Function of a mitogen-activated protein kinase pathway in N gene-mediated resistance in tobacco

The active defense of plants against pathogens often includes rapid and localized cell death known as hypersensitive response (HR). Protein phosphorylation and dephosphorylation are implicated in this event based on studies using protein kinase and phosphatase inhibitors. Recent transient gain-of-function studies demonstrated that the activation of salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two tobacco mitogen-activated protein kinases (MAPKs) by their upstream MAPK kinase (MAPKK), NtMEK2 leads to HR-like cell death. Here, we report that the conserved kinase interaction motif (KIM) in MAPKKs is required for NtMEK2 function. Mutation of the conserved basic amino acids in this motif, or the deletion of N-terminal 64 amino acids containing this motif significantly compromised or abolished the ability of NtMEK2DD to activate SIPK/WIPK in vivo. These mutants were also defective in interacting with SIPK and WIPK, suggesting protein-protein interaction is required for the functional integrity of this MAPK cascade. To eliminate Agrobacterium that is known to activate a number of defense responses in transient transformation experiments, we generated permanent transgenic plants. Induction of NtMEK2DD expression by dexamethasone induced HR-like cell death in both T1 and T2 plants. In addition, by using PVX-induced gene silencing, we demonstrated that the suppression of all three known components in the NtMEK2-SIPK/WIPK pathway attenuated N gene-mediated TMV resistance. Together with previous report that SIPK and WIPK are activated by TMV in a gene-for-gene-dependent manner, we conclude that NtMEK2-SIPK/WIPK pathway plays a positive role in N gene-mediated resistance, possibly through regulating HR cell death.     

Breeding tobacco for disease resistance

Resistance toward eight fungal, four virus, two nematode and one parasitic seed-plant disease have almost exclusively occupied the attention of tobacco breeders, and improvement in quality has so far received only negligible consideration.     

Expression of the baculovirus p35 protein in tobacco affects cell death progression and compromises N gene-mediated disease resistance response to Tobacco mosaic virus

The p35 protein from baculovirus is a broad-range caspase inhibitor and suppresses programmed cell death in animals. We report here the effects of transgenic expression in tobacco of the p35 protein during the hypersensitive response (HR). Expression of p35 causes partial inhibition of nonhost HR triggered by bacteria and gene-for-gene HR triggered by virus. Infection of p35-expressing tobacco plants with Tobacco mosaic virus (TMV) disrupts N-mediated disease resistance, causing systemic spreading of the virus within a resistant background. Mutant variants altered in aspartate residues within the loop region of p35 are inefficient substrates for caspases in vitro, and they do not suppress caspase proteolytic activity in animal systems. Tobacco plants expressing these mutant variants of the p35 protein do not show inhibition of HR cell death or enhanced virus systemic movement. Thus, HR inhibition and TMV systemic spreading phenotype in p35-expressing plants correlate with the ability of the p35 protein to suppress caspase activity in animal systems. In addition, a C-terminal truncated variant of p35 is unable to suppress cell death in animals as well as HR cell death in transgenic tobacco. Our results provide evidence for the participation of caspase-like proteases during the HR. In addition, they suggest that timely activation of cell death is necessary for effective TMV containment within the primary infection site.     

Lipid droplet binding and oligomerization properties of the Parkinson's disease protein alpha-synuclein.

alpha-Synuclein is a major component of the fibrillary lesion known as Lewy bodies and Lewy neurites that are the pathologic hallmarks of Parkinson's disease (PD). In addition, point mutations in the alpha-synuclein gene imply alpha-synuclein dysfunction in the pathology of inherited forms of PD. alpha-Synuclein is a member of a family of proteins found primarily in the brain and is concentrated within presynaptic terminals. Here, we address the localization and membrane binding characteristics of wild type and PD mutants of alpha-synuclein in cultured cells. In cells treated with high concentrations of fatty acids, wild type alpha-synuclein accumulated on phospholipid monolayers surrounding triglyceride-rich lipid droplets and was able to protect stored triglycerides from hydrolysis. PD mutant synucleins showed variable distributions on lipid droplets and were less effective in regulating triglyceride turnover. Chemical cross-linking demonstrated that synuclein formed small oligomers within cells, primarily dimers and trimers, that preferentially associated with lipid droplets and cell membranes. Our results suggest that the initial phases of synuclein aggregation may occur on the surfaces of membranes and that pathological conditions that induce cross-linking of synuclein may enhance the propensity for subsequent synuclein aggregation.     

