MARTX Toxin-Stimulated Interplay between Human Cells and Vibrio vulnificus.

To understand toxin-stimulated host-pathogen interactions, we performed dual-transcriptome sequencing experiments using human epithelial (HT-29) and differentiated THP-1 (dTHP-1) immune cells infected with the sepsis-causing pathogen Vibrio vulnificus (either the wild-type [WT] pathogen or a multifunctional-autoprocessing repeats-in-toxin [MARTX] toxin-deficient strain). Gene set enrichment analyses revealed MARTX toxin-dependent responses, including negative regulation of extracellular related kinase 1 (ERK1) and ERK2 (ERK1/2) signaling and cell cycle regulation in HT-29 and dTHP-1 cells, respectively. Further analysis of the expression of immune-related genes suggested that the MARTX toxin dampens immune responses in gut epithelial cells but accelerates inflammation and nuclear factor κB (NF-κB) signaling in immune cells. With respect to the pathogen, siderophore biosynthesis genes were significantly more highly expressed in WT V. vulnificus than in the MARTX toxin-deficient mutant upon infection of dTHP-1 cells. Consistent with these results, iron homeostasis genes that limit iron levels for invading pathogens were overexpressed in WT V. vulnificus-infected dTHP-1 cells. Taken together, these results suggest that MARTX toxin regulates host inflammatory responses during V. vulnificus infection while also countering host defense mechanisms such as iron limitation.IMPORTANCEV. vulnificus is an opportunistic human pathogen that can cause life-threatening sepsis in immunocompromised patients via seafood poisoning or wound infection. Among the toxic substances produced by this pathogen, the MARTX toxin greatly contributes to disease progression by promoting the dysfunction and death of host cells, which allows the bacteria to disseminate and colonize the host. In response to this, host cells mount a counterattack against the invaders by upregulating various defense genes. In this study, the gene expression profiles of both host cells and V. vulnificus were analyzed by RNA sequencing to gain a comprehensive understanding of host-pathogen interactions. Our results suggest that V. vulnificus uses the MARTX toxin to subvert host cell immune responses as well as to oppose host counterattacks such as iron limitation.

Molecular Insights into Toluene Sensing in the TodS/TodT Signal Transduction System.

TodS is a sensor kinase that responds to various monoaromatic compounds, which either cause an agonistic or antagonistic effect on phosphorylation of its cognate response regulator TodT, and controls tod operon expression in Pseudomonas putida strains. We describe a molecular sensing mechanism of TodS that is activated in response to toluene. The crystal structures of the TodS Per-Arnt-Sim (PAS) 1 sensor domain (residues 43-164) and its complex with toluene (agonist) or 1,2,4-trimethylbenzene (antagonist) show a typical ß2α3ß3 PAS fold structure (residues 45-149), forming a hydrophobic ligand-binding site. A signal transfer region (residues 150-163) located immediately after the canonical PAS fold may be intrinsically flexible and disordered in both apo-PAS1 and antagonist-bound forms and dramatically adapt an α-helix upon toluene binding. This structural change in the signal transfer region is proposed to result in signal transmission to activate the TodS/TodT two-component signal transduction system. Site-directed mutagenesis and ß-galactosidase assays using a P. putida reporter strain system verified the essential residues involved in ligand sensing and signal transfer and suggest that the Phe(46) residue acts as a ligand-specific switch.

Arabidopsis TTR1 causes LRR-dependent lethal systemic necrosis, rather than systemic acquired resistance, to Tobacco ringspot virus.

