Genetic resistance to Peronospora tabacina in Nicotiana langsdorffii, a South American wild tobacco.

Several accessions of Nicotiana langsdorffii, a wild tobacco relative native to South America, express an incompatible interaction in response to infection by Peronospora tabacina, an oömycete that causes blue mold disease of tobacco (N. tabacum) and many other species of Nicotiana. In resistant accessions of N. langsdorffii, such as S-4-4, incompatibility takes the form of necrotic lesions that appear 48 h after pathogen inoculation, restricting pathogen growth, and suppressing subsequent asexual sporulation. Significantly elevated levels of salicylic acid and expression of a defense-related gene (HSR203J) were observed in S-4-4 leaves following blue mold infection. Genetic segregation analysis in F(2) and modified backcross populations showed that blue mold resistance is determined by a single dominant gene (NlRPT) present in S-4-4. Further characterization of this unique host-pathogen interaction has revealed that (i) necrotic lesion resistance is due to the hypersensitive response (HR), (ii) HR-mediated resistance is present in 7 of 10 N. langsdorffii accessions examined, but not in closely related species, (iii) in some accessions of N. langsdorffii, resistance is expressed in cotyledon tissue and seedling leaves as well as in adult plants, and (iv) several resistant accessions including S-4-4 express an unregulated ("runaway") HR in response to P. tabacina infection.

Necrotic Lesion Resistance Induced by Peronospora tabacina on Leaves of Nicotiana obtusifolia.

ABSTRACT Infection of Nicotiana obtusifolia plant introduction (PI) #555573 by the downy mildew pathogen Peronospora tabacina resulted in a compatible interaction, in which P. tabacina penetrated and freely colonized host leaf tissue. This interaction became incompatible 5 to 6 days later, with the appearance of necrotic lesions (NLs) and inhibition of pathogen growth and subsequent sporulation. NL development depended upon the presence of P. tabacina in host tissue, was not due to the effects of other microbes, and occurred co-incident in time with the pathogen's ability to produce asexual sporangia on a susceptible N. obtusifolia genotype. Inhibition of the necrotic response by CoCl(2) (a calcium channel blocker) and pathogen-induced transcription of a defense-related gene (PR-1a) suggested that necrosis was due to hypersensitive cell death in the host. In contrast, N. obtusifolia PI#555543 did not exhibit hypersensitivity upon infection by P. tabacina, but rather developed characteristic symptoms of tobacco blue mold disease: chlorotic lesions accompanied by abundant pathogen sporulation. Disease reactions scored on PI#555573 x PI#555543 F(2) progeny inoculated with P. tabacina sporangia indicated that the resistance phenotype was due to the action of a single gene from N. obtusifolia PI#555573, which we have named Rpt1. To date, Rpt1 is the only gene known to confer a hypersensitive response (HR) to P. tabacina infection in any species of Nicotiana. A survey of wild N. obtusifolia revealed that the HR to P. tabacina was expressed in the progeny of 7 of 21 (33%) plants collected in southern Arizona, but not in the progeny of plants originating from Death Valley National Park in California and the Big Bend National Park in west Texas.

Targeted identification of markers linked to malaria and filarioid nematode parasite resistance genes in the mosquito Aedes aegypti.

Quantitative trait loci (QTL) have been identified for competence of the mosquito Aedes aegypti to transmit the avian malaria parasite Plasmodium gallinaceum and the human filarial parasite Brugia malayi. Efforts towards the map-based cloning of the associated genes are limited by the availability of genetic markers for fine-scale mapping of the QTL positions. Two F2 mosquito populations were subjected to bulked segregant analysis to identify random amplified polymorphic DNA (RAPD)-PCR fragments linked with the major QTL determining susceptibility to both parasites. Individual mosquitoes for the bulks were selected on the basis of their genotypes at restriction fragment length polymorphism (RFLP) loci tightly linked with the QTL. Pool-positive RAPD fragments were cloned and evaluated as RFLP markers. Of the 62 RAPD/RFLP fragments examined, 10 represented low-copy number sequences. Five of these clones were linked with the major QTL for P. gallinaceum susceptibility (pgs1), of which one clone mapped within the flanking markers that define the QTL interval. The remaining five clones were linked with the major QTL for B. malayi susceptibility (fsb1), and again one clone mapped within the flanking markers that define the QTL interval. In addition, nine RAPD/RFLP fragments were isolated that seem to be of non-mosquito origin.

Linkage of rhm, a recessive gene for resistance to southern corn leaf blight, to RFLP marker loci in maize (Zea mays) seedlings.

The recessive gene rhm confers chlorotic-lesion resistance to Bipolaris maydis race O, the southern corn leaf blight pathogen, in otherwise susceptible maize plants. Because of inconsistencies encountered in scoring the disease on mature plants in the field, an assay was developed to monitor the expression of this gene in maize seedlings under controlled conditions. One hundred and two related F3 families from the cross RH95rhm x B73 were inoculated with conidia of B. maydis race O, and the genotype at the rhm locus of each F2 parent was deduced from the reactions observed in the progeny seedlings. The F2 genomes were reconstituted by extracting DNA from leaf tissue pooled from 30-36 F3 progeny plants per family. The seedling disease ratings were analyzed together with the segregation scores for 14 single-copy DNA probes. Our results indicate that rhm is tightly linked to two restriction fragment length polymorphism (RFLP) marker loci (UMC85 and p144) that map to the short arm of chromosome 6. In addition, a rapid assay based on the polymerase chain reaction was used to confirm the linkage between rhm and the p144 RFLP marker locus in a second unrelated F2 population.

