We have established a protocol for the isolation of highly purified peroxisomes from mature Arabidopsis thaliana leaves and analyzed the proteome by complementary gel-based and gel-free approaches. Seventy-eight nonredundant proteins were identified, of which 42 novel proteins had previously not been associated with plant peroxisomes. Seventeen novel proteins carried predicted peroxisomal targeting signals (PTS) type 1 or type 2; 11 proteins contained PTS-related peptides. Peroxisome targeting was supported for many novel proteins by in silico analyses and confirmed for 11 representative fulllength fusion proteins by fluorescence microscopy. The targeting function of predicted and unpredicted signals was investigated and SSL>, SSI>, and ASL> were established as novel functional PTS1 peptides. In contrast with the generally accepted confinement of PTS2 peptides to the N-terminal domain, the bifunctional transthyretin-like protein was demonstrated to carry internally a functional PTS2. The novel enzymes include numerous enoyl-CoA hydratases, short-chain dehydrogenases, and several enzymes involved in NADP and glutathione metabolism. Seven proteins, including b-glucosidases and myrosinases, support the currently emerging evidence for an important role of leaf peroxisomes in defense against pathogens and herbivores. The data provide new insights into the biology of plant peroxisomes and improve the prediction accuracy of peroxisome-targeted proteins from genome sequences.

ABSTRACTPlant xylem fluid is considered a nutrient-poor environment, but the bacterial wilt pathogenRalstonia solanacearumis well adapted to it, growing to 108to 109 CFU/g tomato stem. To better understand howR. solanacearumsucceeds in this habitat, we analyzed the transcriptomes of two phylogenetically distinctR. solanacearumstrains that both wilt tomato, strains UW551 (phylotype II) and GMI1000 (phylotype I). We profiled bacterial gene expression at ~6 × 108 CFU/ml in culture or in plant xylem during early tomato bacterial wilt pathogenesis. Despite phylogenetic differences, these two strains expressed their 3,477 common orthologous genes in generally similar patterns, with about 12% of their transcriptomes significantly alteredin plantaversus in rich medium. Several primary metabolic pathways were highly expressed during pathogenesis. These pathways included sucrose uptake and catabolism, and components of these pathways were encoded by genes in thescrABYcluster. A UW551scrAmutant was significantly reduced in virulence on resistant and susceptible tomato as well as on potato and the epidemiologically important weed hostSolanum dulcamara. FunctionalscrAcontributed to pathogen competitive fitness during colonization of tomato xylem, which contained ~300 µM sucrose.scrAexpression was induced by sucrose, but to a much greater degree by growthin planta. Unexpectedly, 45% of the genes directly regulated by HrpB, the transcriptional activator of the type 3 secretion system (T3SS), were upregulatedin plantaat high cell densities. This result modifies a regulatory model based on bacterial behavior in culture, where this key virulence factor is repressed at high cell densities. The active transcription of these genes in wilting plants suggests that T3SS has a biological role throughout the disease cycle.IMPORTANCERalstonia solanacearumis a widespread plant pathogen that causes bacterial wilt disease. It inflicts serious crop losses on tropical farmers, with major economic and human consequences. It is also a model for the many destructive microbes that colonize the water-conducting plant xylem tissue, which is low in nutrients and oxygen. We extracted bacteria from infected tomato plants and globally identified the biological functions thatR. solanacearumexpresses during plant pathogenesis. This revealed the unexpected presence of sucrose in tomato xylem fluid and the pathogen’s dependence on host sucrose for virulence on tomato, potato, and the common weed bittersweet nightshade. Further,R. solanacearumwas highly responsive to the plant environment, expressing several metabolic and virulence functions quite differently in the plant than in pure culture. These results reinforce the utility of studying pathogens in interaction with hosts and suggest that selecting for reduced sucrose levels could generate wilt-resistant crops.

