Polygalacturonase gene FaPG1 downregulation is related to increased strawberry fruit resistance to fungal decay

Plant health is a major target in breading programs because crops are under constant biotic stress, and climate change is exacerbating pests and disease negative impacts in agriculture. Obtaining crop varieties armed with better defences is a potential strategy to reduce losses from biotic attacks. Plant cell walls perform crucial roles on many physiological processes, and under biotic stress, play crucial defensive roles as protecting barrier, as well as a source of integrity signalling molecules. Plant immunity has evolved a complex multi-layered system which first line of defence is initiated by conserved molecular patterns coming from pathogens, named pathogen-associated molecular patterns or PAMPs, or from their own corrupted cell walls due to pathogen invasion, named damaged-associated molecular patterns or DAMPs. Accumulating evidence from cell wall mutants has unveiled several components and mechanisms of plant innate immunity under biotic stresses, mostly in Arabidopsis, but still little is known from species with agronomic interest as strawberry. Our group has an established strawberry transgenic collection of cell wall mutants. Among them, RNAseq expression profiles of FaPG1 mutants has shown downregulation of other cell wall related genes than PG [1], but the mechanisms underneath required further investigation. FaPG genes code for enzymes with endo-PG activity related to oligogalacturonic acid (OGA) release, which would be associated to the changes in gene expression of other cell wall genes than FaPG. In this work, postharvest assays of FaPG1 fruits showed not only the increased fruit firmness typical of this mutant, but a better resistance to fungal infections by Botrytis cinerea, enhancing fruit shelf life in comparison with control fruits.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Partial Activation of SA- and JA-Defensive Pathways in Strawberry upon Colletotrichum acutatum Interaction

[EN] Understanding the nature of pathogen host interaction may help improve strawberry (Fragaria x anahassa) cultivars. Plant resistance to pathogenic agents usually operates through a complex network of defense mechanisms mediated by a diverse array of signaling molecules. In strawberry, resistance to a variety of pathogens has been reported to be mostly polygenic and quantitatively inherited, making it difficult to associate molecular markers with disease resistance genes. Colletotrichum acutaturn spp. is a major strawberry pathogen, and completely resistant cultivars have not been reported. Moreover, strawberry defense network components and mechanisms remain largely unknown and poorly understood. Assessment of the strawberry response to C. acutatum included a global transcript analysis, and acidic hormones SA and JA measurements were analyzed after challenge with the pathogen. Induction of transcripts corresponding to the SA and JA signaling pathways and key genes controlling major steps within these defense pathways was detected. Accordingly, SA and JA accumulated in strawberry after infection. Contrastingly, induction of several important SA, JA, and oxidative stress-responsive defense genes, including FaPR1-1, FaLOX2, FaJAR1, FaPDF1, and FaGST1, was not detected, which suggests that specific branches in these defense pathways (those leading to FaPR1-2, FaPR2-1, FaPR2-2, FaAOS, FaPR5, and FaPR10) were activated. Our results reveal that specific aspects in SA and JA dependent signaling pathways are activated in strawberry upon interaction with C. acutatum. Certain described defense-associated transcripts related to these two known signaling pathways do not increase in abundance following infection. This finding suggests new insight into a specific putative molecular strategy for defense against this pathogen.Authors are grateful to Dr. JM Lopez-Aranda (IFAPA-Centro de Churriana) for providing micropropagated strawberry plants and to Nicolas Garcia-Caparros for technical assistance. Authors also want to thank Kevin M. Folta for his insightful comments on the paper. This work was supported by Junta de Andalucia, Spain [Proyectos de Excelencia P07-AGR-02482/P12-AGR-2174, and grants to Grupo-BIO278].Amil-Ruiz, F.; Garrido-Gala, J.; Gadea Vacas, J.; Blanco-Portales, R.; Munoz-Merida, A.; Trelles, O.; De Los Santos, B.... (2016). Partial Activation of SA- and JA-Defensive Pathways in Strawberry upon Colletotrichum acutatum Interaction. Frontiers in Plant Science. 7(1036). https://doi.org/10.3389/fpls.2016.01036S71036Acosta, I. F., & Farmer, E. E. (2010). Jasmonates. The Arabidopsis Book, 8, e0129. doi:10.1199/tab.0129Al-Shahrour, F., Diaz-Uriarte, R., & Dopazo, J. (2004). 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FaMYB123 interacts with FabHLH3 to regulate the late steps of anthocyanin and flavonol biosynthesis during ripening.

