Purification and expression of a protein elicitor from Alternaria tenuissima and elicitor-mediated defence responses in tobacco

A new protein elicitor, PeaT1, was purified from the mycelium of Alternaria tenuissima by column chromatography. PeaT1 was identified as a heat-stable and acidic protein. It induced systemic acquired resistance to tobacco mosaic virus (TMV) in tobacco plants but did not cause hypersensitive response. The elicitor-encoding gene was cloned by rapid amplification of cDNA ends method. Sequence analysis revealed that the cDNA is 624 bp in length and the open reading frame encodes for a polypeptide of 207 amino acids with a nascent polypeptide-associated complex domain. The peaT1 gene was cloned into the expression vector pET-28a and transformed into Escherichia coli BL21 (DE3). The recombinant elicitor also triggered defence responses in intact tobacco plants. The availability of the pure protein offers the possibility to isolate the corresponding receptor and links it to the downstream signalling pathway.     

MAP kinase cascades in elicitor signal transduction

 Protein kinases play important roles in elicitor signal transduction. In this article, I describe the current view of the role of mitogen-activated protein kinase (MAPK) cascades in elicitor signal transduction of plant cells based on our own research and recent developments in this field. In the past several years, it has become apparent that MAPK cascades play important roles in elicitor signal transduction in plants. Our early studies demonstrated the identification of p47 MAPK in tobacco as an elicitor-responsive protein kinase and possible involvement of p47 MAPK in elicitor signal transduction to induce defense responses, including defense gene expression and hypersensitive cell death. However, the molecular identity of p47 MAPK is still unclear. Recent important studies suggest that tobacco MAPK cascades that include SIPK, and/or WIPK, and NtMEK2, an upstream kinase for both SIPK and WIPK, have a crucial function in induction of defense responses and hypersensitive cell death. The orthologs of these protein kinases in Arabidopsis and alfalfa are also suggested to have similar functions. Furthermore, the identification of loss-of-function mutation in Arabidopsis reveals a negative regulatory role for putative MAPK cascades in plant defense mechanisms. Received: February 7, 2002 / Accepted: February 25, 2002     

<Emphasis Type="SmallCaps">l</Emphasis>-Alanine augments rhizobacteria-induced systemic resistance in cucumber

Bacillus vallismortis strain EXTN-1 is a proven biotic elicitor of systemic resistance in many crops against various pathogens. l-Alanine (Ala) was tested in cucumber as a chemical elicitor of induced systemic resistance (ISR) against Colletotrichum orbiculare. In the greenhouse, both Ala and EXTN-1 induced significant levels of disease suppression in cucumber against anthracnose. When cucumber plants were treated with EXTN-1 and Ala together, augmentative disease suppression was observed. Experiments with transgenic tobacco plants carrying pathogenesis-related genes fused with the β-glucuronidase (GUS) reported gene (PR-1a::GUS & PDF 1.2::GUS) showed an enhanced activation of both PR-1a and PDF 1.2 genes upon combined treatment with Ala and EXTN-1. RT-PCR analysis with transgenic (PR-1a or PDF 1.2 over expressing) Arabidopsis plant showed more enhanced expression of resistance genes PR-1a and PDF 1.2 upon combined treatment with Ala and EXTN-1 than either alone. An augmentative ISR effect, when the bacterial elicitor and chemical elicitor were combined together, was confirmed.     

The purification and characterization of a novel hypersensitive-like response-inducing elicitor from Verticillium dahliae that induces resistance responses in tobacco

PevD1, a novel protein elicitor from the pathogenic cotton verticillium wilt fungus, Verticillium dahliae, induced a hypersensitive response in tobacco plants. In this paper, the elicitor was purified and analyzed using de novo sequencing. The protein-encoding pevD1 gene consists of a 468-bp open reading frame that produces a polypeptide of 155 amino acids, with a theoretical molecular weight of 16.23 kDa. The sequence of elicitor protein PevD1 was matched to the genomic sequence (GenBank accession no. ABJE 01000445.1) of a putative protein from V. dahliae strain vdls.17, but a function had not yet been reported. The pevD1 gene was expressed in Escherichia coli, and the recombinant protein was characterized for its ability to confer systemic acquired resistance to tobacco mosaic virus (TMV). Recombinant PevD1-treated plants exhibited enhanced systemic resistance compared to control, including a significant reduction in the number and size of TMV lesions on tobacco leaves. The elicitor protein-induced hydrogen peroxide production, extracellular-medium alkalization, callose deposition, phenolics metabolism, and lignin synthesis in tobacco. Our results demonstrate that elicitor-PevD1 triggers defense responses in intact tobacco plants.     

