The involvement of thaumatin-like proteins in plant food cross-reactivity: a multicenter study using a specific protein microarray.

Cross-reactivity of plant foods is an important phenomenon in allergy, with geographical variations with respect to the number and prevalence of the allergens involved in this process, whose complexity requires detailed studies. We have addressed the role of thaumatin-like proteins (TLPs) in cross-reactivity between fruit and pollen allergies. A representative panel of 16 purified TLPs was printed onto an allergen microarray. The proteins selected belonged to the sources most frequently associated with peach allergy in representative regions of Spain. Sera from two groups of well characterized patients, one with allergy to Rosaceae fruit (FAG) and another against pollens but tolerant to food-plant allergens (PAG), were obtained from seven geographical areas with different environmental pollen profiles. Cross-reactivity between members of this family was demonstrated by inhibition assays. Only 6 out of 16 purified TLPs showed noticeable allergenic activity in the studied populations. Pru p 2.0201, the peach TLP (41%), chestnut TLP (24%) and plane pollen TLP (22%) proved to be allergens of probable relevance to fruit allergy, being mainly associated with pollen sensitization, and strongly linked to specific geographical areas such as Barcelona, Bilbao, the Canary Islands and Madrid. The patients exhibited mayor que50% positive response to Pru p 2.0201 and to chestnut TLP in these specific areas. Therefore, their recognition patterns were associated with the geographical area, suggesting a role for pollen in the sensitization of these allergens. Finally, the co-sensitizations of patients considering pairs of TLP allergens were analyzed by using the co-sensitization graph associated with an allergen microarray immunoassay. Our data indicate that TLPs are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy.

Alt a 1 from Alternaria interacts with PR5 thaumatin-like proteins

Alt a 1 is a protein found in Alternaria alternata spores related to virulence and pathogenicity and considered to be responsible for chronic asthma in children. We found that spores of Alternaria inoculated on the outer surface of kiwifruits did not develop hyphae. Nevertheless, the expression of Alt a 1 gene was upregulated, and the protein was detected in the pulp where it co-localized with kiwi PR5. Pull-down assays demonstrated experimentally that the two proteins interact in such a way that Alt a 1 inhibits the enzymatic activity of PR5. These results are relevant not only for plant defense, but also for human health as patients with chronic asthma could suffer from an allergic reaction when they eat fruit contaminated with Alternaria.

Sensitization profiles and cross-reactivity among plant allergens. Molecular mechanisms of food allergy development and the role of enhancers

