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Beyond Xylella, Integrated Management Strategies for Mitigating Xylella fastidiosa Impact in Europe (BeXyl) (Grant Agreement 101060593). Partner/Coordinador principal: Blanca B. Landa del Castillo, Investigadora Científica del Instituto de Agricultura Sostenible (IAS-CSIC)., Xylella fastidiosa is a plant pathogenic bacterium that has been introduced in the European Union (EU), causing significant yield losses in economically important Mediterranean crops. Almond leaf scorch (ALS) is currently one of the most relevant diseases observed in Spain, and no cure has been found to be effective for this disease. In previous reports, the peptide BP178 has shown a strong bactericidal activity in vitro against X. fastidiosa and to other plant pathogens, and to trigger defense responses in tomato plants. In the present work, BP178 was applied by endotherapy to almond plants of cultivar Avijor using preventive and curative strategies. The capacity of BP178 to reduce the population levels of X. fastidiosa and to decrease disease symptoms and its persistence over time were demonstrated under greenhouse conditions. The most effective treatment consisted of a combination of preventive and curative applications, and the peptide was detected in the stem up to 60 days posttreatment. Priming plants with BP178 induced defense responses mainly through the salicylic acid pathway, but also overexpressed some genes of the jasmonic acid and ethylene pathways. It is concluded that the bifunctional peptide is a promising candidate to be further developed to manage ALS caused by X. fastidiosa.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., This work was supported by grants from Spain Ministerio de Ciencia e Innovación (MCIU)/AEI and EU FEDER (RTI2018-099410-B-C21, and FPU19/01434 to L. Moll) and from the European Commission XF-ACTORS (grant 727987) and BeXyL (grant 101060593). A. Bar o was recipient of a research grantfrom the Secretaria d’Universitats i Recerca, Departament d'Innovaci o, Universitatsi Empresa, Generalitat de Catalunya (grant 2018 FI B00334).

The aggressiveness of Spanish isolates of Xylella fastidiosa, representing different sequence types, were studied in almond plants of several cultivars by means of the dynamics of the population levels and symptoms, colonization and spread, and dose–effect relationships. Pathogen dynamics in almond plants under greenhouse conditions showed doubling times of 2.1 to 2.5 days during the exponential growth phase, with a maximum population size of about 35 days postinoculation (dpi). Differences in patterns in population dynamics were observed between sap and xylem tissue after the exponential growth, as population levels in the xylem tissue remained stable while viable cells in sap decreased. Population levels were higher in two upward zones than in the downward zone with respect to the inoculation area. The first symptoms were observed between 20 and 60 dpi, and disease severity increased over time at doubling times of 30 days, with a maximum observed at 120 dpi. Strains tested showed differences in population levels in the cultivars studied and were able to spread with different intensity from contaminated plant parts to new growing shoots after pruning. Two almond isolates showed different performance in dose–effect relationships when inoculated in cultivar Avijor. Whereas IVIA 5387.2 reached high population levels but showed high median effective dose (ED50) and minimal infective dose (MID) values, IVIA 5901.2 showed low population levels and low ED50 and MID values. This study has implications for the epidemiology of X. fastidiosa in almond crops, estimating doubling times of the pathogen in planta and of symptom development and showing differences in aggressiveness between strains.

The control of plant diseases caused by bacteria that seriously compromise crop productivity around the world is still one of the most important challenges in food security. Integrated approaches for disease control generally lack plant protection products with high efficacy and low environmental and health adverse effects. Functional peptides, either from natural sources or synthetic, are considered as novel candidates to develop biopesticides. Synthetic peptides can be obtained based on the structure of natural compounds or de novo designed, considering the features of antimicrobial peptides. The advantage of this approach is that analogues can be conveniently prepared, enabling the identification of sequences with improved biological properties. Several peptide libraries have been designed and synthetized, and the best sequences showed strong bactericidal activity against important plant pathogenic bacteria, with a good profile of biodegradability and low toxicity. Among these sequences, there are bacteriolytic or antibiofilm peptides that work against the target bacteria, plant defense elicitor peptides, and multifunctional peptides that display several of these properties. Here, we report the research performed by our groups during the last twenty years, as well as our ongoing work. We also highlight those peptides that can be used as candidates to develop novel biopesticides, and the main challenges and prospects This research has been funded in the last five years by different grants from Spain, including those by the Ministerio de Ciencia, Innovación y Universidades RTI2018-099410-B-C21; RTI2018-099410-B-C22; and ERTA INIA 2017-00004-C06-03 and from the European Union H2020 Program XF-Actors cod. 727987

