Avian H6 Influenza Viruses in Vietnamese Live Bird Markets during 2018-2021.

Avian influenza viruses of the H6 subtype are prevalent in wild ducks and likely play an important role in the ecology of influenza viruses through reassortment with other avian influenza viruses. Yet, only 152 Vietnamese H6 virus sequences were available in GISAID (Global Initiative on Sharing All Influenza Data) prior to this study with the most recent sequences being from 2018. Through surveillance in Vietnamese live bird markets from 2018 to 2021, we identified 287 samples containing one or several H6 viruses and other influenza A virus subtypes, demonstrating a high rate of co-infections among birds in Vietnamese live bird markets. For the 132 H6 samples with unique influenza virus sequences, we conducted phylogenetic and genetic analyses. Most of the H6 viruses were similar to each other and closely related to other H6 viruses; however, signs of reassortment with other avian influenza viruses were evident. At the genetic level, the Vietnamese H6 viruses characterized in our study encode a single basic amino acid at the HA cleavage site, consistent with low pathogenicity in poultry. The Vietnamese H6 viruses analyzed here possess an amino acid motif in HA that confers binding to both avian- and human-type receptors on host cells, consistent with their ability to infect mammals. The frequent detection of H6 viruses in Vietnamese live bird markets, the high rate of co-infections of birds with different influenza viruses, and the dual receptor-binding specificity of these viruses warrant their close monitoring for potential infection and spread among mammals.

Characterization of a human H3N8 influenza virus.

BACKGROUND: In 2022 and 2023, novel reassortant H3N8 influenza viruses infected three people, marking the first human infections with viruses of this subtype. METHODS: Here, we generated one of these viruses (A/Henan/4-10CNIC/2022; hereafter called A/Henan/2022 virus) by using reverse genetics and characterized it. FINDINGS: In intranasally infected mice, reverse genetics-generated A/Henan/2022 virus caused weight loss in all five animals (one of which had to be euthanized) and replicated efficiently in the respiratory tract. Intranasal infection of ferrets resulted in minor weight loss and moderate fever but no mortality. Reverse genetics-generated A/Henan/2022 virus replicated efficiently in the upper respiratory tract of ferrets but was not detected in the lungs. Virus transmission via respiratory droplets occurred in one of four pairs of ferrets. Deep-sequencing of nasal swab samples from inoculated and exposed ferrets revealed sequence polymorphisms in the haemagglutinin protein that may affect receptor-binding specificity. We also tested 90 human sera for neutralizing antibodies against reverse genetics-generated A/Henan/2022 virus and found that some of them possessed neutralizing antibody titres, especially sera from older donors with likely exposure to earlier human H3N2 viruses. INTERPRETATION: Our data demonstrate that reverse genetics-generated A/Henan/2022 virus is a low pathogenic influenza virus (of avian influenza virus descent) with some antigenic resemblance to older human H3N2 viruses and limited respiratory droplet transmissibility in ferrets. FUNDING: This work was supported by the Japan Program for Infectious Diseases Research and Infrastructure (JP23wm0125002), and the Japan Initiative for World-leading Vaccine Research and Development Centers (JP233fa627001) from the Japan Agency for Medical Research and Development (AMED).

Assessment of the antigenic evolution of a clade 6B.1 human H1N1pdm influenza virus revealed differences between ferret and human convalescent sera.

BACKGROUND: Influenza viruses continually acquire mutations in the antigenic epitopes of their major viral antigen, the surface glycoprotein haemagglutinin (HA), allowing evasion from immunity in humans induced upon prior influenza virus infections or vaccinations. Consequently, the influenza strains used for vaccine production must be updated frequently. METHODS: To better understand the antigenic evolution of influenza viruses, we introduced random mutations into the HA head region (where the immunodominant epitopes are located) of a pandemic H1N1 (H1N1pdm) virus from 2015 and incubated it with various human sera collected in 2015-2016. Mutants not neutralized by the human sera were sequenced and further characterized for their haemagglutination inhibition (HI) titers with human sera and with ferret sera raised to H1N1pdm viruses from 2009 to 2015. FINDINGS: The largest antigenic changes were conferred by mutations at HA amino acid position 187; interestingly, these antigenic changes were recognized by human, but not by ferret serum. H1N1pdm viruses with amino acid changes at position 187 were very rare until the end of 2018, but have become more frequent since; in fact, the D187A amino acid change is one of the defining changes of clade 6B.1A.5a.1 viruses, which emerged in 2019. INTERPRETATION: Our findings indicate that amino acid substitutions in H1N1pdm epitopes may be recognized by human sera, but not by homologous ferret sera. FUNDING: This project was supported by funding from the NIAID-funded Center for Research on Influenza Pathogenesis (CRIP, HHSN272201400008C).

