Category: Publications

Hult EF, Tobe SS, Chang BS

PLoS ONE 2011;6(8):e23416

PubMed PMID: 21901121

Abstract

Ultraspiracle protein/retinoid X receptor (USP/RXR) is a nuclear receptor and transcription factor which is an essential component of a heterodimeric receptor complex with the ecdysone receptor (EcR). In insects this complex binds ecdysteroids and plays an important role in the regulation of growth, development, metamorphosis and reproduction. In some holometabolous insects, including Lepidoptera and Diptera, USP/RXR is thought to have experienced several important shifts in function. These include the acquisition of novel ligand-binding properties and an expanded dimerization interface with EcR. In light of these recent hypotheses, we implemented codon-based likelihood methods to investigate if the proposed shifts in function are reflected in changes in site-specific evolutionary rates across functional and structural motifs in insect USP/RXR sequences, and if there is any evidence for positive selection at functionally important sites. Our results reveal evidence of positive selection acting on sites within the loop connecting helices H1 and H3, the ligand-binding pocket, and the dimer interface in the holometabolous lineage leading to the Lepidoptera/Diptera/Trichoptera. Similar analyses conducted using EcR sequences did not indicate positive selection. However, analyses allowing for variation across sites demonstrated elevated non-synonymous/synonymous rate ratios (d(N)/d(S)), suggesting relaxed constraint, within the dimerization interface of both USP/RXR and EcR as well as within the coactivator binding groove and helix H12 of USP/RXR. Since the above methods are based on the assumption that d(S) is constant among sites, we also used more recent models which relax this assumption and obtained results consistent with traditional random-sites models. Overall our findings support the evolution of novel function in USP/RXR of more derived holometabolous insects, and are consistent with shifts in structure and function which may have increased USP/RXR reliance on EcR for cofactor recruitment. Moreover, these findings raise important questions regarding hypotheses which suggest the independent activation of USP/RXR by its own ligand.

Cai R, Lewis J, Yan S, Liu H, Clarke CR, Campanile F, Almeida NF, Studholme DJ, Lindeberg M, Schneider D, Zaccardelli M, Setubal JC, Morales-Lizcano NP, Bernal A, Coaker G, Baker C, Bender CL, Leman S, Vinatzer BA

PLoS Pathog. 2011 Aug;7(8):e1002130

PubMed PMID: 21901088

Abstract

Recently, genome sequencing of many isolates of genetically monomorphic bacterial human pathogens has given new insights into pathogen microevolution and phylogeography. Here, we report a genome-based micro-evolutionary study of a bacterial plant pathogen, Pseudomonas syringae pv. tomato. Only 267 mutations were identified between five sequenced isolates in 3,543,009 nt of analyzed genome sequence, which suggests a recent evolutionary origin of this pathogen. Further analysis with genome-derived markers of 89 world-wide isolates showed that several genotypes exist in North America and in Europe indicating frequent pathogen movement between these world regions. Genome-derived markers and molecular analyses of key pathogen loci important for virulence and motility both suggest ongoing adaptation to the tomato host. A mutational hotspot was found in the type III-secreted effector gene hopM1. These mutations abolish the cell death triggering activity of the full-length protein indicating strong selection for loss of function of this effector, which was previously considered a virulence factor. Two non-synonymous mutations in the flagellin-encoding gene fliC allowed identifying a new microbe associated molecular pattern (MAMP) in a region distinct from the known MAMP flg22. Interestingly, the ancestral allele of this MAMP induces a stronger tomato immune response than the derived alleles. The ancestral allele has largely disappeared from today’s Pto populations suggesting that flagellin-triggered immunity limits pathogen fitness even in highly virulent pathogens. An additional non-synonymous mutation was identified in flg22 in South American isolates. Therefore, MAMPs are more variable than expected differing even between otherwise almost identical isolates of the same pathogen strain.

