Combining hexanoic acid plant priming with Bacillus thuringiensis insecticidal activity against Colorado potato beetle.

Interaction between insect herbivores and host plants can be modulated by endogenous and exogenous compounds present in the source of food and might be successfully exploited in Colorado potato beetle (CPB) pest management. Feeding tests with CPB larvae reared on three solanaceous plants (potato, eggplant and tomato) resulted in variable larval growth rates and differential susceptibility to Bacillus thuringiensis Cry3Aa toxin as a function of the host plant. An inverse correlation with toxicity was observed in Cry3Aa proteolytic patterns generated by CPB midgut brush-border membrane vesicles (BBMV) from Solanaceae-fed larvae, being the toxin most extensively proteolyzed on potato, followed by eggplant and tomato. We found that CPB cysteine proteases intestains may interact with Cry3Aa toxin and, in CPB BBMV from larvae fed all three Solanaceae, the toxin was able to compete for the hydrolysis of a papain substrate. In response to treatment with the JA-dependent plant inducer Hexanoic acid (Hx), we showed that eggplant reduced OPDA basal levels and both, potato and eggplant induced JA-Ile. CPB larvae feeding on Hx-induced plants exhibited enhanced Cry3Aa toxicity, which correlated with altered papain activity. Results indicated host-mediated effects on B. thuringiensis efficacy against CPB that can be enhanced in combination with Hx plant induction.

Does Plant Breeding for Antioxidant-Rich Foods Have an Impact on Human Health?

Given the general beneficial effects of antioxidants-rich foods on human health and disease prevention, there is a continuous interest in plant secondary metabolites conferring attractive colors to fruits and grains and responsible, together with others, for nutraceutical properties. Cereals and Solanaceae are important components of the human diet, thus, they are the main targets for functional food development by exploitation of genetic resources and metabolic engineering. In this review, we focus on the impact of antioxidants-rich cereal and Solanaceae derived foods on human health by analyzing natural biodiversity and biotechnological strategies aiming at increasing the antioxidant level of grains and fruits, the impact of agronomic practices and food processing on antioxidant properties combined with a focus on the current state of pre-clinical and clinical studies. Despite the strong evidence in in vitro and animal studies supporting the beneficial effects of antioxidants-rich diets in preventing diseases, clinical studies are still not sufficient to prove the impact of antioxidant rich cereal and Solanaceae derived foods on human.

Dual Antimicrobial and Antiproliferative Activity of TcPaSK Peptide Derived from a Tribolium castaneum Insect Defensin.

Antimicrobial peptides (AMPs) found in the innate immune system of a wide range of organisms might prove useful to fight infections, due to the reported slower development of resistance to AMPs. Increasing the cationicity and keeping moderate hydrophobicity of the AMPs have been described to improve antimicrobial activity. We previously found a peptide derived from the Tribolium castaneum insect defensin 3, exhibiting antrimicrobial activity against several human pathogens. Here, we analyzed the effect against Staphyloccocus aureus of an extended peptide (TcPaSK) containing two additional amino acids, lysine and asparagine, flanking the former peptide fragment in the original insect defensin 3 protein. TcPaSK peptide displayed higher antimicrobial activity against S. aureus, and additionally showed antiproliferative activity against the MDA-MB-231 triple negative breast cancer cell line. A SWATH proteomic analysis revealed the downregulation of proteins involved in cell growth and tumor progression upon TcPaSK cell treatment. The dual role of TcPaSK peptide as antimicrobial and antiproliferative agent makes it a versatile molecule that warrants exploration for its use in novel therapeutic developments as an alternative approach to overcome bacterial antibiotic resistance and to increase the efficacy of conventional cancer treatments.

Hexanoic acid-induced resistance against Botrytis cinerea in tomato plants.

We have demonstrated that root treatment with hexanoic acid protects tomato plants against Botrytis cinerea. Hexanoic acid-induced resistance (Hx-IR) was blocked in the jasmonic acid (JA)-insensitive mutant jai1 (a coi1 homolog) and in the abscisic acid (ABA)-deficient mutant flacca (flc). Upon infection, the LoxD gene as well as the oxylipin 12-oxo-phytodienoic acid and the bioactive molecule JA-Ile were clearly induced in treated plants. However, the basal ABA levels were not altered. Hexanoic acid primed callose deposition against B. cinerea in a cultivar-dependent manner. Treated plants from Ailsa Craig, Moneymaker, and Rheinlands Ruhm showed increased callose deposition but not from Castlemart. Hexanoic acid did not prime callose accumulation in flc plants upon B. cinerea infection; therefore, ABA could act as a positive regulator of Hx-IR by enhancing callose deposition. Furthermore, although hexanoic acid protected the JA-deficient mutant defensless1 (def1), the priming for callose was higher than in the wild type. This suggests a link between JA and callose deposition in tomato. Hence, the obtained results support the idea that callose, oxylipins, and the JA-signaling pathway are involved in Hx-IR against B. cinerea. Moreover our data support the relevance of JA-signaling for basal defense against this necrotroph in tomato. Hexanoic acid also protected against Pseudomonas syringae, indicating a broad-spectrum effect for this new inducer.

