Author: biota

Importance of the alphaC-helix in the cyclic nucleotide binding domain for the stable channel regulation and function of cyclic nucleotide gated ion channels in Arabidopsis

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Chin K, Moeder W, Abdel-Hamid H, Shahinas D, Gupta D, Yoshioka K

J. Exp. Bot. 2010 May;61(9):2383-93

PubMed PMID: 20378667

Abstract

The involvement of cyclic nucleotide gated ion channels (CNGCs) in the signal transduction of animal light and odorant perception is well documented. Although plant CNGCs have recently been revealed to mediate multiple stress responses and developmental pathways, studies that aim to elucidate their structural and regulatory properties are still very much in their infancy. The structure-function relationship of plant CNGCs was investigated here by using the chimeric Arabidopsis AtCNGC11/12 gene that induces multiple defence responses in the Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22) for the identification of functionally essential residues. A genetic screen for mutants that suppress cpr22-conferred phenotypes identified over 20 novel mutant alleles in AtCNGC11/12. One of these mutants, suppressor S58 possesses a single amino acid substitution, arginine 557 to cysteine, in the alphaC-helix of the cyclic nucleotide-binding domain (CNBD). The suppressor S58 lost all cpr22 related phenotypes, such as spontaneous cell death formation under ambient temperature conditions. However, these phenotypes were recovered at 16 degrees C suggesting that the stability of channel function is affected by temperature. In silico modelling and site-directed mutagenesis analyses suggest that arginine 557 in the alphaC-helix of the CNBD is important for channel regulation, but not for basic function. Furthermore, another suppressor mutant, S136 that lacks the entire alphaC-helix due to a premature stop codon, lost channel function completely. Our data presented here indicate that the alphaC-helix is functionally important in plant CNGCs.

Allele-specific virulence attenuation of the Pseudomonas syringae HopZ1a type III effector via the Arabidopsis ZAR1 resistance protein

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Lewis JD, Wu R, Guttman DS, Desveaux D

PLoS Genet. 2010 Apr;6(4):e1000894

PubMed PMID: 20368970

Abstract

Plant resistance (R) proteins provide a robust surveillance system to defend against potential pathogens. Despite their importance in plant innate immunity, relatively few of the approximately 170 R proteins in Arabidopsis have well-characterized resistance specificity. In order to identify the R protein responsible for recognition of the Pseudomonas syringae type III secreted effector (T3SE) HopZ1a, we assembled an Arabidopsis R gene T-DNA Insertion Collection (ARTIC) from publicly available Arabidopsis thaliana insertion lines and screened it for plants lacking HopZ1a-induced immunity. This reverse genetic screen revealed that the Arabidopsis R protein HOPZ-activated resistance 1 (ZAR1; At3g50950) is required for recognition of HopZ1a in Arabidopsis. ZAR1 belongs to the coiled-coil (CC) class of nucleotide binding site and leucine-rich repeat (NBS-LRR) containing R proteins; however, the ZAR1 CC domain phylogenetically clusters in a clade distinct from other related Arabidopsis R proteins. ZAR1-mediated immunity is independent of several genes required by other R protein signaling pathways, including NDR1 and RAR1, suggesting that ZAR1 possesses distinct signaling requirements. The closely-related T3SE protein, HopZ1b, is still recognized by zar1 Arabidopsis plants indicating that Arabidopsis has evolved at least two independent R proteins to recognize the HopZ T3SE family. Also, in Arabidopsis zar1 plants HopZ1a promotes P. syringae growth indicative of an ancestral virulence function for this T3SE prior to the evolution of recognition by the host resistance protein ZAR1. Our results demonstrate that the Arabidopsis resistance protein ZAR1 confers allele-specific recognition and virulence attenuation of the Pseudomonas syringae T3SE protein HopZ1a.

Evolution of characterized phosphorylation sites in budding yeast

Posted on by biota in Publications

Ba AN, Moses AM

Mol. Biol. Evol. 2010 Sep;27(9):2027-37

PubMed PMID: 20368267

Abstract

Phosphorylation is one of the most studied and important regulatory mechanisms that modulate protein function in eukaryotic cells. Recently, several studies have investigated the evolution of phosphorylation sites identified by high-throughput methods. These studies have revealed varying degrees of evidence for constraint and plasticity, and therefore, there is currently no consensus as to the evolutionary properties of this important regulatory mechanism. Here, we present a study of high-confidence annotated sites from budding yeast and show that these sites are significantly constrained compared with their flanking region in closely related species. We show that this property does not change in structured or unstructured regions. We investigate the birth, death and compensation rates of the phosphorylation sites and test if sites are more likely to be gained or lost in proteins with greater numbers of sites. Finally, we also show that this evolutionary conservation can yield significant improvement for kinase target predictions when the kinase recognition motif is known, and can be used to infer the recognition motif when a set of targets is known. Our analysis indicates that phosphorylation sites are under selective constraint, consistent with their functional importance. We also find that a small fraction of phosphorylation sites turnover during evolution, which may be an important process underlying the evolution of regulatory networks.