Peek J, Lee J, Hu S, Senisterra G, Christendat D

Biochemistry 2011 Oct;50(40):8616-27

PubMed PMID: 21846128

Abstract

Shikimate dehydrogenase (SDH) catalyzes the reversible NADPH-dependent reduction of 3-dehydroshikimate to shikimate. This reaction represents the fourth step of the shikimate pathway, the essential route for the biosynthesis of the aromatic amino acids in plants, fungi, bacteria, and apicomplexan parasites. The absence of this pathway in animals makes it an attractive target for herbicides and antimicrobials. At least four functionally distinct enzyme classes, AroE, YdiB, SDH-like (SdhL), and AroE-like1 (Ael1), utilize shikimate as a substrate in vitro and form the SDH family. Crystal structures have been determined for AroE, YdiB, and SdhL. In this study, we have determined the first representative crystal structure of an Ael1 enzyme. We demonstrate that Ael1 shares a similar overall structure with the other members of the SDH family. This high level of structural conservation extends to the active sites of the enzymes. In particular, an ionizable active site lysine and aspartate are present in all SDH homologues. Two distinct biochemical roles have been reported for this Lys-Asp pair: as binding residues in YdiB and as a catalytic dyad in AroE and SdhL. Here, we establish that the residues function as a catalytic dyad in Ael1 and, interestingly, in at least one YdiB homologue. The conservation of three-dimensional fold, active site architecture, and catalytic mechanism among members of the SDH family will facilitate the design of drugs targeting the shikimate pathway.

Ckurshumova W, Caragea AE, Goldstein RS, Berleth T

Mol Plant 2011 Sep;4(5):794-804

PubMed PMID: 21772029

Abstract

Since the hallmark discovery of Aequorea victoria’s Green Fluorescent Protein (GFP) and its adaptation for efficient use in plants, fluorescent protein tags marking expression profiles or genuine proteins of interest have been used to recognize plant tissues and cell types, to monitor dynamic cell fate selection processes, and to obtain cell type-specific transcriptomes. Fluorescent tagging enabled visualization in living tissues and the precise recordings of dynamic expression pattern changes. The resulting accurate recording of cell fate acquisition kinetics in space and time has strongly stimulated mathematical modeling of self-organizing feedback mechanisms. In developmental studies, the use of fluorescent proteins has become critical, where morphological markers of tissues, cell types, or differentiation stages are either not known or not easily recognizable. In this review, we focus on the use of fluorescent markers to identify and illuminate otherwise invisible cell states in plant development.

Raj S, Bräutigam K, Hamanishi ET, Wilkins O, Thomas BR, Schroeder W, Mansfield SD, Plant AL, Campbell MM

Proc. Natl. Acad. Sci. U.S.A. 2011 Jul;108(30):12521-6

PubMed PMID: 21746919

Abstract

Just as animal monozygotic twins can experience different environmental conditions by being reared apart, individual genetically identical trees of the genus Populus can also be exposed to contrasting environmental conditions by being grown in different locations. As such, clonally propagated Populus trees provide an opportunity to interrogate the impact of individual environmental history on current response to environmental stimuli. To test the hypothesis that current responses to an environmental stimulus, drought, are contingent on environmental history, the transcriptome- level drought responses of three economically important hybrid genotypes-DN34 (Populus deltoides × Populus nigra), Walker [P. deltoides var. occidentalis × (Populus laurifolia × P. nigra)], and Okanese [Walker × (P. laurifolia × P. nigra)]-derived from two different locations were compared. Strikingly, differences in transcript abundance patterns in response to drought were based on differences in geographic origin of clones for two of the three genotypes. This observation was most pronounced for the genotypes with the longest time since establishment and last common propagation. Differences in genome-wide DNA methylation paralleled the transcriptome level trends, whereby the clones with the most divergent transcriptomes and clone history had the most marked differences in the extent of total DNA methylation, suggesting an epigenomic basis for the clone history-dependent transcriptome divergence. The data provide insights into the interplay between genotype and environment in the ecologically and economically important Populus genus, with implications for the industrial application of Populus trees and the evolution and persistence of these important tree species and their associated hybrids.

