A Nomadic Subtelomeric Disease Resistance Gene Cluster in Common Bean

The B4 resistance (R) gene cluster is one of the largest clusters known in common bean (Phaseolus vulgaris [Pv]). It is located in a peculiar genomic environment in the subtelomeric region of the short arm of chromosome 4, adjacent to two heterochromatic blocks (knobs). We sequenced 650 kb spanning this locus and annotated 97 genes, 26 of which correspond to Coiled-Coil-Nucleotide-Binding-Site-Leucine-Rich-Repeat (CNL). Conserved microsynteny was observed between the Pv B4 locus and corresponding regions of Medicago truncatula and Lotus japonicus in chromosomes Mt6 and Lj2, respectively. The notable exception was the CNL sequences, which were completely absent in these regions. The origin of the Pv B4-CNL sequences was investigated through phylogenetic analysis, which reveals that, in the Pv genome, paralogous CNL genes are shared among nonhomologous chromosomes (4 and 11). Together, our results suggest that Pv B4-CNL was derived from CNL sequences from another cluster, the Co-2 cluster, through an ectopic recombination event. Integration of the soybean (Glycine max) genome data enables us to date more precisely this event and also to infer that a single CNL moved from the Co-2 to the B4 cluster. Moreover, we identified a new 528-bp satellite repeat, referred to as khipu, specific to the Phaseolus genus, present both between B4-CNL sequences and in the two knobs identified at the B4 R gene cluster. The khipu repeat is present on most chromosomal termini, indicating the existence of frequent ectopic recombination events in Pv subtelomeric regions. Our results highlight the importance of ectopic recombination in R gene evolution.In the human genome, extensive cytogenetic and sequence analyses have revealed that subtelomeres are hot spots of interchromosomal recombination and segmental duplications (Linardopoulou et al., 2005). This peculiar dynamic activity of subtelomeres has been reported in such diverse organisms as yeast and the malaria parasite Plasmodium (Louis, 1995; Freitas-Junior et al., 2000, 2005). As expected for a plastic region of the genome subject to reshuffling through recombination events, subtelomeres exhibit unusually high levels of within-species structural and nucleotide polymorphism (Mefford and Trask, 2002). In plants, this plasticity of subtelomeres has not been identified in Arabidopsis (Arabidopsis thaliana; Heacock et al., 2004; Kuo et al., 2006) and, to our knowledge, has not yet been investigated at a large scale for other plant species with full genome sequences available. Regarding Arabidopsis, the apparent lack of high subtelomeric recombination may reflect its small and simple subtelomeres, mirroring its small genome size and relative paucity of repetitive sequences (Heacock et al., 2004; Kuo et al., 2006).Repetitive sequences, such as satellite DNA and retroelements, constitute an important fraction of every eukaryotic genome and therefore constitute the environment in which genes are expressed. Satellite DNA can be defined as highly reiterated noncoding DNA sequences, organized as long arrays of head-to-tail linked repeats of 150- to 180-bp or 300- to 360-bp monomers located in the constitutive heterochromatin (Plohl et al., 2008). Despite their ubiquity in eukaryotic genomes, little is known about the mechanisms that allow these elements to accumulate. Early hypotheses considered them to be nonfunctional “selfish” or “junk” DNA segments that increase or decrease their frequency without any advantage or disadvantage for an organism (Ohno, 1972; Orgel and Crick, 1980). However, identification of satellite DNA at structurally important parts of chromosomes, such as centromeres, has suggested functional roles of satellite DNA (Ma and Jackson, 2006; Kawabe and Charlesworth, 2007). Satellite DNA can also be localized in knobs, which are cytologically visible regions of highly condensed chromatin (heterochromatin) that are distinct from pericentromeric regions in pachytene chromosomes (Fransz et al., 2000; Gaut et al., 2007; Lamb et al., 2007).The survival of most organisms depends on the presence of specific genetic systems that maintain diversity in order to respond to changing environments. Plants, like animals, are continually challenged by a large array of pathogens. To perceive and counter pathogen attack, plants have evolved disease resistance (R) genes. The largest class of R genes encodes proteins containing a central Nucleotide-Binding Site (NBS) domain, a C-terminal Leucine-Rich Repeat (LRR) domain, and a variable N-terminal domain. These R proteins detect the presence of disease-causing bacteria, oomycetes, fungi, nematodes, insects, and viruses by sensing either specific pathogen effector molecules produced during the infection process or key molecules in the plant cell that may be attacked by pathogen effectors (Dangl and McDowell, 2006). The evolution of new R genes serves to counteract the evolution of novel virulence