Expression of the Hypersensitive Response-assisting Protein in <Emphasis Type="Italic">Arabidopsis</Emphasis> Results in Harpin-dependent Hypersensitive Cell Death in Response to <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

Active defense mechanisms of plants against pathogens often include a rapid plant cell death known as the hypersensitive cell death (HCD). Hypersensitive response-assisting protein (HRAP) isolated from sweet pepper intensifies the harpin_Pss-mediated HCD. Here we demonstrate that constitutive expression of the hrap gene in Arabidopsis results in an enhanced disease resistance towards soft rot pathogen, E. carotovora subsp. carotovora. This resistance was due to the induction of HCD since different HCD markers viz. Athsr3, Athsr4, ion leakage, H₂O₂ and protein kinase were induced. One of the elicitor harpin proteins, HrpN, from Erwinia carotovora subsp. carotovora was able to induce a stronger HCD in hrap-Arabidopsis than non-transgenic controls. To elucidate the role of HrpN, we used E. carotovora subsp. carotovora defective in HrpN production. The hrpN⁻ mutant did not induce disease resistance or HCD markers in hrap-Arabidopsis. These results imply that the disease resistance of hrap-Arabidopsis against a virulent pathogen is harpin dependent.     

Expression of a stilbene synthase gene in Nicotiana tabacum results in synthesis of the phytoalexin resveratrol

A gene from groundnut (Arachis hypogaea) coding for stilbene synthase was transferred together with a chimaeric kanamycin resistance gene. It was found to be rapidly expressed after induction with UV light and elicitor in tobacco cells (Nicotiana tabacum). Comparative studies of stilbene synthase mRNA synthesis in groudnut and transgenic tobacco suspension cultures revealed the same kinetics of gene expression. Stilbene synthase specific mRNA was detectable 30 minutes after elicitor induction and 10 minutes after UV irradiation. The maximum of mRNA accumulation was between 2 and 8 hours post induction. 24 hours after induction stilbene synthase mRNA accumulation ceased. Furthermore, in transgenic tobacco plants, the gene was found to be inducible in sterile roots, stems and leaves. Stilbene synthase was demonstrated in crude protein extracts from transgenic tobacco cell cultures using specific antibodies. Resveratrol, the product of stilbene synthase, was identified by HPLC and antisera raised against resveratrol.     

The fungal subtilase AsES elicits a PTI-like defence response in Arabidopsis thaliana plants independently of its enzymatic activity

Acremonium strictum elicitor subtilisin (AsES) is a 34-kDa serine-protease secreted by the strawberry fungal pathogen A. strictum. On AsES perception, a set of defence reactions is induced, both locally and systemically, in a wide variety of plant species and against pathogens of alternative lifestyles. However, it is not clear whether AsES proteolytic activity is required for triggering a defence response or if the protein itself acts as an elicitor. To investigate the necessity of the protease activity to activate the defence response, AsES coding sequences of the wild-type gene and a mutant on the active site (S226A) were cloned and expressed in Escherichia coli. Our data show that pretreatment of Arabidopsis plants with inactive proteins, i.e. inhibited with phenylmethylsulphonyl fluoride (PMSF) and mutant, resulted in an increased systemic resistance to Botrytis cinerea and expression of defence-related genes in a temporal manner that mimics the effect already reported for the native AsES protein. The data presented in this study indicate that the defence-eliciting property exhibited by AsES is not associated with its proteolytic activity. Moreover, the enhanced expression of some immune marker genes, seedling growth inhibition and the involvement of the co-receptor BAK1 observed in plants treated with AsES suggests that AsES is being recognized as a pathogen-associated molecular pattern by a leucine-rich repeat receptor. The understanding of the mechanism of action of AsES will contribute to the development of new breeding strategies to confer durable resistance in plants.     

