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Pamela C Ronald

Publications and source records attributed to Pamela C Ronald.

16 recordsLinked to original sources
PubMed2026

The rice cellulose synthase-like D4 gene (OsCSLD4) is required for resistance to Xanthomonas pv. oryzae.

Plant cell walls serve as a physical support and a barrier to pathogen invasion. Cellulose is the main component of cell walls. The cellulose synthase-like D (CSLD) subfamily genes are required for plant normal development. In rice, Oscsld4 mutant plants are dwarfed and have narrow, rolled leaves. The role of OsCSLD4 in rice immune responses is unclear. We carried out a forward genetic screen using rice mutants expressing the XA21 immune receptor to identify components required for the resistance to Xanthomonas oryzae pv. oryzae (Xoo). One mutant from the screen carries a loss-of-function mutation in OsCSLD4. OsCSLD4 is required for resistance to Xoo mediated by both the XA21 and XA26 immune receptors and also participates in the basal resistance to Xoo. Hallmarks of the XA21-mediated immune response, including induction of the defense marker gene KO5, reactive oxygen species (ROS) burst and the phosphorylation of mitogen-activated protein kinases (MAPKs), are not compromised in the Oscsld4 mutant. These findings suggest that OsCSLD4 does not function as a core signaling component of the immune receptor pathway, but rather maintains the structural integrity of the cell wall as an effective physical barrier. This structural defense is essential for the full manifestation of both innate and receptor-mediated immunity.

Oryza↗
PubMed2007

Use of Nipponbare BAC clones for physical mapping of an R gene locus in rice.

Major advances in rice genomics during the last few years have made positional cloning in rice much more efficient. Nipponbare is a model rice genotype being sequenced by the International Rice Genome Sequencing Project Consortium. Here, we describe an efficient procedure of the construction of physical map for positional cloning of resistance gene (R) using the Nipponbare genetic resources. This advanced strategy should be useful for the efficient identification of agronomic important R genes from many resistant rice genotypes, including wild rice species.

Chromosomes, Artificial, Bacterial↗
PubMed2007

Yeast two-hybrid approaches to dissecting the plant defense response.

We describe a reliable GAL4-based yeast two-hybrid system for identifying and isolating clones encoding proteins interacting with a protein of interest. This two-hybrid system gives extremely low background and few false-positive clones, making it ideal for library screening purposes. We have successfully used it not only to isolate Arabidopsis NPR1-interactors from rice but also to pull out the rice NPR1 ortholog using one of the interactors as bait.

Arabidopsis↗
PubMed2006

Rice XA21 binding protein 3 is a ubiquitin ligase required for full Xa21-mediated disease resistance.

XA21 is a receptor-like kinase protein in rice (Oryza sativa) that confers gene-for-gene resistance to specific races of the causal agent of bacterial blight disease, Xanthomonas oryzae pv oryzae. We identified XA21 binding protein 3 (XB3), an E3 ubiquitin ligase, as a substrate for the XA21 Ser and Thr kinase. The interaction between XB3 and the kinase domain of XA21 has been shown in yeast and in vitro, and the physical association between XB3 and XA21 in vivo has also been confirmed by coimmunoprecipitation assays. XB3 contains an ankyrin repeat domain and a RING finger motif that is sufficient for its interaction with the kinase domain of XA21 and for its E3 ubiquitin ligase activity, respectively. Transgenic plants with reduced expression of the Xb3 gene are compromised in resistance to the avirulent race of X. oryzae pv oryzae. Furthermore, reduced levels of Xb3 lead to decreased levels of the XA21 protein. These results indicate that Xb3 is necessary for full accumulation of the XA21 protein and for Xa21-mediated resistance.

Amino Acid Sequence↗
PubMed2006

From the Academy: Colloquium review. Unique characteristics of Xanthomonas oryzae pv. oryzae AvrXa21 and implications for plant innate immunity.