Continued Circulation of Highly Pathogenic H5 Influenza Viruses in Vietnamese Live Bird Markets in 2018-2021.

We isolated 77 highly pathogenic avian influenza viruses during routine surveillance in live poultry markets in northern provinces of Vietnam from 2018 to 2021. These viruses are of the H5N6 subtype and belong to HA clades 2.3.4.4g and 2.3.4.4h. Interestingly, we did not detect viruses of clade 2.3.4.4b, which in recent years have dominated in different parts of the world. The viruses isolated in this current study do not encode major determinants of mammalian adaptation (e.g., PB2-E627K or PB1-D701N) but possess amino acid substitutions that may affect viral receptor-binding, replication, or the responses to human antiviral factors. Several of the highly pathogenic H5N6 virus samples contained other influenza viruses, providing an opportunity for reassortment. Collectively, our study demonstrates that the highly pathogenic H5 viruses circulating in Vietnam in 2018-2021 were different from those in other parts of the world, and that the Vietnamese H5 viruses continue to evolve through mutations and reassortment.

Highly Pathogenic H5 Influenza Viruses Isolated between 2016 and 2017 in Vietnamese Live Bird Markets.

Routine surveillance in live poultry markets in the northern regions of Vietnam from 2016 to 2017 resulted in the isolation of 27 highly pathogenic avian H5N1 and H5N6 viruses of 3 different clades (2.3.2.1c, 2.3.4.4f, and 2.3.4.4g). Sequence and phylogenetic analysis of these viruses revealed reassortment with various subtypes of low pathogenic avian influenza viruses. Deep-sequencing identified minor viral subpopulations encoding variants that may affect pathogenicity and sensitivity to antiviral drugs. Interestingly, mice infected with two different clade 2.3.2.1c viruses lost body weight rapidly and succumbed to virus infection, whereas mice infected with clade 2.3.4.4f or 2.3.4.4g viruses experienced non-lethal infections.

Validating Methods To Eradicate Plant-Pathogenic Ralstonia Strains Reveals that Growth In Planta Increases Bacterial Stress Tolerance.

Plant-pathogenic bacteria in the Ralstonia solanacearum species complex (RSSC) cause highly destructive bacterial wilt disease of diverse crops. Wilt disease prevention and management is difficult because RSSC persists in soil, water, and plant material. Growers need practical methods to kill these pathogens in irrigation water, a common source of disease outbreaks. Additionally, the R. solanacearum race 3 biovar 2 (R3bv2) subgroup is a quarantine pest in many countries and a highly regulated select agent pathogen in the United States. Plant protection officials and researchers need validated protocols to eradicate R3bv2 for regulatory compliance. To meet these needs, we measured the survival of four R3bv2 and three phylotype I RSSC strains following treatment with hydrogen peroxide, stabilized hydrogen peroxide (Huwa-San), active chlorine, heat, UV radiation, and desiccation. No surviving RSSC cells were detected after cultured bacteria were exposed for 10 min to 400 ppm hydrogen peroxide, 50 ppm Huwa-San, 50 ppm active chlorine, or temperatures above 50°C. RSSC cells on agar plates were eradicated by 30 s of UV irradiation and killed by desiccation on most biotic and all abiotic surfaces tested. RSSC bacteria did not survive the cell lysis steps of four nucleic acid extraction protocols. However, bacteria in planta were more difficult to kill. Stems of infected tomato plants contained a subpopulation of bacteria with increased tolerance of heat and UV light, but not oxidative stress. This result has significant management implications. We demonstrate the utility of these protocols for compliance with select agent research regulations and for management of a bacterial wilt outbreak in the field. IMPORTANCE Bacteria in the Ralstonia solanacearum species complex (RSSC) are globally distributed and cause destructive vascular wilt diseases of many high-value crops. These aggressive pathogens spread in diseased plant material and via contaminated soil, tools, and irrigation water. A subgroup of the RSSC, race 3 biovar 2, is a European and Canadian quarantine pathogen and a U.S. select agent subject to stringent and constantly evolving regulations intended to prevent pathogen introduction or release. We validated eradication and inactivation methods that can be used by (i) growers seeking to disinfest water and manage bacterial wilt disease outbreaks, (ii) researchers who must remain in compliance with regulations, and (iii) regulators who are expected to define containment practices. Relevant to all these stakeholders, we show that while cultured RSSC cells are sensitive to relatively low levels of oxidative chemicals, desiccation, and heat, more aggressive treatment, such as autoclaving or incineration, is required to eradicate plant-pathogenic Ralstonia growing inside plant material.