Production of the AVR9 elicitor from the fungal pathogen Cladosporium fulvum in transgenic tobacco and tomato plants

Three constructs were used to study the expression of the avirulence gene Avr9 from the fungal tomato pathogen Cladosporium fulvum in plants. They include pAVIR1, pAVIR2 and pAVIR21, encoding the wild-type AVR9 protein and two hybrid AVR9 proteins containing the signal sequences of the pathogenesis-related proteins PR-S and PR-1a, respectively. Transgenic tobacco plants obtained with the three constructs showed a normal phenotype and produced AVR9 elicitor with the same specific necrosis-inducing activity as the wild-type AVR9 elicitor produced in planta by isolates of C. fulvum containing the Avr9 gene. Level of expression was not correlated with number of T-DNA integrations, but plants homozygous for the Avr9 gene produced more elicitor protein than heterozygous plants. The amino acid sequence of the processed AVR9 peptide present in apoplastic fluid (AF) of pAVIR1 transformed plants producing the wild-type AVR9 elicitor was identical to that of the wild-type AVR9 peptide isolated from C. fulvum-infected tomato leaves. Transgenic Cf0 genotypes of tomato, obtained by transformation with construct pAVIR21, showed a normal phenotype. However, transgenic F1 plants expressing the Avr9 gene, obtained from crossing transgenic Cf0 genotypes with wild-type Cf9 genotypes, showed delayed growth, necrosis and complete plant death indicating that the AVR9 peptide produced in plants carrying the Cf9 gene is deleterious. The necrotic defence response observed in Cf9 genotypes expressing the Avr9 gene support the potential to apply avirulence genes in molecular resistance breeding.     

Overexpression of AtMHX in tobacco causes increased sensitivity to Mg<Superscript>2+</Superscript>, Zn<Superscript>2+</Superscript>, and Cd<Superscript>2+</Superscript> ions,induction of V-ATPase expression,and a reduction in plant size

AtMHX is a vacuolar transporter encoded by a single gene in Arabidopsis. Electrophysiological analysis showed that it exchanges protons with Mg²⁺, Zn²⁺, and Fe²⁺ ions. The physiological impact of AtMHX was examined so far only in tissue-culture grown seedlings of tobacco plants overexpressing this transporter. Here we investigated the impact of AtMHX on growth, response to different metals, and metal accumulation of mature tobacco plants, as well as Arabidopsis plants in which we overexpressed this transporter. The analyses were carried out in hydroponic growth-systems, in which the mineral composition could be effectively controlled, and the metal content of roots could be examined. Transformed tobacco plants showed necrotic lesions and apical burnings upon growth with increased levels of Mg²⁺, Zn²⁺, and Cd²⁺ ions. This suggested that AtMHX can carry in planta not only Mg²⁺ and Zn²⁺ ions, as previously deduced based on observations in tissue-culture, but also Cd²⁺ ions. Transformed plants of both tobacco and Arabidopsis showed a reduction in plant size. However, the overall response of Arabidopsis to AtMHX overexpression was minor. No change was detected in the mineral content of any organ of the transgenic tobacco or Arabidopsis plants. The necrotic lesions in tobacco resembled those seen in plants with perturbed proton balancing, raising the assumption that AtMHX can affect the proton homeostasis of cells. In agreement with this assumption, the transformed tobacco plants had increased expression and activity of the vacuolar H⁺-ATPase. The relative significance of AtMHX for metal and proton homeostasis still has to be elucidated.     

Differences in the processing of DNA ends in Arabidopsis thaliana and tobacco: possible implications for genome evolution

Surprising species-specific differences in non-homologous end-joining (NHEJ) of genomic double-strand breaks (DSBs) have been reported for the two dicotyledonous plants Arabidopsis thaliana and Nicotiana tabacum. In Arabidopsis deletions were, on average, larger than in tobacco and not associated with insertions. To establish the molecular basis of the phenomenon we analysed the fate of free DNA ends in both plant species by biolistic transformation of leaf tissue with linearized plasmid molecules. Southern blotting indicated that, irrespective of the nature of the ends (blunt, 5 or 3 overhangs), linearized full-length DNA molecules were, on average, more stable in tobacco than in Arabidopsis. The relative expression of a -glucuronidase gene encoded by the plasmid was similar in both plant species when the break was distant from the marker gene. However, if a DSB was introduced between the promoter and the open reading frame of the marker, transient expression was halved in Arabidopsisas compared to tobacco. These results indicate that free DNA ends are more stable in tobacco than in Arabidopsis, either due to lower DNA exonuclease activity or due to a better protection of DNA break ends or both. Exonucleolytic degradation of DNA ends might be a driving force in the evolution of genome size as the Arabidopsis genome is more than twenty times smaller than the tobacco genome.     

