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Background: New technologies for the early detection of tuberculosis (TB) are urgently needed. Pathological changes within an organ might be reflected in proteomic patterns in serum. The aim of the present study was to screen for the potential protein biomarkers in serum for the diagnosis of TB using proteomic fingerprint technology., Methods: Proteomic fingerprint technology combining protein chips with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) was used to profile the serum proteins from 50 patients with TB, 25 patients with lung disease other than TB, and 25 healthy volunteers. The protein fingerprint expression of all the serum samples and the resulting profiles between TB and control groups were analyzed with the Biomarker Wizard system., Results: A total of 30 discriminating m/z peaks were detected that were related to TB (p<0.01). The model of biomarkers constructed by the Biomarker Patterns Software based on the three biomarkers (2024, 8007, and 8598 Da) generated excellent separation between the TB and control groups. The sensitivity was 84.0% and the specificity was 86.0%. Blind test data indicated a sensitivity of 80.0% and a specificity of 84.2%., Conclusions: The data suggested a potential application of SELDI-TOF MS as an effective technology to profile serum proteome, and with pattern analysis, a diagnostic model comprising three potential biomarkers was indicated to differentiate people with TB and healthy controls rapidly and precisely.

Jasmonate (JA) and ethylene (ET) are two major plant hormones that synergistically regulate plant development and tolerance to necrotrophic fungi. Both JA and ET induce the expression of several pathogenesis-related genes, while blocking either signaling pathway abolishes the induction of these genes by JA and ET alone or in combination. However, the molecular basis of JA/ET coaction and signaling interdependency is largely unknown. Here, we report that two Arabidopsis ET-stabilized transcription factors (EIN3 and EIL1) integrate ET and JA signaling in the regulation of gene expression, root development, and necrotrophic pathogen defense. Further studies reveal that JA enhances the transcriptional activity of EIN3/EIL1 by removal of JA-Zim domain (JAZ) proteins, which physically interact with and repress EIN3/EIL1. In addition, we find that JAZ proteins recruit an RPD3-type histone deacetylase (HDA6) as a corepressor that modulates histone acetylation, represses EIN3/EIL1-dependent transcription, and inhibits JA signaling. Our studies identify EIN3/EIL1 as a key integration node whose activation requires both JA and ET signaling, and illustrate transcriptional derepression as a common mechanism to integrate diverse signaling pathways in the regulation of plant development and defense.

Plant cell surface-localized receptor kinases such as FLS2, EFR, and CERK1 play a crucial role in detecting invading pathogenic bacteria. Upon stimulation by bacterium-derived ligands, FLS2 and EFR interact with BAK1, a receptor-like kinase, to activate immune responses. A number of Pseudomonas syringae effector proteins are known to block immune responses mediated by these receptors. Previous reports suggested that both FLS2 and BAK1 could be targeted by the P. syringae effector AvrPto to inhibit plant defenses. Here, we provide new evidence further supporting that FLS2 but not BAK1 is targeted by AvrPto in plants. The AvrPto-FLS2 interaction prevented the phosphorylation of BIK1, a downstream component of the FLS2 pathway.

Pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) are recognized by host cell surface-localized pattern-recognition receptors (PRRs) to activate plant immunity. PAMP-triggered immunity (PTI) constitutes the first layer of plant immunity that restricts pathogen proliferation. PTI signaling components often are targeted by various Pseudomonas syringae virulence effector proteins, resulting in diminished plant defenses and increased bacterial virulence. Some of the proteins targeted by pathogen effectors have evolved to sense the effector activity by associating with cytoplasmic immune receptors classically known as resistance proteins. This allows plants to activate a second layer of immunity termed effector-triggered immunity (ETI). Recent studies on PTI regulation and P. syringae effector targets have uncovered new components in PTI signaling. Although MAP kinase (MAPK) cascades have been considered crucial for PTI, emerging evidence indicates that a MAPK-independent pathway also plays an important role in PTI signaling.

Background: Wheat (Triticum aestivum L.) O-methyltransferase (TaOMT2) catalyzes the sequential methylation of the flavone, tricetin, to its 3'-methyl- (selgin), 3',5'-dimethyl- (tricin) and 3',4',5'-trimethyl ether derivatives. Tricin, a potential multifunctional nutraceutical, is the major enzyme reaction product. These successive methylations raised the question as to whether they take place in one, or different active sites. We constructed a 3-D model of this protein using the crystal structure of the highly homologous Medicago sativa caffeic acid/5-hydroxyferulic acid O-methyltransferase (MsCOMT) as a template with the aim of proposing a mechanism for multiple methyl transfer reactions in wheat., Results: This model revealed unique structural features of TaOMT2 which permit the stepwise methylation of tricetin. Substrate binding is mediated by an extensive network of H-bonds and van der Waals interactions. Mutational analysis of structurally guided active site residues identified those involved in binding and catalysis. The partly buried tricetin active site, as well as proximity and orientation effects ensured sequential methylation of the substrate within the same pocket. Stepwise methylation of tricetin involves deprotonation of its hydroxyl groups by a His262-Asp263 pair followed by nucleophilic attack of SAM-methyl groups. We also demonstrate that Val309, which is conserved in a number of graminaceous flavone OMTs, defines the preference of TaOMT2 for tricetin as the substrate., Conclusions: We propose a mechanism for the sequential methylation of tricetin, and discuss the potential application of TaOMT2 to increase the production of tricin as a nutraceutical. The single amino acid residue in TaOMT2, Val309, determines its preference for tricetin as the substrate, and may define the evolutionary differences between the two closely related proteins, COMT and flavone OMT.

The two-component system RhpRS was identified in Pseudomonas syringae as a regulator of the genes encoding the type III secretion system and type III effector proteins (together called the T3 genes). In the absence of the sensor kinase RhpS, the response regulator RhpR represses the induction of the T3 gene regulatory cascade consisting of hrpRS, hrpL, and the T3 genes in a phosphorylation-dependent manner. The repressor activity of RhpR is inhibited by RhpS, which presumably acts as a phosphatase under the T3 gene inducing conditions. Here, we show that RhpR binds and induces its own promoter in a phosphorylation-dependent manner. Deletion and mutagenesis analyses revealed an inverted repeat (IR) element, GTATC-N(6)-GATAC, in the rhpR promoter that confers the RhpR-dependent induction. Computational search of the P. syringae genomes for the putative IR elements and Northern blot analysis of the genes with a putative IR element in the promoter region uncovered five genes that were upregulated and two genes that were downregulated in an RhpR-dependent manner. Two genes that were strongly induced by RhpR were assayed for the IR element activity in gene regulation and, in both cases, the IR element mediated the RhpR-dependent gene induction. Chromatin immunoprecipitation assays indicated that RhpR binds the promoters containing a putative IR element but not the hrpR and hrpL promoters that do not have an IR element, suggesting that RhpR indirectly regulates the transcriptional cascade of hrpRS, hrpL, and the T3 genes.