Most Arabidopsis ecotypes display tolerance to the Tobacco ringspot virus (TRSV), but a subset of Arabidopsis ecotypes, including Estland (Est), develop lethal systemic necrosis (LSN), which differs from the localized hypersensitive responses (HRs) or systemic acquired resistance (SAR) characteristic of incompatible reactions. Neither viral replication nor the systemic movement of TRSV was restricted in tolerant or sensitive Arabidopsis ecotypes; therefore, the LSN phenotype shown in the sensitive ecotypes might not be due to viral accumulation. In the present study, we identified the Est TTR1 gene (tolerance to Tobacco ringspot virus 1) encoding a TIR-NBS-LRR protein that controls the ecotype-dependent tolerant/sensitive phenotypes by a map-based cloning method. The tolerant Col-0 ecotype Arabidopsis transformed with the sensitive Est TTR1 allele developed an LSN phenotype upon TRSV infection, suggesting that the Est TTR1 allele is dominant over the tolerant ttr1 allele of Col-0. Multiple sequence alignments of 10 tolerant ecotypes from those of eight sensitive ecotypes showed that 10 LRR amino acid polymorphisms were consistently distributed across the TTR1/ttr1 alleles. Site-directed mutagenesis of these amino acids in the LRR region revealed that two sites, L956S and K1124Q, completely abolished the LSN phenotype. VIGS study revealed that TTR1 is dependent on SGT1, rather than EDS1. The LSN phenotype by TTR1 was shown to be transferred to Nicotiana benthamiana, demonstrating functional conservation of TTR1 across plant families, which are involved in SGT-dependent defense responses, rather than EDS1-dependent signaling pathways.

An HrpB-dependent but type III-independent extracellular aspartic protease is a virulence factor of Ralstonia solanacearum.

The host specificity of Ralstonia solanacearum, the causal organism of bacterial wilt on many solanaceous crops, is poorly understood. To identify a gene conferring host specificity of the bacterium, SL341 (virulent to hot pepper but avirulent to potato) and SL2029 (virulent to potato but avirulent to hot pepper) were chosen as representative strains. We identified a gene, rsa1, from SL2029 that confers avirulence to SL341 in hot pepper. The rsa1 gene encoding an 11.8-kDa protein possessed the perfect consensus hrp(II) box motif upstream of the gene. Although the expression of rsa1 was activated by HrpB, a transcriptional activator for hrp gene expression, Rsa1 protein was secreted in an Hrp type III secretion-independent manner. Rsa1 exhibited weak homology with an aspartic protease, cathepsin D, and possessed protease activity. Two specific aspartic protease inhibitors, pepstatin A and diazoacetyl-d,l-norleucine methyl ester, inhibited the protease activity of Rsa1. Substitution of two aspartic acid residues with alanine at positions 54 and 59 abolished protease activity. The SL2029 rsa1 mutant was much less virulent than the wild-type strain, but did not induce disease symptoms in hot pepper. These data indicate that Rsa1 is an extracellular aspartic protease and plays an important role for the virulence of SL2029 in potato.

Visualizing cellular dynamics in plant-microbe interactions using fluorescent-tagged proteins.

Interactions between plant cells and microbial pathogens involve highly dynamic processes of cellular trafficking and reorganization. Substantial advances in imaging technologies, including the discovery and widespread use of fluorescent proteins as tags as well as advances in laser-based confocal microscopy have provided the first glimpses of the dynamic nature of the processes of defense and pathogenicity. Prior to the development of these techniques, high resolution imaging by electron microscopy gave only a static picture of these dynamic events and live cell imaging was significantly limited in resolution as well as the availability of relevant stains and markers. The incorporation of fluorescent protein fusions and laser-based confocal microscopy into studies of plant-microbe interactions has opened the door to fascinating new questions about the cellular response to attempted infection. Additionally, studies of cellular responses to pathogen infection may lead to new knowledge about fundamental processes in plant cells, such as mechanisms underlying subcellular trafficking and targeting of proteins and other molecules.

A novel light-dependent selection marker system in plants.

Photosensitizers are common in nature and play diverse roles as defense compounds and pathogenicity determinants and as important molecules in many biological processes. Toxoflavin, a photosensitizer produced by Burkholderia glumae, has been implicated as an essential virulence factor causing bacterial rice grain rot. Toxoflavin produces superoxide and H2O2 during redox cycles under oxygen and light, and these reactive oxygen species cause phytotoxic effects. To utilize toxoflavin as a selection agent in plant transformation, we identified a gene, tflA, which encodes a toxoflavin-degrading enzyme in the Paenibacillus polymyxa JH2 strain. TflA was estimated as 24.56 kDa in size based on the amino acid sequence and is similar to a ring-cleavage extradiol dioxygenase in the Exiguobacterium sp. 255-15; however, unlike other extradiol dioxygenases, Mn(2+) and dithiothreitol were required for toxoflavin degradation by TflA. Here, our results suggested toxoflavin is a photosensitizer and its degradation by TflA serves as a light-dependent selection marker system in diverse plant species. We examined the efficiencies of two different plant selection systems, toxoflavin/tflA and hygromycin/hygromycin phosphotransferase (hpt) in both rice and Arabidopsis. The toxoflavin/tflA selection was more remarkable than hygromycin/hpt selection in the high-density screening of transgenic Arabidopsis seeds. Based on these results, we propose the toxoflavin/tflA selection system, which is based on the degradation of the photosensitizer, provides a new robust nonantibiotic selection marker system for diverse plants.

