Showing papers by "Pamela C. Ronald published in 2017"
TL;DR: This work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations and reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes.
Abstract: The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a single-nucleotide resolution facilitate functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake (Oryza sativa ssp japonica), which completes its life cycle in 9 weeks. We sequenced 1504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, i.e., 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single-base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportion of loss-of-function mutations. We identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line. This result reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks. This population complements other available mutant collections and gene-editing technologies. This work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations.
121 citations
TL;DR: It is suggested that RaxX serves as a molecular mimic of PSY peptides to facilitate Xoo infection and that XA21 has evolved the ability to recognize and respond specifically to the microbial form of the peptide.
Abstract: Summary
The biotrophic pathogen Xanthomonas oryzae pv. oryzae (Xoo) produces a sulfated peptide named RaxX, which shares similarity to peptides in the PSY (plant peptide containing sulfated tyrosine) family. We hypothesize that RaxX mimics the growth-stimulating activity of PSY peptides.
Root length was measured in Arabidopsis and rice treated with synthetic RaxX peptides. We also used comparative genomic analyses and reactive oxygen species burst assays to evaluate the activity of RaxX and PSY peptides.
Here we found that a synthetic sulfated RaxX derivative comprising 13 residues (RaxX13-sY), highly conserved between RaxX and PSY, induces root growth in Arabidopsis and rice in a manner similar to that triggered by PSY. We identified residues that are required for activation of immunity mediated by the rice XA21 receptor but that are not essential for root growth induced by PSY. Finally, we showed that a Xanthomonas strain lacking raxX is impaired in virulence.
These findings suggest that RaxX serves as a molecular mimic of PSY peptides to facilitate Xoo infection and that XA21 has evolved the ability to recognize and respond specifically to the microbial form of the peptide.
58 citations
TL;DR: Integration of diverse genomics data based on the Bayesian statistics framework has been successfully applied to the construction of genome-scale functional networks for major crop species, but functional genomics studies on bread wheat have lagged behind those on other crops.
18 citations
TL;DR: It is reported that XA21 is an auxilin-like protein predicted to function in clathrin-mediated endocytosis and in regulation of cell death, and the up-regulation of genes controlling ‘vesicle-mediated transport’ in XB21 overexpression lines is consistent with a functional role for XB 21 as an Auxilin.
Abstract: The rice immune receptor XA21 confers resistance to the bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo). To elucidate the mechanism of XA21-mediated immunity, we previously performed a yeast two-hybrid screening for XA21 interactors and identified XA21 binding protein 21 (XB21). Here, we report that XB21 is an auxilin-like protein predicted to function in clathrin-mediated endocytosis. We demonstrate an XA21/XB21 in vivo interaction using co-immunoprecipitation in rice. Overexpression of XB21 in rice variety Kitaake and a Kitaake transgenic line expressing XA21 confers a necrotic lesion phenotype and enhances resistance to Xoo. RNA sequencing reveals that XB21 overexpression results in the differential expression of 8735 genes (4939 genes up- and 3846 genes down-regulated) (≥2-folds, FDR ≤0.01). The up-regulated genes include those predicted to be involved in ‘cell death’ and ‘vesicle-mediated transport’. These results indicate that XB21 plays a role in the plant immune response and in regulation of cell death. The up-regulation of genes controlling ‘vesicle-mediated transport’ in XB21 overexpression lines is consistent with a functional role for XB21 as an auxilin.
18 citations
TL;DR: Results indicate that LRR1 is required for wild-type Xa21 transcript expression and XA21-mediated immunity, and compromises resistance to Xoo compared with control XA 21 plants.
Abstract: The rice immune receptor XA21 confers resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight. We previously demonstrated that an auxilin-like protein, XA21 BINDING PROTEIN 21 (XB21), positively regulates resistance to Xoo. To further investigate the function of XB21, we performed a yeast two-hybrid screen. We identified 22 unique XB21 interacting proteins, including LEUCINE-RICH REPEAT PROTEIN 1 (LRR1), which we selected for further analysis. Silencing of LRR1 in the XA21 genetic background (XA21-LRR1Ri) compromises resistance to Xoo compared with control XA21 plants. XA21-LRR1Ri plants have reduced Xa21 transcript levels and reduced expression of genes that serve as markers of XA21-mediated activation. Overexpression of LRR1 is insufficient to alter resistance to Xoo in rice lines lacking XA21. Taken together, our results indicate that LRR1 is required for wild-type Xa21 transcript expression and XA21-mediated immunity.