Pathogens induce pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) in plants. PAMPs are microbial molecules recognized by host plants as nonself signals, whereas pathogen effectors are evolved to aid in parasitism but are sometimes recognized by specific intracellular resistance proteins. In the absence of detectable ETI determining classical incompatible interactions, basal resistance exists during compatible and nonhost interactions. What triggers the basal resistance has remained elusive. Here, we provide evidence that ETI contributes to basal resistance during both compatible and nonhost Arabidopsis-Pseudomonas syringae interactions. Mutations in RAR1 and NDR1, two genes required for ETI, compromise basal resistance in both compatible and nonhost interactions. Complete nonhost resistance to P. syringae pv. tabaci required a functional type III secretion system. PTI appears to play a greater role in nonhost resistance than basal resistance during compatible interactions, because abrogation of PTI compromises basal resistance during nonhost but not compatible interactions. Strikingly, simultaneous abrogation of ETI and flagellin-induced PTI rendered plants completely susceptible to the nonadapted bacterium P. syringae pv. tabaci, indicating that ETI and PTI act synergistically during nonhost resistance. Thus, both nonhost resistance and basal resistance to virulent bacteria can be unified under PTI and ETI.

The successful recognition of pathogen-associated molecular patterns (PAMPs) as a danger signal is crucial for plants to fend off numerous potential pathogenic microbes. The signal is relayed through mitogen-activated protein kinase (MPK) cascades to activate defenses. Here, we show that the Pseudomonas syringae type III effector HopF2 can interact with Arabidopsis thaliana MAP KINASE KINASE5 (MKK5) and likely other MKKs to inhibit MPKs and PAMP-triggered immunity. Inhibition of PAMP-induced MPK phosphorylation was observed when HopF2 was delivered naturally by the bacterial type III secretion system. In addition, HopF2 Arg-71 and Asp-175 residues that are required for the interaction with MKK5 are also necessary for blocking MAP kinase activation, PAMP-triggered defenses, and virulence function in plants. HopF2 can inactivate MKK5 and ADP-ribosylate the C terminus of MKK5 in vitro. Arg-313 of MKK5 is required for ADP-ribosylation by HopF2 and MKK5 function in the plant cell. Together, these results indicate that MKKs are important targets of HopF2.

Cell-surface-localized plant immune receptors, such as FLS2, detect pathogen-associated molecular patterns (PAMPs) and initiate PAMP-triggered immunity (PTI) through poorly understood signal-transduction pathways. The pathogenic Pseudomonas syringae effector AvrPphB, a cysteine protease, cleaves the Arabidopsis receptor-like cytoplasmic kinase PBS1 to trigger cytoplasmic immune receptor RPS5-specified effector-triggered immunity (ETI). Analyzing the function of AvrPphB in plants lacking RPS5, we find that AvrPphB can inhibit PTI by cleaving additional PBS1-like (PBL) kinases, including BIK1, PBL1, and PBL2. In unstimulated plants, BIK1 and PBL1 interact with FLS2 and are rapidly phosphorylated upon FLS2 activation by its ligand flg22. Genetic and molecular analyses indicate that BIK1, and possibly PBL1, PBL2, and PBS1, integrate immune signaling from multiple immune receptors. Whereas AvrPphB-mediated degradation of one of these kinases, PBS1, is monitored by RPS5 to initiate ETI, this pathogenic effector targets other PBL kinases for PTI inhibition., (Copyright 2010 Elsevier Inc. All rights reserved.)

Pathogen-associated molecular patterns (PAMPs) trigger plant defenses when perceived by surface-localized immune receptors. PAMP-triggered immunity (PTI) plays a vital role in the resistance of plants to numerous potential pathogens. MicroRNA (miRNA) biogenesis is known to be important for PTI, but miRNA species involved in this process have not been fully explored. Here we show that the Arabidopsis (Arabidopsis thaliana) miRNA effector protein, Argonaute1 (AGO1), is required for a number of PTI responses including PAMP-induced callose deposition, gene expression, and seedling growth inhibition. Deep sequencing of AGO1-bound small RNAs led to the identification of a number of miRNAs that are up- or down-regulated by flg22, a well-studied PAMP. Overexpression of selected miRNAs in stable transgenic plants demonstrated that miR160a positively regulate PAMP-induced callose deposition, whereas miR398b and miR773 negatively regulate PAMP-induced callose deposition and disease resistance to bacteria, suggesting a complexity of the miRNA regulation in plant innate immunity.

Pathogenic microbes often modulate phytohormone physiology in the host to their advantage. We previously showed that the Pseudomonas syringae effector protein AvrB perturbs hormone signaling, as exemplified by upregulated expression of jasmonic acid response genes, and enhances plant susceptibility. Here we show that these effects of AvrB require the Arabidopsis mitogen-activated protein kinase MAP kinase 4 (MPK4), HSP90 chaperone components, and the AvrB-interacting protein, RIN4. AvrB interacts with MPK4 and the HSP90 chaperone, and AvrB induces MPK4 activation in a manner promoted by HSP90; RIN4 likely acts downstream of MPK4. These findings link Arabidopsis proteins MPK4, HSP90, and RIN4 into a pathway that P. syringae AvrB activates for the benefit of the bacterium, perturbing hormone signaling and enhancing plant susceptibility., (2010 Elsevier Inc. All rights reserved.)

Phytopathogenic bacteria inject an array of effector proteins into host cells to alter host physiology and assist the infection process. Some of these effectors can also trigger disease resistance as a result of recognition in the plant cell by cytoplasmic immune receptors. In addition to effector-triggered immunity, plants immunity can be triggered upon the detection of Pathogen/Microbe-Associated Molecular Patterns by surface-localized immune receptors. Recent progress indicates that many bacterial effector proteins use a variety of biochemical properties to directly attack key components of PAMP-triggered immunity and effector-triggered immunity, providing new insights into the molecular basis of plant innate immunity. Emerging evidence indicate that the evolution of disease resistance in plants is intimately linked to the mechanism by which bacterial effectors promote parasitism. This review focuses on how these studies have conceptually advanced our understanding of plant-pathogen interactions.

Pseudomonas syringae bacteria utilize the type III secretion system (T3SS) to deliver effector proteins into host cells. The T3SS and T3 effector genes (together called the T3 genes hereafter) are repressed in nutrient-rich medium but rapidly induced after the bacteria are transferred into minimal medium or infiltrated into plants. The induction of the T3 genes is mediated by HrpL, an alternative sigma factor that recognizes the conserved hrp box motif in the T3 gene promoters. The induction of hrpL is mediated by HrpR and HrpS, two homologous proteins that bind the hrpL promoter. To identify additional genes involved in regulation of the T3 genes, we screened for the P. syringae pv. phaseolicola NPS3121 transposon-tagged mutants with reduced induction of avrPto-luc and hrpL-luc, reporter genes for promoters of effector gene avrPto and hrpL, respectively. Determination of the transposon-insertion sites revealed genes with putative functions in signal transduction and transcriptional regulation, protein synthesis, and basic metabolism. A transcriptional regulator (AefR(NPS3121)) was identified in our screen that is homologous to AefR of P. syringae pv. syringae strain B728a, a regulator of the quorum-sensing signal and epiphytic traits, but was not known to regulate the T3 genes. AefR(NPS3121) in P. syringae pv. phaseolicola NPS3121 and AefR in P. syringae pv. syringae B728a behave similarly in regulating the quorum-sensing signal in liquid medium but differ in regulating the epiphytic traits, including swarming motility, leaf entry, and epiphytic survival.

Pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) trigger plant immunity that forms the first line inducible defenses in plants. The regulatory mechanism of MAMP-triggered immunity, however, is poorly understood. Here, we show that Arabidopsis thaliana transcription factors ETHYLENE INSENSITIVE3 (EIN3) and ETHYLENE INSENSITIVE3-LIKE1 (EIL1), previously known to mediate ethylene signaling, also negatively regulate PAMP-triggered immunity. Plants lacking EIN3 and EIL1 display enhanced PAMP defenses and heightened resistance to Pseudomonas syringae bacteria. Conversely, plants overaccumulating EIN3 are compromised in PAMP defenses and exhibit enhanced disease susceptibility to Pseudomonas syringae. Microarray analysis revealed that EIN3 and EIL1 negatively control PAMP response genes. Further analyses indicated that SALICYLIC ACID INDUCTION DEFICIENT2 (SID2), which encodes isochorismate synthase required for pathogen-induced biosynthesis of salicylic acid (SA), is a key target of EIN3 and EIL1. Consistent with this, the ein3-1 eil1-1 double mutant constitutively accumulates SA in the absence of pathogen attack, and a mutation in SID2 restores normal susceptibility in the ein3 eil1 double mutant. EIN3 can specifically bind SID2 promoter sequence in vitro and in vivo. Taken together, our data provide evidence that EIN3/EIL1 directly target SID2 to downregulate PAMP defenses.

Phytopathogens invade into plant apoplast and proliferate by assimilating nutrition from plant cells. Plants depend on sophisticated defensive strategies to resist this invasion. Therefore, pathogenic disease and plant disease resistance are two opposite phases. Fascinating molecular mechanisms uncovered that interactions between plant and pathogen are multilevel and dynamic processes. On one side, plant immunity system contains multiple layers mainly including the perception of common pathogen- associated molecular patterns (PAMPs) using distinct cell-surface pattern-recognition receptors (PRRs) to activate intracellular signaling pathways for broad-spectrum immunity, and the recognition of pathogen virulence proteins by the specific intracellular disease resistance (R) proteins for cultivar-specific immunity. On the opposite side, the bacterial pathogens employ virulence factors, such as phytotoxin and type III effectors (T3SEs) to interfere with the host immunity in different levels. Meanwhile, natural selection drives plants and pathogens to evolve new strategies to confront with each other constantly. The present review highlights recent insights about Arabidopsis immunity and mechanisms for Pseudomonas syringae to counteract this immunity to give a full understanding of plant-pathogen interactions.

A wheat (Triticum aestivum L., near isogenic line of Hamlet) O-methyltransferase (OMT) was previously reported as a putative caffeic acid OMT (TaCOMT1), involved in lignin biosynthesis, based on its high sequence similarity with a number of graminaceous COMTs. The fact that the putative TaCOMT1 exhibits a significantly high sequence homology to another recently characterized wheat flavone-specific OMT (TaOMT2), and that molecular modeling studies indicated several conserved amino acid residues involved in substrate binding and catalysis of both proteins, prompted an investigation of its appropriate substrate specificity. We report here that TaCOMT1 exhibits highest preference for the flavone tricetin, and lowest activity with the lignin precursors, caffeic acid/5-hydroxyferulic acid as the methyl acceptor molecules, indicating that it is not involved in lignin biosynthesis. We recommend its reannotation to a flavone-specific TaOMT1 that is distinct from TaOMT2.

Surface water and shallow groundwater within the flow of an electroplating factory was analyzed in order to study the resulting impact. The analysis method of ICP-AES was used to analyze content of zinc, manganese, chromium, copper and nickel in surface water and groundwater samples. The results indicate acidic pollutants of zinc, manganese, chromium, copper and nickel were discharged from the factory with concentrations of 1.34, 3.77, 28.1, 6.40 and 9.37 mg x L(-1), respectively; and pH was 2.32. They all exceeded permissible levels according to Integrated Wastewater Discharge Standard except zinc. Factory discharge is responsible for the longitudinal distribution characteristics of heavy metals in the stream water downstream from the factory. Heavy metals variations in the well water do not suggest they were affected by heavy metals in the stream, indicating that the migration rates of heavy metals in soils were relatively low. Risk assessment shows surface water quality significantly deteriorated. Nickel and manganese in the stream water exceeded the standard levels seriously, and chromium and copper in some samples were also above Grade III standard levels according to Environmental Quality Standard for Surface Water. Moreover, all studied heavy metals in 14 groundwater samples measured within drinking water standard, except manganese in 4 groundwater samples, which were Grade IV according to Quality Standard for Ground water.

Determination the bioavailability of contaminants in different fertilizer management soils can be of great theoretical significance for predicting and decreasing the effect of pollutants on eco-environment. In this study, three different fertilization soils were employed in a 16-year field experiment: no fertilization (CK), composted wheat straw and oil cakes (OM) and NPK fertilizer (NPK). The influences of Cu2+ (100 mg/kg), Cd2+ (5 mg/kg) and cypermethrin (l0 mg/kg) and their combinations on microbial functional diversity were observed in different fertilization soils. The results showed that the highest microbial functional diversity was observed in the NPK treatment, followed by CK, and the lowest in the OM soil. At 72 h incubation, AWCD (average well color development) of the NPK soil was 1.19 times than that of the CK soil and 1.62 times than that of the OM soil. After addition of contaminants into soils, microbial functional diversity decreased to a different extent. The greatest decrease extent was recorded in the NPK soil, followed by CK and the least in OM. Combination of Cd and cypermethrin had the biggest influence on the microbial functional diversity. AWCD was decreased by 48%, six substrate groups and microbial diversity indices inhibited significantly in this treatment of the NPK soil. Although cypermethrin had little effect on soil microbial utilization of carbon source,utilization potential decreased greatly in combination pollution treatments, especially in combination with Cd2+. The effect of Cu2+, Cd2+ and cypermethrin and their combinations on soil microbes not only affected by the soil organic matter content, likely also by the constitution of organic matter and microbial community.