factors from the pathogens (McDowell and Simon, 2008). Among this prevalent class of R gene, two subclasses, corresponding to two ancient lineages (Bai et al., 2002; Meyers et al., 2003; Ameline-Torregrosa et al., 2008), have been identified based on the N-terminal domain of the R protein: the Coiled-Coil (CC)-NBS-LRR (CNL) and the Toll-Interleukin receptor (TIR)-NBS-LRR (TNL). Genome studies have demonstrated that NBS-LRR (NL) sequences are abundant in any plant genome. For example, annotation of the Arabidopsis, rice (Oryza sativa), poplar (Populus trichocarpa), Medicago truncatula (Mt), grape (Vitis vinifera), Lotus japonicus (Lj), and papaya (Carica papaya) genomes identified at least 149, 480, 317, 333, 233, 229, and 55 genes encoding NL proteins, respectively (Bai et al., 2002; Meyers et al., 2003; Zhou et al., 2004; Tuskan et al., 2006; Velasco et al., 2007; Ameline-Torregrosa et al., 2008; Kohler et al., 2008; Ming et al., 2008; Sato et al., 2008). NL sequences are often located at complex loci (Smith et al., 2004), as exemplified by Arabidopsis, where two-thirds of them are organized in tightly linked clusters (Meyers et al., 2003; Leister, 2004; McDowell and Simon, 2006). Evolution of NL sequences in the Arabidopsis genome has been investigated according to their phylogenetic positions and physical locations. Although tandem duplications explain the origin of a large fraction of NLs, it seems that ectopic recombination has also played a role in Arabidopsis NL evolution, since mixed clusters comprising evolutionarily distant NL exist. Ectopic recombination is also evident when phylogenetically close R genes are physically dispersed on different chromosomes (Leister, 2004; McDowell and Simon, 2006). These results confirm pioneer macrosynteny studies between related monocot species suggesting the existence of NL movement in plant genomes. Indeed, extensive loss of collinearity between NL sequences between rice and barley (Hordeum vulgare), which diverged 50 million years ago (Mya), has suggested rapid reorganization of NL sequences (Leister et al., 1998; Leister, 2004). However, our knowledge of the molecular evolution of R genes remains limited due to the still small number of complete plant genome sequences available to date. Detailed comparative study across taxa at different evolutionary distances is needed to see how R gene clusters evolve at various time scales.Legumes (Fabaceae) constitute the third largest family of flowering plants and represent the second most important family of agronomically important plants after Poaceae (Graham and Vance, 2003). As a result of recent sequencing efforts, legumes are one of the few plant families with extensive genome sequences in different species, since the soybean (Glycine max [Gm]) genome sequence is complete (http://www.phytozome.net/soybean.php) and both Mt and Lj genome sequences are nearly complete (Young et al., 2005; Sato et al., 2008). Consequently, the legume family is extremely well adapted for comparative phylogenomic approaches, in which phylogenetic inference is combined with structural genomic analyses (Ammiraju et al., 2008). Common bean (Phaseolus vulgaris [Pv]) is the most important grain legume for direct human consumption (Broughton et al., 2003). Pv is a selfing species and has a small diploid genome (2n = 22) of 588 Mb (Bennett and Leitch, 1995). Conservation of genome macrostructure (macrosynteny) has been reported between several legumes, including common bean and the two model legume species Mt and Lj genomes (Zhu et al., 2005; Hougaard et al., 2008). However, the extent of gene order conservation at the DNA sequence level has not yet been evaluated within orthologous chromosome segments between Pv and the two model legume species.In the genome of common bean, many disease R genes are clustered at complex loci located at the ends (rather than the centers) of linkage groups (LGs; Vallejos et al., 2006; Geffroy et al., 2008). For example, Colletotrichum lindemuthianum Co-2 R specificity maps at one end of LG B11 (Adam-Blondon et al., 1994). Molecular analysis has revealed that this locus consists of a tandem array of CNL sequences (Geffroy et al., 1998; Creusot et al., 1999). Another CNL-rich region has been identified at the end of LG B4 in the vicinity of R specificities and R quantitative trait loci against a large selection of pathogens, including C. lindemuthianum, Uromyces appendiculatus, and the bacterium Pseudomonas syringae (Geffroy et al., 1998, 1999; Miklas et al., 2006). Recently, fluorescence in situ hybridization (FISH) analysis revealed that this complex R cluster is located in the subtelomeric region of the short arm of chromosome 4 and includes two knobs (Geffroy et al., 2009). In a sequencing effort focused on CNL sequences, we have previously identified 17 CNL sequences of the B4 locus (referred to as B4-CNL) from Pv genotype BAT93 (Ferrier Cana et al., 2003, 2005; Geffroy et al., 2009). In the BAT93 genotype, these B4-CNL sequences