Gastrodia anti-fungal protein from the orchid Gastrodia elata confers disease resistance to root pathogens in transgenic tobacco

Diseases of agricultural crops are caused by pathogens from several higher-order phylogenetic lineages including fungi, straminipila, eubacteria, and metazoa. These pathogens are commonly managed with pesticides due to the lack of broad-spectrum host resistance. Gastrodia anti-fungal protein (GAFP; gastrodianin) may provide a level of broad-spectrum resistance due to its documented anti-fungal activity in vitro and structural similarity to insecticidal lectins. We transformed tobacco (Nicotiana tabacum cv. Wisconsin 38) with GAFP-1 and challenged transformants with agriculturally important plant pathogens from several higher-order lineages including Rhizoctonia solani (fungus), Phytophthora nicotianae (straminipile), Ralstonia solanacearum (eubacterium), and Meloidogyne incognita (metazoan). Quantitative real-time PCR and western blotting analysis indicated that GAFP-1 was transcribed and translated in transgenic lines. When challenged by R. solani and P. nicotianae, GAFP-1 expressing lines had reduced symptom development and improved plant vigor compared to non-transformed and empty vector control lines. These lines also exhibited reduced root galling when challenged by M. incognita. Against R. solanacearum expression of GAFP-1 neither conferred resistance, nor exacerbated disease development. These results indicate that heterologous expression of GAFP-1 can confer enhanced resistance to a diverse set of plant pathogens and may be a good candidate gene for the development of transgenic, root-disease-resistant crops.     

Hantavirus nucleocapsid protein oligomerization

Hantaviruses are enveloped, negative-strand RNA viruses which can be lethal to humans, causing either a hemorrhagic fever with renal syndrome or a hantaviral pulmonary syndrome. The viral genomes consist of three RNA segments: the L segment encodes the viral polymerase, the M segment encodes the viral surface glycoproteins G1 and G2, and the S segment encodes the nucleocapsid (N) protein. The N protein is a 420- to 430-residue, 50-kDa protein which appears to direct hantavirus assembly, although mechanisms of N protein oligomerization, RNA encapsidation, budding, and release are poorly understood. We have undertaken a biochemical and genetic analysis of N protein oligomerization. Bacterially expressed N proteins were found by gradient fractionation to associate not only as large multimers or aggregates but also as dimers or trimers. Chemical cross-linking of hantavirus particles yielded N protein cross-link products with molecular masses of 140 to 150 kDa, consistent with the size of an N trimer. We also employed a genetic, yeast two-hybrid method for monitoring N protein interactions. Analyses showed that the C-terminal half of the N protein plus the N-terminal 40 residues permitted association with a full-length N protein fusion. These N-terminal 40 residues of seven different hantavirus strains were predicted to form trimeric coiled coils. Our results suggest that coiled-coil motifs contribute to N protein trimerization and that nucleocapsid protein trimers are hantavirus particle assembly intermediates.     

NRG1, a CC-NB-LRR protein, together with N, a TIR-NB-LRR protein, mediates resistance against tobacco mosaic virus

In animals and plants, innate immunity is regulated by nucleotide binding domain and leucine-rich repeat (NB-LRR) proteins that mediate pathogen recognition and that activate host-cell defense responses. Plant NB-LRR proteins, referred to as R proteins, have amino-terminal domains that contain a coiled coil (CC) or that share similarity with animal Toll and interleukin 1 receptors (TIR). To investigate R protein function, we are using the TIR-NB-LRR protein N that mediates resistance against tobacco mosaic virus (TMV) through recognition of the TMV p50 protein. Here, we describe N requirement gene 1 (NRG1), a novel N-resistance component that was identified by a virus-induced gene silencing (VIGS) screen of a cDNA library. Surprisingly, NRG1 encodes an NB-LRR type R protein that, in contrast to N, contains a CC rather than a TIR domain. Our findings support emerging evidence that many disease-resistance pathways each recruit more than a single NB-LRR protein. The results also indicate that, in addition to the previously recognized role in elicitor recognition, NB-LRR proteins may also function in downstream signaling pathways.     