Bacterial effectors are double-edged swords that enhance bacterial virulence in susceptible plants while trigger resistance in plants carrying cognate resistance proteins. A well-known example of this is Pseudomonas syringae protein AvrPto that is delivered into plant cells through the type III secretion system. AvrPto inhibits immune responses in Arabidopsis plants but triggers resistance in some tomato plants carrying cognate resistance proteins Pto, a serine/threonine kinase, and Prf, a nucleotide-binding leucine-rich repeat protein. In a recent structural study we showed that AvrPto is an inhibitor of the Pto protein kinase. Because Pto closely resemble the kinase domain of receptor kinases, which include pattern recognition receptors (PRRs) crucial for plants to detect invading pathogens, we tested the possibility that PRRs such as FLS2 and EFR are targeted by AvrPto in susceptible plants. Indeed, AvrPto is capable of binding the FLS2 and EFR kinases to block plant immune responses when expressed in protoplasts. In Arabidopsis plants containing FLS2, the P. syringae strain lacking avrPto is compromised in its ability to multiply. However, the defect of the avrPto-deletion strain was alleviated in fls2 plants, indicating a role of AvrPto in overcoming FLS2-mediated resistance. Interestingly, the FLS2-AvrPto and Pto-AvrPto interactions share significant similarity, raising the tantalizing possibility that Pto has evolved as a molecular decoy of the intended targets of AvrPto.

Infrared photoacoustic spectroscopy (PAS) is a new style to obtain data based on photoacoustic theory. Photoacoustic thoeory is based on the absorption of electromagnetic radiation by analyte molecules, and the absorbed energy is measured by detecting pressure fluctuations in the form of sound waves or shock pulses. In contrast to conventional absorption spectroscopy, PAS allows the determination of absorption coefficients over several orders of magnitude, even in very black and strongly scattering soil samples. Red soil, fulvic soil and paddy soil were collected from Fengqiu National Ecological Experimental Station, Yingtan Red Soil Experimental Station, and Changshu Ecological Experimental Station, respectively. These soil samples were used as experimental materials to characterize the Fourier transform mid-infrared photoacoustic spectra (FTIR-PAS). Compared with infrared transmittance spectra and reflectance spectra, the testing of FTIR-PAS spectra was very fast and convenient without any pr-treatment, and there were more abundant absorptions as well as appropriate absorption values in the spectra; The main soil components (kaolin, bentonite, sand and CaCO3) also showed several strong absorptions with specific characteristics in the spectra; Further more, the interference of water with the PAS spectra was significantly smaller than that with reflectance spectra. Therefore, the soil properties could be better characterized by FTIR-PAS. The principal components analysis based on the FTIR-PAS spectra indicated that there were two main principal components (PCA1, PCA2) which contained 98.17% variance of the spectra, and the two-dimensionol distribution was made by plotting these two principal components to classify the soil type, and the results indicated that this distribution could be applied to distinguish soil type, which provided new technique for soil identification as well as further quantitative analysis in soil science.

Wheat flavone O-methyltransferase catalyzes three sequential methylations of the flavone tricetin. Like other flavonoid O-methyltransferases, the protein is a homodimer. We demonstrate, using analytical ultracentrifugation, that perchlorate dissociates the dimer into monomers. The resulting monomers retain all their catalytic capacity, including the ability to catalyze the three successive methylations. We show, using isothermal titration calorimetry, that the binding constant for S-adenosyl-L-methionine does not change significantly as the protein dissociates. The second substrate, tricetin, binds to the dimers but could not be tested with the monomers. CD, UV and fluorescence spectroscopy show that there are substantial changes in the structure of the protein as it dissociates. The fact that there are differences between the monomers and dimers even as the monomers maintain activity may be the result of the very low catalytic capacity of this enzyme. Maximal turnover numbers for the dimers and monomers are only about 6-7 per minute. Even though the binding pockets for S-adenosyl-L-methionine, tricetin, selgin and tricin are intact, selection of a catalytically competent structure may be a very slow step during catalysis.

Like animals, plants sense bacterial pathogens through surface-localized pattern recognition receptors (PRRs) and intracellular nucleotide-binding leucine-rich repeat proteins (NB-LRR) and trigger defense responses. Many plant-pathogenic bacteria secrete a large repertoire of effector proteins into host cells to modulate host responses, enabling successful infection and multiplication in plants. A number of these effector proteins target plant innate immunity signaling pathways, while others induce specific host genes to enhance plant susceptibility. Substantial progress has been made in the past two years concerning biochemical function of effectors and their host targets. These advances provide new insights into regulatory mechanisms of plant immunity and host-pathogen co-evolution.

The development of sensitive and versatile techniques to detect protein-protein interactions in vivo is important for understanding protein functions. The previously described techniques, fluorescence resonance energy transfer and bimolecular fluorescence complementation, which are used widely for protein-protein interaction studies in plants, require extensive instrumentation. To facilitate protein-protein interaction studies in plants, we adopted the luciferase complementation imaging assay. The amino-terminal and carboxyl-terminal halves of the firefly luciferase reconstitute active luciferase enzyme only when fused to two interacting proteins, and that can be visualized with a low-light imaging system. A series of plasmid constructs were made to enable the transient expression of fusion proteins or generation of stable transgenic plants. We tested nine pairs of proteins known to interact in plants, including Pseudomonas syringae bacterial effector proteins and their protein targets in the plant, proteins of the SKP1-Cullin-F-box protein E3 ligase complex, the HSP90 chaperone complex, components of disease resistance protein complex, and transcription factors. In each case, strong luciferase complementation was observed for positive interactions. Mutants that are known to compromise protein-protein interactions showed little or much reduced luciferase activity. Thus, the assay is simple, reliable, and quantitative in detection of protein-protein interactions in plants.

Plants use receptor kinases, such as FLS2 and EFR, to perceive bacterial pathogens and initiate innate immunity. This immunity is often suppressed by bacterial effectors, allowing pathogen propagation. To counteract, plants have evolved disease resistance genes that detect the bacterial effectors and reinstate resistance. The Pseudomonas syringae effector AvrPto promotes infection in susceptible plants but triggers resistance in plants carrying the protein kinase Pto and the associated resistance protein Prf. Here we show that AvrPto binds receptor kinases, including Arabidopsis FLS2 and EFR and tomato LeFLS2, to block plant immune responses in the plant cell. The ability to target receptor kinases is required for the virulence function of AvrPto in plants. The FLS2-AvrPto interaction and Pto-AvrPto interaction appear to share similar sequence requirements, and Pto competes with FLS2 for AvrPto binding. The results suggest that the mechanism by which AvrPto recognizes virulence targets is linked to the evolution of Pto, which, in association with Prf, recognizes the bacterium and triggers strong resistance.

Salmonella SpvC belongs to a new enzyme family designated phosphothreonine lyases that irreversibly inactivate mitogen-activated protein kinases. The crystal structure of SpvC reported here reveals that the two phosphorylated residues in the substrate peptide predominantly mediate its recognition by SpvC. Substrate-induced conformational changes in SpvC sequester the phosphothreonine in a completely solvent-free environment, preventing the hydrolysis of the phosphate group and facilitating the elimination reaction.

