The fields were chosen with no previous information regarding their sanitation status, and samples were taken from plants showing viruslike symptoms. Samples were analyzed using molecular hybridization to detect Beet pseudo-yellows virus (BPYV), Cucurbit aphid-borne yellows virus (CABYV), Cucumber mosaic virus (CMV), Cucumber vein yellowing virus (CVYV), Cucurbit yellow stunting disorder virus (CYSDV), Melon necrotic spot virus (MNSV), Papaya ringspot virus (PRSV), Watermelon mosaic virus (WMV), and Zucchini yellow mosaic virus (ZYMV). We collected 924 samples from 48 field plots. Out of these, almost 90% were infected by at least one of the viruses considered, usually CABYV, which was present in 83 and 66% of the melon and squash samples, respectively. In the case of melon, CYSDV, BPYV, and WMV followed CABYV in relative importance, with frequencies of around 20 to 30%, while in squash, CVYV and BPYY showed frequencies between 28 and 21%. The number of multiple infections was very high, 66 and 56% of the infected samples of melon and squash, respectively, being afflicted. CABYV was present in all multiple infections. The high incidence of CABYV in single and multiple infections suggests that this virus may well become an important threat for cucurbit crops in the region. Restriction fragment length polymorphism (RFLP) analysis revealed that CABYV isolates can be grouped into two genetic types, both of which seemed to be present during the 2003 epidemic episode, but only one of the types was found in 2004.

Role of the "YxGG/A" motif of Phi29 DNA polymerase in protein-primed replication.

We have analyzed the functional significance of the phi29 DNA polymerase "YxGG/A" motif in initiation and replication reactions involving the terminal protein (TP) as a primer. This motif, located between the proposed limits of the polymerase and exonuclease domains, has been shown to be very important for the coordination between synthesis and degradation in phi29 DNA polymerase. Mutations in this region affected the polymerization/exonucleolysis (pol/exo) balance, due to its importance for DNA template binding stability at both active sites. Here, we show that the YxGG/A motif of phi29 DNA polymerase is necessary for the formation of a stable complex between TP and phi29 DNA polymerase, affecting initiation and transition during replication of phi29 TP-DNA. The phenotypes in TP-primed reactions in nine of 11 mutant polymerases, showed reduced initiation and/or replication activities using TP-DNA as template. High dATP concentrations allowed the reduced initiation activities of some of these mutant polymerases to reach the wild-type level. The reduction in their affinity for the initiating nucleotide is likely due to their reduced interaction with the TP. Besides, the YxGG/A motif of phi29 DNA polymerase controls the pol/exo balance in the transition step immediately after TP-primed initiation, before DNA polymerase and TP dissociate. Thus, from the first elongation step, the phenotypes of the mutant polymerases parallel those obtained in DNA-primed replication: wild-type, high and low pol/exo balance. A detailed analysis of different transition intermediates suggests that mutants at the YxGG/A motif switch from interaction with TP to DNA once the TP has been extended with six nucleotides.

Analysis of O29 DNA polymerase by partial proteolysis: binding of terminal protein in the double-stranded DNA channel.

ø29 DNA polymerase, which belongs to the family of the eukaryotic type DNA polymerases, is able to use two kinds of primers to initiate DNA replication: DNA and terminal protein (TP). By partial proteolysis we have studied the regions of ø29 DNA polymerase involved in primer binding. With proteinase K, no change in the proteolytic pattern was observed upon DNA binding, suggesting that it does not induce a global conformational change in ø29 DNA polymerase. Conversely, two of the three main cleavage sites obtained by partial digestion of free ø29 DNA polymerase with endoproteinase LysC were protected upon DNA binding, indicating that the DNA could be occluding these cleavage sites to the protease either directly by itself and/or indirectly by induction of local conformational changes affecting their exposure. Partial proteolysis with endoproteinase LysC of ø29 DNA polymerase/TP heterodimer resulted in a protection and digestion pattern similar to that obtained with DNA, suggesting that both primers, DNA and TP, fit in the same double-stranded DNA-binding channel and protect the same regions of ø29 DNA polymerase.

