Activation Of Disease Resistance Research Articles

Activation tagging of ADR2 conveys a spreading lesion phenotype and resistance to biotrophic pathogens

An Arabidopsis PR1::luciferase (LUC) transgenic line was transformed with activation T-DNA tags and the resulting population screened for dominant gain-of-function mutants exhibiting constitutive LUC activity. LUC imaging identified activated disease resistance 2 (adr2), which exhibited slowly spreading lesions in the absence of pathogen challenge. Molecular, genetic and histochemical analysis was employed to characterize this mutant in detail. adr2 plants constitutively expressed defence-related and antioxidant genes. Moreover, this line accrued increased quantities of salicylic acid (SA) and exhibited heightened mitogen-activated protein kinase activity. adr2 plants exhibited increased resistance against numerous biotrophic but not necrotrophic pathogens. The adr2 phenotype resulted from the overexpression of a Toll interleukin receptor (TIR) nucleotide binding site (NBS) leucine rich repeat (LRR) gene (At1g56510). Constitutive PR1 expression was completely abolished in adr2 nahG, adr2 npr1 and adr2 eds1 double mutants. Furthermore, heightened resistance against Hyaloperonospora arabidopsis Noco2 was compromised in adr2 nahG and adr2 eds1 double mutants but not in adr2 npr1, adr2 coi1 or adr2 etr1 plants. These data imply that adr2-mediated resistance operates through an Enhanced Disease Susceptibility (EDS) and SA-dependent defence signalling network which functions independently from COI1 or ETR1.

A Series of Novel rps2 Alleles Disrupt the RPS2-avrRpt2 Gene-for-gene Interaction

The Arabidopsis resistance (R) RPS2 gene confers resistance to Pseudomonas syringae (P. syringae) strains that express the avirulence (avr) avrRpt2 gene [Kunkel et al., 1993]. When Arabidopsis plants containing RPS2 are challenged with P. syringae bacteria carrying avrRpt2, the plants activate disease resistance responses, including a specialized type of programmed cell death known as the hypersensitive response (HR) [Kunkel et al., 1993; Hammond-Kosack and Jones, 1997]. The interaction between RPS2 and avrRpt2 products leads to an excellent model for pathogen-induced resistance responses and signal transduction events. As a typical resistance protein of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class, the RPS2 protein, consisting of 909 amino acids, contains several possible functional peptide motifs, including an Nterminal hydrophobic region, a putative leucine zipper (LZ), a NBS and a LRR (Fig. 1A) [Hammond-Kosack and Jones, 1997]. Additional motifs found in NBS-LRR resistance genes, such as GLPL and KMH motifs, are present in the RPS2 sequence [Traut, 1994]. The LZ and LRR domains have been implicated in regards to proteinprotein interactions (Alber, 1992; Kobe and Deisenhofer, 1994). The conserved central NBS domain is hypothesized to bind either to ATP or GTP involving kinase activities, containing 3 motifs: the P loop (or kninase-1a), kinase-2 and less conserved kinase-3a [Traut, 1994]. These structural features regarding the RPS2 protein suggest that it is involved in signal transduction processes for disease resistance responses. For studies of the molecular function of RPS2 and signaling by RPS2-avrRpt2 interaction, about 20 mutants including rps2-101C have so far been isolated [Kunkel et al., 1993; Mindrinos et al., 1994; Axtell et al., 2001]. In order to study on the spatial structure and function of RPS2, we tried to isolate numerous additional mutants, defective in RPS2-avrRpt2 dependent cell death, by using Axtell et al. [2001]’s method with minor modifications. Transgenic Col-0 Arabidopsis seeds, possessing a dexamethasone-inducible avrRpt2 transgene (pTA7001avrRpt2) [McNellis et al., 1998], were obtained from Dr. Brian J. Staskawicz (University of California, Berkeley, CA), and then mutagenized with 0.1% ethyl methane sulfonate (Sigma, St. Louis, MO). M2 plants based on the self-fertilization of M1 seeds were grown on soil. When dexamethasone (30 μM) was sprayed onto 2-week-old M2 plants, almost were yellowish in color, had aborted growth rates and eventually died a few days later (Fig. 1B). To identify the presence of the avrRpt2 transgene in the surviving plants, genomic DNAs were isolated from leaves, then the polymerase chain reaction (PCR) was performed by using two specific primers (data not shown). The upstream and downstream sequences were 5'-ATGAAAATTGCTCCAGTTGCCAT-3' and 5'-TTAG CGGTAGAGCATTGCGTGT-3', respectively. Twenty-six mutants out of the surviving plants were screened by PCR. Some mutations were expected to eliminate expression of the avrRpt2 transgene due to mutations in the dexamethasone-inducible expression system (pTA7001 expression cassette). To test this possibility, we performed a Northern blot analysis (Fig. 1C). Total RNA was isolated from leaves taken 1 day after application of dexamethasone. The accumulation of avrRpt2 transcripts was detected in 7 out of 26 mutants as well as rps2-101C as a positive control line. To examine whether 7 mutants showed the ability to mount an HR in response to avrRpt2, we performed a secondary screening procedure by inoculating mutant leaves with the P. syringae pv. maculicola strain DG6 (carrying avrRpt2) at OD600=0.1 [Guttman and Greenberg, 2001]. The typical resistance response to this interaction *Corresponding author Phone: +82-53-950-7753; Fax: +82-53-958-6880 E-mail: jtsong68@knu.ac.kr