Overexpression of a modified eIF4E regulates potato virus Y resistance at the transcriptional level in potato.

BACKGROUND: Potato virus Y (PVY) is a major pathogen of potatoes with major impact on global agricultural production. Resistance to PVY can be achieved by engineering potatoes to express a recessive, resistant allele of eukaryotic translation initiation factor eIF4E, a host dependency factor essential to PVY replication. Here we analyzed transcriptome changes in eIF4E over-expressing potatoes to shed light on the mechanism underpinning eIF4E-mediated recessive PVY resistance. RESULTS: As anticipated, modified eIF4E-expressing potatoes demonstrated a high level of resistance, eIF4E expression, and an unexpected suppression of the susceptible allele transcript, likely explaining the bulk of the potent antiviral phenotype. In resistant plants, we also detected marked upregulation of genes involved in cell stress responses. CONCLUSIONS: Our results reveal a previously unanticipated second layer of signaling attributable to eIF4E regulatory control, and potentially relevant to establishment of a broader, more systematic antiviral host defense.

Inappropriate Intrusion of an Axonal Mitochondrial Anchor into Dendrites Causes Neurodegeneration.

Syntaphilin (SNPH) is a major mitochondrial anchoring protein targeted to axons and excluded from dendrites. In this study, we provide in vivo evidence that this spatial specificity is lost in Shiverer (Shi) mice, a model for progressive multiple sclerosis (MS), resulting in inappropriate intrusion of SNPH into dendrites of cerebellar Purkinje cells with neurodegenerative consequences. Thus, reconstituting dendritic SNPH intrusion in SNPH-KO mice by viral transduction greatly sensitizes Purkinje cells to excitotoxicity when the glutamatergic climbing fibers are stimulated. Finally, we demonstrate in vitro that overexpression of SNPH in dendrites compromises neuronal viability by inducing N-methyl-D-aspartate (NMDA) excitotoxicity, reducing mitochondrial calcium uptake, and interfering with quality control of mitochondria by blocking somal mitophagy. Collectively, we propose that inappropriate immobilization of dendritic mitochondria by SNPH intrusion produces excitotoxicity and suggest that interception of dendritic SNPH intrusion is a therapeutic strategy to combat neurodegeneration.

Optimization of an isothermal recombinase polymerase amplification method for real-time detection of Potato virus Y O and N types in potato.

Potato virus Y (PVY) is a global challenge for potato production and the leading cause of seed crop downgrading and rejection for certification. Accurate and timely diagnosis is key to effective control of PVY. Here we optimized the isothermal recombinase polymerase amplification (RPA) for accurate detection of different PVY O and N types that were tested, present in different tissues of potato plants including tubers with a primer set that specifically targets the highly conserved pipo region within the viral genome. Combined with a simplified preparation of the template by tissue homogenization, we established a rapid RPA procedure, which can allow real time detection in less than 10 min with a fluorescent probe. Specificity of the reaction was determined by the lack of cross-reactivity with other common potato viruses. Although RPA reagents remain more expensive than PCR reagents, RPA technology is equivalent in that results can be visualized by gel electrophoresis or with a fluorescent probe with greater sensitivity; and it is superior to the common PCR-based assay in its versatility, speed, and lack of need for a highly purified RNA template.