La prevalencia de las alergias está aumentando desde mediados del siglo XX, y se estima que actualmente afectan a alrededor del 2-8 % de la población, pero las causas de este aumento aún no están claras. Encontrar el origen del mecanismo por el cual una proteína inofensiva se convierte en capaz de inducir una respuesta alérgica es de vital importancia para prevenir y tratar estas enfermedades. Aunque la caracterización de alérgenos relevantes ha ayudado a mejorar el manejo clínico y a aclarar los mecanismos básicos de las reacciones alérgicas, todavía queda un largo camino para establecer el origen de la alergenicidad y reactividad cruzada. El objetivo de esta tesis ha sido caracterizar las bases moleculares de la alergenicidad tomando como modelo dos familias de panalergenos (proteínas de transferencia de lípidos –LTPs- y taumatinas –TLPs-) y estudiando los mecanismos que median la sensibilización y la reactividad cruzada para mejorar tanto el diagnóstico como el tratamiento de la alergia. Para ello, se llevaron a cabo dos estrategias: estudiar la reactividad cruzada de miembros de familias de panalérgenos; y estudiar moléculas-co-adyuvantes que pudieran favorecer la capacidad alergénica de dichas proteínas. Para estudiar la reactividad cruzada entre miembros de la misma familia de proteínas, se seleccionaron LTPs y TLPs, descritas como alergenos, tomando como modelo la alergia a frutas. Por otra parte, se estudiaron los perfiles de sensibilización a alérgenos de trigo relacionados con el asma del panadero, la enfermedad ocupacional más relevante de origen alérgico. Estos estudios se llevaron a cabo estandarizando ensayos tipo microarrays con alérgenos y analizando los resultados por la teoría de grafos. En relación al estudiar moléculas-co-adyuvantes que pudieran favorecer la capacidad alergénica de dichas proteínas, se llevaron a cabo estudios sobre la interacción de los alérgenos alimentarios con células del sistema inmune humano y murino y el epitelio de las mucosas, analizando la importancia de moléculas co-transportadas con los alérgenos en el desarrollo de una respuesta Th2. Para ello, Pru p 3(LTP y alérgeno principal del melocotón) se selección como modelo para llevarlo a cabo. Por otra parte, se analizó el papel de moléculas activadoras del sistema inmune producidas por patógenos en la inducción de alergias alimentarias seleccionando el modelo kiwi-alternaria, y el papel de Alt a 1, alérgeno mayor de dicho hongo, en la sensibilización a Act d 2, alérgeno mayor de kiwi. En resumen, el presente trabajo presenta una investigación innovadora aportando resultados de gran utilidad tanto para la mejora del diagnóstico como para nuevas investigaciones sobre la alergia y el esclarecimiento final de los mecanismos que caracterizan esta enfermedad. ABSTRACT Allergies are increasing their prevalence from mid twentieth century, and they are currently estimated to affect around 2-8% of the population but the underlying causes of this increase remain still elusive. The understanding of the mechanism by which a harmless protein becomes capable of inducing an allergic response provides us the basis to prevent and treat these diseases. Although the characterization of relevant allergens has led to improved clinical management and has helped to clarify the basic mechanisms of allergic reactions, it seems justified in aspiring to molecularly dissecting these allergens to establish the structural basis of their allergenicity and cross-reactivity. The aim of this thesis was to characterize the molecular basis of the allergenicity of model proteins belonging to different families (Lipid Transfer Proteins –LTPs-, and Thaumatin-like Proteins –TLPs-) in order to identify mechanisms that mediate sensitization and cross reactivity for developing new strategies in the management of allergy, both diagnosis and treatment, in the near future. With this purpose, two strategies have been conducted: studies of cross-reactivity among panallergen families and molecular studies of the contribution of cofactors in the induction of the allergic response by these panallergens. Following the first strategy, we studied the cross-reactivity among members of two plant panallergens (LTPs , Lipid Transfer Proteins , and TLPs , Thaumatin-like Proteins) using the peach allergy as a model. Similarly, we characterized the sensitization profiles to wheat allergens in baker's asthma development, the most relevant occupational disease. These studies were performed using allergen microarrays and the graph theory for analyzing the results. Regarding the second approach, we analyzed the interaction of plant allergens with immune and epithelial cells. To perform these studies , we examined the importance of ligands and co-transported molecules of plant allergens in the development of Th2 responses. To this end, Pru p 3, nsLTP (non-specific Lipid Transfer Protein) and peach major allergen, was selected as a model to investigate its interaction with cells of the human and murine immune systems as well as with the intestinal epithelium and the contribution of its ligand in inducing an allergic response was studied. Moreover, we analyzed the role of pathogen associated molecules in the induction of food allergy. For that, we selected the kiwi- alternaria system as a model and the role of Alt a 1 , major allergen of the fungus, in the development of Act d 2-sensitization was studied. In summary, this work presents an innovative research providing useful results for improving diagnosis and leading to further research on allergy and the final clarification of the mechanisms that characterize this disease.

Characterization of four bifunctional plant IAM/PAM-amidohydrolases capable of contributing to auxin biosynthesis.

Amidases [EC 3.5.1.4] capable of converting indole-3-acetamide (IAM) into the major plant growth hormone indole-3-acetic acid (IAA) are assumed to be involved in auxin de novo biosynthesis. With the emerging amount of genomics data, it was possible to identify over forty proteins with substantial homology to the already characterized amidases from Arabidopsis and tobacco. The observed high conservation of amidase-like proteins throughout the plant kingdom may suggest an important role of theses enzymes in plant development. Here, we report cloning and functional analysis of four, thus far, uncharacterized plant amidases from Oryza sativa, Sorghum bicolor, Medicago truncatula, and Populus trichocarpa. Intriguingly, we were able to demonstrate that the examined amidases are also capable of converting phenyl-2-acetamide (PAM) into phenyl-2-acetic acid (PAA), an auxin endogenous to several plant species including Arabidopsis. Furthermore, we compared the subcellular localization of the enzymes to that of Arabidopsis AMI1, providing further evidence for similar enzymatic functions. Our results point to the presence of a presumably conserved pathway of auxin biosynthesis via IAM, as amidases, both of monocot, and dicot origins, were analyzed.