Activation of the Indole-3-Acetic Acid–Amido Synthetase GH3-8 Suppresses Expansin Expression and Promotes Salicylate- and Jasmonate-Independent Basal Immunity in Rice

New evidence suggests a role for the plant growth hormone auxin in pathogenesis and disease resistance. Bacterial infection induces the accumulation of indole-3-acetic acid (IAA), the major type of auxin, in rice (Oryza sativa). IAA induces the expression of expansins, proteins that loosen the cell wall. Loosening the cell wall is key for plant growth but may also make the plant vulnerable to biotic intruders. Here, we report that rice GH3-8, an auxin-responsive gene functioning in auxin-dependent development, activates disease resistance in a salicylic acid signaling- and jasmonic acid signaling-independent pathway. GH3-8 encodes an IAA-amino synthetase that prevents free IAA accumulation. Overexpression of GH3-8 results in enhanced disease resistance to the rice pathogen Xanthomonas oryzae pv oryzae. This resistance is independent of jasmonic acid and salicylic acid signaling. Overexpression of GH3-8 also causes abnormal plant morphology and retarded growth and development. Both enhanced resistance and abnormal development may be caused by inhibition of the expression of expansins via suppressed auxin signaling.

Identification of New Recurrent Lesions and Clinical Subsets by Genome-Wide DNA Profiling in Chronic Lymphocytic Leukemia with 17p Deletion.

Chronic lymphocytic leukemia (CLL) with deletion of 17p(p53) locus (17p- CLL) identifies the most aggressive CLL subset, due to active disease course and resistance to current treatments. Intrinsic mechanisms of the poor prognosis of 17p- CLL are scarcely known. In order to identify additional lesions occurring in 17p- CLL which contribute to prognosis, we performed genome-wide DNA profiling in 18 cases of 17p- CLL (median follow-up 25 months, range 4–107), using high-resolution single-nucleotide polymorphism (SNP) arrays (Affymetrix Human Mapping Nsp 250 k arrays). Chromosomal 11q, 13q or 17p deletions and trisomy 12 were determined by FISH. Tumor IGHV usage and mutational status were determined by PCR/sequencing of the tumor gDNA/cDNA. Median interval from diagnosis to progression (11 months, p=0.000122) and treatment (16 months, p=0.000827) were representative of the bad behavior of our 17p- CLL cases if compared to 95 non17p- CLL patients from our internal cohort (68 and 69 months, respectively). Interestingly, 3/18 (16%) 17p- CLL expressed the IGHV3–74 segment which is rare in normal B-cells (1,4%, p=0.02) and in non17p- CLL (2.1%, p=0.02). Tumor IGHV genes were unmutated in 10/18 and mutated in 8/18 17p- CLL. All cases carried 17p13.1 deletion in more than 60% nuclei, as assessed by FISH. Genome-wide DNA profiling was 99% concordant with FISH (71/72 data-points; one 13q deletion detected by FISH in 20% of the nuclei was missed by the microarray) and confirmed 17p deletion in all cases. Deletions at the 13q14.3 locus recurred in 50% cases. Only one case had an extra-copy of chromosome 12. No cases carried ATM deletion. Other recurrent lesions identified by genome-wide DNA profiling were: gains of 2p16.1-pter (30%), 3q24-qter (20%), 8q23.3-qter (30%), 17q21.2-q22 (30%), 14q32 (30%); losses of 8p12-pter (30%), 9q21.33-q22.1 (20%), 14q32 (40%), 20p12.1-pter (20%). Approximately half of the cases had concomitant losses of 8p or 17p and gains of the 8q or 17q regions, respectively, suggesting the presence of isochromosomes 8 or 17. Indeed, isochromosome 17 was evident in one case analyzed by conventional cytogenetics. Six patients had one or more regions of loss of heterozygosity (LOH) longer than 5 Mb in the absence of copy number reduction, suggestive of uniparental disomy. Several lesions, such as those involving chromosomes 3, 8 and 9, associate with poor clinical behavior in other B-cell tumors. Thus, we investigated the prognostic significance of the lesions identified in our series. Despite the small number of cases, we found that 17p- CLLs with loss of 8p12-pter, which contains tumor necrosis factor and defensin family genes, associated with worse treatment free interval from diagnosis (12 vs 46 months in 8p- vs non 8p- CLL, p=0.04). Overall, our analysis identified new recurrent genomic abnormalities at high frequencies that may specifically contribute to a different biologic and clinical behavior within 17p- CLL. Among these, isochromosomes 8 and 17 were particularly common, and 8p loss might identify a new aggressive subentity within 17p- CLL.