Pathogenic microbes use effectors to enhance susceptibility in host plants. However, plants have evolved a sophisticated immune system to detect these effectors using cognate disease resistance proteins, a recognition that is highly specific, often elicits rapid and localized cell death, known as a hypersensitive response, and thus potentially limits pathogen growth. Despite numerous genetic and biochemical studies on the interactions between pathogen effector proteins and plant resistance proteins, the structural bases for such interactions remain elusive. The direct interaction between the tomato protein kinase Pto and the Pseudomonas syringae effector protein AvrPto is known to trigger disease resistance and programmed cell death through the nucleotide-binding site/leucine-rich repeat (NBS-LRR) class of disease resistance protein Prf. Here we present the crystal structure of an AvrPto-Pto complex. Contrary to the widely held hypothesis that AvrPto activates Pto kinase activity, our structural and biochemical analyses demonstrated that AvrPto is an inhibitor of Pto kinase in vitro. The AvrPto-Pto interaction is mediated by the phosphorylation-stabilized P+1 loop and a second loop in Pto, both of which negatively regulate the Prf-mediated defences in the absence of AvrPto in tomato plants. Together, our results show that AvrPto derepresses host defences by interacting with the two defence-inhibition loops of Pto.

The environmental risk of chelating agents such as EDTA application to the heavy metals polluted soils and the stress on plant roots due to the abrupt increase metals concentration limit the wide commercial use of chelate-induced phytoextraction. Chelating agent ethylenediaminetetraacetic acid (EDTA) and nitrilotriacetic acid (NTA) and auxin indole-3-acetic acid (IAA) were used for enhancing heavy metals uptake from soils by Zea mays L. (corn) in pot experiments. The metals content in plant tissues was quantified using an inductively coupled plasma mass spectrometer (ICP-MS). The results showed that the combination of IAA and EDTA increased the biomass by about 40.0% and the contents of Cu, Zn, Cd and Pb in corn shoots by 27.0%, 26.8%, 27.5% and 32.8% respectively, as compared to those in EDTA treatment. While NTA&IAA treatment increased the biomass by about 29.9% and the contents of Cu, Zn, Cd and Pb in corn shoots by 31.8%, 27.6%, 17.0% and 26.9% respectively, as compared to those in NTA treatment. These results indicated that corn growth was promoted, and the biomass and the accumulation of heavy metals in plant shoots were increased significantly with the addition of IAA, which probably helps to change the cell membrane properties and the biomass distribution, resulting in the alleviation of the phytotoxicity of metals and the chelating agents.

The bacterial Lon protease participates in a variety of biological processes. In Pseudomonas syringae, mutation of lon is known to activate hrpL and a few hrpL-regulated genes in rich medium. The elevated expression of hrpL and hrpL-regulated genes results from increased stability of HrpR, the transcriptional activator of hrpL, in the lon mutant. Here, we conducted a microarray analysis to identify genes that are differentially expressed in a lon- mutant of P. syringae pv. tomato DC3000 grown in the rich medium King's B (KB). Most genes induced in the lon- mutant belong to the HrpL regulon or are related to transcription, protein synthesis, and energy metabolism. A major group of genes reduced in the lon- mutant are related to cell wall biogenesis. The HrpL-regulated genes exhibit different induction patterns in the lon- mutant, suggesting that additional regulators other than HrpL are likely to be involved in regulation of these genes. Compared with the wild-type bacteria, lon- mutants of P. syringae pv. tomato DC3000 and P. syringae pv. phaseolicola NPS3121 strains exhibit elevated hrpL expression in KB medium, but reduced hrpL expression in minimal medium (MM). The reduced hrpL RNA is correlated with reduced hrpR and hrpS RNAs, suggesting that the Lon-mediated regulation of hrpL involves different mechanisms in KB and MM. The lon- mutation also reduced bacterial pathogenicity.

Pathogen-associated molecular patterns (PAMPs) elicit basal defense responses in plants, and, in turn, pathogens have evolved mechanisms to overcome these PAMP-induced defenses. To suppress immunity, the phytopathogenic bacterium Pseudomonas syringae secretes effector proteins, the biochemical function and virulence targets of which remain largely unknown. We show that HopAI1, an effector widely conserved in both plant and animal bacterial pathogens, inhibits the Arabidopsis mitogen-activated protein kinases (MAPKs) activated by exposure to PAMPs. HopAI1 inactivates MAPKs by removing the phosphate group from phosphothreonine through a unique phosphothreonine lyase activity, which is required for HopAI1 function. The inhibition of MAPKs by HopA1 suppresses two independent downstream events, namely the reinforcement of cell wall defense and transcriptional activation of PAMP response genes. The MAPKs MPK3 and MPK6 physically interact with HopAI1 indicating that they are direct targets of HopAI1. These findings uncover a mechanism by which Pseudomonas syringae overcomes host innate immunity to promote pathogenesis.

The Pseudomonas syringae type III secretion system (T3SS) is induced during interaction with the plant or culture in minimal medium (MM). How the bacterium senses these environments to activate the T3SS is poorly understood. Here, we report the identification of a novel two-component system (TCS), RhpRS, that regulates the induction of P. syringae T3SS genes. The rhpR and rhpS genes are organized in an operon with rhpR encoding a putative TCS response regulator and rhpS encoding a putative biphasic sensor kinase. Transposon insertion in rhpS severely reduced the induction of P. syringae T3SS genes in the plant as well as in MM and significantly compromised the pathogenicity on host plants and hypersensitive response-inducing activity on nonhost plants. However, deletion of the rhpRS locus allowed the induction of T3SS genes to the same level as in the wild-type strain and the recovery of pathogenicity upon infiltration into plants. Overexpression of RhpR in the deltarhpRS deletion strain abolished the induction of T3SS genes. However, overexpression of RhpR in the wild-type strain or overexpression of RhpR(D70A), a mutant of the predicted phosphorylation site of RhpR, in the deltarhpRS deletion strain only slightly reduced the induction of T3SS genes. Based on these results, we propose that the phosphorylated RhpR represses the induction of T3SS genes and that RhpS reverses phosphorylation of RhpR under the T3SS-inducing conditions. Epistasis analysis indicated that rhpS and rhpR act upstream of hrpR to regulate T3SS genes.

Pathogenic bacteria use the type III secretion system to deliver effector proteins into host cells to modulate the host signaling pathways. In this study, the Shigella type III effector OspF was shown to inactivate mitogen-activated protein kinases (MAPKs) [extracellular signal-regulated kinases 1 and 2 (Erk1/2), c-Jun N-terminal kinase, and p38]. OspF irreversibly removed phosphate groups from the phosphothreonine but not from the phosphotyrosine residue in the activation loop of MAPKs. Mass spectrometry revealed a mass loss of 98 daltons in p-Erk2, due to the abstraction of the alpha proton concomitant with cleavage of the C-OP bond in the phosphothreonine residue. This unexpected enzymatic activity, termed phosphothreonine lyase, appeared specific for MAPKs and was shared by other OspF family members.