Phi 29 DNA polymerase requires the N-terminal domain to bind terminal protein and DNA primer substrates.

A 44 kDa C-terminal fragment of phi 29 DNA polymerase has been separately expressed and purified from Escherichia coli cells. As expected, the truncated protein lacked the 3'-5' exonuclease activity and strand-displacement capacity, previously mapped in the N-terminal domain of phi 29 DNA polymerase. On the other hand, the 44 kDa C-terminal fragment retained polymerase activity when using Mn2+ as metal activator, although the catalytic efficiency was greatly reduced with respect to that of the complete enzyme. Moreover, and in contrast to the high processivity exhibited by phi 29 DNA polymerase (> 70 kb), polymerization by its C-terminal domain was completely distributive. All these polymerization defects were related to a strong impairment of DNA binding, suggesting that additional contacts present in the N-terminal domain are important for an optimal stabilization and translocation of the DNA during polymerization. Moreover, the C-terminal domain showed a very reduced capacity to initiate terminal protein (TP)-primed DNA replication, as a consequence of a weakened interaction with the TP primer, and a lack of activation by protein p6, the initiator of phi 29 DNA replication. We conclude that the C-terminal portion of phi 29 DNA polymerase (residues 188 to 575), although having a structural entity as the domain responsible for the synthetic activities, requires the N-terminal domain to provide important contacts for the two different substrates, DNA and TP, that prime DNA synthesis. These results support the hypothesis of a modular organization of enzymatic activities in DNA-dependent DNA polymerases, but emphasize the functional coordination required for coupling DNA synthesis and proofreading, and for the more specific functions (TP-priming, high processivity and strand-displacement) inherent to phi 29 DNA polymerase.

Glycerol uptake in Escherichia coli is sensitive to membrane lipid composition.

In Escherichia coli, a functional GlpF protein is necessary for efficient uptake of glycerol at low concentrations. Here we show that GlpF-mediated glycerol uptake was sensitive to a variety of lipid alterations. Overproduction or mutation of the genes coding for enzymes involved in lipid biosynthesis resulted in changed membrane composition and fluidity. The strains with altered lipid composition had a substrate affinity for glycerol (Km) similar to that of wild-type cells, but the Vmax for glycerol uptake was affected. Experiments with glpF::lacZ and glpK::lacZ protein fusions showed that the expression of these two genes was not changed under these conditions. In addition, we observed that mutations in glpF were accompanied by reduced membrane permeability for compounds unrelated to glycerol. Passive diffusion across the membranes of glpF mutants for o-nitrophenyl galactoside was 5-fold slower than in glpF+ cells. The mutants were more resistant to the hydrophobic antibiotic tetracycline, as well as to the membrane perturbants ethanol and dimethylsulphoxide and to the stress of low-osmolarity medium.

First Report of Cucurbit aphid-borne yellows virus in Spain.