Nitrate Reductase is Responsible for Elicitin-induced Nitric Oxide Production in Nicotiana benthamiana

Recent works have established a key role for nitric oxide (NO) in activating disease resistance in plants. Nitrate reductase (NR) is one of the enzymes that are capable of producing NO in plants. In a previous study, we reported that pathogen signals induce expression of NR genes in potato, suggesting the involvement of NR in NO production induced by pathogen signals. In this study, we cloned NR genes from Nicotiana benthamiana and investigated their involvement in NO production induced by INF1, a major elicitin secreted by Phytophthora infestans. Treatment of protoplasts prepared from N. benthamiana leaves with INF1 elevated NO production to a maximum level 1-3 h after treatment. INF1-induced NO generation was suppressed completely by an NO-specific scavenger, but partially by a nitric oxide synthase inhibitor. To investigate the involvement of NR in INF1-induced NO production, NR genes were silenced by virus-induced gene silencing. The NR-silenced plants showed yellowish leaves which resemble the characteristic of Arabidopsis NR double mutants. Silencing of NR genes significantly decreased both NO(2) (-)-producing activity and INF1-induced NO production, indicating that NR is involved in INF1-induced NO production. In contrast, overexpression of NbNR1 encoding N. benthamiana NR by Agrobacterium-mediated transient expression elevated NO(2) (-)-producing activity nine times over the control; however, INF1-induced NO production in protoplasts overexpressing NbNR1 was comparable with that in control protoplasts. These results suggest that NR is involved in INF1-induced NO production, and post-translational modification of NR or availability of substrate NO(2) (-) may be a rate-limiting step of NO production by NR.

Rapid determination of methyl salicylate, a plant-signaling compound, in tomato leaves by direct sample introduction and thermal desorption followed by GC-MS

Many plants infested by herbivores or viruses can rapidly produce and accumulate a plant-signaling compound, methyl salicylate (MeSA), in their leaves to activate disease resistance. In the present work, a simple, rapid, and sensitive method was developed for the determination of MeSA in tomato leaves by direct sample introduction and thermal desorption followed by GC-MS. Results show that the proposed method has a low detection limit (0.08 ng mg(-1)) and good precision (RSD = 8.9%). The present method was applied to the investigation of tomato plant defense response to tobacco mosaic virus (TMV) by rapid analysis of volatile compounds in plant leaves. It was found that tomato plants can produce large amounts of MeSA as a defense response to TMV. This indicates that MeSA may be a plant-signaling compound in tomato plant defense response to TMV.

