A Point Mutation in the Ethylene-Inducing Xylanase Elicitor Inhibits the β-1-4-Endoxylanase Activity But Not the Elicitation Activity

Ethylene-inducing xylanase (EIX) elicits plant defense responses in certain tobacco (Nicotiana tabacum) and tomato cultivars in addition to its xylan degradation activity. It is not clear, however, whether elicitation occurs by cell wall fragments released by the enzymatic activity or by the xylanase protein interacting directly with the plant cells. We cloned the gene encoding EIX protein and overexpressed it in insect cells. To determine the relationship between the two activities, substitution of amino acids in the xylanase active site was performed. Substitution at glutamic acid-86 or -177 with glutamine (Gln), aspartic acid (Asp), or glycine (Gly) inhibited the β-1-4-endoxylanase activity. Mutants having Asp-86 or Gln-177 also lost the ability to induce the hypersensitive response and ethylene biosynthesis. However, mutants having Gln-86, Gly-86, Asp-177, or Gly-177 retained ability to induce ethylene biosynthesis and the hypersensitive response. Our data show that the xylanase activity of EIX elicitor can be separated from the elicitation process, as some of the mutants lack the former but retain the latter.     

The Elicitation of Ethylene Biosynthesis by a Trichoderma Xylanase Is Not Related to the Cell Wall Degradation Activity of the Enzyme

A [beta]-1,4-endoxylanase (EIX) isolated from Trichoderma viride elicits plant defense responses in certain tobacco (Nicotiana tabacum L.) cultivars in addition to its xylan degradation activity. It was not clear whether elicitation occurs by cell wall fragments released by the enzymic activity or by the xylanase protein interacting directly with the plant cells. We used protoplasts isolated from tobacco leaves to test whether the cell wall is required for the stimulation of ethylene biosynthesis by EIX. Protoplasts of tobacco (cv Xanthi) responded to treatment with the EIX, as indicated by an increased production of ethylene and the loss of protoplast viability. Protoplasts prepared from ethylene-pretreated leaves produced more ethylene and had higher rates of cell death in response to EIX than protoplasts prepared from nonethylene-treated leaves. Protoplasts of an EIX-insensitive cultivar of tobacco (Hicks) were insensitive to high concentrations of EIX. The addition of a crude cell wall preparation to protoplasts during incubation with EIX did not enhance the induction of ethylene biosynthesis by nonsaturating as well as saturating concentrations of EIX. These data indicate that the xylanase activity of EIX is unrelated to the elicitation of ethylene biosynthesis through the production of some cell wall fragment, since the protein per se appears capable of eliciting ethylene biosynthesis in protoplasts.     

Identification of an essential component of the elicitation active site of the EIX protein elicitor

Defense mechanisms of plants against pathogens often entail cell wall strengthening, ethylene biosynthesis, expression of pathogen-related proteins and hypersensitive responses (HR). Pathogen-derived elicitors trigger these defense responses. The Elicitor Ethylene-inducing Xylanase (EIX) elicits HR and other plant defense responses in some tobacco and tomato cultivars independently of its xylan degradation activity. The elicitation epitope on the EIX protein responsible for inducing the HR response has been elucidated. Through the generation of EIX-specific polyclonal antibodies and screening of combinatorial phage display peptide libraries an essential sequence of the EIX elicitation activity has been identified. This sequence consists of the pentapeptide TKLGE mapped to an exposed beta-strand of the EIX protein. Substitution of the pentapeptide TKLGE to VKGT inhibited the elicitation activity but not the beta-1-4-endoxylanase activity of the EIX protein further demonstrating that elicitation and enzyme activity are independent properties. Elucidation of a peptide sequence that is essential for elicitation of HR creates the opportunity to understand the control and signaling of plant defense.     