To identify unknown proteins from plant peroxisomes, the Arabidopsis genome was screened for proteins with putative major or minor peroxisome targeting signals type 1 or 2 (PTS1 or PTS2), as defined previously (Reumann S [2004] Plant Physiol 135: 783-800). About 220 and 60 proteins were identified that carry a putative PTS1 or PTS2, respectively. To further support postulated targeting to peroxisomes, several prediction programs were applied and the putative targeting domains analyzed for properties conserved in peroxisomal proteins and for PTS conservation in homologous plant expressed sequence tags. The majority of proteins with a major PTS and medium to high overall probability of peroxisomal targeting represent novel nonhypothetical proteins and include several enzymes involved in b-oxidation of unsaturated fatty acids and branched amino acids, and 2-hydroxy acid oxidases with a predicted function in fatty acid a-oxidation, as well as NADP-dependent dehydrogenases and reductases. In addition, large protein families with many putative peroxisomal isoforms were recognized, including acyl-activating enzymes, GDSL lipases, and small thioesterases. Several proteins are homologous to prokaryotic enzymes of a novel aerobic hybrid degradation pathway for aromatic compounds and proposed to be involved in peroxisomal biosynthesis of plant hormones like jasmonic acid, auxin, and salicylic acid. Putative regulatory proteins of plant peroxisomes include protein kinases, small heat shock proteins, and proteases. The information on subcellular targeting prediction, homology, and in silico expression analysis for these Arabidopsis proteins has been compiled in the public database AraPerox to accelerate discovery and experimental investigation of novel metabolic and regulatory pathways of plant peroxisomes.

Ralstonia solanacearum, which causes bacterial wilt of diverse plants, produces copious extracellular polysaccharide (EPS), a major virulence factor. The function of EPS in wilt disease is uncertain. Leading hypotheses are that EPS physically obstructs plant water transport, or that EPS cloaks the bacterium from host plant recognition and subsequent defense. Tomato plants infected with R. solanacearum race 3 biovar 2 strain UW551 and tropical strain GMI1000 upregulated genes in both the ethylene (ET) and salicylic acid (SA) defense signal transduction pathways. The horizontally wilt-resistant tomato line Hawaii7996 activated expression of these defense genes faster and to a greater degree in response to R. solanacearum infection than did susceptible cultivar Bonny Best. However, EPS played different roles in resistant and susceptible host responses to R. solanacearum. In susceptible plants the wild-type and eps− mutant strains induced generally similar defense responses. But in resistant Hawaii7996 tomato plants, the wild-type pathogens induced significantly greater defense responses than the eps− mutants, suggesting that the resistant host recognizes R. solanacearum EPS. Consistent with this idea, purified EPS triggered significant SA pathway defense gene expression in resistant, but not in susceptible, tomato plants. In addition, the eps− mutant triggered noticeably less production of defense-associated reactive oxygen species in resistant tomato stems and leaves, despite attaining similar cell densities in planta. Collectively, these data suggest that bacterial wilt-resistant plants can specifically recognize EPS from R. solanacearum.

Small heat-shock proteins (sHsps) are widespread molecular chaperones for which a peroxisomal localization has not yet been reported. The Arabidopsis (Arabidopsis thaliana) genome encodes two sHsps with putative peroxisomal targeting signals type 1 or 2 (PTS1 or PTS2). As demonstrated by double-labeling experiments using full-length fusion proteins with enhanced yellow fluorescent protein and deletion constructs lacking the putative targeting domains, AtHsp15.7 (At5g37670) and AtAcd31.2 (At1g06460) are targeted to the peroxisome matrix by a functional PTS1 (SKL.) and a functional PTS2 (RLx 5 HF), respectively. The peroxisomal localization of AtAcd31.2 was further confirmed by isolation of leaf peroxisomes from Arabidopsis by two successive sucrose density gradients, protein separation by one-and two-dimensional gel electrophoresis, and mass spectrometric protein identification. When AtHsp15.7 and AtAcd31.2 were heterologously expressed in yeast (Saccharomyces cerevisiae) and directed to the cytosol by deletion of the PTSs, both sHsps were able to complement the morphological phenotype of yeast mutants deficient in the cytosolic homologs ScHsp42 or ScHsp26. According to expression studies by reverse transcription-PCR, AtAcd31.2 is constitutively expressed, whereas AtHsp15.7 is hardly expressed under normal conditions but strongly induced by heat and oxidative stress, the latter of which was triggered by the catalase inhibitor 3-aminotriazole or the herbicide methyl viologen applied by watering of whole plants or infiltration of rosette leaves. Thus, plants are exceptional among eukaryotes in employing sHsps in the peroxisome matrix to prevent unspecific aggregation of partially denatured proteins under both physiological and stress conditions.

The PhoPQ two-component system regulates virulence factors in Erwinia chrysanthemi, a pectinolytic enterobacterium that causes soft rot in several plant species. We characterized the effect of a mutation in phoQ, the gene encoding the sensor kinase PhoQ of the PhoPQ two-component regulatory system, on the global transcriptional profile of E. chrysanthemi using cDNA microarrays and further confirmed our results by quantitative reverse transcription-PCR analysis. Our results indicate that a mutation in phoQ affects transcription of at least 40 genes, even in the absence of inducing conditions. Enhanced expression of several genes involved in iron metabolism was observed in the mutant, including that of the acs operon that is involved in achromobactin biosynthesis and transport. This siderophore is required for full virulence of E. chrysanthemi, and its expression is governed by the global repressor protein Fur. Changes in gene expression were also observed for membrane transporters, stress-related genes, toxins, and transcriptional regulators. Our results indicate that the PhoPQ system governs the expression of several additional virulence factors and may also be involved in interactions with other regulatory systems.