Salt JN, Yoshioka K, Moeder W, Goring DR

PLoS ONE 2011;6(6):e21321

PubMed PMID: 21738636

Abstract

BACKGROUND: In plants, the ubiquitin-proteasome system is emerging as a significant regulatory system throughout the plant lifecycle. The ubiquitination of a target protein requires the sequential actions of the E1, E2 and E3 enzymes, with the latter E3 enzyme conferring target selection in this process. There are a large number of predicted E3 enzymes in plant genomes, and very little is known about the functions of many of these predicted genes. Here we report here an analysis of two closely-related members of the Arabidopsis Plant U-box (PUB) family of E3 ubiquitin ligases, PUB43 and PUB44.

PRINCIPAL FINDINGS: Homozygous pub44/pub44 mutant seedlings were found displayed a seedling lethal phenotype and this corresponded with widespread cell death lesions throughout the cotyledons and roots. Interestingly, heterozygous PUB44/pub44 seedlings were wild-type in appearance yet displayed intermediate levels of cell death lesions in comparison to pub44/pub44 seedlings. In contrast, homozygous pub43/pub43 mutants were viable and did not show any signs of cell death despite the PUB43 gene being more highly expressed than PUB44. The PUB44 mutants are not classical lesion mimic mutants as they did not have increased resistance to plant pathogens. We also observed increased germination rates in mutant seeds for both PUB44 and PUB43 under inhibitory concentrations of abscisic acid. Finally, the subcellular localization of PUB44 was investigated with transient expression assays in BY-2 cells. Under varying conditions, PUB44 was observed to be localized to the cytoplasm, plasma membrane, or nucleus.

CONCLUSIONS: Based on mutant plant analyses, the Arabidopsis PUB43 and PUB44 genes are proposed to function during seed germination and early seedling growth. Given PUB44’s ability to shuttle from the nucleus to the plasma membrane, PUB44 may be active in different subcellular compartments as part of these biological functions.

Lewis JD, Lee A, Ma W, Zhou H, Guttman DS, Desveaux D

Mol. Plant Pathol. 2011 Dec;12(9):928-37

PubMed PMID: 21726386

Abstract

Bacterial pathogens employ the type III secretion system to secrete and translocate effector proteins into their hosts. The primary function of these effector proteins is believed to be the suppression of host defence responses or innate immunity. However, some effector proteins may be recognized by the host and consequently trigger a targeted immune response. The YopJ/HopZ/AvrRxv family of bacterial effector proteins is a widely distributed and evolutionarily diverse family, found in both animal and plant pathogens, as well as plant symbionts. How can an effector family effectively promote the virulence of pathogens on hosts from two separate kingdoms? Our understanding of the evolutionary relationships among the YopJ superfamily members provides an excellent opportunity to address this question and to investigate the functions and virulence strategies of a diverse type III effector family in animal and plant hosts. In this work, we briefly review the literature on YopJ, the archetypal member from Yersinia pestis, and discuss members of the superfamily in species of Pseudomonas, Xanthomonas, Ralstonia and Rhizobium. We review the molecular and cellular functions, if known, of the YopJ homologues in plants, and highlight the diversity of responses in different plant species, with a particular focus on the Pseudomonas syringae HopZ family. The YopJ superfamily provides an excellent foundation for the study of effector diversification in the context of wide-ranging, co-evolutionary interactions.

