Activate Plant Defense Research Articles

Autoimmunity in plants.

Attenuation in the activity of the negative regulators or the hyperactivity of plant innate immune receptors often causes ectopic defense activation manifested in severe growth retardation and spontaneous lesion formations, referred to as autoimmunity. In this review, we have described the cellular and molecular basis of the development of autoimmune responses for their useful applications in plant defense. Plants are exposed to diverse disease-causing pathogens, which bring infections by taking over the control on host immune machineries. To counter the challenges of evolving pathogenic races, plants recruit specific types of intracellular immune receptors that mostly belong to the family of polymorphic nucleotide-binding oligomerization domain-containing leucine-rich repeat (NLR) proteins. Upon recognition of effector molecules, NLR triggers hyperimmune signaling, which culminates in the form of a typical programmed cell death, designated hypersensitive response. Besides, few plant NLRs also guard certain host proteins known as 'guardee' that are modified by effector proteins. However, this fine-tuned innate immune system can be lopsided upon knock-out of the alleles that correspond to the host guardees, which mimick the presence of pathogen. The absence of pathogens causes inappropriate activation of the respective NLRs and results in the constitutive activation of plant defense and exhibiting autoimmunity. In plants, autoimmune mutants are readily scorable due to their dwarf phenotype and development of characteristic macroscopic disease lesions. Here, we summarize recent reports on autoimmune response in plants, how it is triggered, and phenotypic consequences associated with this phenomenon.

Postharvest Quality of Cut Roses With the Application of Salicylic Acid

The objective of this work was to evaluate the effect of the application of salicylic acid (SA) on the maintenance of quality and longevity of cut roses cv. Vega. Cut roses were kept in a vase solution of SA and water at concentrations of 0; 0.5; 1.0; 1.5 and 2.0 mM. All treatments were kept at 8±2 oC for 96 hours, simulating storage in flower shops; the flowers were then evaluated regarding loss of fresh matter and leaf chlorophyll content and were transferred to beakers containing distilled water at 25±2 ºC for more 144 hours, simulating shelf life. At 24, 48, 72 and 96 hours from the beginning of the experiment, biochemical analyses of total proteins and the activity of the phenylalanine ammonia-lyase (PAL) and peroxidases (PO) were performed. Visual analyses were performed (stem curvature, turgescence and petal darkening) every 48 hour intervals until the end of the experiment. The treatments with SA allowed the maintenance of post-harvest quality, reducing the loss of fresh matter mass, lower stem curvature, greater turgescence and less darkening of the petals. The results showed that the application of SA increased total protein contents and FAL, which characterizes the activation of plant defense mechanisms to the senescence process.

Induced Systemic Resistance Against Citrus Canker Disease by Rhizobacteria

Citrus canker, caused by Xanthomonas citri subsp. citri, is an important citrus disease that causes significant economic losses worldwide. All commercial citrus varieties are susceptible to citrus canker. Currently, chemical control with copper based products is the main approach to control X. citri subsp. citri dispersal and plant colonization. However, extensive use of copper compounds can result in copper-resistant strains and cause adverse effects on the environment. Alternatives to chemical control involve the activation of citrus immunity to control the disease. Here, we investigated the ability of multiple rhizobacteria to induce a systemic defense response in cultivar Duncan grapefruit. Burkholderia territorii strain A63, Burkholderia metallica strain A53, and Pseudomonas geniculata strain 95 were found to effectively activate plant defense and significantly reduce symptom development in leaves challenged with X. citri subsp. citri. In the priming phase, root application of P. geniculata induced the expression of salicylic acid (SA)-signaling pathway marker genes (PR1, PR2, PR5, and salicylic acid carboxyl methyltransferase [SAM-SACM]). Gene expression analyses after X. citri subsp. citri challenge showed that root inoculation with P. geniculata strain 95 increased the relative levels of phenylalanine ammonia lyase 1 and SAM-SACM, two genes involved in the phenylpropanoid pathway as well as the biosynthesis of SA and methyl salicylate (MeSA), respectively. However, hormone analyses by UPLC-MS/MS showed no significant difference between SA in P. geniculata-treated plants and control plants at 8 days post-beneficial bacteria root inoculation. Moreover, P. geniculata root-treated plants contained higher reactive oxygen species levels in aerial tissues than control plants 8 days post-treatment application. This study demonstrates that rhizobacteria can modulate citrus immunity resulting in a systemic defense response against X. citri subsp. citri under greenhouse conditions.

