Category: Publications

Interactions between the S-domain receptor kinases and AtPUB-ARM E3 ubiquitin ligases suggest a conserved signaling pathway in Arabidopsis

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Samuel MA, Mudgil Y, Salt JN, Delmas F, Ramachandran S, Chilelli A, Goring DR

Plant Physiol. 2008 Aug;147(4):2084-95

PubMed PMID: 18552232

Abstract

The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses.

Patterns of molecular evolution in Caenorhabditis preclude ancient origins of selfing

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Cutter AD, Wasmuth JD, Washington NL

Genetics 2008 Apr;178(4):2093-104

PubMed PMID: 18430935

Abstract

The evolution of self-fertilization can mediate pronounced changes in genomes as a by-product of a drastic reduction in effective population size and the concomitant accumulation of slightly deleterious mutations by genetic drift. In the nematode genus Caenorhabditis, a highly selfing lifestyle has evolved twice independently, thus permitting an opportunity to test for the effects of mode of reproduction on patterns of molecular evolution on a genomic scale. Here we contrast rates of nucleotide substitution and codon usage bias among thousands of orthologous groups of genes in six species of Caenorhabditis, including the classic model organism Caenorhabditis elegans. Despite evidence that weak selection on synonymous codon usage is pervasive in the history of all species in this genus, we find little difference among species in the patterns of codon usage bias and in replacement-site substitution. Applying a model of relaxed selection on codon usage to the C. elegans and C. briggsae lineages suggests that self-fertilization is unlikely to have evolved more than approximately 4 million years ago, which is less than a quarter of the time since they shared a common ancestor with outcrossing species. We conclude that the profound changes in mating behavior, physiology, and developmental mechanisms that accompanied the transition from an obligately outcrossing to a primarily selfing mode of reproduction evolved in the not-too-distant past.

Endocrine and reproductive differences and genetic divergence in two populations of the cockroach Diploptera punctata

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Lenkic LE, Wolfe JM, Chang BS, Tobe SS

J. Insect Physiol. 2008 Jun;54(6):931-8

PubMed PMID: 18406421

Abstract

The viviparous cockroach, Diploptera punctata, has been a valuable model organism for studies of the regulation of reproduction by juvenile hormone (JH) in insects. As a result of its truly viviparous mode of reproduction, precise regulation of JH biosynthesis and reproduction is required for production of offspring, providing a model system for the study of the relationship between JH production and oocyte growth and maturation. Most studies to date have focused on individuals isolated from a Hawaiian population of this species. A new population of this cockroach was found in Nakorn Pathom, Thailand, which demonstrated striking differences in cuticle pigmentation and mating behaviours, suggesting possible physiological differences between the two populations. To better characterize these differences, rates of JH release and oocyte growth were measured during the first gonadotrophic cycle. The Thai population was found to show significantly earlier increases in the rate of JH release, and oocyte development as compared with the Hawaiian population. Breeding experiments to determine the degree of interfertility between the two populations demonstrated greatly reduced fertility in crosses between the two populations. Additionally, levels of genetic divergence between the two populations estimated by sequencing a fragment of the mitochondrial 16S rRNA gene were surprisingly high. The significant differences in physiology and mating behaviours, combined with the reduced interfertility and high levels of sequence divergence, suggest that these two populations of D. punctata are quite distinct, and may even be in the process of speciation. Moreover, these studies have important implications for the study of JH function in the reproductive cycle of insects, as differences in timing of rates of JH biosynthesis may suggest a process of heterochrony in reproduction between the two populations.

Elucidating the germination transcriptional program using small molecules

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Bassel GW, Fung P, Chow TF, Foong JA, Provart NJ, Cutler SR

Plant Physiol. 2008 May;147(1):143-55

PubMed PMID: 18359847

Abstract

The transition from seed to seedling is mediated by germination, a complex process that starts with imbibition and completes with radicle emergence. To gain insight into the transcriptional program mediating germination, previous studies have compared the transcript profiles of dry, dormant, and germinating after-ripened Arabidopsis (Arabidopsis thaliana) seeds. While informative, these approaches did not distinguish the transcriptional responses due to imbibition, shifts in metabolism, or breaking of dormancy from those triggered by the initiation of germination. In this study, three mechanistically distinct small molecules that inhibit Arabidopsis seed germination (methotrexate, 2, 4-dinitrophenol, and cycloheximide) were identified using a small-molecule screen and used to probe the germination transcriptome. Germination-responsive transcripts were defined as those with significantly altered transcript abundance across all inhibitory treatments with respect to control germinating seeds, using data from ATH1 microarrays. This analysis identified numerous germination regulators as germination responsive, including the DELLA proteins GAI, RGA, and RGL3, the abscisic acid-insensitive proteins ABI4, ABI5, ABI8, and FRY1, and the gibberellin receptor GID1A. To help visualize these and other publicly available seed microarray data, we designed a seed mRNA expression browser using the electronic Fluorescent Pictograph platform. An overall decrease in gene expression and a 5-fold greater number of transcripts identified as statistically down-regulated in drug-inhibited seeds point to a role for mRNA degradation or turnover during seed germination. The genes identified in our study as responsive to germination define potential uncharacterized regulators of this process and provide a refined transcriptional signature for germinating Arabidopsis seeds.