Modification of disease resistance of tobacco callus tissues by cytokinins

The effects of differing cytokinin and auxin concentrations on resistance of tobacco (Nicotiana tabacum L.) tissue cultures to race 0 of Phytophthora parasitica var. nicotianae were examined. With 1 micromolar kinetin and either 11.5 micromolar indoleacetic acid or 1 micromolar 2,4-dichlorophen-oxyacetic acid, tissues from resistant cultivars exhibited a “hypersensitive” reaction to zoospores of the fungus and subsequently were colonized only slightly. With susceptible cultivars or with tissues from resistant cultivars supplied with higher cytokinin levels (e.g. 10 micromolar kinetin), this hypersensitive reaction did not occur and tissues were heavily colonized. Benzylaminopurine and kinetin were particularly effective in eliminating both the hypersensitive reaction and disease resistance. Zeatin and 6-(3-methyl-2-butenylamino)purine were less effective. Increases in indoleacetic acid levels reversed the effects of high cytokinin concentrations. The balance of phytohormones apparently controls the host response to the fungus; thus, in this system, resistance or susceptibility can be studied without changing either host or fungal genotype.     

Bacterial lipopolysaccharides as inducers of disease resistance in tobacco.

The cell wall component of Pseudomonas solanacearum that induces disease resistance in tobacco was highly heat stable at neutral or alkaline pH but highly labile at acid pH. Activity was unaffected by nucleases and proteases but destroyed by a mixture of beta-glycosidases. Washing of bacterial cell walls released a lipopolysaccharide (LPS) fraction with high inducer activity. Purified LPS, extracted by a variety of procedures from whole cells, isolated cell walls, and culture filtrates of both smooth and rough forms of P. solanacearum, induced disease resistance in tobacco at concentrations as low as 50 microgram/ml. The LPS from the non-plant pathogens Escherichia coli B, E. coli K, and Serratia marcescens was also active. Cell wall protein, free phospholipid, and nucleic acids were not necessary for activity. Moreover, since LPS from rough forms was active, the O-specific polysaccharide of the LPS was not required for activity. Hydrolysis of the remaining core-lipid A linkage or deacylation of lipid A destroyed inducer activity. When injected into tobacco leaves, purified LPS attached to tobacco mesophyll cell walls and induced ultrastructural changes in the host cell similar to those induced by attachment of whole heat-killed bacteria.     

Bacterial lipopolysaccharides as inducers of disease resistance in tobacco.

The cell wall component of Pseudomonas solanacearum that induces disease resistance in tobacco was highly heat stable at neutral or alkaline pH but highly labile at acid pH. Activity was unaffected by nucleases and proteases but destroyed by a mixture of beta-glycosidases. Washing of bacterial cell walls released a lipopolysaccharide (LPS) fraction with high inducer activity. Purified LPS, extracted by a variety of procedures from whole cells, isolated cell walls, and culture filtrates of both smooth and rough forms of P. solanacearum, induced disease resistance in tobacco at concentrations as low as 50 microgram/ml. The LPS from the non-plant pathogens Escherichia coli B, E. coli K, and Serratia marcescens was also active. Cell wall protein, free phospholipid, and nucleic acids were not necessary for activity. Moreover, since LPS from rough forms was active, the O-specific polysaccharide of the LPS was not required for activity. Hydrolysis of the remaining core-lipid A linkage or deacylation of lipid A destroyed inducer activity. When injected into tobacco leaves, purified LPS attached to tobacco mesophyll cell walls and induced ultrastructural changes in the host cell similar to those induced by attachment of whole heat-killed bacteria.     