Effects of manganese toxicity on the protein profile of tomato (Solanum lycopersicum) roots as revealed by two complementary proteomic approaches, two-dimensional electrophoresis and shotgun analysis

The aim of this work was to assess the effects of manganese (Mn) toxicity on the proteome of tomato roots using two proteomic approaches, shotgun and two-dimensional electrophoresis. The shotgun approach yielded 367 reliable proteins, whereas the 2-DE approach detected 340 consistent spots. The 2-DE method found 54 proteins changing in relative abundance in the excess Mn treatment, whereas the shotgun detected changes in 118 proteins. Only 7% of the differential proteins were found by both methods, illustrating their complementary nature. Metabolic pathways most affected were protein metabolism, oxido-reductases and signaling. Results support that Mn toxicity alters the protein turnover and impairs energy production in roots, leading to changes in glycolysis, pyruvate metabolism, TCA and oxidative phosphorylation. Excess Mn also induced changes in peroxidases and hydrolases participating in cell wall lignification and suberization and activated plant defense mechanisms, with changes occurring via pathogenesis-related proteins as well as peroxidases. Finally, Mn toxicity elicited regulatory mechanisms and affected the abundance of root nutrient reservoir proteins. The overall analysis of the differential root proteome upon Mn toxicity suggests a general slowdown of metabolic activities, especially energy production, cell wall integrity and protein turnover, which occurs in parallel with increases in stress related proteins.

English

Secondary metabolites may play a number of roles, among them, is the activation of plant defense mechanisms, such as phytoalexins and the synthesis of antioxidative enzymes. This study aimed to test the eliciting activity of phytoalexins of Lippia sidoides Cham. and six endophytic fungi associated with it and to verify the synthesis of the enzymes, as a response to the antioxidative and antifungal system. The assays were performed using spectrophotometric methods. Of the concentrations of essential oil used, 7.5 mg mL-1 was 66.48% higher than the Acorda® positive control for the induction of glyceollin. The endophytic fungus, Verticillium sp. showed the best result for 3-deoxythianocyanidine production. Excellent results were demonstrated by the LS-14 fungus for superoxide dismutase enzyme activity (130 U g-1 E-1 min-1); by L. sidoides for the activity of ascorbate peroxidase enzyme (3821 μmol ASA g-1 E-1 min-1); by L. sidoides for the activity of catalase enzyme (2429 μmol H2O2 g-1 E-1 min-1); by Fusarium sp. 2 for the activity of phenol peroxidase (87 μmol H2O2 g-1 E-1 min-1) and by Fusarium sp. 1 for the activity of chitinase (0.214 U min-1). Both the essential oil of L. sidoides and the extracts of the endophytic fungi had a positive response to the induction of phytoalexin and presence of enzymes related to antioxidative activity were verified and shown. Key words: Catalase, superoxide dismutase, peroxidase phenols, ascorbate peroxidase, chitinase.

Antibacterial potential of secondary metabolites produced by Aspergillus sp., an endophyte of Mitrephora wangii.

Mitrephora wangii, an ethnomedicinal plant, has been used as a natural antibiotic and immunity booster in Thailand. A total of 22 fungi were isolated from M. wangii flowers. The fungal isolates were categorized into six genera including Agrocybe, Aspergillus, Colletotrichum, Nigrospora, Puccinia and Ustilago. Most extracts exhibited antibacterial activity against at least one of the test bacteria. Aspergillus sp. MFLUCC16-0845 was identified as the most bioactive fungus. Chemical composition of Aspergillus sp. MFLUCC16-0845 investigated using gas chromatography-mass spectrometry indicated that the major antibacterial compound was β-thujaplicin. Moreover, the newly isolated Aspergillus sp. MFLUCC16-0845 could be exploited as a potential source of bioactive compounds and plant defense activators. In addition, it is the first time that strain of Aspergillus sp. isolated and cultured from M. wangii flowers could produce β-thujaplicin at high yield with strong antimicrobial spectrum, which may lead to wide utilization in producing cosmetics and clinical products.