This article provides a brief overview of some of the major concepts and molecular features of plant and animal innate immune systems. The rice pathogen recognition receptor, XA21, confers resistance to Xanthomonas oryzae pv. oryzae strains producing the AvrXa21 elicitor. Xa21 codes for a receptor-like kinase consisting of an extracellular leucine-rich repeat domain, a transmembrane domain, and a cytoplasmic kinase domain. We show that AvrXa21 activity requires the presence of rax (required for AvrXa21) A, raxB, and raxC genes that encode components of a type one secretion system. In contrast, an hrpC(-) strain deficient in type three secretion maintains AvrXa21 activity. Xanthomonas campestris pv. campestris can express AvrXa21 activity if raxST, encoding a putative sulfotransferase, and raxA are provided in trans. Expression of rax genes depends on population density and other functioning rax genes. This and other data suggest that the AvrXa21 pathogen-associated molecule is involved in quorum sensing. Together these data suggest that AvrXa21 represents a previously uncharacterized class of Gram-negative bacterial signaling molecules. These results from our studies of the XA21/AvrXa21 interaction call for some modifications in the way we think about innate immunity strategies.

Animals↗
PubMed2006

Sub1A is an ethylene-response-factor-like gene that confers submergence tolerance to rice.

Most Oryza sativa cultivars die within a week of complete submergence--a major constraint to rice production in south and southeast Asia that causes annual losses of over US 1 billion dollars and affects disproportionately the poorest farmers in the world. A few cultivars, such as the O. sativa ssp. indica cultivar FR13A, are highly tolerant and survive up to two weeks of complete submergence owing to a major quantitative trait locus designated Submergence 1 (Sub1) near the centromere of chromosome 9 (refs 3, 4, 5-6). Here we describe the identification of a cluster of three genes at the Sub1 locus, encoding putative ethylene response factors. Two of these genes, Sub1B and Sub1C, are invariably present in the Sub1 region of all rice accessions analysed. In contrast, the presence of Sub1A is variable. A survey identified two alleles within those indica varieties that possess this gene: a tolerance-specific allele named Sub1A-1 and an intolerance-specific allele named Sub1A-2. Overexpression of Sub1A-1 in a submergence-intolerant O. sativa ssp. japonica conferred enhanced tolerance to the plants, downregulation of Sub1C and upregulation of Alcohol dehydrogenase 1 (Adh1), indicating that Sub1A-1 is a primary determinant of submergence tolerance. The FR13A Sub1 locus was introgressed into a widely grown Asian rice cultivar using marker-assisted selection. The new variety maintains the high yield and other agronomic properties of the recurrent parent and is tolerant to submergence. Cultivation of this variety is expected to provide protection against damaging floods and increase crop security for farmers.

Alleles↗
PubMed2006

A variable cluster of ethylene response factor-like genes regulates metabolic and developmental acclimation responses to submergence in rice.

Submergence-1 (Sub1), a major quantitative trait locus affecting tolerance to complete submergence in lowland rice (Oryza sativa), contains two or three ethylene response factor (ERF)-like genes whose transcripts are regulated by submergence. In the submergence-intolerant japonica cultivar M202, this locus encodes two ERF genes, Sub1B and Sub1C. In the tolerant near-isogenic line containing the Sub1 locus from the indica FR13A, M202(Sub1), the locus additionally encodes the ERF gene Sub1A. During submergence, the tolerant M202(Sub1) displayed restrained leaf and internode elongation, chlorophyll degradation, and carbohydrate consumption, whereas the enzymatic activities of pyruvate decarboxylase and alcohol dehydrogenase were increased significantly compared with the intolerant M202. Transcript levels of genes associated with carbohydrate consumption, ethanolic fermentation, and cell expansion were distinctly regulated in the two lines. Sub1A and Sub1C transcript levels were shown to be upregulated by submergence and ethylene, with the Sub1C allele in M202 also upregulated by treatment with gibberellic acid (GA). These findings demonstrate that the Sub1 region haplotype determines ethylene- and GA-mediated metabolic and developmental responses to submergence through differential expression of Sub1A and Sub1C. Submergence tolerance in lowland rice is conferred by a specific allele variant of Sub1A that dampens ethylene production and GA responsiveness, causing quiescence in growth that correlates with the capacity for regrowth upon desubmergence.