An Ethylene Biosynthesis-Inducing Endoxylanase Elicits Electrolyte Leakage and Necrosis in Nicotiana tabacum cv Xanthi Leaves

We have previously demonstrated that a protein purified from xylan-induced culture filtrates of Trichoderma viride contains β-1,4-endoxylanase activity and induces ethylene biosynthesis in tobacco (Nicotiana tabacum cv Xanthi) leaf discs. When the ethylene biosynthesis-inducing xylanase (EIX) was applied to cut petioles of detached tobacco leaves, it induced ethylene biosynthesis within 1 hour and extensive electrolyte leakage and necrosis were observed in tobacco leaf tissue within 5 hours. Ethylene-pretreatment (120 microliters per liter ethylene for 14 hours) of tobacco leaves enhanced ethylene biosynthesis in response to EIX by more than threefold and accelerated development of cellular leakage and necrosis. In intact plants, similar symptoms could be induced in leaves that were distant from the point of the enzyme application. The evidence suggests that EIX is translocated via the vascular system and elicits plant responses similar to those observed in a hypersensitive response.     

EHD2 inhibits ligand-induced endocytosis and signaling of the leucine-rich repeat receptor-like protein LeEix2

Plants are constantly being challenged by aspiring pathogens. In order to protect themselves, plants have developed numerous defense mechanisms that are either specific or non-specific to the pathogen. Pattern recognition receptors can trigger plant defense responses in response to specific ligands or patterns. EIX (ethylene-inducing xylanase) triggers a defense response via the LeEix2 receptor, while bacterial flagellin triggers plant innate immunity via the FLS2 receptor. Endocytosis has been suggested to be crucial for the process in both cases. Here we show that the EIX elicitor triggers internalization of the LeEix2 receptor. Treatment with endocytosis, actin or microtubule inhibitors greatly reduced the internalization of LeEix2. Additionally, we demonstrate that plant EHD2 binds to LeEix2 and is an important factor in its internalization and in regulation of the induction of defense responses such as the hypersensitive response, ethylene biosynthesis and induction of pathogenesis-related protein expression in the case of EIX/LeEix2 (an LRR receptor lacking a kinase domain), but does not appear to be involved in the FLS2 system (an LRR receptor possessing a kinase domain). Our results suggest that various endocytosis pathways are involved in the induction of plant defense responses.     

Escherichia coli H(+)-ATPase: role of the delta subunit in binding Fl to the Fo sector.

The roles of the Escherichia coli H(+)-ATPase (FoFl) delta subunit (177 amino acid residues) was studied by analyzing mutants. The membranes of nonsense (Gln-23----end, Gln-29----end, Gln-74----end) and missense (Gly-150----Asp) mutants had very low ATPase activities, indicating that the delta subunit is essential for the binding of the Fl portion to Fo. The Gln-176----end mutant had essentially the same membrane-bound activity as the wild type, whereas in the Val-174----end mutant most of the ATPase activity was in the cytoplasm. Thus Val-174 (and possibly Leu-175 also) was essential for maintaining the structure of the subunit, whereas the two carboxyl terminal residues Gln-176 and Ser-177 were dispensable. Substitutions were introduced at various residues (Thr-11, Glu-26, Asp-30, Glu-42, Glu-82, Arg-85, Asp-144, Arg-154, Asp-161, Ser-163), including apparently conserved hydrophilic ones. The resulting mutants had essentially the same phenotypes as the wild type, indicating that these residues do not have any significant functional role(s). Analysis of mutations (Gly-150----Asp, Pro, or Ala) indicated that Gly-150 itself was not essential, but that the mutations might affect the structure of the subunit. These results suggest that the overall structure of the delta subunit is necessary, but that individual residues may not have strict functional roles.     

Isolation of a novel SUMO protein from tomato that suppresses EIX-induced cell death

Challenging tomato or tobacco varieties with ethylene-inducing xylanase (EIX) from the fungus Trichoderma viride causes rapid induction of plant defence responses leading to programmed cell death. Using the yeast two-hybrid system, we isolated a novel protein, tomato small ubiquitin-related modifier protein (T-SUMO), which specifically interacts with EIX. T-SUMO, a cytoplasmic protein, is a member of the ubiquitin-like protein family. It shows homology to human protein sentrin/SUMO1, which suppresses tumour necrosis factor-induced cell death. Transgenic plants that express T-SUMO in the sense orientation suppress EIX induction of ethylene biosynthesis and cell death, while in the antisense orientation they enhance EIX-induced ethylene biosynthesis. These results indicate that T-SUMO is involved in mediating the signal generated by EIX that leads to induction of plant defence responses.     

