Rhizogenic Fe–C redox cycling: a hypothetical biogeochemical mechanism that drives crustal weathering in upland soils

Field-scale observations of two upland soils derived from contrasting granite and basalt bedrocks are presented to hypothesize that redox activity of rhizospheres exerts substantial effects on mineral dissolution and colloidal translocation in many upland soils. Rhizospheres are redox-active microsites and in the absence of O₂, oxidation of rhizodeposits can be coupled by reduction of redox-active species such as Fe, a biogenic reduction that leads to Fe translocation and oxidation, accompanied by substantial proton flux. Not only do rhizogenic Fe–C redox cycles demonstrate a process by which the rhizosphere affects an environment well outside the near-root zone, but these redox processes are also hypothesized to be potent weathering systems, such that rhizogenic redox-reactions complement acid- and ligand-promoted reactions as major biogeochemical processes that control crustal weathering. The potential significance of Fe–C redox cycling is underscored by the deep and extensive rooting and mottling of upland subsoils across a wide range of plant communities, lithologies, and soil-moisture and temperature regimes.     

Expression and immunoaffinity purification of recombinant dengue virus 2 NS1 protein as a cleavable SUMOstar fusion

Dengue virus (DENV) encoded nonstructural one (NS1) is a 352 amino acid protein that exists in multiple oligomeric states and is conserved within the flavivirus family. Although NS1 has been heavily researched for its diagnostic utility, there is a gap in the understanding of its role in a range of viral processes, including replication and development of clinical pathologies such as vascular leakage. Many of these functions involve unknown interactions with viral and host proteins. This study describes the generation of a mouse monoclonal antibody (mAb 56.2) that reacts with NS1 from DENV1 and 2, and the expression of recombinant SUMOstar-tagged DENV2 NS1 (DENV2 S^∗-NS1) in baculovirus. This is the first time dengue NS1 has been produced as a SUMOstar fusion with the S^∗-tag increasing protein solubility and secretion compared with a non-S^∗-tagged NS1 construct. The protein was readily purified using a mAb 56.2 immunoaffinity column and untagged NS1 was obtained by treatment with tobacco etch virus protease to remove the S^∗-tag. Size exclusion chromatography and glycosylation assays showed that both secreted S^∗-NS1, and cleaved NS1, are hexameric and glycosylated, and will be useful tools in elucidating dengue NS1 protein interactions and functions.     

Extracellular Interaction between hCD98 and the PDZ Class II Domain of hCASK in Intestinal Epithelia

The extracellular domain of the glycoprotein-associated integrin hCD98 protrudes into the basolateral extracellular space of the intestine and contains a PDZ class II-binding domain (GLLLRFPYAA, amino acids 520-529). Protein-protein interaction studies in vitro as well as in human colonic sections and Caco2-BBE cells have revealed that hCD98 coimmunoprecipitated with the basolateral membrane-associated guanylate kinase hCASK and that this interaction occurred in a PDZ domain-dependent manner. These novel results, which provide the first evidence for a PDZ domain-dependent interaction between a membrane protein and an extracellular protein, open a new field of investigation related to extracellular signaling in cell biology.     

