Size, acetylation and concentration of chitooligosaccharide elicitors determine the switch from defence involving PAL activation to cell death and water peroxide production in Arabidopsis cell suspensions

Chitosan oligomers are known elicitors of plant defence mechanisms. In this work, chitooligosaccharides of different degrees of polymerization and degrees of acetylation were prepared and characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The effect of the degree of polymerization (DP), degree of acetylation and concentration of these chitooligosaccharides on defence activation in Arabidopsis thaliana suspension-cultured cells was studied. Our study results show that fully deacetylated chitooligosaccharides (chitosan oligomers) induce, depending on their DP and concentration, phenylalanine ammonia-lyase (PAL) activation, H₂O₂ synthesis and cell death in A. thaliana cell suspensions. The progressive reacetylation of the chitosan oligomer elicitors progressively impaired their ability to enhance H₂O₂ accumulation and cell death, but did not affect the activation of PAL.     

Activation of the tobacco SIP kinase by both a cell wall-derived carbohydrate elicitor and purified proteinaceous elicitins from Phytophthora spp.

Two purified proteinaceous fungal elicitors, parasiticein (an alpha elicitin) and cryptogein (a beta elicitin), as well as a fungal cell wall-derived carbohydrate elicitor all rapidly activated a 48-kD kinase in tobacco suspension cells. The maximum activation of this kinase paralleled or preceded medium alkalization and activation of the defense gene phenylalanine ammonia-lyase (PAL). In addition, the two elicitins, which also induced hypersensitive cell death, activated a 44- and a 40-kD kinase with delayed kinetics. By contrast, the cell wall-derived elicitor only weakly activated the 44-kD kinase and failed to activate the 40-kD kinase. The size and substrate preference of the 48-kD kinase are reminiscent of the recently purified and cloned salicylic acid-induced protein (SIP) kinase, which is a member of the mitogen-activated protein kinase family. Antibodies raised against a peptide corresponding to the unique N terminus of SIP kinase immunoreacted with the 48-kD kinase activated by all three elicitors from Phytophthora spp. In addition, the cell wall elicitor and the salicylic acid-activated 48-kD kinase copurified through several chromatography steps and comigrated on two-dimensional gels. Based on these results, all three fungal elicitors appear to activate the SIP kinase. In addition, inhibition of SIP kinase activation by kinase inhibitors correlated with the suppression of cell wall elicitor-induced medium alkalization and PAL gene activation, suggesting a regulatory function for the SIP kinase in these defense responses.     

Down-regulation of T cell activation following inhibition of dipeptidyl peptidase IV/CD26 by the N-terminal part of the thromboxane A2 receptor

Using synthetic inhibitors, it has been shown that the ectopeptidase dipeptidyl peptidase IV (DP IV) (CD26) plays an important role in the activation and proliferation of T lymphocytes. The human immunodeficiency virus-1 Tat protein, as well as the N-terminal nonapeptide Tat(1-9) and other peptides containing the N-terminal sequence XXP, also inhibit DP IV and therefore T cell activation. Studying the effect of amino acid exchanges in the N-terminal three positions of the Tat(1-9) sequence, we found that tryptophan in position 2 strongly improves DP IV inhibition. NMR spectroscopy and molecular modeling show that the effect of Trp(2)-Tat(1-9) could not be explained by significant alterations in the backbone structure and suggest that tryptophan enters favorable interactions with DP IV. Data base searches revealed the thromboxane A2 receptor (TXA2-R) as a membrane protein extracellularly exposing N-terminal MWP. TXA2-R is expressed within the immune system on antigen-presenting cells, namely monocytes. The N-terminal nonapeptide of TXA2-R, TXA2-R(1-9), inhibits DP IV and DNA synthesis and IL-2 production of tetanus toxoid-stimulated peripheral blood mononuclear cells. Moreover, TXA2-R(1-9) induces the production of the immunosuppressive cytokine transforming growth factor-beta1. These data suggest that the N-terminal part of TXA2-R is an endogenous inhibitory ligand of DP IV and may modulate T cell activation via DP IV/CD26 inhibition.     

Molecular cloning of rat prostate transglutaminase complementary DNA. The major androgen-regulated protein DP1 of rat dorsal prostate and coagulating gland.