The receptor for the fungal elicitor ethylene-inducing xylanase is a member of a resistance-like gene family in tomato

An ethylene-inducing xylanase (EIX) is a potent elicitor of plant defense responses in specific cultivars of tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum). The LeEix locus in tomatoes was characterized by map-based cloning, which led to the identification of a novel gene cluster from which two members (LeEix1 and LeEix2) were isolated. Similar to the tomato Ve resistance genes in tomato plants, the deduced amino acid sequences encoded by LeEix1 and LeEix2 contain a Leu zipper, an extracellular Leu-rich repeat domain with glycosylation signals, a transmembrane domain, and a C-terminal domain with a mammalian endocytosis signal. Silencing expression of the LeEix genes prevented the binding of EIX to cells of an EIX-responsive plant and thus inhibited the hypersensitive response. Overexpression of either LeEix1 or LeEix2 genes in EIX-nonresponsive tobacco plants enabled the binding of EIX, although only LeEix2 could transmit the signal that induced the hypersensitive response. Overexpressing LeEix2 in mammalian COS-7 cells enables binding of EIX, indicating physical interaction between the EIX elicitor and LeEix2 gene product. Structural analysis of the LeEix proteins suggests that they belong to a class of cell-surface glycoproteins with a signal for receptor-mediated endocytosis. Mutating the endocytosis signal in LeEix2 (Tyr 993 to Ala) abolished its ability to induce the hypersensitive response, suggesting that endocytosis plays a key role in the signal transduction pathway.     

High-affinity binding site for ethylene-inducing xylanase elicitor on Nicotiana tabacum membranes

Challenge of Nicotiana tabacum cv Xanthi with the ethylene-inducing xylanase (EIX) from Trichoderma viride causes rapid induction of plant defense responses leading to hypersensitive necrosis. This phenomenon is cultivar-specific; no response is detected when N. tabacum cv Hicks is similarly treated. The responsiveness is determined in tobacco and tomato by a single dominant gene. EIX was labeled with fluorescein-isothiocyanate and incubated with cell suspension cultures, protoplasts or microsomal membranes. Binding of EIX to the microsomal membranes was found to be specific and saturable, with a dissociation constant of 6.2 nM. Using confocal laser microscopy, the EIX binding site was localized to the plasma membrane. Binding of EIX to its high-affinity site occurred in responsive species. These results demonstrate the existence of a high-affinity binding site for EIX on the plasma membrane of responsive cultivars. Chemical cross-linking of EIX to microsomal membranes from responding plants revealed a 66 kDa protein complex. This protein may function as the receptor that mediates the hypersensitive response induced by EIX binding.     

Ethylene Biosynthesis-Inducing Xylanase : III. Product Characterization

Induction of ethylene biosynthesis in tobacco (Nicotiana tabacum cv Xanthi) leaf discs by the ethylene biosynthesis-inducing xylanase (EIX) isolated from Cellulysin or xylan-grown cultures of Trichoderma viride was dependent upon the concentration of xylanase applied and upon the length of incubation. Arrhenius activation energies of 9,100 and 10,500 calories for the Cellulysin and T. viride EIX xylanase activities, respectively, were derived from the K_m and V_max values determined for each enzyme at several temperatures. The two xylanases digested xylan in a strictly endo fashion, releasing neither xylobiose nor free xylose, and no debranching activity was associated with either enzyme. The xylanases released polysaccharides from ground corn cobs, but little or no carbohydrate was released from tobacco mesophyll cell walls incubated with EIX. No heat-stable products capable of inducing ethylene biosynthesis in tobacco leaf discs were found in EIX digests of purified xylans.     

