Map-based cloning of a protein kinase gene conferring disease resistance in tomato.

The Pto gene in tomato confers resistance to races of Pseudomonas syringae pv. tomato that carry the avirulence gene avrPto. A yeast artificial chromosome clone that spans the Pto region was identified and used to probe a leaf complementary DNA (cDNA) library. A cDNA clone was isolated that represents a gene family, at least six members of which genetically cosegregate with Pto. When susceptible tomato plants were transformed with a cDNA from this family, they were resistant to the pathogen. Analysis of the amino acid sequence revealed similarity to serine-threonine protein kinases, suggesting a role for Pto in a signal transduction pathway.

The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression.

The complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single-copy region. The genes for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15 genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to components of the respiratory-chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes are sufficient to read all codons if the ;two out of three' and ;U:N wobble' mechanisms operate in the chloroplast. Two sequences which autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of the chloroplast genes.

Saturated molecular map of the rice genome based on an interspecific backcross population.

A molecular map has been constructed for the rice genome comprised of 726 markers (mainly restriction fragment length polymorphisms; RFLPs). The mapping population was derived from a backcross between cultivated rice, Oryza sativa, and its wild African relative, Oryza longistaminata. The very high level of polymorphism between these species, combined with the use of polymerase chain reaction-amplified cDNA libraries, contributed to mapping efficiency. A subset of the probes used in this study was previously used to construct an RFLP map derived from an inter subspecific cross, providing a basis for comparison of the two maps and of the relative mapping efficiencies in the two crosses. In addition to the previously described PstI genomic rice library, three cDNA libraries from rice (Oryza), oat (Avena) and barley (Hordeum) were used in this mapping project. Levels of polymorphism detected by each and the frequency of identifying heterologous sequences for use in rice mapping are discussed. Though strong reproductive barriers isolate O. sativa from O. longistaminata, the percentage of markers showing distorted segregation in this backcross population was not significantly different than that observed in an intraspecific F2 population previously used for mapping. The map contains 1491 cM with an average interval size of 4.0 cM on the framework map, and 2.0 cM overall. A total of 238 markers from the previously described PstI genomic rice library, 250 markers from a cDNA library of rice (Oryza), 112 cDNA markers from oat (Avena), and 20 cDNA markers from a barley (Hordeum) library, two genomic clones from maize (Zea), 11 microsatellite markers, three telomere markers, eleven isozymes, 26 cloned genes, six RAPD, and 47 mutant phenotypes were used in this mapping project. Applications of a molecular map for plant improvement are discussed.

A tobacco chloroplast DNA sequence possibly coding for a polypeptide similar to E. coli RNA polymerase beta-subunit.

DNA sequencing has revealed a long open reading frame (ORF) in the large single-copy region of tobacco chloroplast DNA. This ORF consists of 1070 codons and its deduced amino acid sequence shows about 39% homology to that of the beta-subunit of E. coli RNA polymerase. This finding raises a possibility that some of the chloroplast RNA polymerase subunits are coded for by the chloroplast genome.

Pre-germination genotypic screening using PCR amplification of half-seeds.

A simple and rapid PCR-based method has been developed for determining the genotype of seeds before germination. Single half-seeds of rice (Oryza sativa L.) and wheat (Triticum aestivum L. em. Thell.) were preincubated, without grinding, in an aqueous extraction buffer. The resulting supernatants were then used in polymerase chain reaction (PCR) with oligonucleotide primers corresponding to rice single-copy sequences or a wheat microsatellite repeat. PCR products of identical size were amplified using either the half-seed extract or DNA isolated from leaf tissue. The remnant half-seeds can be maintained in ordered arrays using microtiter plates allowing the recovery of selected genotypes. Pre-germination genotypic screening of seed populations as described in this report should be useful for a variety of applications in plant breeding and genetics studies.

Rapid splicing and stepwise processing of a transcript from the psbB operon in tobacco chloroplasts: determination of the intron sites in petB and petD.

Expression of the psbB gene cluster in tobacco chloroplasts has been studied. This cluster contains the genes for the 51 kDa chlorophyll a apoprotein (psbB) and the 10 kDa phosphoprotein (psbH) of the photosystem II, and cytochrome b6 (petB) and subunit IV (petD) of the cytochrome b/f complex in this order. Northern blot hybridization and reverse transcription analyses have revealed that petB and petD contain single introns and the psbB gene cluster is transcribed as a single polycistronic unit. The primary transcript seems to be spliced very rapidly and then processed into several small RNA species. The exact splice sites have been located by cDNA sequencing. The transcriptional initiation site of the psbB operon has been determined by S1 mapping with in vitro capped chloroplast RNA. The stepwise processing of chloroplast RNA precursors is discussed.

Chromosomal localization and molecular-marker tagging of the powdery mildew resistance gene (Lv) in tomato.

We report the tagging of a powdery mildew [Leveillula taurica (Lév.) Arnaud.] resistance gene (Lv) in tomato using RAPD and RFLP markers. DNA from a resistant (cv Laurica) and a susceptible cultivar were screened with 300 random primers that were used to amplify DNA of resistant and susceptible plants. Four primers yielded fragments that were unique to the resistant line and linked to the resistance gene in an F2 population. One of these amplified fragments, OP248, with a molecular weight of 0.7 kb, was subsequently mapped to chromosome 12, 1 cM away from CT134. Using RFLP markers located on chromosome 12, it was shown that approximately one half of chromosome 12 (about 42 cM), in the resistant variety is comprised of foreign DNA, presumably introgressed with the resistance gene from the wild species L. chilense. Further analysis of a backcross population revealed that the Lv gene lies in the 5.5-cM interval between RFLP markers, CT211 and CT219. As a prelude to map-based cloning of the Lv gene, we are currently enriching the density of markers in this region by a combination of RAPD primers and other techniques.

A member of the tomato Pto gene family confers sensitivity to fenthion resulting in rapid cell death.

Leaves of tomato cultivars that contain the Pto bacterial resistance locus develop small necrotic lesions within 24 hr after exposure to fenthion, an organophosphorous insecticide. Recently, the Pto gene was isolated and shown to be a putative serine/threonine protein kinase. Pto is one member of a multigene family that is clustered within a 400-kb region on chromosome 5. Here, we report that another member of this gene family, termed Fen, is responsible for the sensitivity to fenthion. Fen was isolated by map-based cloning using closely linked DNA markers to identify a yeast artificial chromosome clone that spanned the Pto region. After transformation with the Fen gene under control of the cauliflower mosaic virus (CaMV) 35S promoter, tomato plants that are normally insensitive to fenthion rapidly developed extensive necrotic lesions upon exposure to fenthion. Two related insecticides, fensulfothion and fenitrothion, also elicited necrotic lesions specifically on Fen-transformed plants. Transgenic tomato plants harboring integrated copies of the Pto gene under control of the CaMV 35S promoter displayed sensitivity to fenthion but to a lesser extent than did wild-type fenthion-sensitive plants. The Fen protein shares 80% identity (87% similarity) with Pto but does not confer resistance to Pseudomonas syringae pv tomato. These results suggest that Pto and Fen participate in the same signal transduction pathway.