Motifs specific for the ADR1 NBS–LRR protein family in Arabidopsis are conserved among NBS–LRR sequences from both dicotyledonous and monocotyledonous plants

The activated disease resistance (ADR) 1 gene encodes a protein that possesses an N-terminal coiled-coil (CC) motif, nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. ADR1 belongs to a small, atypical Arabidopsis thaliana sub-class containing four CC-NBS-LRR genes. The NBS region of most NBS-LRR proteins possesses numerous conserved motifs. In contrast, the LRR domain, which is subject to positive selection, is highly variable. Surprisingly, sequence analysis revealed that the LRR domain of the ADR1 sub-class was more conserved than the NBS region. Sequence analysis identified two novel conserved motifs, termed TVS and PKAE, specific for this CC-NBS-LRR sub-class. The TVS motif is adjacent to the P-loop, whereas the PKAE motif corresponded to the inter-domain region termed the NBS-LRR linker, which was conserved within the different CC-NBS-LRR classes but varied among classes. These ADR1-specific motifs were employed to identify putative ADR1 homologs in phylogenetically distant and agronomically important plant species. Putative ADR1 homologs were identified in 11 species including rice and in 3 further Poaceae species. The ADR1 sub-class of CC-NBS-LRR proteins is therefore conserved in both monocotyledonous and dicotyledonous plant species.

Drought tolerance established by enhanced expression of the CC-NBS-LRR gene, ADR1, requires salicylic acid, EDS1 and ABI1.

An activation-tagged allele of activated disease resistance 1 (ADR1) has previously been shown to convey broad spectrum disease resistance. ADR1 was found to encode a coiled-coil (CC)-nucleotide-binding site (NBS)-leucine-rich repeat (LRR) protein, which possessed domains of homology with serine/threonine protein kinases. Here, we show that either constitutive or conditional enhanced expression of ADR1 conferred significant drought tolerance. This was not a general feature of defence-related mutants because cir (constitutive induced resistance)1, cir2 and cpr (constitutive expressor of PR genes)1, which constitutively express systemic acquired resistance (SAR), failed to exhibit this phenotype. Cross-tolerance was not a characteristic of adr1 plants, rather they showed increased sensitivity to thermal and salinity stress. Hence, adr1-activated signalling may antagonise some stress responses. Northern analysis of abiotic marker genes revealed that dehydration-responsive element (DRE)B2A but not DREB1A, RD (response to dehydration)29A or RD22 was expressed in adr1 plant lines. Furthermore, DREB2A expression was salicylic acid (SA) dependent but NPR (non-expressor of PR genes)1 independent. In adr1/ADR1 nahG (naphthalene hydroxylase G), adr1/ADR1 eds (enhanced disease susceptibility)1 and adr1/ADR1 abi1 double mutants, drought tolerance was significantly reduced. Microarray analyses of plants containing a conditional adr1 allele demonstrated that a significant number of the upregulated genes had been previously implicated in responses to dehydration. Therefore, biotic and abiotic signalling pathways may share multiple nodes and their outputs may have significant functional overlap.

Involvement of nitric oxide generation in hypersensitive cell death induced by elicitin in tobacco cell suspension culture

Recent studies suggest that nitric oxide (NO), an important signaling and defense molecule in mammals, plays a key role in activating disease resistance in plants. We characterized NO production by tobacco Bright Yellow-2 cells pharmacologically after treatment with INF1, the major elicitin secreted by the late blight pathogen Phytophthora infestans, prepared from Escherichia coli. NO production rapidly occurred within 1 h and reached a maximum level 3–6 h after the addition of INF1. Carboxy-PTIO, a NO-specific scavenger, abolished INF1-induced NO production in a dose-dependent manner. Pretreatment of protein synthesis inhibitor cycloheximide and protein kinase inhibitor K252a blocked NO production 3–12 h after INF1 treatment, indicating that NO production requires de novo protein synthesis and protein phosphorylation. In an investigation of the relations between NO generation and several defense responses induced by INF1, carboxy-PTIO completely suppressed activation of a 41-kDa protein kinase and cell death by INF1. Carboxy-PTIO also suppressed the induction of hypersensitive-related (hsr) genes HSR515 and HSR203J, the expression of which is strongly correlated with the hypersensitive response in plants. The results suggest that NO plays a crucial role in the induction of hypersensitive cell death.

Targeted activation tagging of the Arabidopsis NBS-LRR gene, ADR1, conveys resistance to virulent pathogens.