Performance of plant defence activators against Podosphaera pannosa causing rose powdery mildew

Different plants defence activators were evaluated against the rose powdery mildew (Podosphaera pannosa) under green house conditions. Plant defence activators were tested at two concentrations i.e. 0.050 and 0.075% before the appearance of disease. The highest disease control of 78.48% was recorded in the plants sprayed with dipotassium orthophosphate followed by salicylic acid (74.62%), β-aminobutyric acid (72.16%), potassium silicate (64.69%) and potassium bicarbonate (66.79%) at concentration of 0.075% in two consecutive years (2015, 2016). The least disease control (54.95%) was observed in treatment with calcium silicate. At lower concentration (0.05%) disease control was comparatively lesser than the higher concentration of the plant defence activators. First spray of the SAR chemical was effective for 21 days and after that the disease appeared.

Effect of altering the steric hindrance of the nitrogen atom in 2,5-bis(pyridin-n-yl)-1,3,4 thiadiazole isomers on their ability to elicit tomato defense responses against Verticillium wilt and crown gall diseases

Three isomers of the ligand 2,5-bis(pyridinyl)-1,3,4-thiadiazole, with the N atom of pyridine group in position 2, 3 or 4, named respectively, L2, L3 and L4 were compared for their use as plant defense activators. They were examined for their ability to protect tomato plants from Verticillium dahliae and Agrobacterium tumefaciens in the greenhouse, to induce reactive oxygen species and to activate plant defenses, including antioxidant enzymes. The three ligand isomers exhibited in vitro only slight inhibition of radial growth of V. dahliae, while no significant inhibition was observed for phytopathogenic bacteria. In the greenhouse, the three ligand isomers statistically reduced the severity of Verticillium wilt and crown gall on tomato plants, and the isomers L3 and L4 were the most efficient to control Verticillium wilt. This superiority was reflected in their differential ability to activate H2O2 accumulation, antioxidant enzymes including catalase and ascorbate peroxidase and other defense-related enzymes such as guaiacol peroxidase and polyphenol oxidase. These results demonstrated that the presence of the N atom within the two pyridinyl groups in the position 3 or 4 highly enhanced the activity of plant defense and antioxidant responses as well as their ability to reduce the severity of symptoms caused by V. dahliae on tomato.

Transcriptome-based analyses of phosphite-mediated suppression of rust pathogens Puccinia emaculata and Phakopsora pachyrhizi and functional characterization of selected fungal target genes.

Phosphite (Phi) is used commercially to manage diseases mainly caused by oomycetes, primarily due to its low cost compared with other fungicides and its persistent control of oomycetous pathogens. We explored the use of Phi in controlling the fungal pathogens Puccinia emaculata and Phakopsora pachyrhizi, the causal agents of switchgrass rust and Asian soybean rust, respectively. Phi primes host defenses and efficiently inhibits the growth of P. emaculata, P. pachyrhizi and several other fungal pathogens tested. To understand these Phi-mediated effects, a detailed molecular analysis was undertaken in both the host and the pathogen. Transcriptomic studies in switchgrass revealed that Phi activates plant defense signaling as early as 1 h after application by increasing the expression of several cytoplasmic and membrane receptor-like kinases and defense-related genes within 24h of application. Unlike in oomycetes, RNA sequencing of P. emaculata and P. pachyrhizi did not exhibit Phi-mediated retardation of cell wall biosynthesis. The genes with reduced expression in either or both rust fungi belonged to functional categories such as ribosomal protein, actin, RNA-dependent RNA polymerase, and aldehyde dehydrogenase. A few P. emaculata genes that had reduced expression upon Phi treatment were further characterized. Application of double-stranded RNAs specific to P. emaculata genes encoding glutamate N-acetyltransferase and cystathionine gamma-synthase to switchgrass leaves resulted in reduced disease severity upon P. emaculata inoculation, suggesting their role in pathogen survival and/or pathogenesis.

Induction of systemic tolerance to Tilletia indica in wheat by plant defence activators

Plant defence activators viz., benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester, β-aminobutyric acid and salicylic acid at 1 mM dosage were evaluated to manage Tilletia indica and cent percent protection was achieved under glass house conditions. Further, efforts were made to dissect expression pattern of pathogenesis-related genes which are downstream components of the systemic acquired resistance (SAR) pathway in response to primed compounds and T. indica infection, based on real-time polymerase chain reaction analysis of marker gene expression levels. Maximal accumulation of PDF1.2 transcripts followed by PR1 and PR5 in independently primed wheat spikes with all tested compounds at three days post-inoculation was observed. Additionally, plant defence activators primed the plant for a rapid and intense response to pathogen infection involving augmented activation of salicylic acid (SA) – and jasmonic acid (JA)/ethylene (ET)-mediated defence response and enhanced level of wheat protection. Therefore, the use of plant defence activators to induce SAR could be used as a management strategy for reducing the impact of T. indica for quality and nutritious wheat production.