Field resistance of transgenic burley tobacco lines and hybrids expressing the tobacco vein mottling virus coat protein gene

Fifty transgenic lines expressing the tobacco vein mottling virus (TVMV) coat protein (CP) gene in five genetic backgrounds were evaluated under field conditions for response to mechanic inoculation with TVMV, tobacco etch virus (TEV) and potato virus Y (PVY). TVMV CP transgenic lines conferred resistance to TVMV, TEV and PVY under field conditions. Combining two strategies, coat protein-mediated resistance (CPMR) coupled with an endogenous resistance gene (Virgin A Mutant, VAM) significantly extended the range and magnitude of virus resistance and provided a potential valuable new source of protection against potyviruses. CP transgenic lines lacking the VAM gene had high resistance to TEV, medium resistance to PVY, and a recovery phenotype to TVMV. A series of hybrids involving transgenic lines were generated and tested under field conditions for response to virus inoculation. One copy of TVMV-CP gene presented in lines homozygous for the VAM gene provided effective resistance to all three potyviruses. These studies also suggested that selection of a suitable recipient genotype was critical and that field evaluation was necessary in order to select elite resistant transgenic lines. Engineering viral CP genes into genotypes possessing some level of virus resistance could be critical to achieve an effective level of resistance.     

Adaptor protein controlled oligomerization activates the AAA+ protein ClpC

The AAA+ protein ClpC is not only involved in the removal of misfolded and aggregated proteins but also controls, through regulated proteolysis, key steps of several developmental processes in the Gram-positive bacterium Bacillus subtilis. In contrast to other AAA+ proteins, ClpC is unable to mediate these processes without an adaptor protein like MecA. Here, we demonstrate that the general activation of ClpC is based upon the ability of MecA to participate in the assembly of an active and substrate-recognizing higher oligomer consisting of ClpC and the adaptor protein, which is a prerequisite for all activities of this AAA+ protein. Using hybrid proteins of ClpA and ClpC, we identified the N-terminal and the Linker domain of the first AAA+ domain of ClpC as the essential MecA interaction sites. This new adaptor-mediated mechanism adds another layer of control to the regulation of the biological activity of AAA+ proteins.     

Expression of a novel small antimicrobial protein from the seeds of motherwort (Leonurus japonicus) confers disease resistance in tobacco

Medicinal plants are valuable resources of natural antimicrobial materials. A novel small protein with antimicrobial activities, designated LJAMP1, was purified from the seeds of a medicinal herb, motherwort (Leonurus japonicus Houtt). LJAMP1 is a heat-stable protein with a molecular mass of 7.8 kDa and a determined isoelectric point of 8.2. In vitro assays showed that LJAMP1 inhibits the growth of an array of fungi and bacteria. The hyphal growth inhibition by LJAMP1 was more evident against hyphomycete fungi, such as Alternaria alternata, Cercospora personata, and Aspergillus niger. The N-terminal amino acid sequence of LJAMP1 was determined, and its coding gene was consequently cloned by the rapid amplification of cDNA ends. The gene LJAMP1 has no intron and encodes a polypeptide of 95 amino acids, in which the first 27 residues was deduced as a signal peptide. The mature LJAMP1 shows relatively low identity to plant napin-like storage proteins. Northern blot assays revealed that LJAMP1 is expressed preferentially in seeds. Bioassays in transgenic tobacco demonstrated that that overexpression of LJAMP1 significantly enhanced the resistance of tobacco against not only the fungal pathogen A. alternata but also the bacterial pathogen Ralstonia solanacearum, while no visible alteration in plant growth and development was observed.     

Genetic noise control via protein oligomerization

Background

Biochemical equilibria are usually modeled iteratively: given one or a few fitted models, if there is a lack of fit or over fitting, a new model with additional or fewer parameters is then fitted, and the process is repeated. The problem with this approach is that different analysts can propose and select different models and thus extract different binding parameter estimates from the same data. An alternative is to first generate a comprehensive standardized list of plausible models, and to then fit them exhaustively, or semi-exhaustively.

Results

A framework is presented in which equilibriums are modeled as pairs (g, h) where g = 0 maps total reactant concentrations (system inputs) into free reactant concentrations (system states) which h then maps into expected values of measurements (system outputs). By letting dissociation constants K _d be either freely estimated, infinity, zero, or equal to other K _d , and by letting undamaged protein fractions be either freely estimated or 1, many g models are formed. A standard space of g models for ligand-induced protein dimerization equilibria is given. Coupled to an h model, the resulting (g, h) were fitted to dTTP induced R1 dimerization data (R1 is the large subunit of ribonucleotide reductase). Models with the fewest parameters were fitted first. Thereafter, upon fitting a batch, the next batch of models (with one more parameter) was fitted only if the current batch yielded a model that was better (based on the Akaike Information Criterion) than the best model in the previous batch (with one less parameter). Within batches models were fitted in parallel. This semi-exhaustive approach yielded the same best models as an exhaustive model space fit, but in approximately one-fifth the time.