Three-dimensional structures of H-ras p21 mutants: molecular basis for their inability to function as signal switch molecules

The X-ray structures of the guanine nucleotide binding domains (amino acids 1-166) of five mutants of the H-ras oncogene product p21 were determined. The mutations described are Gly-12----Arg, Gly-12----Val, Gln-61----His, Gln-61----Leu, which are all oncogenic, and the effector region mutant Asp-38----Glu. The resolutions of the crystal structures range from 2.0 to 2.6 A. Cellular and mutant p21 proteins are almost identical, and the only significant differences are seen in loop L4 and in the vicinity of the gamma-phosphate. For the Gly-12 mutants the larger side chains interfere with GTP binding and/or hydrolysis. Gln-61 in cellular p21 adopts a conformation where it is able to catalyze GTP hydrolysis. This conformation has not been found for the mutants of Gln-61. Furthermore, Leu-61 cannot activate the nucleophilic water because of the chemical nature of its side chain. The D38E mutation preserves its ability to bind GAP.     

Sensitivity to an Ethylene Biosynthesis-Inducing Endoxylanase in Nicotiana tabacum L. cv Xanthi Is Controlled by a Single Dominant Gene

The ethylene biosynthesis-inducing xylanase (EIX) is known to be a potent elicitor of ethylene biosynthesis and other responses when applied to leaf tissue of Nicotiana tabacum L. cv Xanthi. In contrast, leaf tissue of the tobacco cultivar Hicks was insensitive to EIX at concentrations 100-fold higher than was needed to elicit responses from Xanthi. Cell-suspension cultures of Xanthi and Hicks showed similar differences in sensitivity to EIX. Equivalent levels of ethylene production were elicited in leaf discs of both cultivars after treatment with CuSO4. The F1 and Xanthi backcross progeny of Hicks and Xanthi crosses were all sensitive to EIX, whereas the F2 and Hicks backcross progeny segregated for sensitivity to EIX. Individual plants from the F2 and Hicks backcross that were insensitive to EIX produced only insensitive progeny when they were self-pollinated. Progeny from sensitive plants either segregated for sensitivity to EIX or produced all sensitive progeny (an F2 plant). Sensitivity to EIX is controlled by a single dominant gene, based on chi-square analysis of segregation ratios.     

Alterations in Nicotiana tabacum L. cv Xanthi Cell Membrane Function following Treatment with an Ethylene Biosynthesis-Inducing Endoxylanase

An ethylene biosynthesis-inducing xylanase (EIX) produced by the fungus Trichoderma viride elicited enhanced ethylene biosynthesis and leakage of potassium and other cellular components when applied to leaf disks of tobacco (Nicotiana tabacum L. cv Xanthi). Suspension-cultured cells of Xanthi tobacco responded to EIX by rapid efflux of potassium, uptake of calcium, alkalization of the medium, inhibition of ethylene biosynthesis, and increased leakage of cellular components. EIX-treated cell suspensions released 1-aminocyclopropane-1-carboxylate (ACC) into the surrounding medium, resulting in a reduction of cellular pools of ACC. The responses of both cell suspensions and leaf disks were inhibited (50-80%) by the preincubation of the tissues with the calcium channel blocker La³⁺. High concentrations of EGTA inhibited the alkalization of the medium by cell suspensions responding to EIX, but EGTA alone caused extensive loss of K⁺ and ACC and inhibited ethylene biosynthesis by tobacco cells. Alterations in membrane function appear to be important in the mode of action of EIX in Xanthi cells.     

Increased N-acylphosphatidylethanolamine biosynthesis in elicitor-treated tobacco cells

