Showing papers by "Pamela C. Ronald published in 2009"

A Type I–Secreted, Sulfated Peptide Triggers XA21-Mediated Innate Immunity

Abstract: The rice Xa21 gene confers immunity to most strains of the bacterium Xanthomonas oryzae pv. oryzae (Xoo). Liquid chromatography–tandem mass spectrometry analysis of biologically active fractions from Xoo supernatants led to the identification of a 194–amino acid protein designated Ax21 (activator of XA21-mediated immunity). A sulfated, 17–amino acid synthetic peptide (axYS22) derived from the N-terminal region of Ax21 is sufficient for activity, whereas peptides lacking tyrosine sulfation are biologically inactive. Using coimmunoprecipitation, we found that XA21 is required for axYS22 binding and recognition. axYS22 is 100% conserved in all analyzed Xanthomonas species, confirming that Ax21 is a pathogen-associated molecular pattern and that XA21 is a pattern recognition receptor.

Rice Pi5-Mediated Resistance to Magnaporthe oryzae Requires the Presence of Two Coiled-Coil–Nucleotide-Binding–Leucine-Rich Repeat Genes

Abstract: Rice blast, caused by the fungus Magnaporthe oryzae, is one of the most devastating diseases of rice. To understand the molecular basis of Pi5-mediated resistance to M. oryzae, we cloned the resistance (R) gene at this locus using a map-based cloning strategy. Genetic and phenotypic analyses of 2014 F2 progeny from a mapping population derived from a cross between IR50, a susceptible rice cultivar, and the RIL260 line carrying Pi5 enabled us to narrow down the Pi5 locus to a 130-kb interval. Sequence analysis of this genomic region identified two candidate genes, Pi5-1 and Pi5-2, which encode proteins carrying three motifs characteristic of R genes: an N-terminal coiled-coil (CC) motif, a nucleotide-binding (NB) domain, and a leucine-rich repeat (LRR) motif. In genetic transformation experiments of a susceptible rice cultivar, neither the Pi5-1 nor the Pi5-2 gene was found to confer resistance to M. oryzae. In contrast, transgenic rice plants expressing both of these genes, generated by crossing transgenic lines carrying each gene individually, conferred Pi5-mediated resistance to M. oryzae. Gene expression analysis revealed that Pi5-1 transcripts accumulate after pathogen challenge, whereas the Pi5-2 gene is constitutively expressed. These results indicate that the presence of these two genes is required for rice Pi5-mediated resistance to M. oryzae.

A Rice Kinase-Protein Interaction Map

Abstract: Plants uniquely contain large numbers of protein kinases, and for the vast majority of the 1,429 kinases predicted in the rice (Oryza sativa) genome, little is known of their functions. Genetic approaches often fail to produce observable phenotypes; thus, new strategies are needed to delineate kinase function. We previously developed a cost-effective high-throughput yeast two-hybrid system. Using this system, we have generated a protein interaction map of 116 representative rice kinases and 254 of their interacting proteins. Overall, the resulting interaction map supports a large number of known or predicted kinase-protein interactions from both plants and animals and reveals many new functional insights. Notably, we found a potential widespread role for E3 ubiquitin ligases in pathogen defense signaling mediated by receptor-like kinases, particularly by the kinases that may have evolved from recently expanded kinase subfamilies in rice. We anticipate that the data provided here will serve as a foundation for targeted functional studies in rice and other plants. The application of yeast two-hybrid and TAPtag analyses for large-scale plant protein interaction studies is also discussed.

Protein-protein interactions of tandem affinity purified protein kinases from rice.

Abstract: Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex.

Analysis of Alternatively Spliced Rice Transcripts Using Microarray Data

Abstract: Alternative splicing creates a diversity of gene products in higher eukaryotes. Twenty-five percent (1,583/6,371) of predicted alternatively spliced transcripts can be detected using the NSF45K rice whole-genome oligonucleotide array. We used the NSF45K array to assess differential expression patterns of 507 loci showing at least a twofold change in expression between light- and dark-grown seedlings. At least 42% of these loci show evidence of alternative splicing in aerial seedling tissue of Oryza sativa ssp. japonica cv. Nipponbare. Most alternative splice forms display the same pattern of regulation as the primary, or most highly expressed, transcript; however, splice forms for ten loci, represented by 35 oligos, display opposite expression patterns in the light vs. dark. We found similar evidence of alternative splicing events in Affymetrix microarray data for Nipponbare rice treated with the causative agent of fungal rice blast, Magnaporthe grisea. This strategy for analyzing alternative splicing in microarray data will enable delineation of the diversity of splicing in rice.

Construction and application of efficient Ac-Ds transposon tagging vectors in rice.

Abstract: Transposons are effective mutagens alternative to T-DNA for the generation of insertional mutants in many plant species including those whose transformation is inefficient. The current strategies of transposon tagging are usually slow and labor-intensive and yield low frequency of tagged lines. We have constructed a series of transposon tagging vectors based on three approaches: (i) AcTPase controlled by glucocorticoid binding domain/VP16 acidic activation domain/Gal4 DNA-binding domain (GVG) chemical-inducible expression system; (ii) deletion of AcTPase via Cre-lox site-specific recombination that was initially triggered by Ds excision; and (iii) suppression of early transposition events in transformed rice callus through a dual-functional hygromycin resistance gene in a novel Ds element (HPT-Ds). We tested these vectors in transgenic rice and characterized the transposition events. Our results showed that these vectors are useful resources for functional genomics of rice and other crop plants. The vectors are freely available for the community.