Functional Analogues of Salicylic Acid and Their Use in Crop Protection

Functional analogues of salicylic acid are able to activate plant defense responses and provide attractive alternatives to conventional biocidal agrochemicals. However, there are many problems that growers must consider during their use in crop protection, including incomplete disease reduction and the fitness cost for plants. High-throughput screening methods of chemical libraries allowed the identification of new compounds that do not affect plant growth, and whose mechanisms of action are based on priming of plant defenses, rather than on their direct activation. Some of these new compounds may also contribute to the discovery of unknown components of the plant immune system.

Evaluation of eliciting activity of peptidil prolyl cys/trans isomerase from Pseudonomas fluorescens encapsulated in sodium alginate regarding plant resistance to viral and fungal pahogens.

Use of chemical pesticides poses a threat for environment and human health, so green technologies of crop protection are of high demand. Some microbial proteins able to activate plant defense mechanisms and prevent the development of resistance in plant pathogens, may be good alternative to chemicals, but practical use of such elicitors is limited due to need to protect them against adverse environment prior their delivery to target receptors of plant cells. In this study we examined a possibility to encapsulate heat resistant FKBP-type peptidyl prolyl cis-trans isomerase (PPIase) from Pseudomonas fluorescens, which possesses a significant eliciting activity in relation to a range of plant pathogens, in sodium alginate microparticles and evaluated the stability of resulted complex under long-term UV irradiation and in the presence of proteinase K, as well as its eliciting activity in three different “plant-pathogen” models comparing to that of free PPIase. The obtained PPIase-containing microparticles consisted of 70% of sodium alginate, 20% of bovine serum albumin, and 10% of PPIase. In contrast to free PPIase, which lost its eliciting properties after 8-h UV treatment, encapsulated PPIase kept its eliciting ability unchanged; after being exposed to proteinase K, its eliciting ability twice exceeded that of free PPIase. Using “tobacco-TMV”, “tobacco-Alternaria longipes”, and “wheat-Stagonospora nodorum” model systems, we showed that encapsulation process did not influence on the eliciting activity of PPIase. In the case of the “wheat-S. nodorum” model system, we also observed a significant eliciting activity of alginate-albumin complex and almost doubled activity of encapsulated PPIase as compared to the free PPIase. As far as we know, this is the first observation of a synergistic interaction between alginate and other compound possessing any bioactive properties. The results of the study show some prospects for a PPIase use in agriculture.

Control of Citrus Huanglongbing via Trunk Injection of Plant Defense Activators and Antibiotics.

Citrus huanglongbing (HLB) or greening is a devastating disease of citrus worldwide and no effective control measure is currently available. Plant defense activators environmentally friendly compounds capable of inducing resistance against many plant pathogens. Earlier studies showed that foliar spray of plant defense inducers could slow down HLB disease progress. In this study, eight plant defense activators and three antibiotics were evaluated in three field trials for their effect to control HLB by trunk injection of young and mature sweet orange trees. Results showed that four trunk injections of several activators, including salicylic acid, oxalic acid, acibenzolar-S-methyl, and potassium phosphate, provided significant control of HLB by suppressing 'Candidatus Liberibacter asiaticus' titer and disease progress. Trunk injection of penicillin, streptomycin, and oxytetracycline hydrochloride resulted in excellent control of HLB. In general, antibiotics were more effective in reduction of 'Ca. L. asiaticus' titer and HLB symptom expressions than plant defense activators. These treatments also resulted in increased yield and better fruit quality. Injection of both salicylic acid and acibenzolar-S-methyl led to significant induction of pathogenesis-related (PR) genes PR-1 and PR-2 genes. Meanwhile, injection of either potassium phosphate or oxalic acid resulted in significant induction of PR-2 or PR-15 gene expression, respectively. These results suggested that HLB diseased trees remained inducible for systemic acquired resistance under field conditions. In summary, this study presents information regarding controlling HLB via trunk injection of plant defense activators and antibiotics, which helps citrus growers in decision making regarding developing an effective HLB management program.

Salicylic acid-dependent immunity contributes to resistance against Rhizoctonia solani, a necrotrophic fungal agent of sheath blight, in rice and Brachypodium distachyon.