Previous studies with tobacco (Nicotiana tabacum L.) cell suspensions indicated that elicitation of defense response (production of phytoalexins) with xylanase (1,4-β-D-xylanxylanohydrolase: EC 3.2.1.8) resulted in a dramatic acylation of phytosterols (Moreau et al. 1994). N-acylphosphatidylethanolamine (NAPE), an acylated derivative of phosphatidylethanolamine (PE), was recently demonstrated to be synthesized in vivo in plant tissues (Chapman and Moore 1993a). Here we report that acylation of PE was increased in elicitor-treated cells. NAPE levels increased 3-fold (from 1.6 to 4.8 mol% of total phospholipids) after a 2-h treatment of cell suspensions with xylanase (1 δg ml^?1). Specific activity of NAPE synthase increased in parallel with NAPE levels. Levels of NAPE and NAPE synthase activity declined during the period of 2–4 h after elicitation while levels of acylated sterolglycosides (ASG) continued to increase. Radiolabeling studies with [2^?14C]-ethanolamine confirmed that three times as much NAPE was synthesized in elicitor-treated cells compared to that in unelicited cells. Patterns of incorporation of [1-¹⁴C]-palmitic acid into membrane phospholipids in elicitor-treated cells suggested that increased acylation of lipids may be a result of changes in the acyl-coenzyme A pool. Treatment of cells with purified ethylene biosynthesis-inducing xylanase (EIX; 1 δg ml^?1 cells) resulted in increased levels of NAPE synthase activity comparable to those observed with the commercial preparations of xylanase. Boiled xylanase did not elicit an increase in the specific activity of NAPE synthase. Collectively our results demonstrate that the accumulation of NAPE in tobacco cells is attributable to increased activity of NAPE synthase. This suggests that NAPE may be specifically synthesized to play a protective role in membranes of plant cells as has been suggested for membranes of damaged animal cells.     

Amino acids of the bacterial toxin SopE involved in G nucleotide exchange on Cdc42

RhoGTPases are central switches in all eukaryotic cells. There are at least two known families of guanine nucleotide exchange factors that can activate RhoGTPases: the Dbl-like eukaryotic G nucleotide exchange factors and the SopE-like toxins of pathogenic bacteria, which are injected into host cells to manipulate signaling. Both families have strikingly different sequences, structures, and catalytic core elements. This suggests that they have emerged by convergent evolution. Nevertheless, both families of G nucleotide exchange factors also share some similarities: (a) both rearrange the G nucleotide binding site of RhoGTPases into virtually identical conformations, and (b) two SopE residues (Gln-109SopE and Asp-124SopE) engage Cdc42 in a similar way as equivalent residues of Dbl-like G nucleotide exchange factors (i.e. Asn-810Dbs and Glu-639Dbs). The functional importance of these observations has remained unclear. Here, we have analyzed the effect of amino acid substitutions at selected SopE residues implicated in catalysis (Asp-124SopE, Gln-109SopE, Asp-103SopE, Lys-198SopE, and Gly-168SopE) on in vitro catalysis of G nucleotide release from Cdc42 and on in vivo activity. Substitutions at Asp-124SopE, Gln-109SopE, and Gly-168SopE severely reduced the SopE activity. Slight defects were observed with Asp-103SopE variants, whereas Lys-198SopE was not found to be required in vitro or in vivo. Our results demonstrate that G nucleotide exchange by SopE involves both catalytic elements unique to the SopE family (i.e. 166GAGA169 loop, Asp-103SopE) and amino acid contacts resembling those of key residues of Dbl-like guanine nucleotide exchange factors. Therefore, besides all of the differences, the catalytic mechanisms of the SopE and the Dbl families share some key functional aspects.     

Nicotiana benthamiana LRR‐RLP NbEIX2 mediates the perception of an EIX‐like protein from Verticillium dahliae

Verticillium wilt diseases caused by the soil‐borne fungus Verticillium dahliae result in devastating yield losses in many economically important crops annually. Here, we identified a novel ethylene‐inducing xylanase (EIX)‐like protein, VdEIX3, from V. dahliae, which exhibits immunity‐inducing activity in Nicotiana benthamiana. In vitro‐purified VdEIX3 can induce strong oxidative burst, activate the expression of defense‐related genes, and increase resistance against oomycete and fungal pathogens in N. benthamiana. VdEIX3 orthologs of other Verticillium pathogens also induce cell death in N. benthamiana, which form a new type of EIX protein family that is distinct from the known EIX proteins. A leucine‐rich repeat receptor‐like protein, NbEIX2, regulates the perception of VdEIX3 in N. benthamiana. Our results demonstrate that VdEIX3 is a novel EIX‐like protein that can be recognized by N. benthamiana NbEIX2, and also suggest that NbEIX2 is a promising receptor‐like protein that is potentially applicable to transgenic breeding for improving resistance to Verticillium wilt diseases.     