OsCPK20 positively regulates Arabidopsis resistance against Pseudomonas syringae pv. tomato and rice resistance against Magnaporthe grisea

Calcium-dependent protein kinases are important decoders of calcium signals in plants, which are involved in plant immunity. We report isolation and functional characterization of a pathogen-responsive OsCPK20 gene in rice. The expression of OsCPK20 in rice was significantly induced following treatment with a Magnaporthe grisea elicitor. Overexpression of constitutively active OsCPK20 in Arabidopsis enhanced the resistance to infection with Pseudomonas syringae pv. tomato, associated with elevated expression of both SA- and JA-related defense genes. Similarly, transgenic rice plants containing constitutively active OsCPK20 exhibited enhanced resistance to blast fungus M. grisea. The enhanced resistance in the transgenic Arabidopsis and rice was associated with activated expression of both SA- and JA-related defense genes. We also found that OsCPK20 was significantly induced by drought stress, indicating that OsCPK20 might be involved in plant response to drought stress. Taken together, our results indicate that rice OsCPK20 positively regulates Arabidopsis resistance against Pseudomonas syringae pv. tomato and rice resistance against M. grisea, and that it may enhance disease resistance by activating both SA- and JA-dependent defense responses.     

Glutamate Fermentation By‐product Activates Plant Defence Responses and Confers Resistance Against Pathogen Infection

In the food industry, glutamate fermentation by‐product (GFB) is generated by purifying glutamate products from microbial fermentation. The potential applications of GFB for upgrading agricultural soil, for foliar fertility, and as plant plankton for shrimp have been studied. We examined the efficacy of GFB foliar application and determined that GFB treatment increased the resistance of Arabidopsis leaves to infection by bacterial pathogens. Microarray gene expression analysis of Arabidopsis leaves after treatment with GFB indicated that the expression of plant defence‐related genes increased. In Corynebacterium fermentation, the active substances for induction of the defence response were extracted or solubilized after treatment with heating under acidic conditions. This extract was also effective in strawberry and grape leaves for the induction of hydrogen peroxide production. These findings suggest that foliar application of GFB that contains elicitor molecules derived from fermentation bacteria is useful for plant protection in agricultural fields.     

Salicylic Acid and a Chitin Elicitor Both Control Expression of the CAD1 Gene Involved in the Plant Immunity of Arabidopsis

The Arabidopsis mutant cad1 (constitutively activated cell death 1) shows a phenotype that mimics hypersensitive response (HR)-like cell death. The CAD1 gene, which encodes a protein containing a domain with significant homology to the MACPF (membrane attach complex and perforin) domain of complement components and perforin, is likely to control plant immunity negatively and has a W-box cis-element in its promoter region. We found that expression of the CAD1 gene and other W-box containing genes, such as NPR1 and PR2, was promoted by salicylic acid (SA) and benzothiadiazole (BTH) as a SA agonist. The CAD1 gene was also stimulated by a purified chitin oligosaccharide elicitor (degree of polymerization = 8). This latter control was not under SA, because CAD1 expression was not suppressed in 35SnahG transgenic plants, which are unable to accumulate SA. These expression profiles were confirmed by promoter analysis using pCAD1::GUS transgenic plants. The CAD1 expression promoted by BTH and the chitin elicitor was not suppressed in the npr1 mutant, which is insensitive to SA signaling. These results indicate that the CAD1 gene is regulated by two distinct pathways involving SA and a chitin elicitor: viz., SA signaling mediated through an NPR1-independent pathway, and chitin elicitor signaling, through an SA-independent pathway. Three CAD1 homologs that have multiple W-box elements in their promoters were also found to be under the control of SA.     