Acclimatization↗
PubMed2006

A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts.

BACKGROUND: Transient assays using protoplasts are ideal for processing large quantities of genetic data coming out of hi-throughput assays. Previously, protoplasts have routinely been prepared from dicot tissue or cell suspension cultures and yet a good system for rice protoplast isolation and manipulation is lacking. RESULTS: We have established a rice seedling protoplast system designed for the rapid characterization of large numbers of genes. We report optimized methods for protoplast isolation from 7-14 day old etiolated rice seedlings. We show that the reporter genes luciferase GL2 and GUS are maximally expressed approximately 20 h after polyethylene glycol (PEG)-mediated transformation into protoplasts. In addition we found that transformation efficiency varied significantly with plasmid size. Five micrograms of a 4.5 kb plasmid resulted in 60-70% transformation efficiency. In contrast, using 50 microg of a 12 kb plasmid we obtained a maximum of 25-30% efficiency. We also show that short interfering RNAs (siRNAs) can be used to silence exogenous genes quickly and efficiently. An siRNA targeting luciferase resulted in a significant level of silencing after only 3 hours and up to an 83% decrease in expression. We have also isolated protoplasts from cells prepared from fully green tissue. These green tissue-derived protoplasts can be transformed to express high levels of luciferase activity and should be useful for assaying light sensitive cellular processes. CONCLUSION: We report a system for isolation, transformation and gene silencing of etiolated rice leaf and stem-derived protoplasts. Additionally, we have extended the technology to protoplasts isolated from fully green tissue. The protoplast system will bridge the gap between hi-throughput assays and functional biology as it can be used to quickly study large number of genes for which the function is unknown.

Journal Article↗
PubMed2005

Overexpression of a rice NPR1 homolog leads to constitutive activation of defense response and hypersensitivity to light.

Arabidopsis NPR1/NIM1 is a key regulator of systemic acquired resistance (SAR), which confers lasting broad-spectrum resistance. Previous reports indicate that rice has a disease-resistance pathway similar to the Arabidopsis SAR pathway. Here we report the isolation and characterization of a rice NPR1 homologue (NH1). Transgenic rice plants overexpressing NH1 (NH1ox) acquire high levels of resistance to Xanthomonas oryzae pv. oryzae. The resistance phenotype is heritable and correlates with the presence of the transgene and reduced bacterial growth. Northern analysis shows that NH1ox rice spontaneously activates defense genes, contrasting with NPR1-overexpressing Arabidopsis, where defense genes are not activated until induction. Wild-type NH1, but not a point mutant corresponding to npr1-1, interacts strongly with the rice transcription factor rTGA2.2 in yeast two-hybrid. Greenhouse-grown NH1ox plants develop lesion-mimic spots on leaves at preflowering stage although no other developmental effects are observed. However, when grown in growth chambers (GCs) under low light, NH1ox plants are dwarfed, indicating elevated sensitivity to light. The GC-grown NH1ox plants show much higher salicylic acid (SA) levels than the wild type, whereas greenhouse-grown NH1ox plants contain lower SA. These results indicate that NH1 may be involved in the regulation of SA in response to environmental changes.

Amino Acid Sequence↗
PubMed2005

Alteration of TGA factor activity in rice results in enhanced tolerance to Xanthomonas oryzae pv. oryzae.