Summary Rhizoctonia solani is a soil‐borne fungus causing sheath blight. In consistent with its necrotrophic life style, no rice cultivars fully resistant to R. solani are known, and agrochemical plant defense activators used for rice blast, which upregulate a phytohormonal salicylic acid (SA)‐dependent pathway, are ineffective towards this pathogen. As a result of the unavailability of genetics, the infection process of R. solani remains unclear.We used the model monocotyledonous plants Brachypodium distachyon and rice, and evaluated the effects of phytohormone‐induced resistance to R. solani by pharmacological, genetic and microscopic approaches to understand fungal pathogenicity.Pretreatment with SA, but not with plant defense activators used in agriculture, can unexpectedly induce sheath blight resistance in plants. SA treatment inhibits the advancement of R. solani to the point in the infection process in which fungal biomass shows remarkable expansion and specific infection machinery is developed. The involvement of SA in R. solani resistance is demonstrated by SA‐deficient NahG transgenic rice and the sheath blight‐resistant B. distachyon accessions, Bd3‐1 and Gaz‐4, which activate SA‐dependent signaling on inoculation.Our findings suggest a hemi‐biotrophic nature of R. solani, which can be targeted by SA‐dependent plant immunity. Furthermore, B. distachyon provides a genetic resource that can confer disease resistance against R. solani to plants.

生物微弱発光(バイオフォトン)を利用した病害抵抗性誘導剤の探索システム

生物微弱発光(バイオフォトン)を利用した病害抵抗性誘導剤の探索システム

Plant elicitor peptides promote plant defences against nematodes in soybean.

Plant elicitor peptides (Peps) are widely distributed among angiosperms, and have been shown to amplify immune responses in multiple plant families. Here, we characterize three Peps from soybean (Glycine max) and describe their effects on plant defences against two damaging agricultural pests, the root-knot nematode (Meloidogyne incognita) and the soybean cyst nematode (Heterodera glycines). Seed treatments with exogenous GmPep1, GmPep2 or GmPep3 significantly reduced the reproduction of both nematodes. Pep treatment also protected plants from the inhibitory effects of root-knot nematodes on above-ground growth, and up-regulated basal expression levels of nematode-responsive defence genes. GmPep1 induced the expression of its propeptide precursor (GmPROPEP1), a nucleotide-binding site leucine-rich repeat protein (NBS-LRR), a pectin methylesterase inhibitor (PMEI), Respiratory Burst Oxidase Protein D (RBOHD) and the accumulation of reactive oxygen species (ROS) in leaves. In addition, GmPep2 and GmPep3 seed treatments up-regulated RBOHD expression and ROS accumulation in roots and leaves. These results suggest that GmPeps activate plant defences through systemic transcriptional reprogramming and ROS signalling, and that Pep seed treatments represent a potential strategy for nematode management.

Toxicity of biosynthetic silver nanoparticles on the growth, cell ultrastructure and physiological activities of barley plant

Silver nanoparticles (AgNPs) were biosynthesized using the cell-free filtrate of bacterium Proteus mirabilis, reacted with 1 mM of AgNO3 solutions at 37 °C. The synthesis of AgNPs was monitored by UV–Vis spectroscopy and transmission electron microscopy (TEM) equipped with selected area electron diffraction (SAED). The results point to formation of spherical to cubical particles of AgNPs ranging in size from 5 to 35 nm with an average of 25 nm in diameter. The toxicity of Ag on barley (Hordeum vulgare L. cv. Gustoe) that was subjected to Ag+ as AgNO3 and AgNPs was explored. The grain germination and seedling growth of barley decreased in the presence of 0.1 mM Ag+ and was inhibited at 1 mM Ag+. In contrast, our results indicated that the AgNPs at low concentration (0.1 mM) could be useful for barley grain germination and seedling growth. However, the higher concentrations of AgNPs (0.5 and 1 mM) reduced grain germination and exhibited a stronger reduction in the root length. A decline in the photosynthetic pigments and disorganization of chloroplast grana thylakoids in Ag+ and AgNPs-treated plants confirmed the leaf chlorosis. An increase of plastoglobuli within chloroplasts was observed in Ag+ and AgNPs-treated leaves. Ag+ caused dense aggregation of nuclear chromatin materials and degeneration of mitochondria. Ag+ and AgNPs increased contents of malondialdehyde, soluble proteins, total phenolic compounds and activity of guaiacol peroxidase in barley leaves; these results point to activation of plant defence mechanisms against oxidative stress in barley.