are located on both sides of the subterminal knob (Geffroy et al., 2009).To investigate the organization and the evolutionary origin of the subtelomeric B4 R gene cluster, we have sequenced approximately 650 kb of the Pv B4 R gene cluster, revealing that, in genotype BAT93, CNL are spread out in four subclusters, separated by non-CNL-encoding genes. This Pv sequence was then compared gene by gene with the sequenced portions of the three sequenced legume genomes, Mt, Lj, and Gm. Conserved microsynteny (conservation of local gene repertoire, order, and orientation) was observed, except for the CNL sequences, which appear to be completely absent in the corresponding regions of Mt and Lj. In this study, by combining genomics, phylogenetic, and cytogenetic approaches, we provide evidence that ectopic recombination in subtelomeric regions between nonhomologous chromosomes (4 and 11), involving a single CNL, gave rise to the Pv B4 R gene cluster. Chromosomal distribution of a new satellite DNA tandem repeat, referred to as khipu, suggests that ectopic recombination events in subtelomeric regions of bean nonhomologous chromosomes are frequent. Our results highlight the importance of ectopic recombination as an important evolutionary mechanism for the evolution of disease resistance genes.     

CrusView: A Java-Based Visualization Platform for Comparative Genomics Analyses in Brassicaceae Species

In plants and animals, chromosomal breakage and fusion events based on conserved syntenic genomic blocks lead to conserved patterns of karyotype evolution among species of the same family. However, karyotype information has not been well utilized in genomic comparison studies. We present CrusView, a Java-based bioinformatic application utilizing Standard Widget Toolkit/Swing graphics libraries and a SQLite database for performing visualized analyses of comparative genomics data in Brassicaceae (crucifer) plants. Compared with similar software and databases, one of the unique features of CrusView is its integration of karyotype information when comparing two genomes. This feature allows users to perform karyotype-based genome assembly and karyotype-assisted genome synteny analyses with preset karyotype patterns of the Brassicaceae genomes. Additionally, CrusView is a local program, which gives its users high flexibility when analyzing unpublished genomes and allows users to upload self-defined genomic information so that they can visually study the associations between genome structural variations and genetic elements, including chromosomal rearrangements, genomic macrosynteny, gene families, high-frequency recombination sites, and tandem and segmental duplications between related species. This tool will greatly facilitate karyotype, chromosome, and genome evolution studies using visualized comparative genomics approaches in Brassicaceae species. CrusView is freely available at http://www.cmbb.arizona.edu/CrusView/.The Brassicaceae (crucifer) plant family contains more than 3,700 species, including the model plant organism Arabidopsis (Arabidopsis thaliana); economically important crop species, such as Brassica rapa and Brassica napus; and close relatives of Arabidopsis used in abiotic stress research, such as Eutrema salsugineum and Schrenkiella parvula. Because Brassicaceae plants have high scientific and economic importance, several whole-genome sequencing projects of the species in this family have been recently launched (http://www.brassica.info). Moreover, Brassicaceae is also a good system for population genomics. The 1001 Arabidopsis Genomes Project (http://www.1001genomes.org/) plans to generate complete genome sequences for 1,001 Arabidopsis strains to study the associations between genetic variation and phenotypic diversity. The Value-directed Evolutionary Genomics Initiative project aims to understand the genome evolution of Brassicaceae species by sequencing several close relatives of Arabidopsis, such as Arabidopsis lyrata and Capsella rubella. Recent advances in high-throughput sequencing technology have greatly expedited these whole-genome sequencing projects of versatile nonmodel organisms. Although increasingly longer reads can now be produced from high-throughput sequencing experiments, de novo assembler tools can only generate contig and/or scaffold sequences from high-throughput sequencing reads. These tools cannot generate complete chromosome sequences without genetic and/or physical maps that typically require years to create. This limitation makes chromosome-scale structural variation (i.e. translocation, inversion, deletion and insertion, and segmental and tandem duplication) and genomic macrosynteny analyses difficult to perform.In both plants and animals, genomes of species within the same family have evolved with conserved karyotype patterns due to the rearrangements of large chromosomal segments. Chromosomal karyotypes can be obtained from comparative chromosomal painting (CCP) experiments by performing in situ hybridization experiments on bacterial artificial chromosome sequences between related species. The genome of each Brassicaceae member is composed of 24 conserved genomic blocks that have been considered as the basic units of chromosomal rearrangement during genome evolution (Lysak et al., 2006). The sizes of these conserved blocks range from several to dozens of megabases. Currently, karyotypes profiled by CCP experiments in approximately 20 Brassicaceae species are available; such karyotypes include those from Arabidopsis (n = 5), Homungia alpine (n = 6), Eutrema spp. (n = 7), A. lyrata (n = 8), B. rapa (n = 10), and Polyctenium fremontii (n = 14). By utilizing the karyotype information in Brassicaceae, we have developed a tool, KGBassembler (for Karyotype-based Genome assembler for Brassicaceae), to finalize the assembly of chromosomes from scaffolds/contigs without relying on a genetic/physical map (Ma et al., 2012).Over the past 2 years, complete whole-genome sequences of several Brassicaceae species have been released, including the aforementioned A. lyrata, S. parvula, B. rapa, and E. salsugineum (Dassanayake et al., 2011; Hu et al., 2011; Wang et al., 2011; Wright and Agren, 2011; Wu et al., 2012; Yang et al., 2013). These genomic resources have opened a new era of comparative genomics in Brassicaceae to better understand the genomic evolution (Cheng et al., 2012). Numerous tools and databases are available for performing comparative genomics analysis in plants. CoGe is a comparative genomics analysis platform that is now a part of the iPlant Collaborative Project (Goff et al., 2011). The CoGe database currently includes nearly 2,000 genome sequences of approximately 1,500 organisms, allowing users to perform online visual analyses of genome synteny and duplication events (Tang and Lyons, 2012). PLAZA and Vista are also Web-based databases that provide comparative analysis services on the genomic data deposited in the databases (Frazer et al., 2004; Van Bel et al., 2012). Other stand-alone bioinformatic applications for comparative genomic analysis, such as Easyfig and genoPlotR, are commonly used to generate synteny plots of given genome segments at a scale ranging from a single gene to one chromosome (Guy et al., 2010; Sullivan et al., 2011).In this work, we present a Java-based bioinformatic application, CrusView, for performing visualized analyses of genome synteny and karyotype evolution in Brassicaceae species. CrusView features a user-friendly graphical user interface (GUI) implemented with Standard Widget Toolkit (SWT)/Swing graphics libraries and a SQLite database used to manage local genomic data. Compared with the most commonly used tools in comparative genomics, one of the unique features of CrusView is that available karyotype data of a Brassicaceae species are incorporated to facilitate karyotype-based chromosome assembly and analyses of chromosomal structural evolution. Compared with Web-based tools, the stand-alone CrusView tool was also designed to give users higher flexibility in analyzing currently unpublished genome data and integrating self-defined genomic information based on the users’ interests, such as gene families, gene duplications, chromosomal break points, Gene Ontology terms, and groups of orthologs/paralogs, with the genomic synteny maps. In addition, CrusView can generate images representing genomic synteny between two compared genomes in PNG/SVG/PDF high-resolution formats that are suitable for publication.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

Large-Scale Reverse Genetics in Arabidopsis: Case Studies from the Chloroplast 2010 Project

Traditionally, phenotype-driven forward genetic plant mutant studies have been among the most successful approaches to revealing the roles of genes and their products and elucidating biochemical, developmental, and signaling pathways. A limitation is that it is time consuming, and sometimes technically challenging, to discover the gene responsible for a phenotype by map-based cloning or discovery of the insertion element. Reverse genetics is also an excellent way to associate genes with phenotypes, although an absence of detectable phenotypes often results when screening a small number of mutants with a limited range of phenotypic assays. The Arabidopsis Chloroplast 2010 Project (www.plastid.msu.edu) seeks synergy between forward and reverse genetics by screening thousands of sequence-indexed Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutants for a diverse set of phenotypes. Results from this project are discussed that highlight the strengths and limitations of the approach. We describe the discovery of altered fatty acid desaturation phenotypes associated with mutants of At1g10310, previously described as a pterin aldehyde reductase in folate metabolism. Data are presented to show that growth, fatty acid, and chlorophyll fluorescence defects previously associated with antisense inhibition of synthesis of the family of acyl carrier