Increasing atmospheric CO2 concentration is generally expected to enhance photosynthesis and growth of agricultural C3 vegetable crops, and therefore results in an increase in crop yield. However, little is known about the combined effect of elevated CO2 and N species on plant growth and development. Two growth-chamber experiments were conducted to determine the effects of NH4+/NO3- ratio and elevated CO2 concentration on the physiological development and water use of tomato seedlings. Tomato was grown for 45 d in containers with nutrient solutions varying in NH4+/NO3- ratios and CO2 concentrations in growth chambers. Results showed that plant height, stem thickness, total dry weight, dry weight of the leaves, stems and roots, G value (total plant dry weight/seedling days), chlorophyll content, photosynthetic rate, leaf-level and whole plant-level water use efficiency and cumulative water consumption of tomato seedlings were increased with increasing proportion of NO3- in nutrient solutions in the elevated CO2 treatment. Plant biomass, plant height, stem thickness and photosynthetic rate were 67%, 22%, 24% and 55% higher at elevated CO2 concentration than at ambient CO2 concentration, depending on the values of NH4+/NO3- ratio. These results indicated that elevating CO2 concentration did not mitigate the adverse effects of 100% NH4(+)-N (in nutrient solution) on the tomato seedlings. At both CO2 levels, NH4+/NO3- ratios of nutrient solutions strongly influenced almost every measure of plant performance, and nitrate-fed plants attained a greater biomass production, as compared to ammonium-fed plants. These phenomena seem to be related to the coordinated regulation of photosynthetic rate and cumulative water consumption of tomato seedlings.

Pathogenic bacterial effectors suppress pathogen-associated molecular pattern (PAMP)-triggered host immunity, thereby promoting parasitism. In the presence of cognate resistance genes, it is proposed that plants detect the virulence activity of bacterial effectors and trigger a defense response, referred to here as effector-triggered immunity (ETI). However, the link between effector virulence and ETI at the molecular level is unknown. Here, we show that the Pseudomonas syringae effector AvrB suppresses PAMP-triggered immunity (PTI) through RAR1, a co-chaperone of HSP90 required for ETI. AvrB expressed in plants lacking the cognate resistance gene RPM1 suppresses cell wall defense induced by the flagellar peptide flg22, a well known PAMP, and promotes the growth of nonpathogenic bacteria in a RAR1-dependent manner. rar1 mutants display enhanced cell wall defense in response to flg22, indicating that RAR1 negatively regulates PTI. Furthermore, coimmunoprecipitation experiments indicated that RAR1 and AvrB interact in the plant. The results demonstrate that RAR1 molecularly links PTI, effector virulence, and ETI. The study supports that both pathogen virulence and plant disease resistance have evolved around PTI.

The type III secretion system (TTSS) is a specialized protein secretion machinery used by numerous gram-negative bacterial pathogens of animals and plants to deliver effector proteins directly into the host cells. In plant-pathogenic bacteria, genes encoding the TTSS were discovered as hypersensitive response and pathogenicity (hrp) genes, because mutation of these genes typically disrupts the bacterial ability to cause diseases on host plants and to elicit hypersensitive response on nonhost plants. The hrp genes and the type III effector genes (collectively called TTSS genes hereafter) are repressed in nutrient-rich media but induced when bacteria are infiltrated into plants or incubated in nutrient-deficient inducing media. Multiple regulatory components have been identified in the plant-pathogenic bacteria regulating TTSS genes under various conditions. In Ralstonia solanacearum, several signal transduction components essential for the induction of TTSS genes in plants are dispensable for the induction in inducing medium. In addition to the inducing signals, recent studies indicated the presence of negative signals in the plant regulating the Pseudomonas syringae TTSS genes. Thus, the levels of TTSS gene expression in plants likely are determined by the interactions of multiple signal transduction pathways. Studies of the hrp regulons indicated that TTSS genes are coordinately regulated with a number of non-TTSS genes.

Flavonoid compounds are ubiquitous in nature. They constitute an important part of the human diet and act as active principles of many medicinal plants. Their O-methylation increases their lipophilicity and hence, their compartmentation and functional diversity. We have isolated and characterized a full-length flavonoid O-methyltransferase cDNA (TaOMT2) from a wheat leaf cDNA library. The recombinant TaOMT2 protein was purified to near homogeneity and tested for its substrate preference against a number of phenolic compounds. Enzyme assays and kinetic analyses indicate that TaOMT2 exhibits a pronounced preference for the flavone, tricetin and gives rise to three methylated enzyme reaction products that were identified by TLC, HPLC and ESI-MS/MS as its mono-, di- and trimethyl ether derivatives. The sequential order of tricetin methylation by TaOMT2 is envisaged to proceed via its 3'-mono--->3',5'-di--->3',4',5'-trimethyl ether derivatives. To our knowledge, this is the first report of a gene product that catalyzes three sequential O-methylations of a flavonoid substrate.

The HopPtoF locus in Pseudomonas syringae pv. tomato DC3000 harbors two genes, ShcF and HopF2 (previously named ShcF(Pto) and HopF(Pto)), that encode a type III chaperone and a cognate effector protein, respectively. The HopF2 gene has a rare initiation codon, ATA that was reported to be functional only in mitochondrial genes. Here, we report that the native HopPtoF locus of DC3000 confers an avirulence function in tobacco W38 plants, indicating that the ATA start codon directs the synthesis of a functional effector. However, disruption of HopF2 in DC3000 genome did not alter the bacterial virulence in tomato plants. The HopPtoF locus displayed a measurable virulence activity in two strains of P. syringae pv. tomato when the ATA start codon was changed to ATG, and this change also elevated the avirulence function in W38 plants. HopF2 contains a putative myristoylation site. Mutational analysis indicated that this site is required for plasma membrane localization and virulence and avirulence activities of HopF2.

The differential centrifugation method was used to study the subcellular distribution of Cu and Zn in the roots, stems and leaves of corn (zea mays) growing on multi-metal contaminated soil with the addition of chelator Nitrilotriacetic Acid (NTA). The results show that the subcellular distributions of Cu and Zn have significant relationship with the ability of metal uptake and accumulation in corn. NTA could evidently promote the uptake and accumulation of Cu and Zn in corn and affect on their distribution in cell wall and vacuole. Most of Cu was bound to the cell wall fraction and secondly cytoplasm fraction and only a small quantity of Cu bound to organelle fractions. For Zn, however, most of Zn was bound to cytoplasm fraction and there was more Zn bound to organelles than Cu. Under the inducement of NTA, there were increasing Cu and Zn bound to cytoplasm fraction, which mostly came from cell wall fraction and partly came from organelle fractions.