Direct calibration of PICKY-designed microarrays

Abstract: Background: Few microarrays have been quantitatively calibrated to identify optimal hybridization conditions because it is difficult to precisely determine the hybridization characteristics of a microarray using biologically variable cDNA samples. Results: Using synthesized samples with known concentrations of specific oligonucleotides, a series of microarray experiments was conducted to evaluate microarrays designed by PICKY, an oligo microarray design software tool, and to test a direct microarray calibration method based on the PICKY-predicted, thermodynamically closest nontarget information. The complete set of microarray experiment results is archived in the GEO database with series accession number GSE14717. Additional data files and Perl programs described in this paper can be obtained from the website http://www.complex.iastate.edu under the PICKY Download area. Conclusion: PICKY-designed microarray probes are highly reliable over a wide range of hybridization temperatures and sample concentrations. The microarray calibration method reported here allows researchers to experimentally optimize their hybridization conditions. Because this method is straightforward, uses existing microarrays and relatively inexpensive synthesized samples, it can be used by any lab that uses microarrays designed by PICKY. In addition, other microarrays can be reanalyzed by PICKY to obtain the thermodynamically closest nontarget information for calibration.

Plant and microbial research seeks biofuel production from lignocellulose

Abstract: A key strategy for biofuel production is making use of the chemical energy stored in plant cell walls. Cell walls are a strong meshwork of sugar chains and other polymers that encircle each plant cell. Collectively known as lignocellulose, cell wall material represents the bulk of plant dry mass. Biofuels can be made by releasing sugars from lignocellulose and converting them into fuel; however, this is currently an energy-intensive process. We summarize the barriers to efficient lignocellulosic biofuel production and highlight scientific research recently funded by the U.S. Department of Agriculture and U.S. Department of Energy, both to understand and harness the mechanisms by which plants build cell walls, and to further develop enzymes and microbes that facilitate sugar release and biofuel production.

Cells Modified or Altered for a Rice-diverged Glycosyltransferase

Abstract: The present invention provides for a cell comprising a modified or altered enzymatic activity of a rice-diverged glycosyltransferase (GT).

Focus: Plant Interactions with Bacterial Pathogens

Abstract: The incorporation of resistance genes into agronomically important crop plants is the most economically effective method for controlling plant disease. This biological disease control strategy is heritable and, therefore, inexpensive and permanently available once introduced ([Keen et al., 1993][1

Genes for enhancing resistance to magnaporthe oryzae and uses thereof

Abstract: The present invention relates to Pi5-1 and Pi5-2 proteins which enhance resistance to Mag-naporthe oryzae, genes which encode the proteins, a recombinant vector comprising the genes, a plant transformed with the recombinant vector and seeds thereof, a method of increasing resistance to a plant pathogen by expressing the genes in a plant, antibodies against the proteins, and a composition comprising the genes which are useful for enhancing resistance to a plant pathogen.

Plant and microbial research seeks biofuel production from lignocellulose - eScholarship

Abstract: A key strategy for biofuel production is making use of the chemical energy stored in plant cell walls. Cell walls are a strong meshwork of sugar chains and other polymers that encircle each plant cell. Collectively known as lignocellulose, cell wall material represents the bulk of plant dry mass. Biofuels can be made by releasing sugars from lignocellulose and converting them into fuel; however, this is currently an energy-intensive process. We summarize the barriers to efficient lignocellulosic biofuel production and highlight scientific research recently funded by the U.S. Department of Agriculture and U.S. Department of Energy, both to understand and harness the mechanisms by which plants build cell walls, and to further develop enzymes and microbes that facilitate sugar release and biofuel production.

A Rice Kinase-Protein Interaction Map 1(W)(OA)

Abstract: Plants uniquely contain large numbers of protein kinases, and for the vast majority of the 1,429 kinases predicted in the rice (Oryza sativa) genome, little is known of their functions. Genetic approaches often fail to produce observable phenotypes; thus, new strategies are needed to delineate kinase function. We previously developed a cost-effective high-throughput yeast twohybrid system. Using this system, we have generated a protein interaction map of 116 representative rice kinases and 254 of their interacting proteins. Overall, the resulting interaction map supports a large number of known or predicted kinaseprotein interactions from both plants and animals and reveals many new functional insights. Notably, we found a potential widespread role for E3 ubiquitin ligases in pathogen defense signaling mediated by receptor-like kinases, particularly by the kinases that may have evolved from recently expanded kinase subfamilies in rice. We anticipate that the data provided here will serve as a foundation for targeted functional studies in rice and other plants. The application of yeast two-hybrid and TAPtag analyses for large-scale plant protein interaction studies is also discussed.

Variants of nrr activate plant disease resistance

Abstract: Methods and compositions for improving plant disease resistance by expression of NPR1-biding domain/transcriptional activation domain fusions are provided.