SummaryAntifungal defensins, MsDef1 and MtDef4, from Medicago spp., inhibit the growth of Fusarium graminearum, which causes head blight disease in cereals. In order to determine the signalling cascades that are modulated by these defensins, we have isolated several insertional mutants of F. graminearum that exhibit hypersensitivity to MsDef1, but not to MtDef4. The molecular characterization of two of these mutants, designated enhanced sensitivity to defensin (esd), has revealed that the Mgv1 and Gpmk1 MAP kinase signalling cascades play a major role in regulating sensitivity of F. graminearum to MsDef1, but not to MtDef4. The Hog1 MAP kinase signalling cascade, which is responsible for adaptation of this fungus to hyperosmotic stress, does not participate in the fungal response to these defensins. Significantly, the esd mutants also exhibit hypersensitivity to other tested defensins and are highly compromised in their pathogenesis on wheat heads and tomato fruits. The studies reported here for the first time implicate two MAP kinase signalling cascades in a plant defensin-mediated alteration of fungal growth. Based on our findings, we propose that specific MAP kinase signalling cascades are essential for protection of a fungal pathogen from the antimicrobial proteins of its host plant.
A large gene family encoding the putative cysteine-rich defensins was discovered in Medicago truncatula. Sixteen members of the family were identified by screening a cloned seed defensin from M. sativa (Gao et al. 2000) against the Institute for Genomic Research's (TIGR) M. truncatula gene index (MtGI version 7). Based on the comparison of their amino acid sequences, M. truncatula defensins fell arbitrarily into three classes displaying extensive sequence divergence outside of the eight canonical cysteine residues. The presence of Class II defensins is reported for the first time in a legume plant. In silico as well as Northern blot and RT-PCR analyses indicated these genes were expressed in a variety of tissues including leaves, flowers, developing pods, mature seed and roots. The expression of these genes was differentially induced in response to a variety of biotic and abiotic stimuli. For the first time, a defensin gene (TC77480) was shown to be induced in roots in response to infection by the mycorrhizal fungus, Glomus versiforme. Northern blot analysis indicated that the tissue-specific expression patterns of the cloned Def1 and Def2 genes differed substantially between M. truncatula and M. sativa. Furthermore, the induction profiles of the Def1 and Def2 genes in response to the signaling molecules methyl jasmonate, ethylene and salicylic acid differed markedly between these two legumes.
Plant defensins are small, highly stable, cysteine-rich antimicrobial proteins that are thought to constitute an important component of plant defense against fungal pathogens. There are a number of such defensins expressed in various plant tissues with differing antifungal activity and spectrum. Relatively little is known about the modes of action and biological roles of these proteins. Our previous work on a virally encoded fungal toxin, KP4, from Ustilago maydis and subsequently with the plant defensin, MsDef1, from Medicago sativa demonstrated that some of these proteins specifically blocked calcium channels in both fungi and animals. The results presented here demonstrate that KP4 and three plant defensins, MsDef1, MtDef2, and RsAFP2, all inhibit root growth in germinating Arabidopsis seeds at low micromolar concentrations. We have previously demonstrated that a fusion protein composed of Rab GTPase (RabA4b) and enhanced yellow fluorescent protein (EYFP) is dependent upon calcium gradients for localization to the tips of the growing root hairs in Arabidopsis thaliana. Using this tip-localized fusion protein, we demonstrate that all four proteins rapidly depolarize the growing root hair and block growth in a reversible manner. This inhibitory activity on root and root hair is not directly correlated with the antifungal activity of these proteins and suggests that plants apparently express targets for these antifungal proteins. The data presented here suggest that plant defensins may have roles in regulating plant growth and development.
