Showing papers by "Pamela C. Ronald published in 2001"

Evidence for a disease‐resistance pathway in rice similar to the NPR1‐mediated signaling pathway in Arabidopsis

Abstract: *† Summary The Arabidopsis NPR1/NIM1 gene is a key regulator of systemic acquired resistance (SAR). Overexpression of NPR1 leads to enhanced resistance in Arabidopsis. To investigate the role of NPR1 in monocots, we over-expressed the Arabidopsis NPR1 in rice and challenged the transgenic plants with Xanthomonas oryzae pv. oryzae (Xoo), the rice bacterial blight pathogen. The transgenic plants displayed enhanced resistance to Xoo. RNA blot hybridization indicates that enhanced resistance requires expression of NPR1 mRNA above a threshold level in rice. To identify components mediating the resistance controlled by NPR1, we used NPR1 as bait in a yeast two-hybrid screen. We isolated four cDNA clones encoding rice NPR1 interactors (named rTGA2.1, rTGA2.2, rTGA2.3 and rLG2) belonging to the bZIP family. rTGA2.1, rTGA2.2 and rTGA2.3 share 75, 76 and 78% identity with Arabidopsis TGA2, respectively. In contrast, rLG2 shares highest identity (81%) to the maize liguleless (LG2) gene product, which is involved in establishing the leaf blade‐sheath boundary. The interaction of NPR1 with the rice bZIP proteins in yeast was impaired by the npr1-1 and npr1-2 mutations, but not by the nim1-4 mutation. The NPR1‐rTGA2.1 interaction was confirmed by an in vitro pull-down experiment. In gel mobility shift assays, rTGA2.1 binds to the rice RCH10 promoter and to a cis-element required sequencespecifically for salicylic acid responsiveness. This is the first demonstration that the Arabidopsis NPR1 gene can enhance disease resistance in a monocot plant. These results also suggest that monocot and dicot plants share a conserved signal transduction pathway controlling NPR1-mediated resistance.

A fast neutron deletion mutagenesis‐based reverse genetics system for plants

Abstract: A new reverse genetics method has been developed to identify and isolate deletion mutants for targeted plant genes. Deletion mutant libraries are generated using fast neutron bombardment. DNA samples extracted from the deletion libraries are used to screen for deletion mutants by polymerase chain reaction (PCR) using specific primers flanking the targeted genes. By adjusting PCR conditions to preferentially amplify the deletion alleles, deletion mutants were identified in pools of DNA samples, each pool containing DNA from 2592 mutant lines. Deletion mutants were obtained for 84% of targeted loci from an Arabidopsis population of 51 840 lines. Using a similar approach, a deletion mutant for a rice gene was identified. Thus we demonstrate that it is possible to apply this method to plant species other than Arabidopsis. As fast neutron mutagenesis is highly efficient, it is practical to develop deletion mutant populations with more complete coverage of the genome than obtained with methods based on insertional mutagenesis. Because fast neutron mutagenesis is applicable to all plant genetic systems, this method has the potential to enable reverse genetics for a wide range of plant species.

Isolation of a Xanthomonas oryzae pv. oryzae Flagellar Operon Region and Molecular Characterization of flhF

Abstract: An 8.1-kb DNA fragment from Xanthomonas oryzae pv. oryzae that contains six open reading frames (ORF) was cloned. The ORF encodes proteins similar to flagellar proteins FlhB, FlhA, FlhF, and FliA, plus two proteins of unknown function, ORF234 and ORF319, from Bacillus subtilis and other organisms. These ORF have a similar genomic organization to those of their homologs in other bacteria. TheflhF gene product, FlhF, has a GTP-binding motif conserved in its homologs. Unlike its homologs, however, X. oryzae pv. oryzae FlhF carries two transmembrane-like domains. Insertional mutations of theflhF gene with the omega cassette or the kanamycin resistance gene significantly retard but do not abolish the motility of the bacteria. Complementation of the mutants with the wild-type flhF gene restored the motility. The X. oryzae pv. oryzae FlhF interacts with itself; the disease resistance gene product XA21; and a protein homologous to the Pill protein of Pseudomonas aeruginosa, XooPilL, in the yeast two-hybrid system. The biological relevance of these interactions remains to be determined.

Use of DNA Markers in introgression and isolation of genes associated with durable resistance to rice blast

Abstract: Rice blast caused by Pyricularia grisea Sacc. (= P. oryzae Cav., teleomorph Magnaporthe grisea Barr.), is one of the most widespread and destructive diseases of rice. Incorporation of blast resistance genes into elite rice cultivars has been a priority in rice breeding for decades in virtually all rice growing countries. Although resistance to blast is often short-lived, some cultivars are considered to possess durable resistance (Johnson 1981). Durable resistance is thought to be associated with partial resistance that is in many cases under oligo-or polygenic control (Higashi and Kushibuchi 1978; Higashi and Saito 1985; Wang et al. 1994; Parlevliet 1988). For example, the rice cultivar Moroberekan displays durable resistance to blast in upland conditions (Bidaux 1978; Ahn 1994; Fomba and Taylor 1994). The use of molecular markers has facilitated studies of the genetic basis of this durable blast resistance (Wang et al. 1994; Chen et al. 1997; Inukai and Aya 1997; Inukai et al. 1997).

Structure, function, and evolution of disease resistance genes in rice.

Abstract: The recent cloning and characterization of several rice genes with resistance to pathogens represent a breakthrough in our understanding of the molecular basis of disease resistance and also provide a starting point for dissecting the resistance pathway in rice. The first resistance gene cloned in rice was Xa21, a gene introgressed from the wild rice Oryza longistaminata. It encodes a putative receptor-like kinase consisting of leucine-rich repeats (LRRs) in the extracellular domain and serine/threonine kinase in the intracellular domain. Sequence analysis of seven members of the gene family at the locus suggests that duplication, recombination, and transposition have occurred during the evolution of this gene family. Experiments with a truncated member indicate that the LRR domain determines race-specific recognition and is subject to adaptive evolution. To identify additional components in the Xa21-mediated resistance pathway, both the yeast two-hybrid screen and mutagenesis approaches are being used. Several defense-related genes were found to interact with the Xa21 protein in yeast when the kinase domain was used in the screen. Using diepoxybutane and fast-neutron mutagenesis, we recovered 31 mutants that have changed from resistant to fully susceptible (10) or partially susceptible (21) to nine races of the bacterial blight pathogen in the Philippines. All fully susceptible mutants showed changes at the Xa21 locus as detected by polymerase chain reaction and Southern hybridization. For the partially susceptible mutants, no detectable changes were found at the Xa21 locus, suggesting that these mutations occur at other loci controlling the Xa21-mediated defense pathway.