proteins can be attributed to a single gene insertion in Acyl Carrier Protein4 (At4g25050). A variety of cautionary examples associated with the use of sequence-indexed T-DNA mutants are described, including the need to genotype all lines chosen for analysis (even when they number in the thousands) and the presence of tagged and untagged secondary mutations that can lead to the observed phenotypes.Decoding of the Arabidopsis (Arabidopsis thaliana) genome sequence earlier this decade (Arabidopsis Genome Initiative, 2000) provided the opportunity to determine the functions of approximately 27,000 protein-coding genes. One or more functions of a small percentage of genes are currently experimentally determined, typically from mutant or transgenic analysis or through biochemistry. However, roles for the vast majority of plant genes are either more or less accurately predicted by DNA sequence homology or unpredictable based upon DNA sequence (Arabidopsis Genome Initiative, 2000; Cho and Walbot, 2001; Rhee et al., 2008; for recent specific examples, see Gao et al., 2009; Schilmiller et al., 2009). Because of the uncertainty associated with homology-based function assessment, high-throughput approaches to gene function identification are needed to expand the universe of genes with experimental annotation.In contrast to organisms amenable to targeted gene replacement, such as bacteria, yeast, and mouse (Wendland, 2003; Wu et al., 2007; Adams and van der Weyden, 2008), obtaining a gene knockout is not as efficient in flowering plants. In Arabidopsis, the conventional way of creating a gene knockout is by insertional mutagenesis via Agrobacterium tumefaciens-mediated transformation (Krysan et al., 1999). Using this technique, a large piece of T-DNA is inserted into the genome in an untargeted manner (Alonso et al., 2003). If it lands within a coding or regulatory region, the T-DNA can influence the expression of the corresponding gene. While the probability of any single insertion element causing a mutation in a gene of interest is low, sequencing of hundreds of thousands of independent insertion sites has led to a collection of mutants in the majority of genes (http://signal.SALK.edu/tabout.html; Alonso et al., 2003).T-DNA mutants can be a valuable tool for forward genetics, in which hundreds or thousands of mutants are subjected to phenotypic assays (Feldmann, 1991; Kuromori et al., 2006), but reverse genetics is the most common way in which these mutant collections are utilized. Typically, a small number of candidate genes are tested for a role in a particular biological process by reducing or increasing gene expression and assaying one or more phenotypes (for review, see Page and Grossniklaus, 2002; Alonso and Ecker, 2006). The availability of a gene-indexed T-DNA mutant collection allows researchers to rapidly obtain mutant lines for their genes of interest (http://signal.SALK.edu/cgi-bin/tdnaexpress). The availability of a large collection of indexed mutant or RNA interference lines in other model organisms has facilitated large-scale reverse genetics studies (Piano et al., 2000; Giaever et al., 2002; Ho et al., 2009).In the course of a large reverse genetics project (The Chloroplast 2010 Project; http://www.plastid.msu.edu/), more than 3,500 T-DNA lines harboring insertions in nuclear genes, most of which were computationally predicted to encode chloroplast-targeted proteins, were subjected to a diverse set of phenotypic screens (Lu et al., 2008). In total, 85 phenotypic observations ranging from quantitative metabolite measurements to qualitative phenotypic observations are collected for each mutant line, and the data are stored in a relational database (http://bioinfo.bch.msu.edu/2010_LIMS). This approach seeks to take advantage of the best features of forward and reverse genetics by screening a large number of lines with mutations in known genes. Unlike conventional genetics screens, where plants are assayed for one or a small number of traits, this project surveys varied phenotypes.In this study, a variety of phenotypic variants were analyzed. In some cases, independent mutants of the same gene were found to have similar phenotypes, revealing new information about those genes. In other examples, a single homozygous mutant allele was found to have a detectable phenotype. These run the gamut from cases where secondary mutations are strongly implicated in causing the phenotype, to an example where an analogous maize (Zea mays) mutant is known to have a similar phenotype, to other instances where the causative mutation is yet to be identified. In several examples of secondary mutations, the phenotype was not due to a T-DNA insertion, reinforcing the idea that these untagged alleles are a cause for concern in conducting large-scale reverse genetics screens (Vitha et al., 2003; Adham et al., 2005; Zolman et al., 2008), while providing opportunities for gene function discovery by map-based cloning or whole genome sequence analysis.     