Hexanoic acid protects tomato plants against Botrytis cinerea by priming defence responses and reducing oxidative stress.

Treatment with the resistance priming inducer hexanoic acid (Hx) protects tomato plants from Botrytis cinerea by activating defence responses. To investigate the molecular mechanisms underlying hexanoic acid-induced resistance (Hx-IR), we compared the expression profiles of three different conditions: Botrytis-infected plants (Inf), Hx-treated plants (Hx) and Hx-treated + infected plants (Hx+Inf). The microarray analysis at 24 h post-inoculation showed that Hx and Hx+Inf plants exhibited the differential expression and priming of many Botrytis-induced genes. Interestingly, we found that the activation by Hx of other genes was not altered by the fungus at this time point. These genes may be considered to be specific targets of the Hx priming effect and may help to elucidate its mechanisms of action. It is noteworthy that, in Hx and Hx+Inf plants, there was up-regulation of proteinase inhibitor genes, DNA-binding factors, enzymes involved in plant hormone signalling and synthesis, and, remarkably, the genes involved in oxidative stress. Given the relevance of the oxidative burst occurring in plant-pathogen interactions, the effect of Hx on this process was studied in depth. We showed by specific staining that reactive oxygen species (ROS) accumulation in Hx+Inf plants was reduced and more restricted around infection sites. In addition, these plants showed higher ratios of reduced to oxidized glutathione and ascorbate, and normal levels of antioxidant activities. The results obtained indicate that Hx protects tomato plants from B. cinerea by regulating and priming Botrytis-specific and non-specific genes, preventing the harmful effects of oxidative stress produced by infection.

Binding of Cyt1Aa and Cry11Aa toxins of Bacillus thuringiensis serovar israelensis to brush border membrane vesicles of Tipula paludosa (Diptera: Nematocera) and subsequent pore formation.

Bacillus thuringiensis serovar israelensis (B. thuringiensis subsp. israelensis) produces four insecticidal crystal proteins (ICPs) (Cry4A, Cry4B, Cry11A, and Cyt1A). Toxicity of recombinant B. thuringiensis subsp. israelensis strains expressing only one of the toxins was determined with first instars of Tipula paludosa (Diptera: Nematocera). Cyt1A was the most toxic protein, whereas Cry4A, Cry4B, and Cry11A were virtually nontoxic. Synergistic effects were recorded when Cry4A and/or Cry4B was combined with Cyt1A but not with Cry11A. The binding and pore formation are key steps in the mode of action of B. thuringiensis subsp. israelensis ICPs. Binding and pore-forming activity of Cry11Aa, which is the most toxic protein against mosquitoes, and Cyt1Aa to brush border membrane vesicles (BBMVs) of T. paludosa were analyzed. Solubilization of Cry11Aa resulted in two fragments, with apparent molecular masses of 32 and 36 kDa. No binding of the 36-kDa fragment to T. paludosa BBMVs was detected, whereas the 32-kDa fragment bound to T. paludosa BBMVs. Only a partial reduction of binding of this fragment was observed in competition experiments, indicating a low specificity of the binding. In contrast to results for mosquitoes, the Cyt1Aa protein bound specifically to the BBMVs of T. paludosa, suggesting an insecticidal mechanism based on a receptor-mediated action, as described for Cry proteins. Cry11Aa and Cyt1Aa toxins were both able to produce pores in T. paludosa BBMVs. Protease treatment with trypsin and proteinase K, previously reported to activate Cry11Aa and Cyt1Aa toxins, respectively, had the opposite effect. A higher efficiency in pore formation was observed when Cyt1A was proteinase K treated, while the activity of trypsin-treated Cry11Aa was reduced. Results on binding and pore formation are consistent with results on ICP toxicity and synergistic effect with Cyt1Aa in T. paludosa.

