Showing papers by "Pamela C. Ronald published in 1999"

A Rapid DNA Minipreparation Method Suitable for AFLP and Other PCR Applications

Abstract: A rapid DNA minipreparation method was developed for rice and other plant species. This method uses an Eppendorf tube and 1-ml pipette tip to grind plant tissues, and requires only one transfer for DNA isolation. In a single day, one person can complete DNA isolation from more than 120 leaf samples. The yields of the DNA samples ranged from 2.3 to 5.2 mg from 25-50 mg fresh leaf tissue. DNA samples extracted using this method from rice were completely digested with five restriction enzymes ( Eco RI ,Eco RV ,Hind III, Mse Ia nd Pst I) and were successfully used for AFLP and other PCR applications.

Short communication: developmental control of Xa21-mediated disease resistance in rice.

Abstract: The rice resistance gene Xa21 confers resistance against the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). The molecular genetic mechanism controlling the integration of the Xa21-mediated disease resistance response with the developmental program in rice is under study in this model system. Reproducible means of infecting plants at certain developmental stages were designed based on the timing of full expansion of the leaf. Xa21-resistance progressively increases from the susceptible juvenile leaf 2 stage through later stages, with 100% resistance at the adult leaf 9/10 stage. We found that Xa21 expression is independent of plant developmental stage, infection with Xoo, or wounding. Expression of the Xa21 gene transcript is not correlated with expression of Xa21 disease resistance indicating that the developmental regulation of Xa21-resistance is either controlled post-transcriptionally or by other factors.

Molecular mapping of the blast resistance gene, Pi44(t), in a line derived from a durably resistant rice cultivar.

Abstract: A recombinant inbred line derived from a cross between CO39 and &Moroberekan', RIL276, was found to be resistant to lineage 44 isolates of Pyricularia grisea in the Philippines. One hundred F 2 individuals were obtained from a backcross of RIL276 and CO39. Phenotypic analysis showed that RIL276 carries a single locus, tentatively named Pi44(t), conferring complete resistance to lineage 44 isolates of P. grisea. RFLP probes, STS primers and AFLP markers were applied to identify DNA markers linked to Pi44(t). Neither RFLP nor STS-PCR analysis gave rise to DNA markers linked to the locus. Using bulk segregant AFLP analysis, however, two dominant AFLP markers (AF 348 and AF 349 ) linked to Pi44(t) were identi"ed. AF 349 and AF 348 were located at 3.3$1.5 cM and 11$3.5 cM from Pi44(t), respective- ly. These markers were mapped on chromosome 11 using an F 2 population derived from a cross between &Labelle' and &Black Gora'. The location of AF 348 on chromosome 11 was con"rmed using another F 2 map- ping population derived from IR40931-26-3-3-5/ PI543851. DNA products at the loci linked to Pi44(t) were ampli"ed from RIL276, &Labelle' and PI543851 using the same primer pairs used to amplify AF 349 and AF 348 . Sequence analysis of these bands showed 100% identity between lines. This result indicates that these AFLP markers could be used for the comparison of maps or assignment of linkage groups to chromosomes.

Particle-bombardment-mediated co-transformation of elite Chinese rice cultivars with genes conferring resistance to bacterial blight and sap-sucking insect pests

Abstract: Transgenic rice plants were generated using particle bombardment to simultaneously introduce the rice Xa21 gene eAective against bacterial blight and the Galanthus nivalis agglutinin (snowdrop lectin; gna) gene eAective against sap-sucking insect pests, specifically the brown plant hopper. Using three plasmids, we co- transformed 5- to 10-d-old, mature seed-derived rice (Oryza sativa L.) callus of two elite Chinese rice cultivars, Eyi 105 and Ewan 5. The plasmids carried a total of four genes. The gna and Xa21 genes were carried on separate plasmids. The selectable marker hygromycin phosphotransferase (hpt) and the reporter gene b-glucu- ronidase (gusA) were linked on the same, co-integrate vector. We recovered over 160 independently derived transgenic rice plants. Over 70% of the transgenic plants carried all four genes, as confirmed by polymerase chain reaction and/or Southern blot analysis. Furthermore, 70% of transgenic plants carrying all four genes also co- expressed all four genes, as confirmed by growth on selection media (hpt), GUS histochemical assays (gusA), western blotting (gna) and reverse transcriptase-poly- merase chain reaction (Xa21) analysis. The co-expres- sion eAciency reported for the four transgenes in our study is the highest ever found in any transgenic plant population generated through co-transformation. The linked genes (hpt and gusA) co-integrated with a frequency of near 100%, and we observed a co-integra- tion frequency greater than 70% for the genes carried on separate plasmids. We observed no preferential integra- tion of any particular gene(s). Genetic analysis con- firmed Mendelian segregation of the transgenes in subsequent generations. We report, for the first time, generation and analysis of transgenic rice lines carrying genes eAective against more than one taxa of pathogen or pest, substantiating that particle bombardment rep- resents an eAective way to introduce unlinked complex multiple traits into plants.

