Jacques Hille

Identification and isolation of the FEEBLY gene from tomato by transposon tagging

TheAc/Ds transposon system from maize was used for insertional mutagenesis in tomato. Marker genes were employed for the selection of plants carrying a total of 471 uniqueDs elements. Three mutants were obtained withDs insertions closely linked to recessive mutations:feebly (fb), yellow jim (yj) anddopey (dp). Thefb seedlings produced high anthocyanin levels, developed into small fragile plants, and were insensitive to the herbicide phosphinothricin. Theyj plants had yellow leaves as a result of reduced levels of chlorophyll. Thedp mutants completely or partially lacked inflorescences. Thefb andyj loci were genetically linked to theDs donor site on chromosome 3. Reactivation of theDs element in thefb mutants by crosses with anAc-containing line resulted in restoration of the wild-type phenotypes. Plant DNA fragments flanking both sides of theDs element in thefb mutant were isolated by the inverse polymerase chain reaction. Molecular analysis showed that phenotypic reversions offb were correlated with excisions ofDs. DNA sequence analysis ofFb reversion alleles showed the characteristicDs footprints. Northern and cDNA sequence analysis indicated that transcription of theFEEBLY (FB) gene was impeded by the insertion ofDs in an intron. Comparison of the predicted amino acid sequence of the FB protein with other database sequences indicated thatFB is a novel gene.

Inheritance and genetic mapping of resistance to Alternaria alternata f. sp. lycopersici in Lycopersicon pennellii

The fungal pathogen Alternaria alternata f. sp. lycopersici produces AAL-toxins that function as chemical determinants of the Alternaria stem canker disease in the tomato (Lycopersicon esculentum). In resistant cultivars, the disease is controlled by the Asc locus on chromosome 3. Our aim was to characterize novel sources of resistance to the fungus and of insensitivity to the host-selective AAL-toxins. To that end, the degree of sensitivity of wild tomato species to AAL-toxins was analyzed. Of all members of the genus Lycopersicon, only L. cheesmanii was revealed to be sensitive to AAL-toxins and susceptible to fungal infection. Besides moderately insensitive responses from some species, L. pennellii and L. peruvianum were shown to be highly insensitive to AAL-toxins as well as resistant to the pathogen. Genetic analyses showed that high insensitivity to AAL-toxins from L. pennellii is inherited in tomato as a single complete dominant locus. This is in contrast to the incomplete dominance of insensitivity to AAL-toxins of L. esculentum. Subsequent classical genetics, RFLP mapping and allelic testing indicated that high insensitivity to AAL-toxins from L. pennellii is conferred by a new allele of the Asc locus.

CHARACTERISATION AND EXPRESSION OF THE MITOCHONDRIAL GENOME OF A NEW TYPE OF CYTOPLASMIC MALE-STERILE SUNFLOWER

A new cytoplasmic male sterile sunflower, CMS3 [44], was characterised in relation to the Petiolaris (PET1) cytoplasmic male-sterile sunflower, CMS89 [25]. Southern blot analysis showed that the mitochondrial genome of CMS3 contains unique rearrangements in at least five loci (atp6, atp9, atpA, nad1+5 and coxIII) compared to the PET1 sterile and the fertile cytoplasms. Transcripts of two (coxIII and atp6) of the five rearranged loci differed in CMS3 when compared to the corresponding loci in the PET1 and fertile cytoplasms. In organello protein synthesis experiments showed that the ca. 15 kDa mitochondrial polypeptide, characteristic of PET1, is not present in the CMS3 line. These data suggest that the molecular basis of male sterility in the CMS3 line differs from that of the PET1 cytoplasm. The nucleotide sequences of the coding and the immediate flanking regions of the coxIII and atp6 genes of CMS3 were compared to the corresponding regions from the fertile sunflower. In CMS3, the ORFB-cox III locus is located immediately 3′ to the atpA gene whereas in the fertile cytoplasm these two loci are ca. 60 kb apart. This DNA rearrangement probably involved a 265 bp repeat which may be implicated in the DNA recombination associated with PET1 CMS. The atp6 gene in CMS3 contains a 5′-terminal extention which results in an extended ORF. The potential involvement of the rearrangements associated with the coxIII and atp6 loci in relation to the CMS phenotype is discussed.