Optimization of a magnetic capture RT-LAMP assay for fast and real-time detection of potato virus Y and differentiation of N and O serotypes.

Potato virus Y (PVY) infection has been a global challenge for potato production and the leading cause of downgrading and rejection of seed crops for certification. Accurate and timely diagnosis is a key for effective disease control. Here, we have optimized a reverse transcription loop-mediated amplification (RT-LAMP) assay to differentiate the PVY O and N serotypes. The RT-LAMP assay is based on isothermal autocyclic strand displacement during DNA synthesis. The high specificity of this method relies heavily on the primer sets designed for the amplification of the targeted regions. We designed specific primer sets targeting a region within the coat protein gene that contains nucleotide signatures typical for O and N coat protein types, and these primers differ in their annealing temperature. Combining this assay with total RNA extraction by magnetic capture, we have established a highly sensitive, simplified and shortened RT-LAMP procedure as an alternative to conventional nucleic acid assays for diagnosis. This optimized procedure for virus detection may be used as a preliminary test for identifying the viral serotype prior to investing time and effort in multiplex RT-PCR tests when a specific strain is needed.

Comparative Transcriptome Analysis Reveals Cool Virulence Factors of Ralstonia solanacearum Race 3 Biovar 2.

While most strains of the plant pathogenic bacterium Ralstonia solanacearum are tropical, the race 3 biovar 2 (R3bv2) subgroup attacks plants in cooler climates. To identify mechanisms underlying this trait, we compared the transcriptional profiles of R. solanacearum R3bv2 strain UW551 and tropical strain GMI1000 at 20°C and 28°C, both in culture and during tomato pathogenesis. 4.2% of the ORFs in the UW551 genome and 7.9% of the GMI1000 ORFs were differentially expressed by temperature in planta. The two strains had distinct transcriptional responses to temperature change. GMI1000 up-regulated several stress response genes at 20°C, apparently struggling to cope with plant defenses. At the cooler temperature, R3bv2 strain UW551 up-regulated a cluster encoding a mannose-fucose binding lectin, LecM; a quorum sensing-dependent protein, AidA; and a related hypothetical protein, AidC. The last two genes are absent from the GMI1000 genome. In UW551, all three genes were positively regulated by the adjacent SolI/R quorum sensing system. These temperature-responsive genes were required for full virulence in R3bv2. Mutants lacking lecM, aidA, or aidC were each significantly more reduced in virulence on tomato at 20°C than at 28°C in both a naturalistic soil soak inoculation assay and when they were inoculated directly into tomato stems. The lecM and aidC mutants also survived poorly in potato tubers at the seed tuber storage temperature of 4°C, and the lecM mutant was defective in biofilm formation in vitro. Together, these results suggest novel mechanisms, including a lectin, are involved in the unique temperate epidemiology of R3bv2.

Transcriptome changes associated with anaerobic growth in Yersinia intermedia (ATCC29909).

BACKGROUND: The yersiniae (Enterobacteriaceae) occupy a variety of niches, including some in human and flea hosts. Metabolic adaptations of the yersiniae, which contribute to their success in these specialized environments, remain largely unknown. We report results of an investigation of the transcriptome under aerobic and anaerobic conditions for Y. intermedia, a non-pathogenic member of the genus that has been used as a research surrogate for Y. pestis. Y. intermedia shares characteristics of pathogenic yersiniae, but is not known to cause disease in humans. Oxygen restriction is an important environmental stimulus experienced by many bacteria during their life-cycles and greatly influences their survival in specific environments. How oxygen availability affects physiology in the yersiniae is of importance in their life cycles but has not been extensively characterized. METHODOLOGY/PRINCIPAL FINDINGS: Tiled oligonucleotide arrays based on a draft genome sequence of Y. intermedia were used in transcript profiling experiments to identify genes that change expression in response to oxygen availability during growth in minimal media with glucose. The expression of more than 400 genes, constituting about 10% of the genome, was significantly altered due to oxygen-limitation in early log phase under these conditions. Broad functional categorization indicated that, in addition to genes involved in central metabolism, genes involved in adaptation to stress and genes likely involved with host interactions were affected by oxygen-availability. Notable among these, were genes encoding functions for motility, chemotaxis and biosynthesis of cobalamin, which were up-regulated and those for iron/heme utilization, methionine metabolism and urease, which were down-regulated. CONCLUSIONS/SIGNIFICANCE: This is the first transcriptome analysis of a non-pathogenic Yersinia spp. and one of few elucidating the global response to oxygen limitation for any of the yersiniae. Thus this study lays the foundation for further experimental characterization of oxygen-responsive genes and pathways in this ecologically diverse genus.