Complementary DNA (cDNA) that codes for a major androgen-dependent secretory protein of rat coagulating gland and dorsal prostate, dorsal protein 1 (DP1), was isolated by molecular cloning. Recombinant DP1 cDNA clones were identified from a bacteriophage lambda gt11 rat coagulating gland expression library using an affinity purified polyclonal antibody. Amino acid sequence deduced from DNA contained sequences identical with several DP1 cyanogen bromide cleavage fragments. Northern blot hybridization of poly(A) RNA isolated from intact rat dorsal prostate and coagulating gland revealed a predominant messenger RNA (mRNA) species of approximately 3200 nucleotides. Tissue-specific expression of DP1 mRNA was indicated by the absence of DP1 mRNA in ventral prostate and other tissues of the rat. Expression of DP1 mRNA was androgen-dependent, decreasing approximately 80% 7 days after castration and increasing rapidly following androgen replacement. Southern blot analysis of restriction enzyme-digested rat DNA indicated that DP1 is encoded by a single gene and that no major genomic rearrangements accounted for its lack of expression in the dorsal prostate-derived rat Dunning tumor. Sequence comparisons revealed that rat prostate DP1 shares sequence identity with Factor XIIIa and tissue transglutaminase, including the active center, GQCWVF, indicating that DP1 is a member of the transglutaminase gene family.     

Plant Defense Response to Fungal Pathogens (II. G-Protein-Mediated Changes in Host Plasma Membrane Redox Reactions)

Elicitor preparations containing the avr5 gene products from races 4 and 2.3 of Cladosporium fulvum, and tomato (Lycopersicon esculentum L.) cells containing the resistance gene Cf5 were used to investigate the involvement of redox processes in the production of active oxygen species associated with the plant response to the fungal elicitors. Here we demonstrate that certain race-specific elicitors of C. fulvum induced an increase in ferricyanide reduction in enriched plasma membrane fractions of tomato cells. The addition of elicitors to plasma membranes also induced increases in NADH oxidase and NADH-dependent cytochrome c reductase activities, whereas ascorbate peroxidase activity was decreased. These results suggest that changes in the host plasma membrane redox processes, transferring electrons from reducing agents to oxygen, could be involved in the increased production of active oxygen species by the race-specific elicitors. Our results also show that the dephosphorylation of enzymes involved in redox reactions is responsible for the race-specific induced redox activity. The effects of guanidine nucleotide analogs and mastoparan on the activation of plasma membrane redox reactions support the role of GTP-binding proteins in the transduction of signals leading to the activation of the defense response mechanisms of tomato against fungal pathogens.     

Structural requirements for catalysis,expression, and dimerization in the CD26/DPIV gene family

Dipeptidyl peptidase IV (DP-IV/CD26), fibroblast activation protein (FAP), DP-like 1 (DPL1), DP8, DP9, and DPL2 comprise the CD26 gene family. CD26/DP-IV has roles in liver disease, T cell costimulation, chemokine biology, type II diabetes, and tumor biology. DPIV substrates include the glucagonlike peptides, neuropeptide Y, and the chemokines CCL3, CCL5, CCL11, CCL22, and CXCL12. We have proposed that the extracellular region of CD26 is analogous to prolyl oligopeptidase in consisting of an alpha/beta hydrolase domain contributed by both N- and C-terminal portions of the polypeptide and a seven-blade beta-propeller domain. Replacing the C-terminal portion of the predicted alpha/beta hydrolase domain of CD26 (residues 501-766) with the homologous portion of DP8 or DP9 produced intact proteins. However, these chimeric proteins lacked dimerization and peptidase activity, suggesting that CD26 dimerization requires the C-terminal portion of the alpha/beta hydrolase domain. Deleting some N-terminal residues of the alpha/beta hydrolase domain of CD26 ablated peptidase activity and greatly diminished cell surface expression. Together with previous data that CD26 peptidase activity requires the C-terminal 20 residues, this suggests that peptidase activity requires the entire alpha/beta hydrolase domain. The catalytic triad of DP8 was shown to be Ser(739)-Asp (817)-His(849). Glu(259) of DP8, a residue distant from the catalytic triad yet greatly conserved in the CD26 gene family, was shown to be required for peptidase activity. These data concord with our predicted CD26 structure, indicate that biosynthesis of a functional fragment of CD26 is difficult, and confirm the functional homology of DP8 with CD26.     

A sphingolipid elicitor-inducible mitogen-activated protein kinase is regulated by the small GTPase OsRac1 and heterotrimeric G-protein in rice 1[w

Mitogen-activated protein kinase (MAPK) cascades are activated in plants during responses to pathogens or to pathogen-derived elicitors and mediate intracellular stress responses. Here, we show that a rice (Oryza sativa) MAPK, OsMAPK6, was posttranslationally activated in a cell culture by a sphingolipid elicitor. Suppression of OsMAPK6 expression by RNA interference resulted in a strong reduction of pathogen-induced Phe ammonia-lyase mRNA, whereas the mRNA level of another rice MAPK, OsMAPK5a, was highly increased. Silencing of a small GTPase, OsRac1, by RNA interference or loss-of-function mutation (d1) of the heterotrimeric G-protein alpha-subunit gene resulted in a strong reduction of the OsMAPK6 protein levels and of kinase activation by a sphingolipid elicitor. Furthermore, coimmunoprecipitation experiments with OsRac1 and OsMAPK6 proteins showed that OsMAPK6 is closely associated with the active form of OsRac1, but not with inactive forms of OsRac1. These results indicate that these two G-proteins regulate an elicitor-inducible MAPK in rice at the protein level.     