Molecular evolution inferences from the C. elegans genome

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Cutter AD

WormBook 2010;:1-14

PubMed PMID: 20213859

Abstract

An understanding of evolution at the molecular level requires the simultaneous consideration of the 5 fundamental evolutionary processes: mutation, recombination, natural selection, genetic drift, and population dynamic effects. Experimental, comparative genomic, and population genetic work in C. elegans has greatly expanded our understanding of these core processes, as well as of C. elegans biology. This chapter presents a brief overview of some of the most salient features of molecular evolution elucidated by the C. elegans system.

Genome-wide evidence for efficient positive and purifying selection in Capsella grandiflora, a plant species with a large effective population size

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Slotte T, Foxe JP, Hazzouri KM, Wright SI

Mol. Biol. Evol. 2010 Aug;27(8):1813-21

PubMed PMID: 20194429

Abstract

Recent studies comparing genome-wide polymorphism and divergence in Drosophila have found evidence for a surprisingly high proportion of adaptive amino acid fixations, but results for other taxa are mixed. In particular, few studies have found convincing evidence for adaptive amino acid substitution in plants. To assess the generality of this finding, we have sequenced 257 loci in the outcrossing crucifer Capsella grandiflora, which has a large effective population size and low population structure. Using a new method that jointly infers selective and demographic effects, we estimate that 40% of amino acid substitutions were fixed by positive selection in this species, and we also infer a low proportion of slightly deleterious amino acid mutations. We contrast these estimates with those for a similar data set from the closely related Arabidopsis thaliana and find significantly higher rates of adaptive evolution and fewer nearly neutral mutations in C. grandiflora. In agreement with results for other taxa, genes involved in reproduction show the strongest evidence for positive selection in C. grandiflora. Taken together, these results imply that both positive and purifying selection are more effective in C. grandiflora than in A. thaliana, consistent with the contrasting demographic history and effective population sizes of these species.

Pollen-pistil interactions regulating successful fertilization in the Brassicaceae

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Chapman LA, Goring DR

J. Exp. Bot. 2010 Apr;61(7):1987-99

PubMed PMID: 20181663

Abstract

In the Brassicaceae, the acceptance of compatible pollen and the rejection of self-incompatible pollen by the pistil involves complex molecular communication systems between the pollen grain and the female reproductive structures. Preference towards species related-pollen combined with self-recognition systems, function to select the most desirable pollen; and thus, increase the plant’s chances for the maximum number of successful fertilizations and vigorous offspring. The Brassicaceae is an ideal group for studying pollen-pistil interactions as this family includes a diverse group of agriculturally relevant crops as well as several excellent model organisms for studying both compatible and self-incompatible pollinations. This review will describe the cellular systems in the pistil that guide the post-pollination events, from pollen capture on the stigmatic papillae to pollen tube guidance to the ovule, with the final release of the sperm cells to effect fertilization. The interplay of other recognition systems, such as the self-incompatibility response and interspecific interactions, on regulating post-pollination events and selecting for compatible pollen-pistil interactions will also be explored.

The lesion-mimic mutant cpr22 shows alterations in abscisic acid signaling and abscisic acid insensitivity in a salicylic acid-dependent manner

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Mosher S, Moeder W, Nishimura N, Jikumaru Y, Joo SH, Urquhart W, Klessig DF, Kim SK, Nambara E, Yoshioka K

Plant Physiol. 2010 Apr;152(4):1901-13

PubMed PMID: 20164209

Abstract

A number of Arabidopsis (Arabidopsis thaliana) lesion-mimic mutants exhibit alterations in both abiotic stress responses and pathogen resistance. One of these mutants, constitutive expresser of PR genes22 (cpr22), which has a mutation in two cyclic nucleotide-gated ion channels, is a typical lesion-mimic mutant exhibiting elevated levels of salicylic acid (SA), spontaneous cell death, constitutive expression of defense-related genes, and enhanced resistance to various pathogens; the majority of its phenotypes are SA dependent. These defense responses in cpr22 are suppressed under high-humidity conditions and enhanced by low humidity. After shifting plants from high to low humidity, the cpr22 mutant, but not the wild type, showed a rapid increase in SA levels followed by an increase in abscisic acid (ABA) levels. Concomitantly, genes for ABA metabolism were up-regulated in the mutant. The expression of a subset of ABA-inducible genes, such as RD29A and KIN1/2, was down-regulated, but that of other genes, like ABI1 and HAB1, was up-regulated in cpr22 after the humidity shift. cpr22 showed reduced responsiveness to ABA not only in abiotic stress responses but also in germination and stomatal closure. Double mutant analysis with nahG plants that degrade SA indicated that these alterations in ABA signaling were attributable to elevated SA levels. Furthermore, cpr22 displayed suppressed drought responses by long-term drought stress. Taken together, these results suggest an effect of SA on ABA signaling/abiotic stress responses during the activation of defense responses in cpr22.