New and sensitive assay for determining Pseudomonas aeruginosa metallo-beta-lactamase resistance to imipenem

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Tan J, Pitout JD, Guttman DS

J. Clin. Microbiol. 2008 May;46(5):1870-2

PubMed PMID: 18337388

Abstract

We present an imipenem lysate metallo-beta-lactamase (MBL) functional assay. This assay eliminates false-positive results due to the bactericidal effects of EDTA, can be performed with inexpensive reagents available in most laboratories, and is as accurate as the MBL Etest. It is appropriate for both high-accuracy screens and laboratories in developing countries with limited resources.

The HopZ family of Pseudomonas syringae type III effectors require myristoylation for virulence and avirulence functions in Arabidopsis thaliana

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Lewis JD, Abada W, Ma W, Guttman DS, Desveaux D

J. Bacteriol. 2008 Apr;190(8):2880-91

PubMed PMID: 18263728

Abstract

Pseudomonas syringae utilizes the type III secretion system to translocate effector proteins into plant cells, where they can contribute to the pathogen’s ability to infect and cause disease. Recognition of these effectors by resistance proteins induces defense responses that typically include a programmed cell death reaction called the hypersensitive response. The YopJ/HopZ family of type III effector proteins is a common family of effector proteins found in animal- and plant-pathogenic bacteria. The HopZ family in P. syringae includes HopZ1a(PsyA2), HopZ1b(PgyUnB647), HopZ1c(PmaE54326), HopZ2(Ppi895A) and HopZ3(PsyB728a). HopZ1a is predicted to be most similar to the ancestral hopZ allele and causes a hypersensitive response in multiple plant species, including Arabidopsis thaliana. Therefore, it has been proposed that host defense responses have driven the diversification of this effector family. In this study, we further characterized the hypersensitive response induced by HopZ1a and demonstrated that it is not dependent on known resistance genes. Further, we identified a novel virulence function for HopZ2 that requires the catalytic cysteine demonstrated to be required for protease activity. Sequence analysis of the HopZ family revealed the presence of a predicted myristoylation sequence in all members except HopZ3. We demonstrated that the myristoylation site is required for membrane localization of this effector family and contributes to the virulence and avirulence activities of HopZ2 and HopZ1a, respectively. This paper provides insight into the selective pressures driving virulence protein evolution by describing a detailed functional characterization of the diverse HopZ family of type III effectors with the model plant Arabidopsis.

A high-throughput chemical screen for resistance to Pseudomonas syringae in Arabidopsis

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Schreiber K, Ckurshumova W, Peek J, Desveaux D

Plant J. 2008 May;54(3):522-31

PubMed PMID: 18248597

Abstract

The study of plant pathogenesis and the development of effective treatments to protect plants from diseases could be greatly facilitated by a high-throughput pathosystem to evaluate small-molecule libraries for inhibitors of pathogen virulence. The interaction between the Gram-negative bacterium Pseudomonas syringae and Arabidopsis thaliana is a model for plant pathogenesis. However, a robust high-throughput assay to score the outcome of this interaction is currently lacking. We demonstrate that Arabidopsis seedlings incubated with P. syringae in liquid culture display a macroscopically visible ‘bleaching’ symptom within 5 days of infection. Bleaching is associated with a loss of chlorophyll from cotyledonary tissues, and is correlated with bacterial virulence. Gene-for-gene resistance is absent in the liquid environment, possibly because of the suppression of the hypersensitive response under these conditions. Importantly, bleaching can be prevented by treating seedlings with known inducers of plant defence, such as salicylic acid (SA) or a basal defence-inducing peptide of bacterial flagellin (flg22) prior to inoculation. Based on these observations, we have devised a high-throughput liquid assay using standard 96-well plates to investigate the P. syringae-Arabidopsis interaction. An initial screen of small molecules active on Arabidopsis revealed a family of sulfanilamide compounds that afford protection against the bleaching symptom. The most active compound, sulfamethoxazole, also reduced in planta bacterial growth when applied to mature soil-grown plants. The whole-organism liquid assay provides a novel approach to probe chemical libraries in a high-throughput manner for compounds that reduce bacterial virulence in plants.