The hybrid peptide BP178 (KKLFKKILKYLAGPAGIGKFLHSAKKDEL-OH), derived from BP100 (KKLFKKILKYL) and magainin (1-10), and engineered for plant expression, had a strong bactericidal activity but not fungicidal. Moreover, the preventive spray of tomato plants with BP178 controlled infections by the plant pathogenic bacteria Pseudomonas syringae pv. tomato and Xanthomonas campestris pv. vesicatoria, as well as the fungus Botrytis cinerea. The treatment of tomato plants with BP178 induced the expression of several genes according to microarray and RT-qPCR analysis. Upregulated genes coded for several pathogenesis-related proteins, including PR1, PR2, PR3, PR4, PR5, PR6, PR7, PR9, PR10, and PR14, as well as transcription factors like ethylene transcription factors, WRKY, NAC and MYB, involved in the salicylic acid, jasmonic acid, and ethylene-signaling pathways. BP178 induced a similar gene expression pattern to flg15 according to RT-qPCR analysis, whereas the parent peptide BP100 did not trigger such as a strong plant defense response. It was concluded that BP178 was a bifunctional peptide protecting the plant against pathogen infection through a dual mechanism of action consisting of antimicrobial activity against bacterial pathogens and plant defense elicitation on plant host We thank the Government of the Generalitat de Catalunya (Xarxa de Referencia en Biotecnología, and Xarxa de Referència en Tecnologia dels Aliments, SGR09626, 2008SGR812, and 2014SGR697) for support. The research group is accredited by SGR 2014-697 and TECNIO net from Catalonia. We also thank the University of Girona for a grant for research improvement 2015–2018

The aggressiveness of Spanish isolates of

The kinetics of cell inactivation and the susceptibility of

The kinetics of cell inactivation and the susceptibility of Xylella fastidiosa subspecies fastidiosa, multiplex, and pauca to synthetic antimicrobial peptides from two libraries (CECMEL11 and CYCLO10) were studied. The bactericidal effect was dependent on the relative concentrations of peptide and bacterial cells, and was influenced by the diluent, either buffer or sap. The most bactericidal and lytic peptide was BP178, an enlarged derivative of the amphipathic cationic linear undecapeptide BP100. The maximum reduction in survivors after BP178 treatment occurred within the first 10 to 20 min of contact and at micromolar concentrations (9 molecules of peptide per bacterial cell was estimated to be necessary to initiate cell inactivation. There was a differential susceptibility to BP178 among strains, with DD1 being the most resistant and CFBP 8173 the most susceptible. Moreover, strains showed a proportion of cells under the viable but nonculturable state, which was highly variable among strains. These findings may have implications for managing the diseases caused by X. fastidiosa.

Recent strong restrictions on the use of pesticides has prompted the search for safer alternatives, being antimicrobial peptides promising candidates. Herein, with the aim of identifying new agents, 15 peptides reported as plant defense elicitors, promiscuous, multifunctional or antimicrobial were selected and tested against six plant pathogenic bacteria of economic importance. Within this set, KSL-W (KKVVFWVKFK-NH2) displayed high antibacterial activity against all the tested pathogens, low hemolysis and low phytotoxicity in tobacco leaves. This peptide was taken as a lead and 49 analogues were designed and synthesized, including N-terminal deletion sequences, peptides incorporating a d-amino acid and lipopeptides. The screening of these sequences revealed that a nine amino acid length was the minimum for activity. The presence of a d-amino acid significantly decreased the hemolysis and endowed KSL-W with the capacity to induce the expression of defense-related genes in tomato plants. The incorporation of an acyl chain led to sequences with high activity against Xanthomonas strains, low hemolysis and phytotoxicity. Therefore, this study demonstrates that KSL-W constitutes an excellent candidate as new agent to control plant diseases and can be considered as a lead to develop derivatives with multifunctional properties, including antimicrobial and plant defense elicitation C.C. was the recipient of a predoctoral fellowship from the MINECO of Spain. This work was financed by grants AGL2012-39880-C02-02, AGL2015-69876-C2-2-R (MINECO/FEDER, EU) and MPCUdG2016/038. The authors acknowledge the Serveis Tècnics de Recerca of the University of Girona for the mass spectrometry analysis