Molecular tools to improve chestnut management: El Bierzo as a case study

The European chestnut (Castanea sativa Mill.) is a multipurpose species that has been widely cultivated around the Mediterranean basin since ancient times. New varieties were brought to the Iberian Peninsula during the Roman Empire, which coexist since then with native populations that survived the last glaciation. The relevance of chestnut cultivation has being steadily growing since the Middle Ages, until the rural decline of the past century put a stop to this trend. Forest fires and diseases were also major factors. Chestnut cultivation is gaining momentum again due to its economic (wood, fruits) and ecologic relevance, and represents currently an important asset in many rural areas of Europe. In this Thesis we apply different molecular tools to help improve current management strategies. For this study we have chosen El Bierzo (Castile and Leon, NW Spain), which has a centenary tradition of chestnut cultivation and management, and also presents several unique features from a genetic perspective (next paragraph). Moreover, its nuts are widely appreciated in Spain and abroad for their organoleptic properties. We have focused our experimental work on two major problems faced by breeders and the industry: the lack of a fine-grained genetic characterization and the need for new strategies to control blight disease. To characterize with sufficient detail the genetic diversity and structure of El Bierzo orchards, we analyzed DNA from 169 trees grafted for nut production covering the entire region. We also analyzed 62 nuts from all traditional varieties. El Bierzo constitutes an outstanding scenario to study chestnut genetics and the influence of human management because: (i) it is located at one extreme of the distribution area; (ii) it is a major glacial refuge for the native species; (iii) it has a long tradition of human management (since Roman times, at least); and (iv) its geographical setting ensures an unusual degree of genetic isolation. Thirteen microsatellite markers provided enough informativeness and discrimination power to genotype at the individual level. Together with an unexpected level of genetic variability, we found evidence of genetic structure, with three major gene pools giving rise to the current population. High levels of genetic differentiation between groups supported this organization. Interestingly, genetic structure does not match with spatial boundaries, suggesting that the exchange of material and cultivation practices have strongly influenced natural gene flow. The microsatellite markers selected for this study were also used to classify a set of 62 samples belonging to all traditional varieties. We identified several cases of synonymies and homonymies, evidencing the need to substitute traditional classification systems with new tools for genetic profiling. Management and conservation strategies should also benefit from these tools. The avenue of high-throughput sequencing technologies, combined with the development of bioinformatics tools, have paved the way to study transcriptomes without the need for a reference genome. We took advantage of RNA sequencing and de novo assembly tools to determine the transcriptional landscape of chestnut in response to blight disease. In addition, we have selected a set of candidate genes with high potential for developing resistant varieties via genetic engineering. Our results evidenced a deep transcriptional reprogramming upon fungal infection. The plant hormones ET and JA appear to orchestrate the defensive response. Interestingly, our results also suggest a role for auxins in modulating such response. Many transcription factors were identified in this work that interact with promoters of genes involved in disease resistance. Among these genes, we have conducted a functional characterization of a two major thaumatin-like proteins (TLP) that belongs to the PR5 family. Two genes encoding chestnut cotyledon TLPs have been previously characterized, termed CsTL1 and CsTL2. We substantiate here their protective role against blight disease for the first time, including in silico, in vitro and in vivo evidence. The synergy between TLPs and other antifungal proteins, particularly endo-p-1,3-glucanases, bolsters their interest for future control strategies based on biotechnological approaches.