Multilocus patterns of polymorphism and selection across the X chromosome of Caenorhabditis remanei

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Cutter AD

Genetics 2008 Mar;178(3):1661-72

PubMed PMID: 18245859

Abstract

Natural selection and neutral processes such as demography, mutation, and gene conversion all contribute to patterns of polymorphism within genomes. Identifying the relative importance of these varied components in evolution provides the principal challenge for population genetics. To address this issue in the nematode Caenorhabditis remanei, I sampled nucleotide polymorphism at 40 loci across the X chromosome. The site-frequency spectrum for these loci provides no evidence for population size change, and one locus presents a candidate for linkage to a target of balancing selection. Selection for codon usage bias leads to the non-neutrality of synonymous sites, and despite its weak magnitude of effect (N(e)s approximately 0.1), is responsible for profound patterns of diversity and divergence in the C. remanei genome. Although gene conversion is evident for many loci, biased gene conversion is not identified as a significant evolutionary process in this sample. No consistent association is observed between synonymous-site diversity and linkage-disequilibrium-based estimators of the population recombination parameter, despite theoretical predictions about background selection or widespread genetic hitchhiking, but genetic map-based estimates of recombination are needed to rigorously test for a diversity-recombination relationship. Coalescent simulations also illustrate how a spurious correlation between diversity and linkage-disequilibrium-based estimators of recombination can occur, due in part to the presence of unbiased gene conversion. These results illustrate the influence that subtle natural selection can exert on polymorphism and divergence, in the form of codon usage bias, and demonstrate the potential of C. remanei for detecting natural selection from genomic scans of polymorphism.

Divergence times in Caenorhabditis and Drosophila inferred from direct estimates of the neutral mutation rate

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Cutter AD

Mol. Biol. Evol. 2008 Apr;25(4):778-86

PubMed PMID: 18234705

Abstract

Accurate inference of the dates of common ancestry among species forms a central problem in understanding the evolutionary history of organisms. Molecular estimates of divergence time rely on the molecular evolutionary prediction that neutral mutations and substitutions occur at the same constant rate in genomes of related species. This underlies the notion of a molecular clock. Most implementations of this idea depend on paleontological calibration to infer dates of common ancestry, but taxa with poor fossil records must rely on external, potentially inappropriate, calibration with distantly related species. The classic biological models Caenorhabditis and Drosophila are examples of such problem taxa. Here, I illustrate internal calibration in these groups with direct estimates of the mutation rate from contemporary populations that are corrected for interfering effects of selection on the assumption of neutrality of substitutions. Divergence times are inferred among 6 species each of Caenorhabditis and Drosophila, based on thousands of orthologous groups of genes. I propose that the 2 closest known species of Caenorhabditis shared a common ancestor <24 MYA (Caenorhabditis briggsae and Caenorhabditis sp. 5) and that Caenorhabditis elegans diverged from its closest known relatives <30 MYA, assuming that these species pass through at least 6 generations per year; these estimates are much more recent than reported previously with molecular clock calibrations from non-nematode phyla. Dates inferred for the common ancestor of Drosophila melanogaster and Drosophila simulans are roughly concordant with previous studies. These revised dates have important implications for rates of genome evolution and the origin of self-fertilization in Caenorhabditis.

Evolution of the type III secretion system and its effectors in plant-microbe interactions

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McCann HC, Guttman DS

New Phytol. 2008;177(1):33-47

PubMed PMID: 18078471

Abstract

Many bacterial plant pathogens require the type III secretion system (T3SS) and its effector proteins (T3SEs) to invade and extract nutrients from their hosts successfully. While the molecular function of this system is being studied intensively, we know comparatively little about the evolutionary and ecological pressures governing its fate over time, and even less about the detailed mechanisms underlying and driving complex T3SS-mediated coevolutionary dynamics. In this review we summarize our current understanding of how host-pathogen interactions evolve, with a particular focus on the T3SS of bacterial plant pathogens. We explore the evolutionary origins of the T3SS relative to the closely related flagellar system, and investigate the evolutionary pressures on this secretion and translocation apparatus. We examine the evolutionary forces acting on T3SEs, and compare the support for vertical descent with modification of these virulence-associated systems (pathoadaptation) vs horizontal gene transfer. We address the evolutionary origins of T3SEs from the perspective of both the evolutionary mechanisms that generate new effectors, and the mobile elements that may be the source of novel genetic material. Finally, we propose a number of questions raised by these studies, which may serve to guide our thinking about these complex processes.