A collection of 36 lipopeptides were designed from the cecropin A-melittin hybrid peptide BP100 (H-Lys-Lys-Leu-Phe-Lys-Lys-Ile-Leu-Lys-Tyr-Leu-NH2) previously described with activity against phytopathogenic bacteria. These lipopeptides were synthesized on solid-phase and screened for their antimicrobial activity, toxicity and proteolytic stability. They incorporated a butanoyl, a hexanoyl or a lauroyl group at the N-terminus or at the side chain of a lysine residue placed at each position of the sequence. Their antimicrobial activity and hemolysis depended on the fatty acid length and its position. In particular, lipopeptides containing a butanoyl or a hexanoyl chain exhibited the best biological activity profile. In addition, we observed that the incorporation of the acyl group did not induce the overexpression of defense-related genes in tomato. Best lipopeptides were BP370, BP378, BP381, BP387 and BP389, which were highly active against all the pathogens tested (minimum inhibitory concentration of 0.8 to 12.5 μM), low hemolytic, low phytotoxic and significantly stable to protease degradation. This family of lipopeptides might be promising functional peptides useful for plant protection The work was supported by the following: Ministerio de Economía y Competitividad (ES); AGL2012-39880-C02-02; LM, EB, EM, LF, MP; Ministerio de Economía y Competitividad (ES); AGL2015-69876-C2-2-R; AO, LM, EB, EM, LF, MP; University of Girona (ES); MPCUDG2016/038; AO, LF, MP

[Background] BP178 peptide is a synthetic BP100-magainin derivative possessing strong inhibitory activity against plant pathogenic bacteria, offering a great potential for future applications in plant protection and other fields. Here we report the production and recovery of a bioactive BP178 peptide using rice seeds as biofactories., [Results] A synthetic gene encoding the BP178 peptide was prepared and introduced in rice plants. The gene was efficiently expressed in transgenic rice under the control of an endosperm-specific promoter. Among the three endosperm-specific rice promoters (Glutelin B1, Glutelin B4 or Globulin 1), best results were obtained when using the Globulin 1 promoter. The BP178 peptide accumulated in the seed endosperm and was easily recovered from rice seeds using a simple procedure with a yield of 21 μg/g. The transgene was stably inherited for at least three generations, and peptide accumulation remained stable during long term storage of transgenic seeds. The purified peptide showed in vitro activity against the bacterial plant pathogen Dickeya sp., the causal agent of the dark brown sheath rot of rice. Seedlings of transgenic events showed enhanced resistance to the fungal pathogen Fusarium verticillioides, supporting that the in planta produced peptide was biologically active., [Conclusions] The strategy developed in this work for the sustainable production of BP178 peptide using rice seeds as biofactories represents a promising system for future production of peptides for plant protection and possibly in other fields., This work was supported by ERA-NET Plant-KBBE program EUI2008-03769 and EUI2008-03572 PROJECTS from the Spanish Ministry of Science and Innovation. We thank Government of the the Generalitat de Catalunya (Xarxa de Referencia en Biotecnología, and Xarxa de Referència en Tecnologia dels Aliments, SGR09626, 2008SGR812 and 2014SGR697) for support. We also thank the University of Girona for a Grant for Research improvement 2015–2018.