Conclusion

Comprehensive model space based biochemical equilibrium model selection methods are realizable. Their significance to systems biology as mappings of data into mathematical models warrants their development.     

转小麦冷胁迫蛋白截短基因烟草及其抗病性分析

TA3-13是克隆于小麦冷胁迫蛋白基因的截短片段。原核表达的TA3-13蛋白能够诱导烟草产生显著的抗烟草花叶病毒(TMV)的作用。文章将TA3-13基因片段克隆到植物表达载体pBI121上,构建成转基因重组体pB-3-13,通过冻融法转化农杆菌EHA105,构建成转基因侵染菌株。采用叶盘法将pB-3-13转化三生烟草,经卡那霉素抗性筛选,获得48株T0代再生植株。通过PCR检测,鉴定出33株转基因单株,收获了20株种子作为T1代株系。PCR-Southern杂交结果显示,PCR阳性条带与TA3-13探针有特异性杂交,说明外源基因被转化到烟草的基因组中。选取两个T1代株系的烟草植株用于各项测定。GUS组织化学活性鉴定和RT-PCR检测结果显示,外源基因可以成功地表达。接种TMV病毒后,转基因烟草抗TMV的能力较转空载体烟草提高3～5倍。转基因烟草具有抗TMV侵入和抗病毒病害发展的作用,同时转基因烟草可以抗细菌软腐病菌的扩展。     

Grb7 protein RA domain oligomerization

The growth factor receptor bound protein 7 (Grb7) is an adaptor protein that is often coamplified with the erythroblastosis oncogene B 2 receptor in 20% to 30% of breast cancer patients. Grb7 overexpression has been linked to increased cell migration and cancer metastasis. The ras associating and pleckstrin homology domain region of Grb7 has been reported to interact with various other downstream signaling proteins such as four and half Lin11, Isl‐1, Mec‐3 (LIM) domains isoform 2 and filamin α. These interactions are believed to play a role in regulating Grb7‐mediated cell migration function. The full‐length Grb7 protein has been shown to dimerize, and the oligomeric state of the Grb7SH2 domain has been extensively studied; however, the oligomerization state of the ras associating and pleckstrin homology domains, and the importance of this oligomerization in Grb7 function, is yet to be fully known. In this study, we characterize the oligomeric state of the Grb7RA domain using size exclusion chromatography, nuclear magnetic resonance, nuclear relaxation studies, glutaraldehyde cross linking, and dynamic light scattering. We report the Grb7RA domain can exist in transient multimeric forms and, based upon modeling results, postulate the potential role of Grb7RA domain oligomerization in Grb7 function.     

Energy migration captures membrane-induced oligomerization of the prion protein

Excitation energy migration via homo-Förster resonance energy transfer (homo-FRET) can serve as an intermolecular proximity ruler within complex biomolecular assemblies. Here we present a unique case to demonstrate that energy migration can be a novel and sensitive readout to capture the membrane-mediated misfolding and oligomerization of the human prion protein (PrP), which is known to undergo an aberrant conformational conversion from an α-helical form into a self-propagating aggregated β-rich state causing deadly transmissible neurodegenerative diseases. Using site-specific energy migration studies by monitoring steady-state and time-resolved fluorescence anisotropy of fluorescently-tagged PrP, we elucidate the molecular details of lipid membrane-induced oligomers. We show that the intrinsically disordered N-terminal segment is critical for lipid-induced conformational sequestration of PrP into higher-order, β-rich oligomeric species that exhibit membrane permeabilization. Our results revealed that the N-terminal regions constitute the central core of the oligomeric architecture, whereas the distal C-terminal ends participate in peripheral association with the lipid membrane. Our study will find applications in the sensitive detection and in the structural characterization of membrane-induced protein misfolding and aggregation in a variety of deadly amyloid diseases.