Flora Sánchez

Cloning and characterization of the isopenicillin N synthetase gene mediating the formation of the β-lactam ring in Aspergillus nidulans

Genomic clones containing an Aspergillus nidulans isopenicillin N synthetase (IPNS) gene have been identified by heterologous hybridization with a Cephalosporium acremonium DNA probe. The open reading frame encodes a 331 amino acid polypeptide with extensive homology with the genes of other beta-lactam-producing fungi. The gene product has been overexpressed in Escherichia coli and shown to have activity of IPNS. This represents the first evidence at the molecular level that the biosynthesis of penicillins in A. nidulans occurs by the same pathway as in other beta-lactam-producing microorganisms. Comparison of available nucleotide sequences from IPNS genes suggests a horizontal transmission of the gene between the prokaryotic beta-lactam producers of the genus Streptomyces and the filamentous fungi.

The cylindrical inclusion gene of Turnip mosaic virus encodes a pathogenic determinant to the brassica resistance gene TuRB01.

The viral component of Turnip mosaic virus (TuMV) determining virulence to the Brassica napus TuRB01 dominant resistance allele has been identified. Sequence comparisons of an infectious cDNA clone of the UK 1 isolate of TuMV (avirulent on TuRB01) and a spontaneous mutant capable of infecting plants possessing TuRB01 suggested that a single nucleotide change in the cylindrical inclusion (CI) protein coding region (gene) of the virus was responsible for the altered phenotype. A second spontaneous mutation involved a different change in the CI gene. The construction of chimeric genomes and subsequent inoculations to plant lines segregating for TuRB01 confirmed the involvement of the CI gene in this interaction. Site-directed mutagenesis of the viral coat protein (CP) gene at the ninth nucleotide was carried out to investigate its interaction with TuRB01. The identity of this nucleotide in the CP gene did not affect the outcome of the viral infection. Both mutations identified in the CI gene caused amino acid changes in the C terminal third of the protein, outside any of the conserved sequences reported to be associated with helicase or cell-to-cell transport activities. This is the first example of a potyvirus CI gene acting as a determinant for a genotype-specific resistance interaction.

Potato virus Y group C isolates are a homogeneous pathotype but two different genetic strains

Potato virus Y group C isolates (PVYC) have been characterized according to biological, molecular and genetic criteria. Two genetic strains, PVYC1 and PVYC2, were identified on the basis of genetic distances (among them and other PVY strains), host range (ability or inability to infect pepper), MAb response (ELISA recognition with MAb 10E3) and coat protein processing site. Some characteristics, such as aphid transmission and ELISA using other MAbs, did not correlate with classification into these two genetic strains. All isolates tested induced a hypersensitive response on potatoes bearing the Nc resistance gene, confirming the nature of PVYC isolates as a homogeneous pathotype.

Nucleotide sequence of Chinese rape mosaic virus (oilseed rape mosaic virus), a crucifer tobamovirus infectious on Arabidopsis thaliana.

The complete nucleotide sequence of Chinese rape mosaic virus has been determined. The virus is a member of the tobamovirus genus of plant virus and is able to infect Arabidopsis thaliana (L.) Heynh systemically. The analysis of the sequence shows a gene array that seems to be characteristic of crucifer tobamoviruses and which is slightly different from the one most frequently found in tobamoviruses. Based on gene organization and on comparisons of sequence homologies between members of the tobamoviruses, a clustering of crucifer tobamoviruses is proposed that groups the presently known crucifer tobamovirus into two viruses with two strains each. A name change of Chinese rape mosaic virus to oilseed rape mosaic virus is proposed.

INFECTIVITY OF TURNIP MOSAIC POTYVIRUS CDNA CLONES AND TRANSCRIPTS ON THE SYSTEMIC HOST ARABIDOPSIS THALIANA AND LOCAL LESION HOSTS

Full-length cDNA clones of turnip mosaic virus were assembled under the control of T7 or 35S promoter. The 35S or nopaline synthase terminator signals were introduced downstream the full length cDNA controlled by 35S promoter. Both the capped in vitro transcripts from T7 controlled template, and the cDNAs from 35S controlled plasmids were infectious on Arabidopis thaliana plants according to systemically induced symptoms and to ELISA assays. The cDNAs from 35S controlled plasmids induced local lesions in Chenopodium amaranticolor and Chenopodium quinoa plants. A spontaneous silent C/T transition, giving rise to an additional SpeI restriction site, not present in the original viral RNA template, was used as a marker of the origin of infection.