Objetivo: Determinar asociación de estrés, ansiedad y riesgo de trabajo de parto pretérmino espontáneo. Metodología: Es un estudio correlacional, longitudinal. El muestreo fue no probabilístico por casos consecutivos. A 124 mujeres embarazadas se les aplicó 2 instrumentos en tres semanas diferentes 20-24, 30-34 y 35-36. Uno evaluó el estado psicosocial de ansiedad con el instrumento "The Spielberger State-Trait Anxiety Inventory" (STAI) α de Cronbach de 0.90. Otro el estrés percibido con "Perceived Stress Questionnaire" (PSQ) α de Cronbach > 0.70. Resultados: La incidencia del parto pretérmino espontáneo (PPE) fue 6.4 %, el estrés percibido no incremento el riesgo PPE (RR 1.01; 95% [IC], 0.24-4.3 p < 0.05; La ansiedad no incremento el riesgo de PPE (RR 0.57; 95% [IC], 0.19-1.7 p < 0.05). Conclusión: El estrés percibido y la ansiedad no incrementaron el riesgo de PPE.

Cecropin A is a natural antimicrobial peptide that exhibits fast and potent activity against a broad spectrum of pathogens and neoplastic cells, and that has important biotechnological applications. However, cecropin A exploitation, as for other antimicrobial peptides, is limited by their production and purification costs. Here, we report the efficient production of this bioactive peptide in rice bran using the rice oleosin 18 as a carrier protein. High cecropin A levels were reached in rice seeds driving the expression of the chimeric gene by the strong embryo-specific oleosin 18 own promoter, and targeting the peptide to the oil body organelle as an oleosin 18-cecropin A fusion protein. The accumulation of cecropin A in oil bodies had no deleterious effects on seed viability and seedling growth, as well as on seed yield. We also show that biologically active cecropin A can be easily purified from the transgenic rice seeds by homogenization and simple flotation centrifugation methods. Our results demonstrate that the oleosin fusion technology is suitable for the production of cecropin A in rice seeds, which can potentially be extended to other antimicrobial peptides to assist their exploitation., This work was supported by the Spanish Ministry of Economy and Competitiveness [Grants EUI2008-03769, EUI2008-03572 and BIO2012-32838], and by the French Research Agency [Grant ANR-08-KBBE-010].

Resumen La gastrosquisis fetal es la malformacion congenita de la pared abdominal mas comun. Esta anomalia es susceptible de una correccion quirurgica posnatal mediante cierre primario de la fascia o diferido mediante una tecnica de silo. Presentamos el caso de una gestante que inicia su primer control de gestacion en la 26 semana, con hallazgo ecografico de gastrosquisis fetal.

Resumen La incidencia global del embarazo ectopico es del 1-2% de las gestaciones. Es infrecuente su diagnostico en el segundo trimestre, ya que por lo general los sintomas aparecen durante el primer trimestre. Presentamos el caso de una mujer que acude a urgencias con dolor en la fosa iliaca de varios dias de evolucion y mediante la ecografia se le diagnostica embarazo tubarico derecho, con feto de biometria concordante con las 13 semanas de amenorrea de la paciente.

Previous studies have shown that erythrocyte cell membranes in patients with schizophrenia contain considerably less omega-3 fatty acids, particularly EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid), reflecting the lower levels present in neuronal and central nervous system membranes. This phenomenon, linked to genetic, metabolic, or dietary factors, has been associated with the development of schizophrenia and the risk of developing and the severity of metabolic syndrome.This study is an observational study conducted in a sample of 31 patients with schizophrenia treated at the Mataró Mental Health Center (Barcelona). Its aim was to relate the erythrocyte levels of omega 3 with the clinical severity of schizophrenia and dietary habits. EPA (eicosapentaenoic acid), DHA (docosahexaenoic acid) and other membrane lipid levels were determined, as well as psychopathology, cognitive, and social functioning measures, previous evolution, and finally a survey of dietary habits.Our results did not show a statistically significant correlation between erythrocyte omega-3 levels and psychopathological and clinical severity variables. Higher, statistically significant, levels were found in the group of women and in subjects with more days of admission to the day hospital. In contrast, lower values were obtained in subjects treated with long-acting antipsychotics and in sunflower oil consumers.Despite not being able to demonstrate our working hypothesis, significant correlations were found that were consistent with published findings in the current literature. The need for studies with larger samples and groups of healthy controls is postulated.