Functional characterization of YODA, a mitogen-activated protein kinase kinase kinase (MAP3K) that regulates a novel innate immunity pathway in Arabidopsis thaliana

Las cascadas de señalización mediadas por proteína quinasas activadas por mitógeno (MAP quinasas) son capaces de integrar y transducir señales ambientales en respuestas celulares. Entre estas señales se encuentran los PAMPs/MAMPs (Pathogen/Microbe-Associated Molecular Patterns), que son moléculas de patógenos o microorganismos, o los DAMPs (Damaged-Associated Molecular Patterns), que son moléculas derivadas de las plantas producidas en respuesta a daño celular. Tras el reconocimiento de los PAMPs/DAMPs por receptores de membrana denominados PRRs (Pattern Recognition Receptors), como los receptores con dominio quinasa (RLKs) o los receptores sin dominio quinasa (RLPs), se activan respuestas moleculares, incluidas cascadas de MAP quinasas, que regulan la puesta en marcha de la inmunidad activada por PAMPs (PTI). Esta Tesis describe la caracterización funcional de la MAP quinasa quinasa quinasa (MAP3K) YODA (YDA), que actúa como un regulador clave de la PTI en Arabidopsis. Se ha descrito previamente que YDA controla varios procesos de desarrollo, como la regulación del patrón estomático, la elongación del zigoto y la arquitectura floral. Hemos caracterizado un alelo mutante hipomórfico de YDA (elk2 o yda11) que presenta una elevada susceptibilidad a patógenos biótrofos y necrótrofos. Notablemente, plantas que expresan una forma constitutivamente activa de YDA (CA-YDA), con una deleción en el dominio N-terminal, presentan una resistencia de amplio espectro frente a diferentes tipos de patógenos, incluyendo hongos, oomicetos y bacterias, lo que indica que YDA juega un papel importante en la regulación de la resistencia de las plantas a patógenos. Nuestros datos indican que esta función es independiente de las respuestas inmunes mediadas por los receptores previamente caracterizados FLS2 y CERK1, que reconocen los PAMPs flg22 y quitina, respectivamente, y que están implicados en la resistencia de Arabidopsis frente a bacterias y hongos. Hemos demostrado que YDA controla la resistencia frente al hongo necrótrofo Plectosphaerella cucumerina y el patrón estomático mediante su interacción genética con la RLK ERECTA (ER), un PRR implicado en la regulación de estos procesos. Por el contrario, la interacción genética entre ER y YDA en la regulación de otros procesos de desarrollo es aditiva en lugar de epistática. Análisis genéticos indicaron que MPK3, una MAP quinasa que funciona aguas abajo de YDA en el desarrollo estomático, es un componente de la ruta de señalización mediada por YDA para la resistencia frente a P. cucumerina, lo que sugiere que el desarrollo de las plantas y la PTI comparten el módulo de transducción de MAP quinasas asociado a YDA. Nuestros experimentos han revelado que la resistencia mediada por YDA es independiente de las rutas de señalización reguladas por las hormonas de defensa ácido salicílico, ácido jasmónico, ácido abscísico o etileno, y también es independiente de la ruta de metabolitos secundarios derivados del triptófano, que están implicados en inmunidad vegetal. Además, hemos demostrado que respuestas asociadas a PTI, como el aumento en la concentración de calcio citoplásmico, la producción de especies reactivas de oxígeno, la fosforilación de MAP quinasas y la expresión de genes de defensa, no están afectadas en el mutante yda11. La expresión constitutiva de la proteína CA-YDA en plantas de Arabidopsis no provoca un aumento de las respuestas PTI, lo que sugiere la existencia de mecanismos de resistencia adicionales regulados por YDA que son diferentes de los regulados por FLS2 y CERK1. En línea con estos resultados, nuestros datos transcriptómicos revelan una sobre-representación en plantas CA-YDA de genes de defensa que codifican, por ejemplo, péptidos antimicrobianos o reguladores de muerte celular, o proteínas implicadas en la biogénesis de la pared celular, lo que sugiere una conexión potencial entre la composición e integridad de la pared celular y la resistencia de amplio espectro mediada por YDA. Además, análisis de fosfoproteómica indican la fosforilación diferencial de proteínas relacionadas con la pared celular en plantas CA-YDA en comparación con plantas silvestres. El posible papel de la ruta ER-YDA en la regulación de la integridad de la pared celular está apoyado por análisis bioquímicos y glicómicos