A regulon conserved in monocot and dicot plants defines a functional module in antifungal plant immunity.

At least two components that modulate plant resistance against the fungal powdery mildew disease are ancient and have been conserved since the time of the monocot-dicot split (≈ 200 Mya). These components are the seven transmembrane domain containing MLO/MLO2 protein and the syntaxin ROR2/PEN1, which act antagonistically and have been identified in the monocot barley (Hordeum vulgare) and the dicot Arabidopsis thaliana, respectively. Additionally, syntaxin-interacting N-ethylmaleimide sensitive factor adaptor protein receptor proteins (VAMP721/722 and SNAP33/34) as well as a myrosinase (PEN2) and an ABC transporter (PEN3) contribute to antifungal resistance in both barley and/or Arabidopsis. Here, we show that these genetically defined defense components share a similar set of coexpressed genes in the two plant species, comprising a statistically significant overrepresentation of gene products involved in regulation of transcription, posttranslational modification, and signaling. Most of the coexpressed Arabidopsis genes possess a common cis-regulatory element that may dictate their coordinated expression. We exploited gene coexpression to uncover numerous components in Arabidopsis involved in antifungal defense. Together, our data provide evidence for an evolutionarily conserved regulon composed of core components and clade/species-specific innovations that functions as a module in plant innate immunity.

Nucleotide sequence and genomic organization of a newly identified member of the genus Carmovirus, soybean yellow mottle mosaic virus, from soybean.

The viral genome of soybean yellow mottle mosaic virus (SYMMV) from infected soybean (Glycine max) in Korea was cloned and sequenced. The complete monopartite single-stranded RNA genome of SYMMV consists of 4009 base pairs with six putative open reading frames and includes 5'- and 3'-untranslated regions of 39 and 229 nucleotides, respectively. The nucleotide and coat protein sequences of SYMMV share the highest sequence identity with those of cowpea mottle virus. Based on its genomic organization, its predicted amino acid sequence, and its phylogenetic relatedness to known carmoviruses, we report that SYMMV is a new member of the genus Carmovirus in the family Tombusviridae.

T-DNA tagged knockout mutation of rice OsGSK1, an orthologue of Arabidopsis BIN2, with enhanced tolerance to various abiotic stresses.

T-DNA-tagged rice plants were screened under cold- or salt-stress conditions to determine the genes involved in the molecular mechanism for their abiotic-stress response. Line 0-165-65 was identified as a salt-responsive line. The gene responsible for this GUS-positive phenotype was revealed by inverse PCR as OsGSK1 (Oryza sativa glycogen synthase kinase3-like gene 1), a member of the plant GSK3/SHAGGY-like protein kinase genes and an orthologue of the Arabidopsis brassinosteroid insensitive 2 (BIN2), AtSK21. Northern blot analysis showed that OsGSK1 was most highly detected in the developing panicles, suggesting that its expression is developmental stage specific. Knockout (KO) mutants of OsGSK1 showed enhanced tolerance to cold, heat, salt, and drought stresses when compared with non-transgenic segregants (NT). Overexpression of the full-length OsGSK1 led to a stunted growth phenotype similar to the one observed with the gain-of-function BIN/AtSK21 mutant. This suggests that OsGSK1 might be a functional rice orthologue that serves as a negative regulator of brassinosteroid (BR)-signaling. Therefore, we propose that stress-responsive OsGSK1 may have physiological roles in stress signal-transduction pathways and floral developmental processes.