English

Thiamethoxam is a neonicotinoid insecticide, which is believed to induce the expression of enzymes and proteins that activate plant defense mechanisms, thus improving the plant resistance to stress factors. The aim of this study was to evaluate the physiological performance of melon and watermelon seeds treated with thiamethoxam. The seed treatment consisted of five doses of thiamethoxam, 0, 2, 4, 6 and 8 mL per kilogram of seeds. The parameters evaluated were measured by a standard germination test, first count of germination test, laboratory vigor tests (accelerated aging and cold tests), and a field vigor test (seedling emergence). Thiamethoxam treatment resulted in increase in the normal seedling percentage in the standard germination, first count of germination, accelerated aging, cold and final seedling emergence tests. The doses of 4.0 to 5.0 mL of thiamethoxam per kilogram of seeds were most efficient in improving the physiological performance of melon and watermelon seeds. Key words: Thiamethoxam, seedling vigor, Cucumis melo, Citrullus lanatus.

Effect of Salicylic Acid on the Growth and Chemical Responses of Pectobacterium carotovorum subsp. carotovorum.

Salicylic acid is a signal molecule which activates plant defense against plant pathogens such as the soft rot enterobacterium Pectobacterium carotovorum subsp. carotovorum. The objectives of study were to determine bactericidal effects of salicylic acid on the growth of P. carotovorum subsp. carotovorum and secondly, assess chemical responses of P. carotovorum subsp. carotovorum to salicylic acid. Pectobacterium carotovorum subsp. carotovorum was grown in lysogeny broth amended with salicylic acid at concentrations of 0, 100, 200, 400, 800 and 1200 mg L-1. The P. carotovorum subsp. carotovorum cultures were incubated at 25°C and sampled at two time points, 0 h (sampled before incubation) and 24 h. Bacterial counts were done at the onset of the incubation (0 h) and after the 24 h incubation. The set which was incubated for 24 h was split into two, one subset was centrifuged and the other was not. From the centrifuged subset the supernatant was recovered and was, together with all the other samples (0 and 24 h not centrifuged), analyzed with1H nuclear magnetic resonance and gas chromatography. Bacterial counts done before and after incubation showed that the lower concentrations of salicylic acid, 0, 100, 200 and 400 mg L-1, supported the growth of P. carotovorum subsp. carotovorum whereas the higher concentrations of 800 and 1200 mg L-1 inhibited the growth of the bacterium completely. Nuclear magnetic resonance results showed either slight or no differences in the metabolite profiles and gas chromatography showed different responses without a clearly defined pattern among the experimental treatments. However, methanethiol was detected by both nuclear magnetic resonance and gas chromatography in all the treatments and was probably formed as a result of the breakdown of lysogeny broth. From the results obtained it was concluded that salicylic acid promotes the growth of P. carotovorum subsp. carotovorum at lower concentrations of 0-400 mg L-1 but higher concentrations of salicylic acid of 800 and 1200 mg L-1 inhibit bacterial growth. All the tested salicylic acid concentrations (0-1200 mg L-1) cause only slight chemical shifts in the bacterial culture. Methanethiol was detected in all treatments and it is probably formed from the breakdown of lysogeny broth.

The Botrytis cinerea elicitor protein BcIEB1 interacts with the tobacco PR5-family protein osmotin and protects the fungus against its antifungal activity.

The broad-range phytopathogenic fungus Botrytis cinerea secretes hundreds of proteins during infection of its plant hosts. One of these proteins, BcIEB1, is abundantly secreted and is able to elicit plant defenses, probably as a pathogen-associated molecular pattern, although its native function in B.cinerea biology remains unknown. Pull-down experiments designed to isolate the molecular target of BcIEB1 in tobacco resulted in the identification of osmotin, a pathogenesis-related protein of family 5 that shows antifungal activity. The expression of osmotin in Escherichia coli allowed the verification of the BcIEB1-osmotin interaction with pure proteins by pull-down and far Western blot experiments, as well as the confirmation of the activity of osmotin against B.cinerea. Interestingly, B.cinerea Δbcieb1 mutants are more susceptible than the wild-type to osmotin, and the external addition of pure BcIEB1 protects the Δbcieb1 mutants, as well as Saccharomyces cerevisiae, from the antifungal action of osmotin, thus pointing at PR5 inhibition as the primary native function of BcIEB1. The question of whether osmotin is also involved in the activation of plant defenses by BcIEB1 is also addressed, and the data suggest that osmotin does not participate in the elicitation process.