Processing Body and Stress Granule Assembly Occur by Independent and Differentially Regulated Pathways in Saccharomyces cerevisiae

A variety of ribonucleoprotein (RNP) granules form in eukaryotic cells to regulate the translation, decay, and localization of the encapsulated messenger RNA (mRNAs). The work here examined the assembly and function of two highly conserved RNP structures, the processing body (P body) and the stress granule, in the yeast Saccharomyces cerevisiae. These granules are induced by similar stress conditions and contain translationally repressed mRNAs and a partially overlapping set of protein constituents. However, despite these similarities, the data indicate that these RNP complexes are independently assembled and that this assembly is controlled by different signaling pathways. In particular, the cAMP-dependent protein kinase (PKA) was found to control P body formation under all conditions examined. In contrast, the assembly of stress granules was not affected by changes in either PKA or TORC1 signalling activity. Both of these RNP granules were also detected in stationary-phase cells, but each appears at a distinct time. P bodies were formed prior to stationary-phase arrest, and the data suggest that these foci are important for the long-term survival of these quiescent cells. Stress granules, on the other hand, were not assembled until after the cells had entered into the stationary phase of growth and their appearance could therefore serve as a specific marker for the entry into this quiescent state. In all, the results here provide a framework for understanding the assembly of these RNP complexes and suggest that these structures have distinct but important activities in quiescent cells.EUKARYOTIC cells contain a number of membrane-bound compartments that partition the cytoplasm into distinct functional units. Proteins that act in similar pathways are often localized to the same compartment whereas those with competing activities are sequestered within different environments. Interestingly, recent data suggest that particular proteins and RNAs are also concentrated in what can be thought of as nontraditional compartments that lack a boundary membrane. These ribonucleoprotein (RNP) complexes, or granules, are more dynamic in nature and are found in both the nucleus and the cytoplasm of the cell (Anderson and Kedersha 2006; Mao et al. 2011; Weber and Brangwynne 2012). The formation of these granules can be induced by a variety of cues, including an exposure to stress or specific developmental transitions. In some cases, the underlying reasons for this reorganization of protein and RNA are known. For example, the polar granules present in germ cells store maternal mRNAs that are translated following fertilization (Schisa et al. 2001; Leatherman and Jongens 2003). However, for most RNP granules, the physiological role of the larger aggregate-like structures remains unclear. Nonetheless, the prevalence and evolutionary conservation of these complexes suggests that they serve important functions in the cell.Two of the better-characterized cytoplasmic RNPs are the processing bodies (P bodies) and stress granules that form in response to a variety of stress conditions. These particles contain translationally repressed messenger RNA (mRNAs) and a partially overlapping set of protein constituents (Kedersha and Anderson 2002; Anderson and Kedersha 2009; Balagopal and Parker 2009). Since a number of factors important for protein translation are also found in stress granules, these structures have been suggested to be sites of mRNA storage (Yamasaki and Anderson 2008). In contrast, P bodies were originally identified as cytoplasmic foci containing proteins important for mRNA decay (Sheth and Parker 2003; Eulalio et al. 2007a). Although this initially led to speculation that these foci were sites of mRNA turnover, more recent studies have found that this decay proceeds normally in cells lacking the larger P body complexes (Stoecklin et al. 2006; Decker et al. 2007; Eulalio et al. 2007b). As a result, the biological functions associated with P body foci remain unclear. However, an intriguing possibility has been suggested by studies demonstrating that P bodies contain proteins that do not appear to have a direct role in mRNA decay. These proteins include the phosphatase, calcineurin, and the catalytic subunits of the cAMP-dependent protein kinase (PKA) (Tudisca et al. 2010; Kozubowski et al. 2011). P bodies may therefore carry out specific functions that are dictated by the particular proteins present within these cytoplasmic structures. These functions may vary depending upon the particular cell type and stress condition used to induce the foci.A significant body of work has linked the induction of both P bodies and stress granules to the inhibition of protein synthesis, but less