In late spring 2003, field-grown melon plants (Cucumis melo L.) showing bright yellowing of older leaves were observed near Valladolises in Campo de Cartagena, Murcia, Spain. Symptoms resembled those caused by viruses of the genus Crinivirus (family Closteroviridae), but absence or very low populations of whiteflies were observed. However, diseased foci showed clear indications of heavy aphid infestations. Later, during the fall of 2003, squash plants (Cucurbita pepo L.) grown in open fields in the same area showed similar symptoms. Tissue print hybridizations to detect Cucurbit yellow stunting disorder virus (CYSDV) and Beet pseudo yellows virus (BPYV) in symptomatic samples were negative. CYSDV and BPYV are two yellowing-inducing criniviruses previously described in Spain. In contrast, standard double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) with antiserum against Cucurbit aphid-borne yellows virus (CABYV; genus Polerovirus, family Luteoviridae) that was kindly provided by H. Lecoq (INRA-Montfavet Cedex, France) were consistently positive. Definitive confirmation of CABYV associated with symptomatic samples was obtained by performing reverse-transcription polymerase chain reaction (RT-PCR) analyses for the CABYV coat protein gene. Total RNA extracts (TRI reagent; Sigma Chemical, St. Louis, MO) were obtained from symptomatic and asymptomatic leaf samples and RT-PCR reactions were carried out using the primers 5'-GAATACGGTCGCGGCTAGAAATC-3' (CE9) and 5'-CTATTTCGGGTTCTGGACCTGGC-3' (CE10) based on the CABYV sequence published by Guilley et al. (2). A single DNA product of approximately 600 bp was obtained only from symptomatic samples. Amplified DNA fragments from two independent samples (samples 36-2 and 37-5) were cloned in E. coli and sequenced (GenBank Accession Nos. AY529653 and AY529654). Sequence comparisons showed a 95% nucleotide sequence identity between the two sequences. A 97% and 94% nucleotide sequence identity was found among 36-2 and 37-5, respectively and the CABYV sequence published by Guilley et al. (2). CABYV seems to be widespread throughout the Mediterranean Basin (1,3) but to our knowledge, it has not previously been described in Spain. Additionally, our data suggest that significant genetic variability might be present in the Spanish CABYV populations. References: (1) Y. Abou-Jawdah et al. Crop Prot. 19:217, 2000. (2) H. Guilley et al. Virology 202:1012, 1994. (3) H. Lecoq et al. Plant Pathol. 41:749, 1992.

Recessive resistance to plant viruses.

About half of the approximately 200 known virus resistance genes in plants are recessively inherited, suggesting that this form of resistance is more common for viruses than for other plant pathogens. The use of such genes is therefore a very important tool in breeding programs to control plant diseases caused by pathogenic viruses. Over the last few years, the detailed analysis of many host/virus combinations has substantially advanced basic research on recessive resistance mechanisms in crop species. This type of resistance is preferentially expressed in protoplasts and inoculated leaves, influencing virus multiplication at the single-cell level as well as cell-to-cell movement. Importantly, a growing number of recessive resistance genes have been cloned from crop species, and further analysis has shown them all to encode translation initiation factors of the 4E (eIF4E) and 4G (eIF4G) families. However, not all of the loss-of-susceptibility mutants identified in collections of mutagenized hosts correspond to mutations in eIF4E and eIF4G. This, together with other supporting data, suggests that more extensive characterization of the natural variability of resistance genes may identify new host factors conferring recessive resistance. In this chapter, we discuss the recent work carried out to characterize loss-of-susceptibility and recessive resistance genes in crop and model species. We review actual and probable recessive resistance mechanisms, and bring the chapter to a close by summarizing the current state-of-the-art and offering perspectives on potential future developments.

Mapping and cloning of gldA, the structural gene of the Escherichia coli glycerol dehydrogenase.

gldA, the structural gene for the NAD(+)-dependent glycerol dehydrogenase, was mapped at 89.2 min on the Escherichia coli linkage map, cotransducible with, but not adjacent to, the glpFKX operon encoding the proteins for the uptake and phosphorylation of glycerol. gldA was cloned, and its position on the physical map of E. coli was determined. The expression of gldA was induced by hydroxyacetone under stationary-phase growth conditions.

Molecular analysis of the glpFKX regions of Escherichia coli and Shigella flexneri.

We have identified a new gene, glpX, belonging to the glp regulon of Escherichia coli, located directly downstream of the glpK gene. The transcription of glpX is inducible with glycerol and sn-glycerol-3-phosphate and is constitutive in a glpR mutant. glpX is the third gene in the glpFKX operon. The function of GlpX remains unknown. GlpX has an apparent molecular weight of 40,000 on sodium dodecyl sulfate-polyacrylamide gels. In addition to determining the E. coli glpX sequence, we also sequenced the corresponding glpFKX region originating from Shigella flexneri, which after transfer into E. coli was instrumental in elucidating the function of glpF in glycerol transport (D. P. Richey and E. C. C. Lin, J. Bacteriol. 112:784-790, 1972). Sequencing of the glpFKX region of this hybrid strain revealed an amber mutation instead of the tryptophan 215 codon in glpF. The most striking difference between the E. coli and S. flexneri DNA was found directly behind glpK, where two repetitive (REP) sequences were present in S. flexneri, but not in the E. coli sequence. The presence or absence of these REP sequences had no effect on transport or on growth on glycerol. Not including the REP sequence-containing region, only 1.1% of a total of 2,167 bp sequenced was different in the two sequences. Comparison of the sequence with those in the EMBL data library revealed a 99% identity between the last third of glpX and the first part of a gene called mvrA. We show that the cloned mvrA gene (M. Morimyo, J. Bacteriol. 170:2136-2142, 1988) originated from the 88-min region of the Escherichia coli chromosome and not, as reported, from the 7-min region and that the gene product identified as MvrA is in fact encoded by a gene distal to glpX.