Absence of the endo-beta-1,4-glucanases Cel1 and Cel2 reduces susceptibility to Botrytis cinerea in tomato.

Cel1 and Cel2 are members of the tomato (Solanum lycopersicum Mill) endo-beta-1,4-glucanase (EGase) family that may play a role in fruit ripening and organ abscission. This work demonstrates that Cel1 protein is present in other vegetative tissues and accumulates during leaf development. We recently reported the downregulation of both the Cel1 mRNA and protein upon fungal infection, suggesting the involvement of EGases in plant-pathogen interactions. This hypothesis was confirmed by assessing the resistance to Botrytis cinerea infection of transgenic plants expressing both genes in an antisense orientation (Anti-Cel1, Anti-Cel2 and Anti-Cel1-Cel2). The Anti-Cel1-Cel2 plants showed enhanced resistance to this fungal necrotroph. Microscopical analysis of infected leaves revealed that tomato plants accumulated pathogen-inducible callose within the expanding lesion. Anti-Cel1-Cel2 plants presented a faster and enhanced callose accumulation against B. cinerea than wild-type plants. The inhibitor 2-deoxy-d-glucose, a callose synthesis inhibitor, showed a direct relationship between faster callose accumulation and enhanced resistance to B. cinerea. EGase activity appears to negatively modulate callose deposition. The absence of both EGase genes was associated with changes in the expression of the pathogen-related genes PR1 and LoxD. Interestingly, Anti-Cel1-Cel2 plants were more susceptible to Pseudomonas syringae, displaying severe disease symptoms and enhanced bacterial growth relative to wild-type plants. Analysis of the involvement of Cel1 and Cel2 in the susceptibility to B. cinerea in fruits was done with the ripening-impaired mutants Never ripe (Nr) and Ripening inhibitor (rin). The data reported in this work support the idea that enzymes involved in cell wall metabolism play a role in susceptibility to pathogens.

Control of the phytopathogen Botrytis cinerea using adipic acid monoethyl ester.

The in vitro and in vivo antifungal activity of adipic acid monoethyl ester (AAME) on the necrotrophic pathogen Botrytis cinerea has been studied. This chemical effectively controlled this important phytopathogen, inhibited spore germination and mycelium development at non-phytotoxic concentrations. The effectiveness of AAME treatment is concentration-dependent and influenced by pH. Spore germination in the presence of AAME is stopped at a very early stage, preventing germ tube development. In addition, cytological changes such as retraction of the conidial cytoplasm in the fungus are observed. AAME was also found to act on membrane integrity, affecting permeability without exhibiting lytic activity, as described previously for other antifungal compounds. Polyamine content in the mycelium of B. cinerea was also affected in response to AAME treatment, resulting in putrescine reduction and spermine accumulation similar to a number of antifungal agents. Microscopic observation of treated conidia after inoculation on tomato leaves suggested that inhibited spores are not able to attach to and penetrate the leaf. Finally, AAME completely suppressed the grey mould disease of tomato fruits under controlled inoculation conditions, providing evidence for its efficacy in a biological context and for the potential use of this chemical as an alternative fungicide treatment.

Characterization of tomato endo-beta-1,4-glucanase Cel1 protein in fruit during ripening and after fungal infection.

The tomato (Lycopersicon esculentum Mill.) endo-beta-1,4-glucanase (EGase) Cel1 protein was characterized in fruit using specific antibodies. Two polypeptides ranging between 51 and 52 kDa were detected in the pericarp, and polypeptides ranging between 49 and 51 kDa were detected in locules. The polypeptides recognized by Cel1 antiserum in fruit are within the size range predicted for Cel1 protein and could be derived from heterogeneous glycosylation. Cel1 protein accumulation was examined throughout fruit ripening. Cel1 protein appears in the pericarp at the stage in which many ripening-related changes start, and remains present throughout fruit ripening. In locules, Cel1 protein is already present at the onset of fruit ripening and remains constant during fruit ripening. This pattern of expression supports a possible role for this EGase in the softening of pericarp tissue and in the liquefaction of locules that takes place during ripening. The accumulation of Cel1 protein was also analyzed after fungal infection. Cel1 protein and mRNA levels are down-regulated in pericarp after Botrytis cinerea infection but are not affected in locular tissue. The same behavior was observed when fruits were infected with Penicillium expansum, another fungal pathogen. Cel1 protein and mRNA levels do not respond to wounding. These results support the idea that the tomato Cel1 EGase responds to pathogen infection and supports a relationship between EGases, plant defense responses and fruit ripening.