Characteristics of ethylene biosynthesis-inducing xylanase movement in tobacco leaves

¹²⁵I-Labeled ethylene biosynthesis-inducing xylanase (EIX) was used to study the movement of this protein in tobacco (Nicotiana tabacum) tissues. A biologically active ¹²⁵I-labeled EIX was obtained using chloramine-T as the oxidizing agent. Labeled EIX was detected in the far most edges of the leaf 5 min after it was applied to the petiole of a detached leaf. EIX was distributed uniformly throughout the leaf, including the mesophyll area within 5 to 15 min, after which there was only little change in the distribution of radioactivity in the leaf. ¹²⁵I-Labeled EIX was extracted from treated leaves, and EIX translocation in the leaf was blocked by preincubation of labeled EIX with anti-EIX antibodies, indicating that the intact peptide moves in the leaf. Injection of anti-EIX antibodies into the intercellular spaces of the leaf mesophyll prevented induction of necrosis by EIX, suggesting the mesophyll as the site of EIX action. EIX was translocated both to upper and lower parts of the plant when applied to a whole plant through the petiole of a cut leaf. Radioactivity was found in all leaves and in the stem, although some leaves accumulated much more EIX than others; EIX was not found in the roots. There was no difference between the accumulation pattern of EIX in fresh and ethylene-treated leaves or between sensitive (Xanthi) and insensitive (Hicks) tobacco cultivars. These data support the hypothesis that intact EIX protein is translocated to the leaf mesophyll, where it directly elicits plant defense responses.     

Ethylene Biosynthesis-Inducing Endoxylanase Is Translocated through the Xylem of Nicotiana tabacum cv Xanthi Plants

Ethylene biosynthesis-inducing xylanase (EIX) from the fungus Trichoderma viride elicits enhanced ethylene production and tissue necrosis in whole tobacco (Nicotiana tabacum cv Xanthi) plants at sites far removed from the point of EIX application when applied through a cut petiole. Symptoms develop in a specific pattern, which appears to be determined by the interconnections of the tobacco xylem. Based on results of tissue printing experiments, EIX enters the xylem of the stem from the point of application and rapidly moves up and down the stem, resulting in localized foliar symptoms on the treated side of the plant above and below the point of EIX application. The observation that a fungal protein that elicits plant defense responses can be translocated through the xylem suggests that plants respond to pathogen-derived extracellular proteins in tissues distant from the invading pathogen.     

CrATP-induced Ca2+ occlusion in mutants of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Use of the nonphosphorylating beta,gamma-bidentate chromium(III) complex of ATP to induce a stable Ca(2+)-occluded form of the sarcoplasmic reticulum Ca(2+)-ATPase was combined with molecular sieve high performance liquid chromatography of detergent-solubilized protein to examine the ability of the Ca(2+)-ATPase mutants Gly-233-->Glu, Gly-233-->Val, Glu-309-->Gln, Gly-310-->Pro, Pro-312-->Ala, Ile-315-->Arg, Leu-319-->Arg, Asp-703-->Ala, Gly-770-->Ala, Glu-771-->Gln, Asp-800-->Asn, and Gly-801-->Val to occlude Ca2+. This provided a new approach to identification of amino acid residues involved in Ca2+ binding and in the closure of the gates to the Ca2+ binding pocket of the Ca(2+)-ATPase. The "phosphorylation-negative" mutant Asp-703-->Ala and mutants of ADP-sensitive phosphoenzyme intermediate type were fully capable of occluding Ca2+, as was the mutant Gly-770-->Ala. Mutants in which carboxylic acid-containing residues in the putative transmembrane segments had been substituted ("Ca(2+)-site mutants") and mutant Gly-801-->Val were unable to occlude either of the two calcium ions. In addition, the mutant Gly-310-->Pro, previously classified as ADP-insensitive phosphoenzyme intermediate type (Andersen, J.P., Vilsen, B., and MacLennan, D.H. (1992). J. Biol. Chem. 267, 2767-2774), was unable to occlude Ca2+, even though Ca(2+)-activated phosphorylation from MgATP took place in this mutant.