Isolation and Characterization of an Elicitor of Necrosis Isolated from Intercellular Fluids of Compatible Interactions of Cladosporium fulvum (Syn. Fulvia fulva) and Tomato

Intercellular fluids of compatible race-cultivar interactions of Cladosporium fulvum and tomato contain specific elicitors of necrosis. These elicitors which are of fungal origin induce chlorosis and necrosis in resistant but not in susceptible plants. With the tomato cultivar Sonatine (carrying resistance gene Cf9, resistant to the fungal races 0, 4, 5, 2, 2.4, and 2.4.5 but susceptible to race 2.4.5.9) as the test plant for assaying necrosis-inducing activity, we isolated and partially characterized an elicitor of necrosis on this cultivar. The elicitor bound to CM-Sephadex but not to DEAE-Sephadex; it was stable to heat (10 minutes at 100°C), HCl (0.01 normal), NaOH (0.01 normal), and NaIO₄ (0.02 molar), sensitive to pronase and protease (from Bacillus polymyxa) but not to other proteases such as α-chymotrypsin and trypsin. After electrophoresis of partially purified elicitor preparations under low pH conditions, the necrosis-inducing activity was association with a peptide with an apparent molecular weight of 5500. Races 0, 4, 5, 2.4, and 2.4.5 but not race 2.4.5.9 produced this elicitor in high yields. The elicitor is probably a product of avirulence gene A9 which is present in all races except in race 2.4.5.9 and induces necrosis in cultivars carrying resistance gene Cf9.     

Determination of the Solution Conformation of HIV-1 Tat(1–9) Peptides by Means of Molecular Dynamics Simulations Considering NMR Data and Docking Studies into an Active Site Model of DP IV

The human immunodeficiency virus 1 Tat protein suppresses antigen-, anti-CD3-and mitogen-induced activation of human T cells when added to T cell cultures. This activity is important for the development of AIDS because lymphocytes from HIV-infected individuals exhibit a similar antigen-specific dysfunction. Moreover, Tat was found to interact with dipeptidyl peptidase IV (DP IV). To find out the amino acid sequence important for the inhibition of the DP IV enzymatic activity we investigated N-terminal Tat(1–9) peptide analogues with amino acid substitutions in different positions. Interestingly, the exchange of Pro⁶ with Leu and Asp⁵ with Ile strongly diminished the DP IV inhibition by Tat(1–9). Based on data derived from one-and two-dimensional ¹H NMR investigations the solution conformations of the three nonapeptides in water were determined by means of molecular dynamics simulations. These conformations were used for studies of the docking behavior of the peptides into a model of the active site of DP IV. The results suggest that several attractive interactions between the native Tat(1–9) and DP IV lead to a stable complex and that the reduced affinity of both L⁶-Tat(1–9) and I⁵-Tat(1–9) derivatives might be caused by conformational alterations in comparison to the parent peptide.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089480040200     

Gene shuffling-generated and natural variants of the tomato resistance gene Cf-9 exhibit different auto-necrosis-inducing activities in Nicotiana species

Tomato Cf genes encode membrane-bound proteins with extracellular leucine-rich repeats, and confer resistance to the fungal tomato pathogen Cladosporium fulvum, and a hypersensitive response (HR) to C. fulvum-derived race-specific elicitors. Several Cf genes, including Cf-4 and Cf-9, are members of the highly homologous Hcr9 (homologues of C. fulvumresistance gene Cf-9) gene family. Hcr9s evolve mainly by sequence exchange between paralogues, by which novel Cf genes may be generated. To mimic this aspect of natural evolution, we generated chimeras between multiple Hcr9s in vitro by gene shuffling. The shufflants were tested for novel specificities by transient expression in Nicotiana benthamiana. Many shufflants induced an HR in the absence of fungal elicitors and were designated auto-activators. We also identified two natural Hcr9 auto-activators in the wild tomato species Lycopersicon peruvianum, which induced an HR upon expression in N. benthamiana. The Hcr9 auto-activators exhibit different auto-necrosis-inducing specificities in five selected species of the Nicotiana genus, and they were shown to function in the same signalling pathway as Cf-9. Auto-activating alleles of nucleotide binding site-leucine-rich repeat genes and the protein kinase Pto were previously described. The auto-activators described here, belonging to the Cf-like structural class of resistance genes, shed light on this important phenotype and may be used as tools to unravel the mechanisms by which this class of resistance proteins function.     

MAPK 级联途径在植物信号转导中的研究进展

促分裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)在植物的胁迫反应应答方面占有重要地位。本文就MAPK的基本组成、分类、激发子和两种可能的机理的最新进展作了综述。并介绍了近年来此领域中的研究方法,包括MAPK活性测定的方法、免疫沉淀法、酵母双杂交、基因突变、RNA干涉和网络模式法。