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Constitutive expression of transgenes encoding derivatives of the synthetic antimicrobial peptide BP100: impact on rice host plant fitness (2012)

Anna Nadal; Maria Montero; Nuri Company; Esther Badosa; Joaquima Messeguer; Laura Montesinos; Emilio Montesinos; Maria Pla

BioMed Central

In: BMC Plant Biology

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Publication Date: 2012-09-05

Description: Background: The Biocontrol Peptide BP100 is a synthetic and strongly cationic a-helical undecapeptide with high, specific antibacterial activity against economically important plant-pathogenic bacteria, and very low toxicity. It was selected from a library of synthetic peptides, along with other peptides with activities against relevant bacterial and fungal species. Expression of the BP100 series of peptides in plants is of major interest to establish disease-resistant plants and facilitate molecular farming. Specific challenges were the small length, peptide degradation by plant proteases and toxicity to the host plant. Here we approached the expression of the BP100 peptide series in plants using BP100 as a proof-of-concept. Results: Our design considered up to three tandemly arranged BP100 units and peptide accumulation in the endoplasmic reticulum (ER), analyzing five BP100 derivatives. The ER retention sequence did not reduce the antimicrobial activity of chemically synthesized BP100 derivatives, making this strategy possible. Transformation with sequences encoding BP100 derivatives (bp100der) was over ten-fold less efficient than that of the hygromycin phosphotransferase (hptII) transgene. The BP100 direct tandems did not show higher antimicrobial activity than BP100, and genetically modified (GM) plants constitutively expressing them were not viable. In contrast, inverted repeats of BP100, whether or not elongated with a portion of a natural antimicrobial peptide (AMP), had higher antimicrobial activity, and fertile GM rice lines constitutively expressing bp100der were produced. These GM lines had increased resistance to the pathogens Dickeya chrysanthemi and Fusarium verticillioides, and tolerance to oxidative stress, with agronomic performance comparable to untransformed lines. Conclusions: Constitutive expression of transgenes encoding short cationic a-helical synthetic peptides can have a strong negative impact on rice fitness. However, GM plants expressing, for example, BP100 based on inverted repeats, have adequate agronomic performance and resistant phenotypes as a result of a complex equilibrium between bp100der toxicity to plant cells, antimicrobial activity and transgene-derived plant stress response. It is likely that these results can be extended to other peptides with similar characteristics.

Electronic ISSN: 1471-2229

Topics: Biology

Published by BioMed Central

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Production of cecropin A antimicrobial peptide in rice seed endosperm (2014)

Bundó Mireia; Montesinos Laura; Izquierdo Esther; Campo Sonia; Mieulet Delphine; Guiderdoni Emmanuel; Rossignol Michel; Badosa Esther; Montesinos Emilio; San Blanca; Coca María

BioMed Central

In: BMC Plant Biology

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Publication Date: 2014-04-23

Description: Background: Cecropin A is a natural antimicrobial peptide that exhibits rapid, potent and long-lasting lytic activity against a broad spectrum of pathogens, thus having great biotechnological potential. Here, we report a system for producing bioactive cecropin A in rice seeds. Results: Transgenic rice plants expressing a codon-optimized synthetic cecropin A gene drived by an endosperm-specific promoter, either the glutelin B1 or glutelin B4 promoter, were generated. The signal peptide sequence from either the glutelin B1 or the glutelin B4 were N-terminally fused to the coding sequence of the cecropin A. We also studied whether the presence of the KDEL endoplasmic reticulum retention signal at the C-terminal has an effect on cecropin A subcellular localization and accumulation. The transgenic rice plants showed stable transgene integration and inheritance. We show that cecropin A accumulates in protein storage bodies in the rice endosperm, particularly in type II protein bodies, supporting that the glutelin N-terminal signal peptides play a crucial role in directing the cecropin A to this organelle, independently of being tagged with the KDEL endoplasmic reticulum retention signal. The production of cecropin A in transgenic rice seeds did not affect seed viability or seedling growth. Furthermore, transgenic cecropin A seeds exhibited resistance to infection by fungal and bacterial pathogens (Fusarium verticillioides and Dickeya dadantii, respectively) indicating that the in planta-produced cecropin A is biologically active. Conclusions: Rice seeds can sustain bioactive cecropin A production and accumulation in protein bodies. The system might benefit the production of this antimicrobial agent for subsequent applications in crop protection and food preservation.