Production of antimicrobial peptides in plants constitutes an approach for obtaining them in high amounts. However, their heterologous expression in a practical and efficient manner demands some structural requirements such as a minimum size, the incorporation of retention signals to assure their accumulation in specific tissues, and the presence of protease cleavage amino acids and of target sequences to facilitate peptide detection. Since any sequence modification may influence the biological activity, peptides that will be obtained from the expression must be screened prior to the synthesis of the genes for plant transformation. We report herein a strategy for the modification of the antimicrobial undecapeptide BP100 that allowed the identification of analogues that can be expressed in plants and exhibit optimum biological properties. We prepared 40 analogues obtained by incorporating repeated units of the antimicrobial undecapeptide, fragments of natural peptides, one or two AGPA hinges, a Gly or Ser residue at the N-terminus, and a KDEL fragment and/or the epitope tag54 at the C-terminus. Their antimicrobial, hemolytic and phytotoxic activities, and protease susceptibility were evaluated. Best sequences contained a magainin fragment linked to the antimicrobial undecapeptide through an AGPA hinge. Moreover, since the presence of a KDEL unit or of tag54 did not influence significantly the biological activity, these moieties can be introduced when designing compounds to be retained in the endoplasmic reticulum and detected using a complementary epitope. These findings may contribute to the design of peptides to be expressed in plants.

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., We thank J.L. La Paz (Centre for Research in Agricultural Genomics) for valuable suggestions and technical assistance, E. Melé (IRTA) for valuable suggestions, the Servei de Microscopia, (Universitat Autònoma de Barcelona) for technical assistance and S. Burgess for revision of the English. This work was financially supported by the Spanish Ministerio de Ciencia e Innovación (projects AGL2010-17181/AGR and PLANT-KBBE EUI2008-03769) and the Generalitat de Catalunya (SGR-2009-12). M.M. received a studentship from the Fundación Ramón Areces.

14-3-3 proteins are found in all eukaryotes where they act as regulators of diverse signalling pathways associated with a wide range of biological processes. In this study the functional characterization of the ZmGF14-6 gene encoding a maize 14-3-3 protein is reported. Gene expression analyses indicated that ZmGF14-6 is up-regulated by fungal infection and salt treatment in maize plants, whereas its expression is down-regulated by drought stress. It is reported that rice plants constitutively expressing ZmGF14-6 displayed enhanced tolerance to drought stress which was accompanied by a stronger induction of drought-associated rice genes. However, rice plants expressing ZmGF14-6 either in a constitutive or under a pathogen-inducible regime showed a higher susceptibility to infection by the fungal pathogens Fusarium verticillioides and Magnaporthe oryzae. Under infection conditions, a lower intensity in the expression of defence-related genes occurred in ZmGF14-6 rice plants. These findings support that ZmGF14-6 positively regulates drought tolerance in transgenic rice while negatively modulating the plant defence response to pathogen infection. Transient expression assays of fluorescently labelled ZmGF14-6 protein in onion epidermal cells revealed a widespread distribution of ZmGF14-6 in the cytoplasm and nucleus. Additionally, colocalization experiments of fluorescently labelled ZmGF14-6 with organelle markers, in combination with cell labelling with the endocytic tracer FM4-64, revealed a subcellular localization of ZmGF14-6 in the early endosomes. Taken together, these results improve our understanding of the role of ZmGF14-6 in stress signalling pathways, while indicating that ZmGF14-6 inversely regulates the plant response to biotic and abiotic stresses., This work was supported by grants BIO2006-05583 and BIO2009-08719 from the Spanish Ministry of Science and Innovation, as well as by the Consolider-Ingenio 2010 Programme CSD2007-00036 ‘‘Centre for Research in Agrigenomics’’. We also thank the ‘‘Departament d’Innovacio, Universitats i Empresa’’ from the Generalitat de Catalunya (Xarxa de Referencia en Biotecnologia and SGR 09626) for substantial support.