de las paredes celulares de plantas er, yda11 y CA-YDA, que revelaron cambios significativos en la composición de la pared celular de estos genotipos en comparación con la de plantas silvestres. En resumen, nuestros datos indican que ER y YDA forman parte de una nueva ruta de inmunidad que regula la integridad de la pared celular y respuestas defensivas, confiriendo una resistencia de amplio espectro frente a patógenos. ABSTRACT Plant mitogen-activated protein kinase (MAPK) cascades transduce environmental signals and developmental cues into cellular responses. Among these signals are the pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) and the damage-associated molecular patterns (DAMPs). These PAMPs/DAMPs, upon recognition by plant pattern recognition receptors (PRRs), such as Receptor-Like Kinases (RLKs) and Receptor-Like Proteins (RLPs), activate molecular responses, including MAPK cascades, which regulate the onset of PAMP-triggered immunity (PTI). This Thesis describes the functional characterization of the MAPK kinase kinase (MAP3K) YODA (YDA) as a key regulator of Arabidopsis PTI. YDA has been previously described to control several developmental processes, such as stomatal patterning, zygote elongation and inflorescence architecture. We characterized a hypomorphic, non-embryo lethal mutant allele of YDA (elk2 or yda11) that was found to be highly susceptible to biotrophic and necrotrophic pathogens. Remarkably, plants expressing a constitutive active form of YDA (CA-YDA), with a deletion in the N-terminal domain, showed broad-spectrum resistance to different types of pathogens, including fungi, oomycetes and bacteria, indicating that YDA plays a relevant function in plant resistance to pathogens. Our data indicated that this function is independent of the immune responses regulated by the well characterized FLS2 and CERK1 RLKs, which are the PRRs recognizing flg22 and chitin PAMPs, respectively, and are required for Arabidopsis resistance to bacteria and fungi. We demonstrate that YDA controls resistance to the necrotrophic fungus Plectosphaerella cucumerina and stomatal patterning by genetically interacting with ERECTA (ER) RLK, a PRR involved in regulating these processes. In contrast, the genetic interaction between ER and YDA in the regulation of other ER-associated developmental processes was additive, rather than epistatic. Genetic analyses indicated that MPK3, a MAP kinase that functions downstream of YDA in stomatal development, also regulates plant resistance to P. cucumerina in a YDA-dependent manner, suggesting that the YDA-associated MAPK transduction module is shared in plant development and PTI. Our experiments revealed that YDA-mediated resistance was independent of signalling pathways regulated by defensive hormones like salicylic acid, jasmonic acid, abscisic acid or ethylene, and of the tryptophan-derived metabolites pathway, which are involved in plant immunity. In addition, we showed that PAMP-mediated PTI responses, such as the increase of cytoplasmic Ca2+ concentration, reactive oxygen species (ROS) burst, MAPK phosphorylation, and expression of defense-related genes are not impaired in the yda11 mutant. Furthermore, the expression of CA-YDA protein does not result in enhanced PTI responses, further suggesting the existence of additional mechanisms of resistance regulated by YDA that differ from those regulated by the PTI receptors FLS2 and CERK1. In line with these observations, our transcriptomic data revealed the over-representation in CA-YDA plants of defensive genes, such as those encoding antimicrobial peptides and cell death regulators, and genes encoding cell wall-related proteins, suggesting a potential link between plant cell wall composition and integrity and broad spectrum resistance mediated by YDA. In addition, phosphoproteomic data revealed an over-representation of genes encoding wall-related proteins in CA-YDA plants in comparison with wild-type plants. The putative role of the ER-YDA pathway in regulating cell wall integrity was further supported by biochemical and glycomics analyses of er, yda11 and CA-YDA cell walls, which revealed significant changes in the cell wall composition of these genotypes compared with that of wild-type plants. In summary, our data indicate that ER and YDA are components of a novel immune pathway that regulates cell wall integrity and defensive responses, which confer broad-spectrum resistance to pathogens.