Arabidopsis NONHOST1 (NHO1) is required for limiting the in planta growth of nonhost Pseudomonas bacteria but completely ineffective against the virulent bacterium Pseudomonas syringae pv. tomato DC3000. However, the molecular basis underlying this observation remains unknown. Here we show that NHO1 is transcriptionally activated by flagellin. The nonhost bacterium P. syringae pv. tabaci lacking flagellin is unable to induce NHO1, multiplies much better than does the wild-type bacterium, and causes disease symptoms on Arabidopsis. DC3000 also possesses flagellin that is potent in NHO1 induction, but this induction is rapidly suppressed by DC3000 in a type III secretion system-dependent manner. Direct expression of DC3000 effectors in protoplasts indicated that at least nine effectors, HopS1, HopAI1, HopAF1, HopT1-1, HopT1-2, HopAA1-1, HopF2, HopC1, and AvrPto, are capable of suppressing the flagellin-induced NHO1 expression. One of the effectors, HopAI1, is conserved in both animal and plant bacteria. When expressed in transgenic Arabidopsis plants, HopAI1 promotes growth of the nonpathogenic hrpL- mutant bacteria. In addition, the purified phytotoxin coronatine, a known virulence factor of P. syringae, suppresses the flagellin-induced NHO1 transcription. These results demonstrate that flagellin-induced defenses play an important role in nonhost resistance. A remarkable number of DC3000 virulence factors act in the plant cell by suppressing the species level defenses, and that contributes to the specialization of DC3000 on Arabidopsis.

The angiogenic mechanism and therapeutic potential of PDGF-CC, a recently discovered member of the VEGF/PDGF superfamily, remain incompletely characterized. Here we report that PDGF-CC mobilized endothelial progenitor cells in ischemic conditions; induced differentiation of bone marrow cells into ECs; and stimulated migration of ECs. Furthermore, PDGF-CC induced the differentiation of bone marrow cells into smooth muscle cells and stimulated their growth during vessel sprouting. Moreover, delivery of PDGF-CC enhanced postischemic revascularization of the heart and limb. Modulating the activity of PDGF-CC may provide novel opportunities for treating ischemic diseases.

The type III effector protein AvrPto acts as a virulence factor in susceptible plants lacking a cognate resistance gene but triggers hypersensitive response and disease resistance in tomato plants carrying the Pto gene or in tobacco plants carrying an unknown resistance gene. To assist the characterization of cellular responses caused by AvrPto in the plant, a pathogen-free system was adopted to isolate genes up-regulated 12 h after induced expression of AvrPto. By using subtraction cloning and transgenic tobacco plants expressing avrPto as a transgene, we isolated 125 nonredundant cDNA clones that represent avrPto-response genes (ARG). In addition to genes that are known to be induced by Pto-avrPto recognition, a number of new genes were also isolated. Most of ARG showed a specific induction in tobacco plants challenged with incompatible or nonhost pathogens. The use of an avrPto mutant that selectively eliminated the avrPto recognition in tobacco demonstrated that the ARG were induced in a highly specific manner by the avirulence, instead of the virulence activity of avrPto.

Pseudomonas syringae relies on type III secretion system to deliver effector proteins into the host cell for parasitism. Type III genes are induced in planta, but host factors affecting the induction are poorly understood. Here we report on the identification of an Arabidopsis mutant, att1 (for aberrant induction of type three genes), that greatly enhances the expression of bacterial type III genes avrPto and hrpL. att1 plants display enhanced disease severity to a virulent strain of P. syringae, suggesting a role of ATT1 in disease resistance. ATT1 encodes CYP86A2, a cytochrome P450 monooxygenase catalyzing fatty acid oxidation. The cutin content is reduced to 30% in att1, indicating that CYP86A2 plays a major role in the biosynthesis of extracellular lipids. att1 has a loose cuticle membrane ultrastructure and shows increased permeability to water vapor, demonstrating the importance of the cuticle membrane in controlling water loss. The enhanced avrPto-luc expression is specific to att1, but not another cuticle mutant, wax2. The results suggest that certain cutin-related fatty acids synthesized by CYP86A2 may repress bacterial type III gene expression in the intercellular spaces.

In this article, influence of monocalcium phosphate and potassium chloride application on nitrogen leaching in paddy soil of Taihu region was studied by soil column leaching. The results showed that nitrate nitrogen was the main component of nitrogen leaching without urea application which accounts for 93.39 percent of total nitrogen leaching (including urea, ammonium, and nitrate nitrogen). Amounts of urea, ammonium, and nitrate nitrogen in leachates increased significantly after urea application which account for 3.95, 15.25, and 80.80 percent of total nitrogen leaching respectively and for 0.26, 0.80, and 2.54 percent of applied nitrogen respectively. Amounts of urea, ammonium, and nitrate nitrogen in leachates were enhanced prominently by monocalcium phosphate or potassium chloride which are 4.54, 24.11, 71.34, and 3.45, 24.53, 72.02 percent of total nitrogen leaching respectively, and which are 0.39, 1.86, 3.34, and 0.32, 2.12, 4.06 percent of applied nitrogen separately. There is interactive influence on nitrogen leaching after application of monocalcium phosphate and potassium chloride together, and amounts of urea, ammonium, and nitrate nitrogen were 4.10, 27.35, and 68.55 percent of total nitrogen leaching, and 0.42, 2.60, and 4.26 percent of total applied nitrogen respectively. The leaching order of different form nitrogen followed as urea N > ammonium N > nitrate N, and leaching amounts of different form nitrogen followed as urea N < ammonium N < nitrate N.

A survey was made of the arbuscular mycorrhizal (AM) status of five dominant wild plants Tamarix chinensis, Phragmites communis, Suaeda glauca, Aeluropus littoralis var. sinensis and Cirsium setosum in saline-alkaline soils of the Yellow River Delta that show low plant diversity. All of the species were colonized and showed typical AM structures (arbuscules, vesicles). The colonization percentage ranged from 0.2% to 9.5%, where C. setosum was the highest. The species richness of AMF at the different sites ranged from 2.00 to 2.40 per 50 ml soil, with an average of 2.16. Species diversity ranged from 1.99 to 2.22 per 50 ml soil, with an average of 2.13. Spore density ranged from 3 to 30 per 50 ml soil, with an average of 12. Glomus was the dominant genus, with a frequency and relative abundance of 88.1% and 68.4%, respectively. G. caledonium, with a frequency and relative abundance of 15.0% and 4.6%, respectively, was the dominant species. Differences were also observed in the distribution of AMF in different soil layers. Although there were still AM fungal spores in the layer 40 cm below the surface, most spores were found at a depth of 0-40 cm.

Gram-negative bacteria use a variety of virulence factors including phytotoxins, exopolysaccharides, effectors secreted by the type III secretion system, and cell-wall-degrading enzymes to promote parasitism in plants. However, little is known about how these virulence factors alter plant cellular responses to promote disease. In this study, we show that virulent Pseudomonas syringae strains activate the transcription of an Arabidopsis ethylene response factor (ERF) gene, RAP2.6, in a coronatine insensitive 1 (COI1)-dependent manner. A highly sensitive RAP2.6 promoter-firefly luciferase (RAP2.6-LUC) reporter line was developed to monitor activities of various bacterial virulence genes. Analyses of P. syringae pv. tomato DC3000 mutants indicated that both type III secretion system and the phytotoxin coronatine are required for RAP2.6 induction. We show that at least five individual type III effectors, avirulence B (AvrB), AvrRpt2, AvrPphB, HopPtoK, and AvrPphEPto, contributed to RAP2.6 induction. Gene-for-gene recognition was not involved in RAP2.6 induction because plants lacking RPM1 and RPS2 responded normally to AvrB and AvrRpt2 in RAP2.6 expression. Interestingly, the role of coronatine in RAP2.6 induction can be partially substituted by the addition of avrB in DC3000, suggesting that AvrB may mimic coronatine. These results suggest that P. syringae type III effectors and coronatine act by augmenting a COI1-dependent pathway to promote parasitism.