The in planta transcriptome of Ralstonia solanacearum: conserved physiological and virulence strategies during bacterial wilt of tomato.

Plant xylem fluid is considered a nutrient-poor environment, but the bacterial wilt pathogen Ralstonia solanacearum is well adapted to it, growing to 10(8) to 10(9) CFU/g tomato stem. To better understand how R. solanacearum succeeds in this habitat, we analyzed the transcriptomes of two phylogenetically distinct R. solanacearum strains that both wilt tomato, strains UW551 (phylotype II) and GMI1000 (phylotype I). We profiled bacterial gene expression at ~6 × 10(8) 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 altered in planta versus 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 the scrABY cluster. A UW551 scrA mutant was significantly reduced in virulence on resistant and susceptible tomato as well as on potato and the epidemiologically important weed host Solanum dulcamara. Functional scrA contributed to pathogen competitive fitness during colonization of tomato xylem, which contained ~300 µM sucrose. scrA expression was induced by sucrose, but to a much greater degree by growth in planta. Unexpectedly, 45% of the genes directly regulated by HrpB, the transcriptional activator of the type 3 secretion system (T3SS), were upregulated in planta at 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. IMPORTANCE Ralstonia solanacearum is 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 that R. solanacearum expresses 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. solanacearum was 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.

Sequencing of K60, type strain of the major plant pathogen Ralstonia solanacearum.

Ralstonia solanacearum is a widespread and destructive plant pathogen. We present the genome of the type strain, K60 (phylotype IIA, sequevar 7). Sequevar 7 strains cause ongoing tomato bacterial wilt outbreaks in the southeastern United States. K60 generally resembles R. solanacearum CFBP2957, a Caribbean tomato isolate, but has almost 360 unique genes.

Evolution of the metabolic and regulatory networks associated with oxygen availability in two phytopathogenic enterobacteria.

BACKGROUND: Dickeya 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. RESULTS: More 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. CONCLUSIONS: The 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 achieved by each organism using different genes and regulatory strategies.

Ralstonia solanacearum extracellular polysaccharide is a specific elicitor of defense responses in wilt-resistant tomato plants.

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.

The proteome map of spinach leaf peroxisomes indicates partial compartmentalization of phylloquinone (vitamin K1) biosynthesis in plant peroxisomes.

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.

Proteome analysis of Arabidopsis leaf peroxisomes reveals novel targeting peptides, metabolic pathways, and defense mechanisms.

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 full-length 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 beta-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.

Proteomic analysis of the carbonate insoluble outer membrane fraction of the soft-rot pathogen Dickeya dadantii (syn. Erwinia chrysanthemi) strain 3937.

We present results of the first comprehensive proteomic analysis of the outer membrane of the bacterial phytopathogen Dickeya dadantii strain 3937 and its response to virulence-contributing factors such as host plant extract, acidic stress, and iron starvation. We analyzed the carbonate-insoluble membrane fractions, which are highly enriched for outer membrane proteins, using two-dimensional electrophoresis and identified the proteins by MALDI-TOF MS. Forty unique proteins were identified, some of which were differentially expressed under the above conditions.

The Erwinia chrysanthemi 3937 PhoQ sensor kinase regulates several virulence determinants.

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.