Single amino acid changes in the turnip mosaic virus viral genome-linked protein (VPg) confer virulence towards Arabidopsis thaliana mutants knocked out for eukaryotic initiation factors eIF(iso)4E and eIF(iso)4G.

Previous resistance analyses of Arabidopsis thaliana mutants knocked out for eukaryotic translation initiation factors showed that disruption of the At-eIF(iso)4E or both the At-eIF(iso)4G1 and At-eIF(iso)4G2 genes resulted in resistance against turnip mosaic virus (TuMV). This study selected TuMV virulent variants that overcame this resistance and showed that two independent mutations in the region coding for the viral genome-linked protein (VPg) were sufficient to restore TuMV virulence in At-eIF(iso)4E and At-eIF(iso)4G1xAt-eIF(iso)4G2 knockout plants. As a VPg-eIF(iso)4E interaction has been shown previously to be critical for TuMV infection, a systematic analysis of the interactions between A. thaliana eIF4Es and VPgs of virulent and avirulent TuMVs was performed. The results suggest that virulent TuMV variants may use an eIF4F-independent pathway.

Strains of Turnip mosaic potyvirus as defined by the molecular analysis of the coat protein gene of the virus.

Turnip mosaic virus (TuMV) is a member of the potyvirus genus with a wide host range and highly variable in its biological characteristics. Analysis of the CP gene sequences from databases, combined with the experimental analysis of the CP gene of further isolates, using data derived from sequence or restriction analysis, has allowed the genetic classification of 60 TuMV isolates or sequences. Two main genetic clusters MB (mostly Brassica isolates) and MR (mostly Radish isolates) were found, together with several apparently independent lineages. Isolates in the latter could be grouped as Intermediate between Brassica and Radish clusters (IBR) or outside Brassica and Radish clusters (OBR), according to their genetic distance to the main clusters. The genetic diversity of TuMV isolates deposited in the databases was increased with the sequences of the CP gene of seven selected isolates, mainly belonging to IBR or OBR groups. There was a correlation between the MR genetic cluster and JPN 1 serotype.

A strain-type clustering of potato virus Y based on the genetic distance between isolates calculated by RFLP analysis of the amplified coat protein gene.

SummaryPotato virus Y (PVY) isolates have been classified into genetic strains by a host-independent criterion using a molecular typing method. The method used extracts from infected tissue, and included immunocapture-RT-PCR-RFLP analysis using 5 different restriction endonucleases (Dde I,Eco RV,Hinf I,Rsa I andTaq I). Genetic distances between the different PVY “restrictotypes” were calculated and used to define the PVY genetic strains. Three main clusters were found: PVYO, PVYN, and non-potato PVY (PVYNP), in good agreement with classical PVY strain definitions that combine different biological criteria. Our approach was incomparably quicker and more reliable and reproducible than biotyping. The potential of this approach for very quick, simple and automatable molecular epidemiological studies is discussed.

Transformation in Penicillium chrysogenum

An auxotrophic mutant of Penicillium chrysogenum with a DNA rearrangement that affects the trpC region has been transformed to the Trp+ phenotype by using a plasmid that contains the trifunctional wild-type gene. A frequency of 40-80 transformants per microgram of input DNA was usually achieved. A low frequency of plasmid integration at the recipient mutated trpC gene was detected; however, most of the transformants integrated the plasmid DNA elsewhere into the genome. Some of the transformants contain multiple rearranged copies of the vector integrated in a tandem fashion.

Transformation of Penicillium chrysogenum to sulfonamide resistance

Penicillium chrysogenum has been transformed to sulfonamide resistance by vectors containing the dihydropteroate synthetase gene from plasmid R388 controlled by the promoter and terminator sequences of the P. chrysogenum trpC gene. Transformation frequencies of four to ten transformants per microgram of vector DNA were obtained.