Plant defense in response to chewing insects: proteome analysis of Arabidopsis thaliana damaged by Plutella xylostella

The interactions between Arabidopsis thaliana and Plutella xylostella have been considered as a model system to unravel the responses of plants to herbivorous insects. Here, we use a 2-DE proteome approach to detect protein expression changes in the leaves of Arabidopsis plants exposed to P. xylostella larval infestation at 27°C within 8?h. Approximately 450 protein spots were reproducibly detected on gels. Of these, comparing healthy and infested leaves, we identified 18 differentially expressed protein spots. Thirteen proteins were successfully identified by MALDI-TOF/MS and LC-ESI-MS/MS. Functional classification analysis indicated that the differentially identified proteins were associated with amino acid, carbohydrate, energy, lipid metabolism, and photosynthesis. In addition, their relative abundances were assessed according to larval pest feeding on Arabidopsis leaves. These data provide valuable new insights for further works in plant-biotic and environmental stress interaction.     

Effects of Foliar and Root Applications of Methanol on the Growth of Arabidopsis, Tobacco, and Tomato Plants

The effects of aqueous methanol solutions applied as a foliar spray or via irrigation were investigated in Arabidopsis, tobacco, and tomato plants. Methanol applied to roots leads to phytotoxic damage in all three species tested. Foliar application causes an increase of fresh and dry weight in Arabidopsis and tobacco plants, but not in tomato plants. The increase in fresh and dry weight of Arabidopsis plants does not correlate with increased levels of soluble sugars, suggesting that increased accumulation of other products is responsible for the differences in the methanol-treated leaves. Foliar application of methanol can induce pectin methylesterase (PME) gene expression in Arabidopsis and tomato plants, activating specific PME genes.     

Developmental regulation and tissue-specific differences of heat shock gene expression in transgenic tobacco and Arabidopsis plants

The heat shock (hs) response during plant growth and development was analyzed in tobacco and Arabidopsis using chimaeric -glucuronidase reporter genes (hs-Gus) driven by a soybean hs promoter. Fluorimetric measurements and histochemical staining revealed high Gus activities in leaves, roots, and flowers exclusively after heat stress. The highest levels of heat-inducible expression were found in the vascular tissues. Without heat stress, a developmental induction of hs-Gus was indicated by the accumulation of high levels of Gus in transgenic tobacco seeds. There was no developmental induction of hs-Gus in Arabidopsis seeds. In situ hybridization to the RNA of the small heat shock protein gene Athsp17.6 in tissue sections revealed an expression in heat-shocked leaves but no expression in control leaves of Arabidopsis. However, a high level of constitutive expression of hs gene was detected in meristematic and provascular tissues of the Arabidopsis embryo. The developmental and tissue-specific regulation of the hs response is discussed.Abbreviations hs heat shock - Hsp heat shock protein(s) - hs Gus: heat-inducible Gus gene(s) - HSE heat shock element(s) - HSF heat shock factor - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronide - Gus -glucuronidase - DAF days after flowering - SAR scaffold attachment region     

Identification and isoprenylation of plant GTP-binding proteins

To identify isoprenylated plant GTP-binding proteins,Arabidopsis thaliana andNicotiana tabacum cDNA expression libraries were screened for cDNA-encoded proteins capable of binding [³²P]GTPin vitro. ATGB2, anArabidopsis homologue of the GTP-binding protein Rab2, was found to bind GTPin vitro and to be a substrate for a geranylgeranyl:protein transferase (GGTase) present in plant extracts. The carboxyl terminus of this protein contains a-GCCG sequence, which has not previously been shown to be recognized by any prenyl:protein transferase (PTase), but which most closely resembles that isoprenylated by the type II GGTase (-XXCC,-XCXC, or-CCXX).In vitro geranylgeranylation of anArabidopsis Rab1 protein containing a carboxyl-terminal-CCGQ sequence contirmed the presence of a type II GGTase-like activity in plant extracts. Several other proteins were also identified byin vitro GTP binding, includingArabidopsis and tobacco homologues of Rab11, ARF (ADP-ribosylation factor) and Sar proteins, as well as a novel 22 kDaArabidopsis protein (ATG81). This 22 kDa protein had consensus GTP-binding motifs and bound GTP with high specificity, but its structure was not closely related to that of any known GTP-binding protein (it most resembled proteins within the ARF/Sar and G protein -subunit superfamilies).