Activation and manipulation of host responses by a Gram-positive bacterium

The interaction between tomato plants and Clavibacter michiganensis subsp. michiganensis (Cmm) represents a model pathosystem to study the interplay between the virulence determinants of a Gram-positive bacterium and the attempt of a crop plant to counteract pathogen invasion. To investigate plant responses activated during this compatible interaction, we recently analyzed gene expression profiles of tomato stems infected with Cmm. This analysis revealed activation of basal defense responses that are typically observed upon plant perception of pathogen-associated molecular patterns. In addition, Cmm infection upregulated the expression of host genes related to ethylene synthesis and response. Further analysis of tomato plants impaired in ethylene perception and production demonstrated an important role for ethylene in the development of disease symptoms. Here we discuss possible molecular strategies used by the plant to recognize Cmm infection and possible mechanisms employed by the pathogen to interfere with the activation of plant defense responses and promote disease.Key words: tomato, Clavibacter michiganensis subsp. michiganensis, ethylene, basal defense, pathogen-associated molecular patternsLittle is known on the strategies employed by Gram-positive phytopathogenic bacteria to sense the presence of the host plant, penetrate and colonize tissue, and counteract plant defense responses. Also largely unexplored are the molecular mechanisms associated with detection of Gram-positive bacteria by the host plant and with the activation of attempted defense responses.Among the most devastating Gram-positive disease agents are actinobacteria of the genus Clavibacter whose subspecies cause systemic infections of the xylem in different plant species.¹ The subspecies Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker of tomato (Solanum lycopersicum), an economically important disease causing yield losses worldwide.¹ In recent years important insight into the molecular mechanism of Cmm pathogenicity has been achieved,¹ and genome sequence of a Cmm strain has been established.² Major Cmm pathogenicity determinants are plasmid borne and include the β-1,4-endocellulase CelA,³ and the putative serine protease Pat-1.⁴ Additional genes important for virulence are located in a pathogenicity island of about 129 kb on the Cmm chromosome which has a relatively low G + C content and is required for effective Cmm colonization of tomato plants.²Tomato is an economically important crop amenable to genetic analysis and transformations. Many resources are available for this plant species, including germplasm collections, natural and induced mutants, an extensive expressed sequence tag database and an ongoing genome sequencing project.⁵ In addition, because of its experimental tractability, tomato plants have been widely used to study plant disease resistance and susceptibility. As genetic and molecular tools for both Cmm and tomato are in place, the tomato-Cmm pathosystem represents an excellent model to study the interplay between virulence determinants of a Gram-positive phytopathogenic bacterium and defense responses of a crop plant.To get insight into host responses occurring during the tomato-Cmm compatible interaction and molecular mechanisms associated with the development of wilt and canker disease symptoms, we recently analyzed gene expression profiles of tomato stems infected with Cmm.⁶ This analysis revealed a clear activation of basal defense responses, which are typically observed upon plant perception of pathogen-associated molecular patterns (PAMPs).⁷ These include production and scavenging of free oxygen radicals, induction of defense-related genes, enhanced protein turnover, and hormone biosynthesis. Interestingly, several tomato genes encoding proteins with characteristics of cell-surface receptors were differentially expressed in response to Cmm infection.⁶ These proteins can be considered as candidate receptors for Cmm PAMPs and include two receptor-like kinases, a homolog of the receptor for the fungal PAMP ethylene-inducing xylanase from Trichoderma viride,⁸ and the Ve1 resistance protein, which confers resistance in tomato to the vascular disease Verticillium wilt.⁹It remains to be elucidated what are the Cmm PAMPs perceived by tomato plants. Cold-shock protein from Gram-positive bacteria and different microbial patterns of Gram-negative bacteria, including lipopolysaccharides, flagellin, and the translational elongation factor EF-TU, were shown to act as PAMPs in plants.¹⁰ Similarly, Cmm cold shock protein or cell wall components, such as peptidoglycan, lipoteichoic acid, and lipopeptides, which function as Gram positive-derived PAMPs in animal systems¹¹, may act as PAMPs during the tomato-Cmm interaction. Additional possible Cmm PAMPs are exopolysaccharides, which are produced in large amounts by the bacterium and may interact directly with surface-exposed plant proteins.¹ The numerous extracellular cell wall degrading enzymes secreted by Cmm may also function as PAMPs, as observed for the fungal ethylene-inducing xylanase.^2,12 Alternatively, by virtue of their hydrolytic activity, these enzymes may release plant cell wall fragments that are recognized by PAMP receptors. Indeed, different β-glucan fragments released from plant cell walls were shown to elicit plant basal defense responses.^13,14How Cmm copes with the activation of basal defense responses is largely unknown. Many potential virulence determinants that might interfere with the plant defense reaction are clustered in the Cmm pathogenicity island, which is essential for effective plant colonization.² Several extracellular serine proteases are encoded in this region and inactivation of part of them by gene replacement drastically reduced Cmm colonization of tomato plants.² Although their targets are still unknown, these proteins might interfere with plant signaling pathways as it was described for certain cysteine proteases that serves in Gram-negative bacteria as suppressors of plant defenses.¹⁵ An additional candidate for interference with plant signaling may be a tomatinase, also encoded in the Cmm pathogenicity island, because hydrolysis products of α-tomatine were shown to suppress plant defense responses in a fungal system.¹⁶In addition to detecting the activation of basal defense responses, host gene expression profiling during the tomato-Cmm interaction unraveled the involvement of ethylene in disease development.⁶ In fact, Cmm infection of tomato stems was found to induce expression of host genes related to ethylene biosynthesis and response (Fig. 1).⁶ Further analysis of ethylene-insensitive Never ripe mutants and transgenic plants with reduced ethylene synthesis indicated that ethylene is required for normal development of wilting symptoms (Fig. 2), but not for the activation of defense-related genes or bacterial colonization.⁶ We hypothesize that during infection ethylene synthesis and response are manipulated by Cmm virulence determinants to promote disease. Alternatively, ethylene is released as part of the host responses activated by bacterial recognition, or as a result of tissue maceration. In line with our first hypothesis, the type III effectors AvrPto and AvrPtoB from Pseudomonas syringae pv. tomato were shown to promote enhanced disease symptoms in tomato leaves, in part, by upregulating genes involved in ethylene production.¹⁷ Interestingly, expression in tomato plants of AvrPto or AvrPtoB, and infection with Cmm resulted in the upregulation of the SlACO1 gene encoding the key enzyme of ethylene biosynthesis ACC oxidase.^6,17Open in a separate windowFigure 1Kinetics of ACC oxidase (ACO) gene expression in tomato plants inoculated with Cmm. Six-week-old tomato plants were infected with a Cmm suspension (10⁸ cfu/ml) or mock-inoculated. Total RNA was extracted from stem samples harvested at the indicated day post-inoculation (dpi) and subjected to Northern blot analysis using as probe a 550 bp fragment of the SlACO1 gene, which shares high homology with other ACO family members (upper). Ethidium bromide staining shows the amount of RNA loaded in each lane (lower).Open in a separate windowFigure 2Effect of impaired ethylene sensitivity on development of wilt symptoms in tomato plants infected with Cmm. Six-week-old plants were infected with a Cmm suspension (10⁸ cfu/ml) and examined for development of wilt symptoms during a 20-day period. The percentage of plants showing wilt symptoms was calculated in a group of at least 30 plants for the ethylene-insensitive mutant Never ripe and wild-type Pearson plants. Data are representative of two independent experiments.In conclusion, future research challenges for understanding how host responses are regulated by the plant and manipulated by a Gram-positive bacterium will be the isolation of Cmm PAMPs and their plant receptors, the identification of Cmm virulence determinants and the elucidation of their mode of action.     