12 citations
TL;DR: It is shown that SnRK2s are clearly divided into four pan-angiosperm clades with those in the traditionally defined Subclass II encompassing two distinct clades, although OsSAPK3 lacks an Arabidopsis ortholog.
Abstract: Members of the sucrose non-fermenting related kinase Group2 (SnRK2) subclasses are implicated in both direct and indirect abscisic acid (ABA) response pathways. We have used phylogenetic, biochemical, and transient in vivo approaches to examine interactions between Triticum tauschii protein kinase 1 (TtPK1) and an interacting protein, Oryza sativa SnRK2-calcium sensor (OsSCS1). Given that TtPK1 has 100% identity with its rice ortholog, osmotic stress/ABA-activated protein kinase (OsSAPK2), we hypothesized that the SCS and TtPK1 interactions are present in both wheat and rice. Here, we show that SnRK2s are clearly divided into four pan-angiosperm clades with those in the traditionally defined Subclass II encompassing two distinct clades (OsSAPK1/2 and OsSAPK3), although OsSAPK3 lacks an Arabidopsis ortholog. We also show that SCSs are distinct from a second lineage, that we term SCSsister, and while both clades pre-date land plants, the SCSsister clade lacks Poales representatives. Our Y2H assays revealed that the removal of the OsSCS1 C-terminal region along with its N-terminal EF-hand abolished its interaction with the kinase. Using transient in planta bimolecular fluorescence complementation experiments, we demonstrate that TtPK1/OsSCS1 dimerization co-localizes with DAPI-stained nuclei and with FM4-64-stained membranes. Finally, OsSCS1- and OsSAPK2-hybridizing transcripts co-accumulate in shoots/coleoptile of drying seedlings, consistent with up-regulated kinase transcripts of PKABA1 and TtPK1. Our studies suggest that interactions between homologs of the SnRK2 and SCS lineages are broadly conserved across angiosperms and offer new directions for investigations of related proteins.
6 citations
TL;DR: The generation of WheatNet, the first genome-scale functional network for T. aestivum and a companion web server, is reported, which was constructed by integrating 20 distinct genomics datasets, including 156,000 wheat-specific co-expression links mined from 1,929 microarray data.
Abstract: Gene networks provide a system-level overview of genetic organizations and enable the dissection of functional modules underlying complex traits. Here we report the generation of WheatNet, the first genome-scale functional network for T. aestivum and a companion web server (www.inetbio.org/wheatnet). WheatNet was constructed by integrating 20 distinct genomics datasets, including 156,000 wheat-specific co-expression links mined from 1,929 microarray data. A unique feature of WheatNet is that each network node represents either a single gene or a group of genes. We computationally partitioned gene groups mimicking homeologous genes by clustering 99,386 wheat genes, resulting in 20,248 gene groups comprising 63,401 genes and 35,985 individual genes. Thus, WheatNet was constructed using 56,233 nodes, and the final integrated network has 20,230 nodes and 567,000 edges. The edge information of the integrated WheatNet and all 20 component networks are available for download.
5 citations
TL;DR: The results suggest that the endodomain of the EFR and XA 21 immune receptors are interchangeable and the XA21 ectodomains is the key determinant conferring robust resistance to Xoo.
Abstract: Rice (Oryza sativa) plants expressing the XA21 cell surface receptor kinase are resistant to Xanthomonas oryzae pv. oryzae (Xoo) infection. We previously demonstrated that expressing a chimeric protein containing the EFR (ELONGATION FACTOR Tu RECEPTOR) ectodomain and the XA21 endodomain (EFR:XA21) in rice does not confer robust resistance to Xoo.
To test if the XA21 ectodomain is required for Xoo resistance, we produced transgenic rice lines expressing a chimeric protein consisting of the XA21 ectodomain and EFR endodomain (XA21:EFR) and inoculated these lines with Xoo. We also tested if the XA21:EFR rice plants respond to a synthetic sulfated 21 amino acid derivative (RaxX21-sY) derived from the activator of XA21-mediated immunity, RaxX.