The type III effector HopF2Pto targets Arabidopsis RIN4 protein to promote Pseudomonas syringae virulence

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Wilton M, Subramaniam R, Elmore J, Felsensteiner C, Coaker G, Desveaux D

Proc. Natl. Acad. Sci. U.S.A. 2010 Feb;107(5):2349-54

PubMed PMID: 20133879

Abstract

Plant immunity can be induced by two major classes of pathogen-associated molecules. Pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) are conserved molecular components of microbes that serve as “non-self” features to induce PAMP-triggered immunity (PTI). Pathogen effector proteins used to promote virulence can also be recognized as “non-self” features or induce a “modified-self” state that can induce effector-triggered immunity (ETI). The Arabidopsis protein RIN4 plays an important role in both branches of plant immunity. Three unrelated type III secretion effector (TTSE) proteins from the phytopathogen Pseudomonas syringae, AvrRpm1, AvrRpt2, and AvrB, target RIN4, resulting in ETI that effectively restricts pathogen growth. However, no pathogenic advantage has been demonstrated for RIN4 manipulation by these TTSEs. Here, we show that the TTSE HopF2(Pto) also targets Arabidopsis RIN4. Transgenic plants conditionally expressing HopF2(Pto) were compromised for AvrRpt2-induced RIN4 modification and associated ETI. HopF2(Pto) interfered with AvrRpt2-induced RIN4 modification in vitro but not with AvrRpt2 activation, suggestive of RIN4 targeting by HopF2(Pto). In support of this hypothesis, HopF2 (Pto) interacted with RIN4 in vitro and in vivo. Unlike AvrRpm1, AvrRpt2, and AvrB, HopF2(Pto) did not induce ETI and instead promoted P. syringae growth in Arabidopsis. This virulence activity was not observed in plants genetically lacking RIN4. These data provide evidence that RIN4 is a major virulence target of HopF2(Pto) and that a pathogenic advantage can be conveyed by TTSEs that target RIN4.

PAMDB, a multilocus sequence typing and analysis database and website for plant-associated microbes

Posted on by biota in Publications

Almeida NF, Yan S, Cai R, Clarke CR, Morris CE, Schaad NW, Schuenzel EL, Lacy GH, Sun X, Jones JB, Castillo JA, Bull CT, Leman S, Guttman DS, Setubal JC, Vinatzer BA

Phytopathology 2010 Mar;100(3):208-15

PubMed PMID: 20128693

Abstract

Although there are adequate DNA sequence differences among plant-associated and plant-pathogenic bacteria to facilitate molecular approaches for their identification, identification at a taxonomic level that is predictive of their phenotype is a challenge. The problem is the absence of a taxonomy that describes genetic variation at a biologically relevant resolution and of a database containing reference strains for comparison. Moreover, molecular evolution, population genetics, ecology, and epidemiology of many plant-pathogenic and plant-associated bacteria are still poorly understood. To address these challenges, a database with web interface was specifically designed for plant-associated and plant-pathogenic microorganisms. The Plant-Associated Microbes Database (PAMDB) comprises, thus far, data from multilocus sequence typing and analysis (MLST/MLSA) studies of Acidovorax citrulli, Pseudomonas syringae, Ralstonia solanacearum, and Xanthomonas spp. Using data deposited in PAMDB, a robust phylogeny of Xanthomonas axonopodis and related bacteria has been inferred, and the diversity existing in the Xanthomonas genus and in described Xanthomonas spp. has been compared with the diversity in P. syringae and R. solanacearum. Moreover, we show how PAMDB makes it easy to distinguish between different pathogens that cause almost identical diseases. The scalable design of PAMDB will make it easy to add more plant pathogens in the future.

PAMDB, a multilocus sequence typing and analysis database and website for plant-associated microbes

Posted on by biota in Publications

Almeida NF, Yan S, Cai R, Clarke CR, Morris CE, Schaad NW, Schuenzel EL, Lacy GH, Sun X, Jones JB, Castillo JA, Bull CT, Leman S, Guttman DS, Setubal JC, Vinatzer BA

Phytopathology 2010 Mar;100(3):208-15

PubMed PMID: 20128693

Abstract

Although there are adequate DNA sequence differences among plant-associated and plant-pathogenic bacteria to facilitate molecular approaches for their identification, identification at a taxonomic level that is predictive of their phenotype is a challenge. The problem is the absence of a taxonomy that describes genetic variation at a biologically relevant resolution and of a database containing reference strains for comparison. Moreover, molecular evolution, population genetics, ecology, and epidemiology of many plant-pathogenic and plant-associated bacteria are still poorly understood. To address these challenges, a database with web interface was specifically designed for plant-associated and plant-pathogenic microorganisms. The Plant-Associated Microbes Database (PAMDB) comprises, thus far, data from multilocus sequence typing and analysis (MLST/MLSA) studies of Acidovorax citrulli, Pseudomonas syringae, Ralstonia solanacearum, and Xanthomonas spp. Using data deposited in PAMDB, a robust phylogeny of Xanthomonas axonopodis and related bacteria has been inferred, and the diversity existing in the Xanthomonas genus and in described Xanthomonas spp. has been compared with the diversity in P. syringae and R. solanacearum. Moreover, we show how PAMDB makes it easy to distinguish between different pathogens that cause almost identical diseases. The scalable design of PAMDB will make it easy to add more plant pathogens in the future.