Sliding clamps tether DNA polymerases to DNA to increase the processivity of synthesis. The Escherichia coli ␥ complex loads the  sliding clamp onto DNA in an ATP-dependent reaction in which ATP binding and hydrolysis modulate the affinity of the ␥ complex for  and DNA. This is the second of two reports (Williams, C. R., Snyder, A. K., Kuzmič , P., O'Donnell, M., and Bloom, L. B. (2004) J. Biol. Chem. 279, 4376 -4385) addressing the question of how ATP binding and hydrolysis regulate specific interactions with DNA and . Mutations were made to an Arg residue in a conserved SRC motif in the ␦ and ␥ subunits that interacts with the ATP site of the neighboring ␥ subunit. Mutation of the ␦ subunit reduced the ATP-dependent  binding activity, whereas mutation of the ␥ subunits reduced the DNA binding activity of the ␥ complex. The ␥ complex containing the ␦ mutation gave a pre-steady-state burst of ATP hydrolysis, but at a reduced rate and amplitude relative to the wild-type ␥ complex. A pre-steady-state burst of ATP hydrolysis was not observed for the complex containing the ␥ mutations, consistent with the reduced DNA binding activity of this complex. The differential effects of these mutations suggest that ATP binding at the ␥ 1 site may be coupled to conformational changes that largely modulate interactions with , whereas ATP binding at the ␥ 2 and/or ␥ 3 site may be coupled to conformational changes that have a major role in interactions with DNA. Additionally, these results show that the "arginine fingers" play a structural role in facilitating the formation of a conformation that has high affinity for  and DNA.Sliding clamps tether DNA polymerases to their templates, enabling the polymerases to rapidly incorporate thousands of nucleotides into a growing polymer without dissociating from DNA. Crystal structures of sliding clamps from bacteria, bacteriophage, and humans have revealed a mechanism by which this is possible (1-4). Sliding clamps are composed of individual subunits that assemble into rings with a central opening large enough to encircle duplex DNA. These ring-shaped clamps must be assembled on DNA by the activity of a clamp loader that uses energy derived from ATP to power its mechanical task (reviewed in Ref. 5).In Escherichia coli, the clamp loader is composed of seven subunits, three copies of the dnaX gene product, ␦, ␦Ј, , and (6 -8). The DnaX proteins form the motor of the clamp loader, and each is capable of binding and hydrolyzing 1 molecule of ATP during the clamp loading process. In vivo, two forms of the DnaX protein are produced: a full-length gene product () and a truncated gene product (␥) with the same sequence, but only about two-thirds the length of (9 -11). The clamp loader at the replication fork most likely contains two copies of the subunit and a single copy of ␥ (7, 8). The unique C-terminal end present in interacts with other enzymes and coordinates the activities present at the replication fork, whereas the N-terminal domain shared by the and ␥ subunits provides the activ...
The Escherichia coli DNA polymerase III ␥ complex loads the  clamp onto DNA, and the clamp tethers the core polymerase to DNA to increase the processivity of synthesis. ATP binding and hydrolysis promote conformational changes within the ␥ complex that modulate its affinity for the clamp and DNA, allowing it to accomplish the mechanical task of assembling clamps on DNA. This is the first of two reports (Snyder, A. K., Williams, C. R., Johnson, A., O'Donnell, M., and Bloom, L. B. (2004) J. Biol. Chem. 279, 4386 -4393) addressing the question of how ATP binding and hydrolysis modulate specific interactions with DNA and . Pre-steady-state rates of ATP hydrolysis were slower when reactions were initiated by addition of ATP than when the ␥ complex was equilibrated with ATP and were limited by the rate of an intramolecular reaction, possibly ATP-induced conformational changes. Kinetic modeling of assays in which the ␥ complex was incubated with ATP for different periods of time prior to adding DNA to trigger hydrolysis suggests a mechanism in which a relatively slow conformational change step (k forward ؍ 6.5 s ؊1 ) produces a species of the ␥ complex that is activated for DNA (and ) binding. In the absence of , 2 of the 3 molecules of ATP are hydrolyzed rapidly prior to releasing DNA, and the 3rd molecule is hydrolyzed slowly. In the presence of , all 3 molecules of ATP are hydrolyzed rapidly. These results suggest that hydrolysis of 2 molecules of ATP may be coupled to conformational changes that reduce interactions with DNA, whereas hydrolysis of the 3rd is coupled to changes that result in release of .Sliding clamps increase the overall efficiency of DNA synthesis by tethering DNA polymerases to the templates. In the absence of the sliding clamp, Escherichia coli DNA polymerase III is capable of incorporating about one dozen nucleotides in a single binding event. However, when it is bound to the  sliding clamp, the processivity of the DNA polymerase III core (composed of the ␣, ⑀, and subunits) increases to thousands of nucleotides (1). Sliding clamps remain tightly associated with template DNA while, at the same time, allowing the polymerase to move rapidly along DNA as it incorporates nucleotides. This is possible because the clamps are composed of protein subunits that are assembled in a ring-shaped structure with a central opening large enough to encircle duplex DNA (2). The E. coli  clamp is composed of two identical crescent-shaped monomers, whereas the human proliferating cell nuclear antigen clamp contains three, but the overall structures of both complexes are strikingly similar (2, 3).The activity of a clamp loader is required to assemble these ring-shaped sliding clamps around DNA. The E. coli DNA polymerase III clamp loader is composed of seven different subunits; three copies of the dnaX gene product; and a single copy each of ␦, ␦Ј, , and (4 -6). The dnaX gene produces two proteins: a full-length protein () and a truncated protein (␥). The ␥ subunit is about two-thirds the length of and is ...
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