Hexaploid wheat (Triticum aestivum) contains triplicated genomes derived from three distinct species. To better understand how different genomes are coordinated in the same nucleus of the hexaploid wheat, we globally compared gene expression of a synthetic hexaploid wheat with its diploid (Aegilops tauschii) and tetraploid (T. turgidum) parents by cDNA-AFLP display. The results suggested that the expression of a significant fraction of genes was altered in the synthetic hexaploid; most appeared to be diminished and some were activated. We characterized nine cDNA clones in details. Cytogenetic as well as genomic sequence analyses indicated that the gene silencing was not due to chromosome/DNA loss but was caused by gene regulation. Northern and RT-PCR divided these genes into three groups: (I) four genes were down-regulated nonspecifically, likely involving both parental orthologues; (II) four genes were down-regulated in an orthologue-dependent manner; (III) one gene was activated specifically in the synthetic hexaploid wheat. These genes were often altered non-randomly in different synthetic hexaploids as well as natural hexaploid wheat, suggesting that many of the gene expression changes were intrinsically associated with polyploidy.

It is poorly understood why a particular plant species is resistant to the vast majority of potential pathogens that infect other plant species, a phenomenon referred to as "nonhost" resistance. Here, we show that Arabidopsis NHO1, encoding a glycerol kinase, is required for resistance to and induced by Pseudomonas syringae isolates from bean and tobacco. NHO1 is also required for resistance to the fungal pathogen Botrytis cinerea, indicating that NHO1 is not limited to bacterial resistance. Strikingly, P. s. pv. tomato DC3000, an isolate fully virulent on Arabidopsis, actively suppressed the NHO1 expression. This suppression is abolished in coi1 plants, indicating that DC3000 required an intact jasmonic acid signaling pathway in the plant to suppress NHO1 expression. Constitutive overexpression of NHO1 led to enhanced resistance to this otherwise virulent bacterium. The presence of avrB in DC3000, which activates a cultivar-specific "gene-for-gene" resistance in Arabidopsis, restored the induction of NHO1 expression. Thus, NHO1 is deployed for both general and specific resistance in Arabidopsis and targeted by the bacterium for parasitism.

Pto confers disease resistance to Pseudomonas syringae pv tomato carrying the cognate avrPto gene. Overexpression of Pto under the cauliflower mosaic virus 35S promoter activates spontaneous lesions and confers disease resistance in tomato (Lycopersicon esculentum) plants in the absence of avrPto. Here, we show that these AvrPto-independent defenses require a functional Prf gene. Several Pto-interacting (Pti) proteins are thought to play a role in Pto-mediated defense pathways. To test if interactions with Pti proteins are required for the AvrPto-independent defense responses by Pto overexpression, we isolated several Pto mutants that were unable to interact with one or more Pti proteins, but retained normal interaction with AvrPto. Overexpression of two mutants, Pto(G50S) and Pto(R150S), failed to activate AvrPto-independent defense responses or confer enhanced resistance to the virulent P. s. pv tomato. When introduced into plants carrying 35S::Pto, 35S::Pto(G50S) dominantly suppressed the AvrPto-independent resistance caused by former transgene. 35S::Pto(G50S) also blocked the induction of a number of defense genes by the wild-type 35S::Pto. However, 35S::Pto(G50S) and 35S::Pto(R150S) plants were completely resistant to P. s. pv tomato (avrPto), indicating a normal gene-for-gene resistance. Furthermore, 35S::Pto(G50S) plants exhibited normal induction of defense genes in recognition of avrPto. Thus, the AvrPto-independent defense activation and gene-for-gene resistance mediated by Pto are functionally separable.

SUMMARY Wheat leaf rust disease, caused by the biotrophic fungus Puccinia triticina, is a result of complex interactions requiring the coordinated activities of the two organisms involved. In an effort to understand the molecular basis of wheat-rust interactions, we isolated and characterized cDNA corresponding to in planta induced fungal genes (PIGs) from susceptible wheat leaves infected with P. triticina by using suppression subtractive hybridization to construct a cDNA library. 350 clones were sequenced, of which 104 were unique. Forty-four cDNA clones encode ribosomal proteins, comprising the single largest category of clones isolated. Twenty-five of these ribosomal protein genes are likely to be of fungal origin, as was suggested by sequence homology. Hybridization of 56 selected non-ribosomal protein clones to rust germling cDNA or genomic DNA probes showed that at least 44 were of fungal origin, demonstrating that the library was highly enriched for fungal cDNA. Differential expression analysis identified 26 non-ribosomal protein genes that were induced in rust-infected leaves. At least 21 of the induced genes were from the rust fungus, indicating that the majority of the induced genes were rust PIGs that are likely to play a role in parasitism. Some of the induced genes share homology to known PIGs or virulence genes in other fungi, suggesting similarities in parasitism among different fungi. Eight clones correspond to novel PIGs that have not been reported in any organism. This paper reports, for the first time, the isolation of P. triticina PIGs and discusses the use of total rust genomic DNA to identify the source of genes.

Tomato plants overexpressing the disease resistance gene Pto (35S::Pto) exhibit spontaneous cell death, accumulation of salicylic acid (SA), elevated expression of pathogenesis-related genes, and enhanced resistance to a broad range of pathogens. Because salicylate plays an important role in the cell death and defense activation in many lesion mimic mutants, we investigated the interaction of SA-mediated processes and the 35S::Pto-mediated defense pathway by introducing the nahG transgene that encodes salicylate hydroxylase. Here, we show that SA is not required for the 35S::Pto-activated microscopic cell death and plays a minor role in defense gene activation and general disease resistance in 35S::Pto plants. In contrast, temperature greatly affects the spontaneous cell death and general resistance in 35S::Pto plants, and high temperature inhibits the cell death. The NahG tomato plants develop spontaneous, unconstrained necrotic lesions on leaves. These lesions also are initiated by the inoculation of a virulent strain of Pseudomonas syringae pv. tomato. However, the NahG-dependent necrotic lesions are inhibited in the NahG/35S::Pto plants. This inhibition is most pronounced under conditions favoring the 35S::Pto-mediated spontaneous cell death development. These results indicate that the signaling pathways activated by Pto overexpression suppress the cellular damage that is caused by SA depletion. We also found that ethylene is dispensable for the 35S::Pto-mediated general defense.