A transgenic Arabidopsis line containing a chimeric PR-1::luciferase (LUC) reporter gene was subjected to mutagenesis with activation tags. Screening of lines via high-throughput LUC imaging identified a number of dominant Arabidopsis mutants that exhibited enhanced PR-1 gene expression. Here, we report the characterization of one of these mutants, designated activated disease resistance (adr) 1. This line showed constitutive expression of a number of key defense marker genes and accumulated salicylic acid but not ethylene or jasmonic acid. Furthermore, adr1 plants exhibited resistance against the biotrophic pathogens Peronospora parasitica and Erysiphe cichoracearum but not the necrotrophic fungus Botrytis cinerea. Analysis of a series of adr1 double mutants suggested that adr1-mediated resistance against P. parasitica was salicylic acid (SA)-dependent, while resistance against E. cichoracearum was both SA-dependent and partially NPR1-dependent. The ADR1 gene encoded a protein possessing a number of key features, including homology to subdomains of protein kinases, a nucleotide binding domain, and leucine-rich repeats. The controlled, transient expression of ADR1 conveyed striking disease resistance in the absence of yield penalty, highlighting the potential utility of this gene in crop protection.

A local accumulation of the Ralstonia solanacearum PopA protein in transgenic tobacco renders a compatible plant-pathogen interaction incompatible.

Plants activate disease resistance responses when they recognize pathogen-derived molecules (elicitors). Frequently, recognition results in a hypersensitive response (HR), which is characterized by local host cell death at the infection site. Here we describe a genetic engineering approach to generate an HR in plants, whether or not an invading micro-organism produces a recognized elicitor. To that aim we created transgenic tobacco plants in which the pathogen-inducible promoter of the hsr203J gene from tobacco controls the expression of the popA elicitor gene from Ralstonia solanacearum. Because PopA itself also induces the hsr203J promoter, transgenic plants rapidly accumulate the bacterial elicitor in the pathogen infection sites. The elicitor becomes converted in plant tissues into its fully active derivatives PopA1-PopA3, showing that the previously observed processing events are not dependent on the bacterial type III secretion system. The outcome of induced PopA accumulation is a localized HR and a high degree of resistance of the transgenic plants to an oomycete pathogen. The system is functional in hybrids between different tobacco varieties, and we show that the engineered resistance, but not the associated cell death, is dependent on the salicylic acid signalling cascade. Although the approach is powerful in generating oomycete resistance, the induced HR might affect plant health. Its application thus requires a careful selection of individual transgenic lines and trials with various pathogens.

Production of 6-methylsalicylic acid by expression of a fungal polyketide synthase activates disease resistance in tobacco.

Salicylic acid (SA) has been shown to act as a signal molecule that is produced by many plants subsequent to the recognition of potentially pathogenic microbes. Increases in levels of SA often trigger the activation of plant defenses and can result in increased resistance to subsequent challenge by pathogens. We observed that the polyketide 6-methylsalicylic acid (6-MeSA), a compound that apparently is not endogenous to tobacco, can mimic SA. Tobacco leaves treated with 6-MeSA show enhanced accumulation of the pathogenesis-related (PR) proteins PR1, beta-1,3-glucanase, and chitinase and also develop increased resistance to tobacco mosaic virus. We transformed tobacco with 6msas, the 6-methylsalicylic acid synthase (6MSAS) gene from Penicillium patulum, to generate plants that constitutively accumulate 6-MeSA. Analysis of primary transformants and the first generation progeny of 6MSAS tobacco revealed that plants can be engineered to accumulate significant amounts of 6-MeSA as a conjugate. Levels of total 6-MeSA increased with plant age. Increased 6-MeSA accumulation correlated with increased levels of PR1 and chitinase proteins and resulted in enhanced resistance of NN genotype 6MSAS tobacco to tobacco mosaic virus. Our results demonstrate that a multistep biosynthetic pathway can be engineered into plants using a single fungal polyketide synthase gene. The functional expression of 6msas can be used to activate disease resistance pathways that normally are induced by SA.

Production of 6-Methylsalicylic Acid by Expression of a Fungal Polyketide Synthase Activates Disease Resistance in Tobacco