Chapman LA, Goring DR

Sex. Plant Reprod. 2011 Dec;24(4):319-26

PubMed PMID: 21691764

Abstract

Phosphoinositides are important lipids involved in membrane identity, vesicle trafficking, and intracellular signaling. In recent years, phosphoinositides have been shown to play a critical role in polarized secretion in plants, as perturbations of phosphoinositide metabolism, through loss of function mutants, result in defects in root hair elongation and pollen tube growth, where polarized secretion occurs rapidly. In the Brassicaceae, responses of stigmatic papillae to compatible pollen are also thought to involve highly regulated secretory events to facilitate pollen hydration and penetration of the pollen tube through the stigmatic surface. We therefore sought to analyze the female sporophyte fertility of the root hair defective4-1 mutant and the PI 4-kinase ?1/?2 double mutant, which differentially affect phosphatidylinositol-4-phosphate (PI4P) levels. Stigmas from both mutants supported slower rates of pollen grain hydration, and the fecundity of these mutants was also diminished as a result of failed pollination events. This study therefore concludes that PI4P is integral to appropriate pistil responses to compatible pollen.

Ckurshumova W, Scarpella E, Goldstein RS, Berleth T

Plant Sci. 2011 Aug;181(2):96-104

PubMed PMID: 21683873

Abstract

Genes expressed in vascular tissues have been identified by several strategies, usually with a focus on mature vascular cells. In this study, we explored the possibility of using two opposite types of altered tissue compositions in combination with a double-filter selection to identify genes with a high probability of vascular expression in early organ primordia. Specifically, we generated full-transcriptome microarray profiles of plants with (a) genetically strongly reduced and (b) pharmacologically vastly increased vascular tissues and identified a reproducible cohort of 158 transcripts that fulfilled the dual requirement of being underrepresented in (a) and overrepresented in (b). In order to assess the predictive value of our identification scheme for vascular gene expression, we determined the expression patterns of genes in two unbiased subsamples. First, we assessed the expression patterns of all twenty annotated transcription factor genes from the cohort of 158 genes and found that seventeen of the twenty genes were preferentially expressed in leaf vascular cells. Remarkably, fifteen of these seventeen vascular genes were clearly expressed already very early in leaf vein development. Twelve genes with published leaf expression patterns served as a second subsample to monitor the representation of vascular genes in our cohort. Of those twelve genes, eleven were preferentially expressed in leaf vascular tissues. Based on these results we propose that our compendium of 158 genes represents a sample that is highly enriched for genes expressed in vascular tissues and that our approach is particularly suited to detect genes expressed in vascular cell lineages at early stages of their inception.

Dean G, Cao Y, Xiang D, Provart NJ, Ramsay L, Ahad A, White R, Selvaraj G, Datla R, Haughn G

Mol Plant 2011 Nov;4(6):1074-91

PubMed PMID: 21653281

Abstract

The seed coat is important for embryo protection, seed hydration, and dispersal. Seed coat composition is also of interest to the agricultural sector, since it impacts the nutritional value for humans and livestock alike. Although some seed coat genes have been identified, the developmental pathways controlling seed coat development are not completely elucidated, and a global genetic program associated with seed coat development has not been reported. This study uses a combination of genetic and genomic approaches in Arabidopsis thaliana to begin to address these knowledge gaps. Seed coat development is a complex process whereby the integuments of the ovule differentiate into specialized cell types. In Arabidopsis, the outermost layer of cells secretes mucilage into the apoplast and develops a secondary cell wall known as a columella. The layer beneath the epidermis, the palisade, synthesizes a secondary cell wall on its inner tangential side. The innermost layer (the pigmented layer or endothelium) produces proanthocyanidins that condense into tannins and oxidize, giving a brown color to mature seeds. Genetic separation of these cell layers was achieved using the ap2-7 and tt16-1 mutants, where the epidermis/palisade and the endothelium do not develop respectively. This genetic ablation was exploited to examine the developmental programs of these cell types by isolating and collecting seed coats at key transitions during development and performing global gene expression analysis. The data indicate that the developmental programs of the epidermis and the pigmented layer proceed relatively independently. Global expression datasets that can be used for identification of new gene candidates for seed coat development were generated. These dataset provide a comprehensive expression profile for developing seed coats in Arabidopsis, and should provide a useful resource and reference for other seed systems.