Isolation and characterization of a complementary DNA clone for an algal pre-apoplastocyanin.

We have isolated a cDNA clone for the Chlamydomonas reinhardtii pre-apoplastocyanin. The sequence contains codons for the complete pre-protein including a two-domain, lumen-targeting transit sequence and the mature apoprotein. The transit sequence (47 amino acids) is the shortest one described for chloroplast lumenal proteins, and like other C. reinhardtii lumen-targeting transit sequences appears to lack an uncharged amino-terminal domain usually present in plant lumen-directing sequences. The mature protein is deduced to be 98 amino acids in length and shows highest primary sequence similarity (74-76% identity) to other unicellular algal plastocyanins. Southern hybridization analysis of C. reinhardtii genomic DNA indicates the presence of a single nuclear gene, as is the case for all other plastocyanin genes characterized to date, although the algal gene might be interrupted. Codon usage in this gene reflects the high GC content of C. reinhardtii nuclear DNA, but is more highly biased than that found in the C. reinhardtii copper-repressible gene for the functionally equivalent pre-apocytochrome c552 (perhaps contributing to the more efficient synthesis in vivo of plastocyanin over cytochrome c552). The deduced physical properties of this plastocyanin are compared to those of the C. reinhardtii plastidic cytochrome c552.

Binding and transcription of relaxed DNA templates by fractions of maize chloroplast extracts.

Preparations of partially purified chloroplast DNA-dependent RNA polymerase from maize and some other plants transcribe cloned chloroplast genes preferentially and much more actively from appropriately negatively supercoiled templates than from relaxed templates. We have found that the polymerase in such fractions does not bind to promoter regions of the maize chloroplast genes psbA and rbcL on small linear DNA fragments but that some protein(s) in unfractionated chloroplast extracts does bind. DEAE chromatography of the extracts has permitted the separation of a DNA-binding fraction from the bulk of the RNA polymerase activity. The binding fraction contains plastid RNA polymerase activity that is relatively independent of template topology.

In vitro expression of chloroplast genes in lysates of higher plant chloroplasts.

A DNA-dependent in vitro-coupled transcription-translation system has been prepared from lysates of isolated chloroplasts. These lysates are comparable to those of Escherichia coli in transcriptional and translational fidelity and efficiency in response to a given template DNA. When Nicotiana tabacum chloroplast DNA is used as template with chloroplast lysates (N. tabacum or spinach) or E. coli lysates, NaDodSO4 gel analysis reveals similar polypeptide patterns that are distinct from the patterns obtained with E. coli DNA. Genes in recombinant plasmids containing chloroplast DNA are also expressed in these in vitro systems. DNA . RNA hybridization experiments show that transcripts are synthesized from most of the chloroplast genome. Newly synthesized large subunit of ribulosebisphosphate carboxylase/oxygenate and a transcript of the large subunit gene (rbcL) are observed in chloroplast lysates using as template chloroplast DNA or cloned fragments of tobacco chloroplast DNA that contain the large subunit gene. Results suggest that differential expression of chloroplast genes occurs in vitro. By using cloned chloroplast DNA templates in this homologous system, it is possible to identify and map structural genes for chloroplast proteins.

Design and development of amplifiable broad-host-range cloning vectors: analysis of the vir region of Agrobacterium tumefaciens plasmid pTiC58.

The construction of a set of new plasmids that are suitable as general cloning vectors in Escherichia coli and Agrobacterium tumefaciens is described. Plasmid pUCD2 is amplifiable in E. coli, replicates in a wide range of gram-negative hosts and contains a number of useful restriction endonuclear cleavage sites and antibiotic resistance genes. This includes unique sites for KpnI, SacI, SacII, PstI, ClaI, SalI, EcoRV, and PvuII and the genes for resistance to kanamycin, tetracycline, ampicillin, and spectinomycin/streptomycin. Derivatives of pUCD2 include pUCD4, which has a unique XbaI site and the cosmid pUCD5, which also contains a unique EcoRI site. Two smaller plasmids pUCD9P and pUCD9X, contain many of the same unique sites as pUCD2 and pUCD4, but carry only the pBR322 replication origin and therefore do not display the extensive host-range of pSa. These plasmids were used to isolate and manipulate fragments of the A. tumefaciens pTiC58 plasmid in both E. coli and A. tumefaciens. Fragments from the virulence (vir) region of pTiC58 inserted immediately upstream of the spectinomycin resistance gene of pUCD2 resulted in spectinomycin resistance levels that varied greatly depending on the particular fragment and its orientation of insertion. Using this property we find that a major portion of the vir region of pTiC58 is transcribed in A. tumefaciens and E. coli from left to right toward the T region.

Characterization of the replication and stability regions of Agrobacterium tumefaciens plasmid pTAR.

A 5.4-kilobase region containing the origin of replication and stability maintenance of the 44-kilobase Agrobacterium tumefaciens plasmid pTAR has been mapped and characterized. Within this region is a 1.3-kilobase segment that is capable of directing autonomous replication. The remaining segment contains the stability locus for maintenance of pTAR during nonselective growth. Approximately 35% of pTAR shares sequence homology with pAg119, a 44-kilobase cryptic plasmid in grapevine strain 1D1119. However, no homology was detected between pTAR DNA and several Ti plasmids or several other small cryptic plasmids in many A. tumefaciens strains. A recombinant plasmid containing the origin of replication and stability maintenance region of pTAR was compatible with pTiC58, pTi15955, and pTi119 and incompatible with pAg119. A new compatibility group, Inc Ag-1, is discussed.