Electronic ISSN: 1471-2229

Topics: Biology

Published by BioMed Central

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Derivatives of the Antimicrobial Peptide BP100 for Expression in Plant Systems (2013)

Badosa, Esther ; Moiset, Gemma ; Montesinos, Laura ; [et al.]

Public Library of Science

In: PLoS ONE. 2013; 8(12): e85515. Published 2013 Dec 23. doi: 10.1371/journal.pone.0085515.

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Publication Date: 2013-12-23

Electronic ISSN: 1932-6203

Topics: Medicine , Natural Sciences in General

Published by Public Library of Science

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Production of cecropin A antimicrobial peptide in rice seed endosperm (2014)

Bundó, Mireia ; Montesinos, Laura ; Izquierdo, Esther ; [et al.]

BioMed Central

In: BMC Plant Biology. 2014; 14(1): 102. Published 2014 Apr 22. doi: 10.1186/1471-2229-14-102.

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Publication Date: 2014-04-22

Electronic ISSN: 1471-2229

Topics: Biology

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Constitutive expression of transgenes encoding derivatives of the synthetic antimicrobial peptide BP100: impact on rice host plant fitness (2012)

Nadal, Anna ; Montero, Maria ; Company, Nuri ; [et al.]

BioMed Central

In: BMC Plant Biology. 2012; 12(1): 159. Published 2012 Sep 04. doi: 10.1186/1471-2229-12-159.

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Publication Date: 2012-09-04

Description: Background The Biopeptide BP100 is a synthetic and strongly cationic α-helical undecapeptide with high, specific antibacterial activity against economically important plant-pathogenic bacteria, and very low toxicity. It was selected from a library of synthetic peptides, along with other peptides with activities against relevant bacterial and fungal species. Expression of the BP100 series of peptides in plants is of major interest to establish disease-resistant plants and facilitate molecular farming. Specific challenges were the small length, peptide degradation by plant proteases and toxicity to the host plant. Here we approached the expression of the BP100 peptide series in plants using BP100 as a proof-of-concept. Results Our design considered up to three tandemly arranged BP100 units and peptide accumulation in the endoplasmic reticulum (ER), analyzing five BP100 derivatives. The ER retention sequence did not reduce the antimicrobial activity of chemically synthesized BP100 derivatives, making this strategy possible. Transformation with sequences encoding BP100 derivatives (bp100der) was over ten-fold less efficient than that of the hygromycin phosphotransferase (hptII) transgene. The BP100 direct tandems did not show higher antimicrobial activity than BP100, and genetically modified (GM) plants constitutively expressing them were not viable. In contrast, inverted repeats of BP100, whether or not elongated with a portion of a natural antimicrobial peptide (AMP), had higher antimicrobial activity, and fertile GM rice lines constitutively expressing bp100der were produced. These GM lines had increased resistance to the pathogens Dickeya chrysanthemi and Fusarium verticillioides, and tolerance to oxidative stress, with agronomic performance comparable to untransformed lines. Conclusions Constitutive expression of transgenes encoding short cationic α-helical synthetic peptides can have a strong negative impact on rice fitness. However, GM plants expressing, for example, BP100 based on inverted repeats, have adequate agronomic performance and resistant phenotypes as a result of a complex equilibrium between bp100der toxicity to plant cells, antimicrobial activity and transgene-derived plant stress response. It is likely that these results can be extended to other peptides with similar characteristics.

Electronic ISSN: 1471-2229

Topics: Biology

Published by BioMed Central

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Electronic Resource

ABC-type neutral amino acid permease N-I is required for optimal diazotrophic growth and is repressed in the heterocysts of Anabaena sp. strain PCC 7120 (2005)

Picossi, Silvia ; Montesinos, María Luz ; Pernil, Rafael ; [et al.]