In dicotyledonous plants broad-spectrum resistance to pathogens is established after the induction of the systemic acquired resistance (SAR) response. In Arabidopsis the NPR1 protein can regulate SAR by interacting with members of the TGA class of basic, leucine-zipper transcription factors to alter pathogenesis-related (PR) gene expression. Overexpression of (At)NPR1 in Arabidopsis enhances resistance to multiple pathogens. Similarly, overexpression of (At)NPR1 in rice enhances resistance to the bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo). These results suggest that components of the (At)NPR1-mediated SAR defense response may be conserved between monocots and dicots. To determine whether or not rice TGA factors are involved in disease resistance responses, the effect of altering the function of rice TGA2.1 was analyzed in transgenic plants. Transgenic rice overexpressing an rTGA2.1 mutant, that can no longer bind DNA, and transgenic rice that have the endogenous rTGA2.1 silenced by dsRNA-mediated silencing were generated. Both types of transgenic rice displayed increased tolerance to Xoo, were dwarfed, and had altered accumulation of PR genes. The results presented in this study suggest that wild-type rTGA2.1 has primarily a negative role in rice basal defense responses to bacterial pathogens.

DNA-Binding Proteins↗
PubMed2005

Rice NRR, a negative regulator of disease resistance, interacts with Arabidopsis NPR1 and rice NH1.

Arabidopsis NPR1/NIM1 is a key regulator of systemic acquired resistance (SAR), which confers lasting broad-spectrum resistance. Over-expression of Arabidopsis NPR1 or the NPR1 homolog 1 (NH1) in rice results in enhanced resistance to the pathogen Xanthomonasoryzae pv. oryzae (Xoo), suggesting the presence of a related defense pathway in rice. We investigated this pathway in rice by identifying proteins that interact with NH1. Here we report the isolation and characterization of a rice cDNA encoding a novel protein, named NRR (for negative regulator of resistance). NRR interacts with NPR1 in the NPR1-interacting domain (NI25) consisting of 25 amino acids. NRR also interacts with NH1; however, NI25 was not sufficient for a strong interaction, indicating a difference between the rice and the Arabidopsis proteins. Silencing of NRR in rice had little effect on resistance to Xoo. When constitutively over-expressed in rice, NRR affected basal resistance, age-related resistance and Xa21-mediated resistance, causing enhanced susceptibility to Xoo. This phenotype was correlated with elevated NRR mRNA and protein levels and increased Xoo growth. Over-expression of NRR suppressed the induction of defense-related genes. NRR:GFP (green fluorescent protein) protein was localized to the nucleus, indicating that NRR may act directly to suppress the activation of defense genes. The fact that NRR compromises Xa21-mediated resistance indicates cross-talk or overlap between NH1- and Xa21-mediated pathways.

Amino Acid Sequence↗
PubMed2004

Overexpression of (At)NPR1 in rice leads to a BTH- and environment-induced lesion-mimic/cell death phenotype.

Systemic acquired resistance (SAR) is an inducible defense response that protects plants against a broad spectrum of pathogens. A central regulator of SAR in Arabidopsis is NPR1 (nonexpresser of pathogenesis-related genes). In rice, overexpression of Arabidopsis NPR1 enhances plant resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae. This report demonstrates that overexpression of (At)NPR1 in rice also triggers a lesion-mimic/cell death (LMD) phenotype. The LMD phenotype is environmentally regulated and heritable. In addition, the development of lesions and death correlates with the expression of rice defense genes and the accumulation of hydrogen peroxide. Application of the salicylic acid (SA) analog, benzo(1,2,3) thiadiazole-7-carbothioc acid S-methyl ester (BTH), potentiates this phenotype Endogenous SA levels are reduced in rice overexpressing (At)NPR1 when compared with wildtype plants, supporting the idea that (At)NPR1 may perceive and modulate the accumulation of SA. The association of (At)NPR1 expression in rice with the development of an LMD phenotype suggests that (At)NPR1 has multiple roles in plant stress responses that may affect its efficacy as a transgenic tool for engineering broad-spectrum resistance.