A DNA binding motif coordinating synthesis and degradation in proofreading DNA polymerases.

The functional significance of the conserved motif 'YxGG/A', located between the 3'-5' exonuclease and polymerization domains of eukaryotic-type DNA polymerases, has been studied by site-directed mutagenesis in phi29 DNA polymerase. Single substitutions at this region were obtained, and 11 phi29 DNA polymerase mutant derivatives were overproduced in Escherichia coli and purified to homogeneity. Nine mutants showed an altered polymerase/3'-5' exonuclease balance on a template/primer DNA structure, giving rise to three different mutant phenotypes: (i) favored polymerization (high pol/exo ratio); (ii) favored exonucleolysis (low pol/exo ratio); and (iii) favored exonucleolysis and null polymerization. Interestingly, these three different phenotypes could be obtained by mutating a single amino acid at the 'YxGG/A' motif. All different phenotypes could be directly related to defects in DNA binding at a particular active site. Thus, a high pol/exo ratio was related to a poor stability at the 3'-5' exonuclease active site. On the contrary, a low pol/exo ratio or null polymerization capacity was related to a poor stability at the polymerization active site and either a normal or an increased accessibility to the exonuclease active site. These results allow us to propose that this motif, located in the connecting region between the N-terminal and C-terminal domains, has a primary role in DNA binding, playing a critical role in the coordination or cross-talk between synthesis and degradation.

Glycerol facilitator of Escherichia coli: cloning of glpF and identification of the glpF product.

The glycerol facilitator is known as the only example of a transport protein that catalyzes facilitated diffusion across the Escherichia coli inner membrane. Here we show that the gene encoding the facilitator, glpF, is the first gene in an operon with glpK, encoding glycerol kinase, at 88 min of the E. coli chromosome. The operon is transcribed counterclockwise. We cloned the glpF gene, demonstrated that it complemented a chromosomal glycerol transport-minus mutation, and identified the gene product. The GlpF protein appeared in the membrane fraction of plasmid-bearing strains and had an apparent Mr of 25,000.

The (I/Y)XGG motif of adenovirus DNA polymerase affects template DNA binding and the transition from initiation to elongation.

Adenovirus DNA polymerase (Ad pol) is a eukaryotic-type DNA polymerase involved in the catalysis of protein-primed initiation as well as DNA polymerization. The functional significance of the (I/Y)XGG motif, highly conserved among eukaryotic-type DNA polymerases, was analyzed in Ad pol by site-directed mutagenesis of four conserved amino acids. All mutant polymerases could bind primer-template DNA efficiently but were impaired in binding duplex DNA. Three mutant polymerases required higher nucleotide concentrations for effective polymerization and showed higher exonuclease activity on double-stranded DNA. These observations suggest a local destabilization of DNA substrate at the polymerase active site. In agreement with this, the mutant polymerases showed reduced initiation activity and increased K(m)(app) for the initiating nucleotide, dCMP. Interestingly, one mutant polymerase, while capable of elongating on the primer-template DNA, failed to elongate after protein priming. Further investigation of this mutant polymerase showed that polymerization activity decreased after each polymerization step and ceased completely after formation of the precursor terminal protein-trinucleotide (pTP-CAT) initiation intermediate. Our results suggest that residues in the conserved motif (I/Y)XGG in Ad pol are involved in binding the template strand in the polymerase active site and play an important role in the transition from initiation to elongation.