We found that five independently transformed XA21:EFR rice lines displayed resistance to Xoo as measured by lesion length analysis, and showed that five lines express markers of the XA21 defense response (generation of reactive oxygen species and defense response gene expression) after treatment with RaxX21-sY.
Our results indicate that expression of the XA21:EFR chimeric receptor in rice confers resistance to Xoo. These results suggest that the endodomain of the EFR and XA21 immune receptors are interchangeable and the XA21 ectodomain is the key determinant conferring robust resistance to Xoo.
4 citations
TL;DR: The findings suggest that RaxX serves as a molecular mimic of PSY peptides to facilitate Xoo infection and that XA21 has evolved the ability to recognize and respond specifically to the microbial form of the peptide.
Abstract: ·The biotrophic pathogen Xanthomonas oryzae pv. oryzae (Xoo) produces a sulfated peptide named RaxX, which shares similarity to peptides in the PSY (plant peptide containing sulfated tyrosine) family. We hypothesize that RaxX functionally mimics the growth stimulating activity of PSY peptides. ·Root length was measured in Arabidopsis and rice treated with synthetic RaxX peptides. We also used comparative genomic analysis and Reactive Oxygen Species (ROS) burst assay to evaluate the activity of RaxX and PSY peptides. ·Here we found that a synthetic sulfated RaxX derivative comprising 13 residues (RaxX13-sY), highly conserved between RaxX and PSY, induces root growth in Arabidopsis and rice in a manner similar to that triggered by PSY. We identified residues that are required for activation of immunity mediated by the rice XA21 receptor but that are not essential for root growth induced by PSY. Finally, we showed that a Xanthomonas strain lacking raxX is impaired in virulence. ·These findings suggest that RaxX serves as a molecular mimic of PSY peptides to facilitate Xoo infection and that XA21 has evolved the ability to recognize and respond specifically to the microbial form of the peptide.
4 citations
TL;DR: A fast-neutron-induced mutant population in the model rice cultivar Kitaake is generated and an inversion affecting a single gene is identified as the causative mutation for the short-grain phenotype in one mutant line with a small segregating population, revealing the usefulness of the resource for efficient identification of genes conferring specific phenotypes.
Abstract: The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a single-nucleotide resolution facilitates functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake (Oryza sativa L. ssp. japonica), which completes its life cycle in 9 weeks. We sequenced 1,504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportion of loss-of-function mutations. Using this mutant population, we identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line with a small segregating population. This result reveals the usefulness of the resource for efficient identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks, enabling rapid functional genomic studies of rice.
3 citations
TL;DR: The discovery of the complete raxX-raxSTAB gene cluster was found only in Xanthomonas species, and its distribution is consistent with multiple gain and loss events during X anthomonas speciation, establishing a foundation for investigating biological roles for tyrosine sulfation in bacteria.
Abstract: Tyrosine sulfation is a post-translational modification that influences interaction specificity between certain receptors and their protein ligands in diverse biological processes. For example, rice XA21 receptor-mediated recognition of the sulfated bacterial protein RaxX activates an immune response and triggers resistance to the phytopathogen Xanthomonas oryzae pv. oryzae (Xoo). A five kb raxX-raxSTAB gene cluster of Xoo encodes RaxX, the RaxST tyrosylprotein sulfotransferasea and the RaxA and RaxB components of a predicted proteolytic maturation and ATP-dependent peptide secretion complex. The complete raxX-raxSTAB gene cluster was found only in Xanthomonas species, and its distribution is consistent with multiple gain and loss events during Xanthomonas speciation. Homologs of the raxST gene are present in genome sequences of diverse bacterial species. Together, these results establish a foundation for investigating biological roles for tyrosine sulfation in bacteria.
TL;DR: Today, virtually everything the authors eat is produced from seeds that have been genetically altered in one way or another, and conventional approaches are often quite crude.
Abstract: For 10,000 years, we have altered the genetic makeup of our crops. Conventional approaches are often quite crude, resulting in new varieties through a combination of trial and error, and without knowledge of the precise function of the genes that are being transferred. Such methods include grafting or forced pollinations between different species, as well as radiation or chemical treatments that induce random mutations in the seed. Today, virtually everything we eat is produced from seeds that have been genetically altered in one way or another.
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