Two potential Petunia hybrida mitochondrial DNA replication origins show structural and in vitro functional homology with the animal mitochondrial DNA heavy and light strand replication origins

SummaryFour Petunia hybrida mitochondrial (mt) DNA fragments have been isolated, sequenced, localized on the physical map and analyzed for their ability to initiate specific DNA synthesis. When all four mtDNA fragments were tested as templates in an in vitro DNA synthesizing lysate system, developed from purified P. hybrida mitochondria, specific initiation of DNA synthesis could only be observed starting within two framents, oriA and oriB. When DNA synthesis incubations were performed with DNA templates consisting of both the A and B origins in the same plasmid in complementary strands, DNA synthesis first initiates in the A-origin, proceeds in the direction of the B-origin after which replication is also initiated in the B-origin. Based on these observations, a replication model for the P. hybrida mitochondrial genome is presented.

Ds read-out transcription in transgenic tomato plants.

To select for Ds transposition in transgenic tomato plants a phenotypic excision assay, based on restoration of hygromycin phosphotransferase (HPT II) gene expression, was employed. Some tomato plants, however, expressed the marker gene even though the Ds had not excised. Read-out transcriptional activity of the Ds element is responsible for the expression of the HPT II gene. Transcription initiation was mapped to multiple positions spanning about 300 by in the subterminal part of the Ds element. In this respect Ds in tomato resembles the maize element Mul, which also promotes transcription outward from the element. Transposon read-out transcription might thus supply an additional general mechanism for controlling plant gene expression.

The Asc locus for resistance to Alternaria stem canker in tomato does not encode the enzyme aspartate carbamoyltransferase

The fungal disease resistance locus Alternaria stem canker (Asc) in tomato has been suggested to encode the enzyme aspartate carbamoyltransferase (AC Tase). To test this hypothesis a segment of the tomato ACTase gene was amplified by the polymerase chain reaction (PCR) using degenerate primers. The PCR product obtained was subsequently used to isolate an ACTase cDNA clone. Restriction fragment length polymorphism (RFLP) linkage analysis showed that the ACTase gene and the Asc locus do not cosegregate. RFLP mapping positioned the ACTase gene on chromosome 11, while the Asc locus is located on chromosome 3. These results exclude the possibility that the ACTase protein is encoded by the Asc locus.

Heterologous transposon tagging as a tool for the isolation of plant genes

Isolation of plant genes by conventional techniques requires knowledge of a gene product. In routine procedures for gene cloning, genomic or expression libraries are screened with probes made of mRNA or protein, respectively. Also, based on the cDNA or protein sequence, oligonucleotides can be designed to apply a PCR approach. However, for genes with unknown products alternative strategies that have a genetic basis are required. Recently, gene isolation techniques have successfully been applied starting with the genomic map position of the gene. These map-based cloning approaches, however, don’t involve mutant phenotypes that may hamper the identification of genes. In order to obtain mutants that allow simultaneous isolation of the corresponding genes, strategies have been developed using transposons. By insertional mutagenesis mutant phenotypes are obtained of which the responsible genes are molecularly tagged and hence can be cloned.

The Interaction of Alternaria Alternata F.Sp. Lycopersici and its AAL-Toxins with Tomato

The Alternaria alternata f.sp. lycopersici-tomato (Lycopersicon esculentum) interaction was studied to characterise AAL-toxins mediated plant cell death. The Alternaria stem canker disease in tomato is controlled by a single locus (Asc) with three alleles on chromosome 3 that determines resistance (Ascl 1 and Ascl 2 ) or susceptibility (asc) to the fungus and its AAL-toxins. The toxicity of AAL-toxins and related fumonisins is generally explained by inhibition of sphingolipid biosynthesis, but no cellular targets of the toxins have been identified yet. Thus, molecular genetic strategies were employed to study and isolate the Asc locus. By EMS mutagenesis of asc and Ascl 1 it was found that only asc could be mutagenised to Ascl 1 . Three independent targeted transposon tagging experiments to inactivate asc and Ascl 1 did not result in transposon induced mutants. Map-based isolation of Asc from yeast artificial chromosome clones by the production of a contig of Lambda clones is in progress. By subsequent complementation it should be possible to capture the gene(s) governing sensitivity and resistance and to show the biochemical principle behind the differential response to AAL-toxins.