Background Xylella fastidiosa is one of the most harmful bacterial plant pathogens worldwide, causing a variety of diseases, with huge economic impact to agriculture and environment. Although it has been extensively studied, there are no therapeutic solutions to suppress disease development in infected plants. In this context, antimicrobial peptides represent promising alternatives to traditional compounds due to their activity against a wide range of plant pathogens, their low cytotoxicity, their mode of action that make resistance more difficult and their availability for being expressed in plants. Results Peptide conjugates derived from the lead peptide BP100 and fragments of cecropin, magainin or melittin were selected and tested against the plant pathogenic bacteria X. fastidiosa. In order to screen the activity of these antimicrobials, and due to the fastidious nature of the pathogen, a methodology consisting of a contact test coupled with the viability-quantitative PCR (v-qPCR) method was developed. The nucleic acid-binding dye PEMAX was used to selectively quantify viable cells by v-qPCR. In addition, the primer set XF16S-3 amplifying a 279 bp fragment was selected as the most suitable for v-qPCR. The performance of the method was assessed by comparing v-qPCR viable cells estimation with conventional qPCR and plate counting. When cells were treated with peptide conjugates derived from BP100, the observed differences between methods suggested that, in addition to cell death due to the lytic effect of the peptides, there was an induction of the viable but non-culturable state in cells. Notably, a contact test coupled to v-qPCR allowed fast and accurate screening of antimicrobial peptides, and led to the identification of new peptide conjugates active against X. fastidiosa. Conclusions Antimicrobial peptides active against X. fastidiosa have been identified using an optimized methodology that quantifies viable cells without a cultivation stage, avoiding underestimation or false negative detection of the pathogen due to the viable but non-culturable state, and overestimation of the viable population observed using qPCR. These findings provide new alternative compounds for being tested in planta for the control of X. fastidiosa, and a methodology that enables the fast screening of a large amount of antimicrobials against this plant pathogenic bacterium.

Xylella fastidiosa (Xf) diseases have emerged in recent years as several outbreaks of considerable intensity and extension in Europe. Their management is currently based on the use of pathogen-free propagation plant material, quarantine and eradication measures, and vector control. However, no cure of the disease exists up to now. Antimicrobial peptides have been identified as potential candidates for plant disease control, and have demonstrated a strong potential for the control of Xf. Specifically, it has been reported that the BP100 derivative peptide, BP178, synthetized for being expressed in plants, show high in vitro antibacterial activity against a collection of Xf strains. Although the application of bactericidal compounds to control xylem and phloem limited bacterial diseases has been tested by spray or trunk injection, the production of transgenic plant lines expressing antimicrobial peptides has been reported as promising alternative in the control of Xf. We have developed a transient-expression system based on Potato virus X (PVX) in Nicotiana benthamiana plants, for the production of the BP178 peptide in plants and evaluation of their potential in controlling Xf. The BP178 sequence was engineered in a PVX viral vector to be expressed in fusion with the NH2 Coat protein terminus. The release of the peptide occurs by the proteolytic activity of a Foot and Mouth Disease Virus 2A sequence. Western blot analysis showed that the BP178-CP fused proteins were expressed in N. benthamiana plants infected with the recombinant PVX-BP178-CP. Co-inoculations of N. benthamiana plants with PVX-BP178-CP and Xff strain IVIA 5387.2 are ongoing with initial results showing efficacy in controlling the pathogen over time., ES; PPT; laura.montesinos@udg.edu

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., This work was supported by SEPSAPE grants (Plant-KBBE programme) EUI2008-03769 and EUI2008-03572 from the Spanish Ministry of Science and Innovation, and ANR-08-KBBE-010 from the French Research Agency. We also thank the Consolider-Ingenio CSD2007-00036 award to CRAG and the Department d’Innovació, Universitats i Empresa of the Generalitat de Catalunya (Xarxa de Referencia en Biotecnología, Xarxa de Referència en Tecnologia dels Aliments, SGR09626, and 2008SGR812) for support. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).