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 57 (2005), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can fix N2 in differentiated cells called heterocysts. The products of Anabaena open reading frames (ORFs) all1046, all1047, all1284, alr1834 and all2912 were identified as putative elements of a neutral amino acid permease. Anabaena mutants of these ORFs were strongly affected (1–12% of the wild-type activity) in the transport of Pro, Phe, Leu and Gly and also impaired (17–30% of the wild-type activity) in the transport of Ala and Ser. These results identified those ORFs as the nat genes encoding the N-I neutral amino acid permease. According to amino acid sequence homologies, natA (all1046) and natE (all2912) encode ATPases, natC (all1047) and natD (all1284) encode transmembrane proteins, and natB (alr1834) encodes a periplasmic substrate-binding protein of an ABC-type uptake transporter. The natA, natC, natD and natE mutants showed defects in Gln and His uptake that were not observed in the natB mutant suggesting that NatB is not a binding protein for Gln or His. The nat mutants released hydrophobic amino acids to the medium, and amino acid release took place at higher levels in cultures incubated in the absence of combined N than in the presence of nitrate. Alanine was the amino acid released at highest levels, and its release was impaired in a mutant unable to develop heterocysts. The nat mutants were also impaired in diazotrophic growth, with natA, natC, natD and natE mutants showing more severe defects than the natB mutant. Expression of natA and natC, which constitute an operon, natCA, as well as of natB was studied and found to take place in vegetative cells but not in the heterocysts. These results indicate that the N-I permease is necessary for normal growth of Anabaena sp. strain PCC 7120 on N2, and that this permease has a role in the diazotrophic filament specifically in the vegetative cells.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1365-2958.2005.04791.x

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Electronic Resource

Osmotically induced trehalose and glycine betaine accumulation improves tolerance to desiccation, survival and efficacy of the postharvest biocontrol agent Pantoea agglomerans EPS125 (2005)

Bonaterra, Anna ; Camps, Jaume ; Montesinos, Emilio

Oxford, UK : Blackwell Publishing Ltd

FEMS microbiology letters 250 (2005), S. 0

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ISSN: 1574-6968

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology

Notes: The application of the biocontrol agent Pantoea agglomerans EPS125 to unwounded fruits was practically ineffective for control of postharvest blue mould caused by Penicillium expansum when the treatment and subsequent wounding and pathogen inoculation were separated by periods of unfavourable conditions. This was due to a rapid decrease in viability of the alocthonous introduced biocontrol agent in the intact peel surface. A system for osmoadaptation of the biocontrol agent was developed by combining saline osmotic stress and osmolyte amendment to the growth medium. Osmoadapted cells accumulated trehalose and glycine betaine (GB) intracellularly and showed a higher tolerance to desiccation than non-osmoadapted cells. Osmoadaptation in NaCl plus GB during inoculum preparation increased considerably survival on the peel surface of apple fruits. This effect was significant under low relative humidity (RH) and fluctuating RH conditions, but was not significant at high RH. Osmoadaptation significantly improved blue mould control under conditions where the standard biological control treatments were ineffective. The rot diameter was significantly reduced in apple fruits which were treated with EPS125 and incubated for several days under low, high or fluctuating RH, followed by wounding and inoculation of P. expansum. Growth of EPS125 with NaCl, either with or without the addition of GB, was an effective osmoadaptation treatment for improving blue mould rot control. However, the addition of GB to the NaCl amended growth medium increased 4–5-fold growth rate and OD of the cultures. This is an advantage for mass production of P. agglomerans EPS125 in a NaCl amended growth medium.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1016/j.femsle.2005.06.028

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Development of a strain-specific quantitative method for monitoring Pseudomonas fluorescens EPS62e, a novel biocontrol agent of fire blight (2005)

Pujol, Marta ; Badosa, Esther ; Cabrefiga, Jordi ; [et al.]

Oxford, UK : Blackwell Publishing Ltd

FEMS microbiology letters 249 (2005), S. 0

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ISSN: 1574-6968

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology

Notes: Pseudomonas fluorescens EPS62e has been selected in a screening procedure for its high efficacy controlling Erwinia amylovora infections in flowers, immature fruits and young pear plants. We developed two monitoring methods which allowed specific detection and quantification of EPS62e by combining classical microbiological techniques with molecular tools. RAPD and unspecific-PCR fingerprints were used to differentiate EPS62e from other P. fluorescens strains. Differential amplified fragments from EPS62e were sequence characterized as SCAR markers and two primer pairs were designed and selected for their specificity against EPS62e. A SCAR primer pair was evaluated and validated for the assessment of population dynamics of EPS62e on pear plants under greenhouse conditions using plating and most probable number assays coupled to PCR. Both techniques were useful in monitoring the biological control agent. The population level of EPS62e after treatment was 7 log CFU (g f.w.)−1, which in turn decreased progressively to 4–5 log CFU (g f.w.)−1 after 17 days and then remained stable until the end of the assay 11 days later. The limit of detection of both monitoring methods developed was around 3 log CFU (g f.w.)−1, thus, providing a reliable tool for the analysis of EPS62e in greenhouse or field trials, and the assessment of threshold population levels for efficient biocontrol of fire blight.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1016/j.femsle.2005.06.029

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