Leaf peroxisomes are fragile, low-abundance plant cell organelles that are difficult to isolate from one of the few plant species whose nuclear genome has been sequenced. Leaf peroxisomes were enriched at high purity from spinach (Spinacia oleracea) and approximately 100 protein spots identified from 2-dimensional gels by a combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and de novo sequencing. In addition to the predominant enzymes involved in photorespiration and detoxification, several minor enzymes were detected, underscoring the high sensitivity of the protein identification. The tryptic peptides of three unknown proteins shared high sequence similarity with Arabidopsis proteins that carry putative peroxisomal targeting signals type 1 or 2 (PTS1/2). The apparent Arabidopsis orthologues are a short-chain alcohol dehydrogenase (SDRa/IBR1, At4g05530, SRL>) and two enoyl-CoA hydratases/isomerases (ECHIa, At4g16210, SKL>; NS/ECHId, At1g60550, RLx(5)HL). The peroxisomal localization of the three proteins was confirmed in vivo by tagging with enhanced yellow fluorescent protein (EYFP), and the targeting signals were identified. The single Arabidopsis isoform of naphthoate synthase (NS) is orthologous to MenB from cyanobacteria, which catalyses an essential reaction in phylloquinone biosynthesis, a pathway previously assumed to be entirely compartmentalized in plastids in higher plants. In an extension of a previous study, the present in vivo targeting data furthermore demonstrate that the enzyme upstream of NS, chloroplastic acyl-CoA activating enzyme isoform 14 (AAE14, SSL>), is dually targeted to both plastids and peroxisomes. This proteomic study, extended by in vivo subcellular localization analyses, indicates a novel function for plant peroxisomes in phylloquinone biosynthesis.

BackgroundDickeya dadantii and Pectobacterium atrosepticum are phytopathogenic enterobacteria capable of facultative anaerobic growth in a wide range of O2 concentrations found in plant and natural environments. The transcriptional response to O2 remains under-explored for these and other phytopathogenic enterobacteria although it has been well characterized for animal-associated genera including Escherichia coli and Salmonella enterica. Knowledge of the extent of conservation of the transcriptional response across orthologous genes in more distantly related species is useful to identify rates and patterns of regulon evolution. Evolutionary events such as loss and acquisition of genes by lateral transfer events along each evolutionary branch results in lineage-specific genes, some of which may have been subsequently incorporated into the O2-responsive stimulon. Here we present a comparison of transcriptional profiles measured using densely tiled oligonucleotide arrays for two phytopathogens, Dickeya dadantii 3937 and Pectobacterium atrosepticum SCRI1043, grown to mid-log phase in MOPS minimal medium (0.1% glucose) with and without O2.ResultsMore than 7% of the genes of each phytopathogen are differentially expressed with greater than 3-fold changes under anaerobic conditions. In addition to anaerobic metabolism genes, the O2 responsive stimulon includes a variety of virulence and pathogenicity-genes. Few of these genes overlap with orthologous genes in the anaerobic stimulon of E. coli. We define these as the conserved core, in which the transcriptional pattern as well as genetic architecture are well preserved. This conserved core includes previously described anaerobic metabolic pathways such as fermentation. Other components of the anaerobic stimulon show variation in genetic content, genome architecture and regulation. Notably formate metabolism, nitrate/nitrite metabolism, and fermentative butanediol production, differ between E. coli and the phytopathogens. Surprisingly, the overlap of the anaerobic stimulon between the phytopathogens is also relatively small considering that they are closely related, occupy similar niches and employ similar strategies to cause disease. There are cases of interesting divergences in the pattern of transcription of genes between Dickeya and Pectobacterium for virulence-associated subsystems including the type VI secretion system (T6SS), suggesting that fine-tuning of the stimulon impacts interaction with plants or competing microbes.ConclusionsThe small number of genes (an even smaller number if we consider operons) comprising the conserved core transcriptional response to O2 limitation demonstrates the extent of regulatory divergence prevalent in the Enterobacteriaceae. Our orthology-driven comparative transcriptomics approach indicates that the adaptive response in the eneterobacteria is a result of interaction of core (regulators) and lineage-specific (structural and regulatory) genes. Our subsystems based approach reveals that similar phenotypic outcomes are sometimes ach...