Characterization of resistance mechanism in transgenic Nicotiana benthamiana containing Turnip crinkle virus coat protein

Two transgenic lines, of Nicotiana benthamiana expressing Turnip crinkle virus (TCV)-coat protein (CP) gene with contrasting phenotype, the highest (#3) and the lowest (#18) CP expressers, were selected and challenged with the homologous TCV. The former, the highest expresser, showed nearly five times more CP expression than the latter. Progenies of #3 and #18 lines showed 30 and 100% infection rates, respectively. The infected progenies of #3 line showed mild and delayed symptom with TCV. This is a coat protein-mediated resistance (CP-MR), and its resistance level is directly proportional to CP transgene expression. However, CP-MR of the transgenic plants was specific only for TCV but not for heterologous viruses. Newly growing leaves of those infected progenies of #3 line did not show any visible symptoms at 4-week post-inoculation (wpi) with TCV, suggesting a reversal from infection. This was confirmed by RT-PCR analysis with the disappearance of the target at 4 wpi. This is a case of RNA-mediated resistance, and a threshold level of transgene expression may be needed to achieve the silent state. To confirm the RNA silencing, we infiltrated Agrobacterium carrying TCV-CP into leaves of progenies of #3 and performed RT-PCR analysis. The results indicate that TCV-CP’s suppressor activity against RNA silencing itself can be silenced by the homologous expression of TCV-CP in the transgenic plants. The transgenic plants containing TCV-CP seem to be a model system to study viral protection mediated by a combination of protein and RNA silencing. Ayyappan Vasudevan and Tae-Kyun Oh have contributed equally in this study.     

Peptide inhibitors of dengue virus NS3 protease. Part 2: SAR study of tetrapeptide aldehyde inhibitors

With the aim of discovering potent and selective dengue NS3 protease inhibitors, we systematically synthesized and evaluated a series of tetrapeptide aldehydes based on lead aldehyde 1 (Bz-Nle-Lys-Arg-Arg-H, K(i)=5.8 microM). In general, we observe that interactions of P(2) side chain are more important than P(1) followed by P(3) and P(4). Tripeptide and dipeptide aldehyde inhibitors also show low micromolar activity. Additionally, an effective non-basic, uncharged replacement of P(1) Arg is identified.     

Lipotropes promote immunobiochemical plasticity and protect fish against low-dose pesticide-induced oxidative stress

An experiment was conducted to evaluate the role of different lipotropes in modulating immunity and biochemical plasticity under conditions of sublethal low-dose pesticide-induced stress in fish. Labeo rohita fish fingerlings were divided in two sets with one set of fish continuously exposed to low-dose endosulfan (1/10th of 96-h LC₅₀) for 21 days, the other was unexposed, and both sets of fish were fed with practical diets supplemented with either 2 % lecithin, 0.5 % betaine, or 0.1 % choline and compared against unsupplemented diet. Low-dose endosulfan exposure had adverse effects (P < 0.05/P < 0.01) on hematological profile (erythrocyte count, hemoglobin, and hematocrit), serum protein (total protein, albumin, and globulin) and lipid profile (cholesterol and triglyceride), anti-oxidative status (ascorbic acid content of muscle, liver, brain, and kidney and activity of anti-oxidative enzymes: catalase and superoxide dismutase), neurotransmission (acetylcholinesterase activity in muscle and brain), immunological attributes (WBC count, albumin to globulin ratio, phagocytic activity, and serum cortisol), and metabolic plasticity as revealed from enzyme activities (muscle lactate dehydrogenase, liver and kidney glucose-6-phosphatase dehydrogenase-G6PDH activity). Dietary lipotropes prevented these effects completely or partially and the effects were lipotrope dependent. Kinetics (maximum velocity value V_max, catalytic efficiency and Michaelis constant K_m) of G6PDH enzyme from crude extracts of liver and kidney indicated inhibition due to endosulfan but lipotropes could protect enzyme and showed a stabilizing effect. The supplements also helped maintain integrity of histoarchitecture of the hepatocytes in endosulfan-exposed fish to a great extent. Feeding lipotropes to fish reared in endosulfan-free water also improved hematological and serum protein and lipid profiles and were immunostimulatory. In conclusion, dietary lipotropes, especially betaine and lecithin at the levels used, improve erythropoiesis, serum protein and lipid profile, anti-oxidant status, immunocompetence, neurotransmission, and protect the livers of L. rohita fingerlings even when continuously exposed to low-dose endosulfan.