EDR2 negatively regulates salicylic acid-based defenses and cell death during powdery mildew infections of Arabidopsis thaliana.

BACKGROUND: The hypersensitive necrosis response (HR) of resistant plants to avirulent pathogens is a form of programmed cell death in which the plant sacrifices a few cells under attack, restricting pathogen growth into adjacent healthy tissues. In spite of the importance of this defense response, relatively little is known about the plant components that execute the cell death program or about its regulation in response to pathogen attack. RESULTS: We isolated the edr2-6 mutant, an allele of the previously described edr2 mutants. We found that edr2-6 exhibited an exaggerated chlorosis and necrosis response to attack by three pathogens, two powdery mildew and one downy mildew species, but not in response to abiotic stresses or attack by the bacterial leaf speck pathogen. The chlorosis and necrosis did not spread beyond inoculated sites suggesting that EDR2 limits the initiation of cell death rather than its spread. The pathogen-induced chlorosis and necrosis of edr2-6 was correlated with a stimulation of the salicylic acid defense pathway and was suppressed in mutants deficient in salicylic acid signaling. EDR2 encodes a novel protein with a pleckstrin homology and a StAR transfer (START) domain as well as a plant-specific domain of unknown function, DUF1336. The pleckstrin homology domain binds to phosphatidylinositol-4-phosphate in vitro and an EDR2:HA:GFP protein localizes to endoplasmic reticulum, plasma membrane and endosomes. CONCLUSION: EDR2 acts as a negative regulator of cell death, specifically the cell death elicited by pathogen attack and mediated by the salicylic acid defense pathway. Phosphatidylinositol-4-phosphate may have a role in limiting cell death via its effect on EDR2. This role in cell death may be indirect, by helping to target EDR2 to the appropriate membrane, or it may play a more direct role.

Show and tell: cell biology of pathogen invasion.

Because the initial stages of pathogen invasion are often confined to a limited number of host cells, measures of host responses that are averaged over attacked and non-attacked cells provide an unsatisfactory view of these events. To identify the earliest and often transient responses to pathogen attack, there is considerable interest in monitoring the subcellular events that occur specifically in living host cells. Recent improvements in live-cell imaging using fluorescent-tagged markers have expanded the scope of the experiments that can be performed. Changes in the subcellular distribution of organelles and of fluorescently tagged proteins can be monitored in real time in living tissues during pathogen attack, and the dynamic nature of such changes across space and over time can be determined. The application of these sensitive imaging methods has extended earlier observations, made with Nomarski microscopy or inferred from static transmission electron micrographs, about the focal accumulation of subcellular organelles at sites of pathogen attack. In addition, recent experiments have demonstrated the focused accumulation and interaction of specific plant proteins at penetration sites, opening a new window on early host responses and raising questions about the underlying plant processes that sense and direct this marshalling of host resources to block pathogen entry.

Arabidopsis thaliana subcellular responses to compatible Erysiphe cichoracearum infections.

Subcellular events of Erysiphe cichoracearum infections of epidermal cells were visualized in living tissues of Arabidopsis plants carrying various green fluorescent protein (GFP)-tagged organelles via laser scanning confocal microscopy. Early in the infection sequence, cytoplasm and organelles moved towards penetration sites and accumulated near penetration pegs. Peroxisomes appeared to accumulate preferentially relative to the cytoplasm at penetration sites. Another early event, which preceded haustorium formation, was the aggregation of some GFP-tagged plasma membrane marker proteins into rings around penetration sites, which extended across cell-wall boundaries into neighboring cells. This feature localized to sites where papillae were deposited. The extrahaustorial membrane (EHM) encases the fungal feeding structure, the haustorium, separating it from the host cytoplasm. Eight plasma membrane markers were excluded from the EHM and remained in a collar-like formation around the haustorial neck. These observations support the suggestions that the EHM is a unique, specialized membrane and is different from the plasma membrane. Our results suggested two possibilities for the origin of the EHM: invagination of the plasma membrane coupled with membrane differentiation; or de novo synthesis of the EHM by targeted vesicle trafficking.

Loss-of-function mutations in chitin responsive genes show increased susceptibility to the powdery mildew pathogen Erysiphe cichoracearum.