Salicylic acid (SA) has been shown to act as a signal molecule that is produced by many plants subsequent to the recognition of potentially pathogenic microbes. Increases in levels of SA often trigger the activation of plant defenses and can result in increased resistance to subsequent challenge by pathogens. We observed that the polyketide 6-methylsalicylic acid (6-MeSA), a compound that apparently is not endogenous to tobacco, can mimic SA. Tobacco leaves treated with 6-MeSA show enhanced accumulation of the pathogenesis-related (PR) proteins PR1, beta-1,3-glucanase, and chitinase and also develop increased resistance to tobacco mosaic virus. We transformed tobacco with 6msas, the 6-methylsalicylic acid synthase (6MSAS) gene from Penicillium patulum, to generate plants that constitutively accumulate 6-MeSA. Analysis of primary transformants and the first generation progeny of 6MSAS tobacco revealed that plants can be engineered to accumulate significant amounts of 6-MeSA as a conjugate. Levels of total 6-MeSA increased with plant age. Increased 6-MeSA accumulation correlated with increased levels of PR1 and chitinase proteins and resulted in enhanced resistance of NN genotype 6MSAS tobacco to tobacco mosaic virus. Our results demonstrate that a multistep biosynthetic pathway can be engineered into plants using a single fungal polyketide synthase gene. The functional expression of 6msas can be used to activate disease resistance pathways that normally are induced by SA.

Production of 6-Methylsalicylic Acid by Expression of a Fungal Polyketide Synthase Activates Disease Resistance in Tobacco

Production of 6-Methylsalicylic Acid by Expression of a Fungal Polyketide Synthase Activates Disease Resistance in Tobacco

The pseudomonas AvrPto protein is differentially recognized by tomato and tobacco and is localized to the plant plasma membrane.

The avrPto gene of Pseudomonas syringae pv tomato triggers race-specific resistance in tomato plants carrying Pto, a resistance gene encoding a protein kinase. When introduced into P. s. tabaci, avrPto triggers resistance in tobacco W38 plants that carry the corresponding R gene. The AvrPto protein is believed to be secreted into host cells through the bacterial type III secretion pathway, where it activates disease resistance in tomato by interacting with Pto. We report here the identification of two distinct regions in AvrPto that determine the recognition specificity of this protein in tomato and tobacco. Point mutations in the central region disrupted the avirulence activity in tomato but not in tobacco. Conversely, point mutations in the C-terminal region abolished the avirulence in tobacco but not in tomato. We further report that AvrPto was localized to the plasma membrane of plant cells. Disrupting the membrane association by mutating a putative myristoylation motif of AvrPto abolished the avirulence activity in both tomato and tobacco. These findings demonstrate that AvrPto is recognized differently by the R genes in tomato and tobacco and that the recognition of AvrPto probably is associated with the plasma membrane.

In vivo imaging of an elicitor-induced nitric oxide burst in tobacco.

A growing body of evidence suggests that nitric oxide (NO), an important signalling and defence molecule in mammals, plays a key role in activating disease resistance in plants, acting as signalling molecule and possibly as direct anti-microbial agent. Recently, a novel fluorophore (diaminofluorescein diacetate, DAF-2 DA) has been developed which allows bio-imaging of NO in vivo. Here we use the cell-permeable DAF-2 DA, in conjunction with confocal laser scanning microscopy, for real-time imaging of NO in living plant cells. Epidermal tobacco cells treated with cryptogein, a fungal elicitor from Phytophthora cryptogea, respond to the elicitor with a strong increase of intracellular NO. NO-induced fluorescence was found in several cellular compartments, and could be inhibited by a NO scavenger and an inhibitor of nitric oxide synthase. The NO burst was triggered within minutes, reminiscent of the oxidative burst during hypersensitive response reactions. These results reveal additional similarities between plant and animal host responses to infection.

Expression of tobacco class II catalase gene activates the endogenous homologous gene and is associated with disease resistance in transgenic potato plants.

We have previously shown that healthy potato plants respond poorly to salicylic acid (SA) for activating disease resistance against the late blight fungal pathogen Phytophthora infestans. However, SA is essential for the establishment of potato systemic acquired resistance (SAR) against P. infestans after treatment with the fungal elicitor arachidonic acid (AA). To understand the molecular mechanisms through which AA induces SA-dependent SAR in potato, we have recently studied the expression of potato class II catalase (Cat2St) in comparison with its tobacco homologue, Cat2Nt, which has previously been shown to bind SA. In the present study, we show that tobacco Cat2Nt is expressed at high levels and accounts for almost half of total SA-binding activity detected in tobacco leaves. In contrast, potato Cat2St is not expressed in healthy leaves, which is associated with the low SA responsiveness of potato plants for activation of disease resistance mechanisms. Upon treatment with AA, expression of potato Cat2St is induced not only in AA-treated leaves, but also in the upper untreated parts of the plants, concomitant with the establishment of SA-dependent SAR to P. infestans. Moreover, expression of the tobacco Cat2Nt gene in transgenic potato plants leads to constitutive expression of the endogenous potato Cat2St gene and is associated with enhanced resistance to P. infestans. These results collectively indicate that plant SA-binding class II catalases may play an important role in the development of disease resistance, possibly by serving as biological targets of SA.