Arabidopsis Proteins↗
PubMed2004

Bacterial genes involved in type I secretion and sulfation are required to elicit the rice Xa21-mediated innate immune response.

Innate immunity to microorganisms relies on the specific sensing of pathogen-associated molecules by host recognition receptors. Whereas studies in animals have largely focused on the recognition of extracellular pathogen-associated molecules by the TLR (toll-like receptor) superfamily, few studies have been carried out in plants, and it is not understood how these molecules are secreted or modified. The rice Xa21 gene encodes a receptor-like kinase that provides immunity against strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae carrying AvrXa21 activity. We identified four X. oryzae pv. oryzae genes that are required for AvrXa21 activity. raxA, raxB, and raxC encode proteins with similarity to a membrane fusion protein, an ATP-binding cassette transporter, and an outer membrane protein, respectively, of bacterial type I secretion systems. The fourth gene, raxST, encodes a sulfotransferase-like protein. Sequence analysis of three naturally occurring X. oryzae pv. oryzae strains no longer recognized by Xa21 revealed alterations in the raxST and raxA genes. The raxC gene complemented an Escherichia coli tolC mutant for secretion of a double glycine-leader peptide confirming the function of raxC in type I secretion. These results indicate that bacterial type I secretion is necessary for Xa21-mediated recognition and immunity and further suggest that type I secretion and modification of pathogen-associated molecules play an important role in triggering the innate immune response in rice.

Amino Acid Sequence↗
PubMed2002

Biochemical characterization of the kinase domain of the rice disease resistance receptor-like kinase XA21.

The rice disease resistance gene, Xa21, encodes a receptor kinase-like protein consisting of leucine-rich repeats in the putative extracellular domain and a serine/threonine kinase in the putative intracellular domain. The putative XA21 kinase domain was expressed as maltose-binding and glutathione S-transferase fusion proteins in Escherichia coli. The fusion proteins are capable of autophosphorylation. Phosphoamino acid analysis of the glutathione S-transferase fusion protein indicates that only serine and threonine residues are phosphorylated. The relative phosphorylation rate of the XA21 kinase against increasing enzyme concentrations follows a first-order rather than second-order kinetics, indicating an intramolecular phosphorylation mechanism. Moreover, the active XA21 kinase cannot phosphorylate a kinase-deficient mutant of XA21 kinase. The enzymatic activity of the XA21 kinase in a buffer containing Mn(2+) is at least 15 times higher than that with Mg(2+). The K(m) and V(max) of XA21 kinase for ATP are 0.3 microm and 8.4 nmol/mg/min, respectively. Tryptic phosphopeptide mapping reveals that multiple sites on the XA21 kinase are phosphorylated. Finally, our data suggest that the region of XA21 kinase corresponding to the RD kinase activation domain is not phosphorylated, revealing a distinct mode of action compared with the tomato Pto serine/threonine kinase conferring disease resistance.

Amino Acid Sequence↗
PubMed2002

Engineering pathogen resistance in crop plants.

As the world population continues to increase, food supplies must also grow to meet nutritional requirements. One means of ensuring the stability and plentitude of the food supply is to mitigate crop loss caused by plant pathogens. Strategies for combating disease include traditional technologies such as plant breeding and chemical applications; current technologies such as generating transgenic plants that express components of known defense signaling pathways; and the adaptation of newer technologies such as RNA silencing of pathogen and plant transcripts. Breeding has been used to pyramid resistance (R) genes into many different plants including rice. Chemical strategies include application of salicylic acid (SA) analogs to stimulate systemic acquired resistance (SAR) responses. Genetic screens in Arabidopsis have identified genes controlling SAR and these genes have been manipulated and used to engineer crop plants. The diseases caused by plant viruses are being thwarted through the initiation of endogenous RNA silencing mechanisms. Many of these strategies show great promise, some limitations, and exciting opportunities to develop many new tools for combating plant pests.

Crops, Agricultural↗