Chitin is a major component of fungal walls and insect exoskeletons. Plants produce chitinases upon pathogen attack and chito-oligomers induce defense responses in plants, though the exact mechanism behind this response is unknown. Using the ATH1 Affymetrix microarrays consisting of about 23,000 genes, we examined the response of Arabidopsis (Arabidopsis thaliana) seedlings to chito-octamers and hydrolyzed chitin after 30 min of treatment. The expression patterns elicited by the chito-octamer and hydrolyzed chitin were similar. Microarray expression profiles for several genes were verified via northern analysis or quantitative reverse transcription-PCR. We characterized T-DNA insertion mutants for nine chito-oligomer responsive genes. Three of the mutants were more susceptible to the fungal pathogen, powdery mildew, than wild type as measured by conidiophore production. These three mutants included mutants of genes for two disease resistance-like proteins and a putative E3 ligase. The isolation of loss-of-function mutants with enhanced disease susceptibility provides direct evidence that the chito-octamer is an important oligosaccharide elicitor of plant defenses. Also, this study demonstrates the value of microarray data for identifying new components of uncharacterized signaling pathways.

AtOPT3, a member of the oligopeptide transporter family, is essential for embryo development in Arabidopsis.

A T-DNA-tagged population of Arabidopsis was screened for mutations in AtOPT3, which encodes a member of the oligopeptide (OPT) family of peptide transporters, and a recessive mutant allele, opt3, was identified. Phenotypic analysis of opt3 showed that most homozygous embryos were arrested at or before the octant stage of embryo development and that none showed the usual periclinal division leading to the formation of the protoderm. This defective phenotype could be reversed by complementation with the full-length, wild-type AtOPT3 gene. A beta-glucuronidase (GUS) fusion to DNA sequences upstream of the putative AtOPT3 ATG start codon was constructed, and the expression pattern was assayed in transgenic plants. AtOPT3 was expressed in the vascular tissues of seedlings and mature plants as well as in pollen. Consistent with the function of AtOPT3 in embryogenesis, AtOPT3::GUS expression also was detected in developing embryos and in the maternal tissues of seeds. These data suggest a critical role for peptide transport in early embryo development.

Peptide transport in plants.

Recent completion of the Arabidopsis genome revealed that this organism has ten times more peptide transporters than any other sequenced organism (prokaryote or eukaryote). These transporters are found in three protein families: the ABC-type transporters; the di- and tripeptide transporters; and the newly described tetra- and pentapeptide oligopetide transporters. The abundance of these transporters suggests that they play diverse and important roles in plant growth and development. Possible substrates for these transporters include glutathione, gamma-glutamyl peptides, hormone-amino acid conjugates, phytosulfokine, peptide-like compounds and peptide phytotoxins. However, the exact role of peptide transport in plants is still undefined.

An oligopeptide transporter gene family in Arabidopsis.

We have identified nine oligopeptide transporter (OPT) orthologs (AtOPT1 to AtOPT9) in Arabidopsis. These proteins show significant sequence similarity to OPTs of Candida albicans (CaOpt1p), Schizosaccharomyces pombe (Isp4p), and Saccharomyces cerevisiae (Opt1p and Opt2p). Hydrophilicity plots of the OPTs suggest that they are integral membrane proteins with 12 to 14 transmembrane domains. Sequence comparisons showed that the AtOPTs form a distinct subfamily when compared with the fungal OPTs. Two highly conserved motifs (NPG and KIPPR) were found among all OPT members. The identification of multiple OPTs in Arabidopsis suggests that they may play different functional roles. This idea is supported by the fact that AtOPTs have a distinct, tissue-specific expression pattern. The cDNAs encoding seven of the AtOPTs were cloned into a yeast vector under the control of a constitutive promoter. AtOPT4 expressed in S. cerevisiae mediated the uptake of KLG-[3H]L. Similarly, expression of five of the seven AtOPT proteins expressed in yeast conferred the ability to uptake tetra- and pentapeptides as measured by growth. This study provides new evidence for multiple peptide transporter systems in Arabidopsis, suggesting an important physiological role for small peptides in plants.