Antigenic Proteins Involved in Occupational Rhinitis 1 and Asthma Caused by Obeche Wood (Triplochiton Scleroxylon)

Background Obeche wood dust is a known cause of occupational asthma where an IgE-mediated mechanism has been demonstrated. Objective To characterize the allergenic profile of obeche wood dust and evaluate the reactivity of the proteins by in vitro, ex vivo and in vivo assays in carpenters with confirmed rhinitis and/or asthma Materials and methods An in-house obeche extract was obtained, and two IgE binding bands were purified (24 and 12 kDa) and sequenced by N-terminal identity. Specific IgE and IgG, basophil activation tests and skin prick tests (SPTs) were performed with whole extract and purified proteins. CCD binding was analyzed by ELISA inhibition studies. Results Sixty-two subjects participated: 12 with confirmed occupational asthma/rhinitis (ORA+), 40 asymptomatic exposed (ORA−), and 10 controls. Of the confirmed subjects, 83% had a positive SPT to obeche. There was a 100% recognition by ELISA in symptomatic subjects vs. 30% and 10% in asymptomatic exposed subjects and controls respectively (p<0.05). Two new proteins were purified, a 24 kDa protein identified as a putative thaumatin-like protein and a 12 kDa gamma-expansin. Both showed allergenic activity in vitro, with the putative thaumatin being the most active, with 92% recognition by ELISA and 100% by basophil activation test in ORA+ subjects. Cross-reactivity due to CCD was ruled out in 82% of cases. Conclusions Two proteins of obeche wood were identified and were recognized by a high percentage of symptomatic subjects and by a small proportion of asymptomatic exposed subjects. Further studies are required to evaluate cross reactivity with other plant allergens.

Identification of thaumatin-like protein and aspartyl protease as new major allergens in lettuce (Lactuca sativa)

Scope: Today, about 2–8% of the population of Western countries exhibits some type of food allergy whose impact ranges from localized symptoms confined to the oral mucosa to severe anaphylactic reactions. Consumed worldwide, lettuce is a Compositae family vegetable that can elicit allergic reactions. To date, however, only one lipid transfer protein has been described in allergic reaction to lettuce. The aim of this study was to identify potential new allergens involved in lettuce allergy. Methods and results: Sera from 42 Spanish lettuce-allergic patients were obtained from pa-tients recruited at the outpatient clinic. IgE-binding proteins were detected by SDS-PAGE and immunoblotting. Molecular characterization of IgE-binding bands was performed by MS. Thaumatin was purified using the Agilent 3100 OFFGEL system. The IgE-binding bands recognized in the sera of more than 50% of patients were identified as lipid transfer protein (9 kDa), a thaumatin-like protein (26 kDa), and an aspartyl protease (35 and 45 kDa). ELISA inhibition studies were performed to confirm the IgE reactivity of the purified allergen. Conclusion: Two new major lettuce allergens—a thaumatin-like protein and an aspartyl protease—have been identified and characterized. These allergens may be used to improve both diagnosis and treatment of lettuce-allergic patients.

Nitrogen fixation by native Bradyrhizobia in symbiosis with Lupinus mariae-josephae requires a T3SS encoding a NopE-like effector

Several bradyrhizobial isolates from L. mariae-josephae root nodules [1] contain a type III secretion system (T3SS) within a cluster of about 30 genes. Among those genes, ttsI codes for the transcriptional activator of the system. Mutation of ttsI resulted in the formation of white, non-fixing nodules with the natural legume host, L. mariae-josephae. The T3SS cluster also contains a gene coding for a NopE-like protein. NopE proteins have been demonstrated to be effectors in the Bradyrhizobium-soybean symbiosis [2] and belong to a small group of poorly characterized proteins from plant-associated bacteria that contain one or two autocleavage motifs known as DUF1521 (Schirrmeister et al. 2011). The amino acid sequence of a NopE-like protein in the L. mariae-josephae strain LmjC contains just one autocatalytic motif. This is unlike NopE1 and NopE2 proteins secreted by the T3SS of B. japonicum, that contain two motifs [3]. The autocleavage of LmjC NopE protein was analyzed after expression in E. coli and purification. Two protein fragments of the predicted sizes appeared in the presence of Ca2+, Cu2+, Cd2+, Zn2+ and Mn2+ cations. In contrast, autocleavage did not take place in the presence of Ni2+, Co2+ or Mg2+. Site-directed mutagenesis of the DUF1521 motif in LmjC NopE abolished self-cleavage in vitro. Symbiotic competence of a NopE- mutant with the L. mariae-josephae host was not affected. Possible roles of NopE are discussed.