Expression of a Gibberellin-Induced Leucine-Rich Repeat Receptor-Like Protein Kinase in Deepwater Rice and Its Interaction with Kinase-Associated Protein Phosphatase

Abstract: We identified in deepwater rice (Oryza sativa L.) a gene encoding a leucine-rich repeat receptor-like transmembrane protein kinase, OsTMK (O. sativa transmembrane kinase). The transcript levels of OsTMK increased in the rice internode in response to gibberellin. Expression of OsTMK was especially high in regions undergoing cell division and elongation. The kinase domain of OsTMK was enzymatically active, autophosphorylating on serine and threonine residues. A cDNA encoding a rice ortholog of a kinase-associated type 2C protein phosphatase (OsKAPP) was cloned. KAPPs are putative downstream components in kinase-mediated signal transduction pathways. The kinase interaction domain of OsKAPP was phosphorylated in vitro by the kinase domain of OsTMK. RNA gel-blot analysis indicated that the expression of OsTMK and OsKAPP was similar in different tissues of the rice plant. In protein-binding assays, OsKAPP interacted with a receptor-like protein kinase, RLK5 of Arabidopsis, but not with the protein kinase domains of the rice and maize receptor-like protein kinases Xa21 and ZmPK1, respectively.

Signal transduction networks and the biology of plant cells.

Abstract: The development of plant transformation in the mid-1980s and of many new tools for cell biology, molecular genetics, and biochemistry has resulted in enormous progress in plant biology in the past decade. With the completion of the genome sequence of Arabidopsis thaliana just around the corner, we can expect even faster progress in the next decade. The interface between cell biology and signal transduction is emerging as a new and important field of research. In the past we thought of cell biology strictly in terms of organelles and their biogenesis and function, and researchers focused on questions such as, how do proteins enter chloroplasts? or, what is the structure of the macromolecules of the cell wall and how are these molecules secreted? Signal transduction dealt primarily with the perception of light (photomorphogenesis) or hormones and with the effect such signals have on enhancing the activity of specific genes. Now we see that the fields of cell biology and signal transduction are merging because signals pass between organelles and a single signal transduction pathway usually involves multiple organelles or cellular structures. Here are some examples to illustrate this new paradigm. How does abscisic acid (ABA) regulate stomatal closure? This pathway involves not only ABA receptors whose location is not yet known, but cation and anion channels in the plasma membrane, changes in the cytoskeleton, movement of water through water channels in the tonoplast and the plasma membrane, proteins with a farnesyl tail that can be located either in the cytosol or attached to a membrane, and probably unidentified ion channels in the tonoplast. In addition there are highly localized calcium oscillations in the cytoplasm resulting from the release of calcium stored in various compartments. The activities of all these cellular structures need to be coordinated during ABA-induced stomatal closure. For another example of the interplay between the proteins of signal transduction pathways and cytoplasmic structures, consider how plants mount defense responses against pathogens. Elicitors produced by pathogens bind to receptors on the plant plasma membrane or in the cytosol and eventually activate a large number of genes. This results in the coordination of activities at the plasma membrane (production of reactive oxygen species), in the cytoskeleton, localized calcium oscillations, and the modulation of protein kinases and protein phosphatases whose locations remain to be determined. The movement of transcription factors into the nucleus to activate the defense genes requires their release from cytosolic anchors and passage through the nuclear pore complexes of the nuclear envelope. This review does not cover all the recent progress in plant signal transduction and cell biology; it is confined to the topics that were discussed at a recent (November 1998) workshop held in Santiago at which lecturers from Chile, the USA and the UK presented recent results from their laboratories.

Nucleic acid encoding an NPR1 interactor from rice and method of use to produce pathogen-resistant plants

Abstract: The present invention provides the identification and characterization of a polynucleotide encoding rice PNI, a proline-rich protein that interacts with Arabidopsis NPR1 in a yeast two-hybrid assay. The present invention also provides methods of enhancing resistance to pathogens by introducing the polynucleotide[s] into plants.