is known about the mechanisms regulating the subsequent formation of the larger aggregate-like assemblies (Franks and Lykke-Andersen 2008). These latter structures appear to form by a self-assembly process that involves the prion-like domains present in a number of granule proteins (Gilks et al. 2004; Decker et al. 2007; Reijns et al. 2008). Some insight into the regulation of this latter process was provided by a recent study of the P bodies that form in response to glucose deprivation in Saccharomyces cerevisiae (Ramachandran et al. 2011). This work showed that the inactivation of the PKA signaling pathway was both a necessary and a sufficient condition for P body foci formation. PKA directly phosphorylates Pat1, a conserved core constituent of these RNP structures, and thereby disrupts Pat1 interactions with a number of P body components, including the RNA helicase Dhh1 (Ramachandran et al. 2011). In contrast, defects in other nutrient-sensing pathways, including those involving the TORC1 or Snf1 protein kinases, did not have a significant effect upon P body formation. This work also suggested that P body foci were important for the long-term survival of cells that had entered into the stationary phase of growth. In particular, mutants that were defective for foci formation lost viability more rapidly during this period of quiescence (Ramachandran et al. 2011). This latter result is of interest in light of other work indicating that as much as 20% of the yeast proteome might relocalize to cytoplasmic foci when cells enter into this G₀-like resting state (Narayanaswamy et al. 2009; Noree et al. 2010). Since this phenomenon might not be restricted to yeast (An et al. 2008; Noree et al. 2010), the concentration of material at discrete sites in the cytoplasm may be generally important for the biology of the quiescent cell. Determining the underlying reasons for this relocalization of protein will therefore be critical for a complete understanding of the physiology of the eukaryotic cell.In S. cerevisiae, PKA is an essential component of one of the key signaling pathways responsible for coordinating cell growth with nutrient availability (Bahn et al. 2007; Zaman et al. 2008). This pathway also involves the GTP-binding Ras proteins Ras1 and Ras2 and is thought to respond, either directly or indirectly, to the levels of glucose present within cells (Santangelo 2006; Slattery et al. 2008; Zaman et al. 2009). The active, GTP-bound forms of the Ras proteins interact with the adenylyl cyclase Cyr1 and stimulate the production of cAMP (Field et al. 1990; Suzuki et al. 1990). This cyclic nucleotide is then bound by Bcy1, the regulatory subunit of the PKA enzyme, leading to the subsequent release of the active catalytic subunits; the basal state of PKA is an inactive heterotetramer made up of two catalytic and two regulatory subunits (Uno et al. 1982; Toda et al. 1987a; Taylor et al. 2008). These catalytic subunits are then free to phosphorylate their respective targets and thereby influence cell growth (Budovskaya et al. 2005). The existing genetic data suggest that this Ras/PKA pathway might play an important role in regulating the entry into stationary phase. For example, mutants that inactivate this pathway result in a growth arrest that resembles stationary phase (Iida and Yahara 1984; Schneper et al. 2004). Conversely, cells with constitutively elevated levels of PKA activity fail to arrest normally in stationary phase when nutrients are limiting (Broek et al. 1985; Broach 1991). The above results with Pat1 suggest that the PKA-mediated control of P body formation is one important component of this regulation of stationary-phase biology.In this study, we examined the regulation of P body and stress granule assembly in response to a variety of environmental cues, including several that can induce both of these RNP foci. This work demonstrated that the PKA pathway has a general role in the regulation of P body foci formation as mutants with constitutive PKA signaling were defective for P body assembly in all conditions tested. In contrast, stress granule formation was not influenced by changes in either PKA or TORC1 signalling activity. The results here also demonstrate that both types of RNP foci are present in stationary-phase cells and provide further support for a role for P bodies in the long-term survival of these quiescent cells. Finally, we show that P bodies and stress granules form at different times during batch culture growth and that stress granules in particular appear only after cells enter into stationary phase. Therefore, stress granule formation could serve as a useful marker for cell entry into this quiescent state. In all, this work indicates that P bodies and stress granules form independently of one another and that each assembly pathway is regulated by distinct signaling mechanisms.