A mechanism for ethylene-induced disease resistance in soybean: enhanced synthesis of an elicitor-releasing factor, β-1,3-endoglucanase

Exogenous ethylene increased the level of β-1,3-endoglucanase (EC 3.2.1.39) activity and the level ofβ-1,3-endoglucanase protein 2- to 4-fold higher than constitutive levels in hypocotyls of soybean seedlings. Ethylene-treated soybean hypocotyls showed partial resistance upon infection with a compatible race of Phytophthora megasperma f. sp. glycinea, accompanied with higher levels of the phytoalexin, glyceollin. Purified soybean β-1,3-endoglucanase had no direct fungitoxic activity to the fungus. The purified glucanase, however, released soluble elicitors from cell walls of the fungus. Higher levels of glyceollin accumulated in hypocotyls of ethylene-treated soybean seedlings than in control hypocotyls when they were treated with native fungal cell walls. Similar levels of glyceollin accumulated when ethylene-treated and control hypocotyls were treated with soluble elicitor released by the glucanase. These results indicate that more efficient elicitor release occurred in the ethylene-treated soybean tissue. In addition, co-treatment of hypocotyls with the purified glucanase and the native cell walls, but not the glucanase-released elicitor, resulted in greater glyceollin accumulation. Exogenous application of the purified glucanase to inoculation sites also increased resistance of soybean hypocotyls against attack by the fungus. In contrast, resistance of soybean hypocotyls to an incompatible race of P. megasperma f. sp. glycinea was partially negated when the seedlings were absorbed with laminarin, which had been shown to inhibit the reaction of elicitor release from fungus cell walls. These results support the idea that the increase in resistance in ethylene-treated soybean tissue may be due, in part, to enhanced β-1, 3-endoglucanase activity which may release elicitor-active molecules from fungal cell walls. The results also suggest that the process of elicitor release mediated by host β-1,3-endoglucanase is an important process leading to active disease resistance in soybean.

Molecular Cloning and Ethylene Induction of mRNA Encoding a Phytoalexin Elicitor-Releasing Factor, β-1,3-Endoglucanase, in Soybean

Soybean (Glycine max) beta-1,3-endoglucanase (EC 3.2. 1.39) is involved in one of the earliest plant-pathogen interactions that may lead to active disease resistance by releasing elicitor-active carbohydrates from the cell walls of fungal pathogens. Ethylene induced beta-1,3-endoglucanase activity to 2- to 3-fold higher levels in cotyledons of soybean seedlings. A specific polyclonal antiserum raised against purified soybean beta-1,3-endoglucanase was used to immunoprecipitate in vitro translation products, demonstrating that ethylene induction increased translatable beta-1,3-endoglucanase mRNA. Several cDNA clones for the endoglucanase gene were obtained by antibody screening of a lambda-gt11 expression library prepared from soybean cotyledons. Hybrid-select translation experiments indicated that the cloned cDNA encoded a 36-kilodalton precursor protein product that was specifically immunoprecipitated with beta-1,3-endoglucanase antiserum. Escherichia coli cells expressing the cloned cDNA also synthesized an immunologically positive protein. Nucleotide sequence of three independent clones revealed a single uninterrupted open reading frame of 1041 nucleotides, corresponding to a polypeptide of 347 residue long. The primary amino acid sequence of beta-1,3-endoglucanase as deduced from the nucleotide sequence was confirmed by direct amino acid sequencing of trypsin digests of the glucanase. The soybean beta-1,3-endoglucanase exhibited 53% amino acid homology to a beta-1,3-glucanase cloned from cultured tobacco cells and 48% homology to a beta-(1,3-1,4)-glucanase from barley. Utilizing the largest cloned cDNA (pEG488) as a hybridization probe, it was found that the increase in translatable beta-1,3-endoglucanase mRNA seen upon ethylene treatment of soybean seedlings was due to 50- to 100-fold increase in steady state mRNA levels, indicating that ethylene regulates gene expression of this enzyme important in disease resistance at the level of gene transcription.