In this work, we identified and functionally characterized the strawberry (Fragaria × ananassa) R2R3 MYB transcription factor FaMYB123. As in most genes associated with organoleptic properties of ripe fruit, FaMYB123 expression is ripening-related, receptacle-specific, and antagonistically regulated by ABA and auxin. Knockdown of FaMYB123 expression by RNAi in ripe strawberry fruit receptacles downregulated the expression of enzymes involved in the late steps of anthocyanin/flavonoid biosynthesis. Transgenic fruits showed a parallel decrease in the contents of total anthocyanin and flavonoid, especially malonyl derivatives of pelargonidin and cyanidins. The decrease was concomitant with accumulation of proanthocyanin, propelargonidins, and other condensed tannins associated mainly with green receptacles. Potential coregulation between FaMYB123 and FaMYB10, which may act on different sets of genes for the enzymes involved in anthocyanin production, was explored. FaMYB123 and FabHLH3 were found to interact and to be involved in the transcriptional activation of FaMT1, a gene responsible for the malonylation of anthocyanin components during ripening. Taken together, these results demonstrate that FaMYB123 regulates the late steps of the flavonoid pathway in a specific manner. In this study, a new function for an R2R3 MYB transcription factor, regulating the expression of a gene that encodes a malonyltransferase, has been elucidated.This work was funded by the Spanish Ministerio de Ciencia e Innovacion (AGL2014-55784-C2-2-R and AGL2017-86531-C2-2-R). FJMR is supported by a ‘Margarita Salas’ post-doctoral fellowship (UCOR02MS) from the University of Cordoba (Requalification of the Spanish university system) from the Ministry of Universities financed by the European Union (NexGenerationEU). FJMH is supported by a ‘Juan de la Cierva-Incorporacion’ fellowship (IJC2020- 045526-I), funded by MCIN/AEI/10.13039/501100011033 and the European Union ‘NextGenerationEU’/PRTR. AR-F and SA are on the European Union’s Horizon 2020 Research and Innovation Program, Project PlantaSYST (SGA-CSA No. 739582 under FPA No. 664620). The authors thank Dr. Gema Garc ıa from the Microscopy Unit of UCAIB-IMIBIC for technical help with the microscope. Funding for open access charge: University of Cordoba/CBUA.S

Transformación vía Agrobacterium tumefaciens para inducir tolerancia a la podredumbre blanca del aguacate

Comunicación presentada en el VII World Avocado Congress, celebrado en Cairns (Australia) del 5 al 9 de septiembre de 2011.[EN] One of the most important limiting factors for avocado production in Spain is the disease caused by the fungus Rosellinia necatrix . Genetic manipulation could be useful for the introduction of fungal resistance traits into this crop. A n efficient Agrobacterium - mediated transformation protocol for avocado using AGL1 Agrobacterium strain and somatic embryos as the target material has been established by our group, although embryo conversion rate into plants needs to be improved. For that reason, we are using the strawberry, another Rosellinia necatrix ́s host, as model species to test the effect of several transgenes (two of fungal origin, chit 42 chitinase and β - 1,3 - glucanase from Trichoderma harzianum , and one of plant origin, At NPR1), on inducing tolerance to this fungus. Strawberry transformation with the β - 1,3 - glucanase gene has allowed the selection of two lines, β6 and β10, with enhanced tolerance to R. necatrix while no positive results were obtained following transformation with the chit - 42 gene. In relation to the At NPR1 gene more than 30 independent transgenic lines have been obtained whose tolerance to R. necatrix is currently under evaluation. Concerning avocado transformation, more than 10 independent transgenic lines (derive d from an embryogenic line of an immature Duke7 zygotic embryo) have been obtained with At NPR1 gene. Plants have been recovered from one line and efforts are underway to recover plants from other lines following micrografting of the transgenic sprouted sho ots onto in vitro germinated seedlings.[ES] Uno de los factores limitantes de la producción de aguacate en España es la enfermedad causada por el hongo R. necatrix. La manipulación genética podría ser de utilidad para introducir caracteres de resistencia en este cultivo. Se ha establecido un sistema eficiente de transformación en aguacate usando la cepa de Agrobacterium AGL1 y células embriogénicas como diana, sin embargo, la conversión en plantas de los embriones transgénicos necesita ser mejorada. Por esta razón, estamos utilizando la fresa, otro huésped de R. necatrix, como especie modelo para testar el efecto de varios transgenes (2 de origen fúngico, la quitinasa chit-42 y la β-1,3-glucanasa de Trichoderma harzianum, y uno derivado de plantas, AtNPR1), en la inducción de tolerancia a este patógeno tras la transformación de esta especie. La transformación de fresa con el gen de β -1,3-glucanasa ha permitido la selección de dos líneas, β6 y β10, con mayor tolerancia a R. necatrix, mientras que no se han obtenido resultados positivos con el gen chit-42. En relación con el gen AtNPR1, se han obtenido más de 30 líneas transgénicas independientes, cuya tolerancia frente a R. necatrix se está evaluando en la actualidad. En relación con la transformación de aguacate, más de 10 líneas transgénicas independientes (derivadas de una línea embriogénica obtenida a partir de un embrión zigótico inmaduro del cv. Duke 7) se han obtenido con el gen AtNPR1. Se han recuperado plantas de una línea y actualmente se está intentando recuperar plantas de otras líneas mediante microinjerto de los embriones transgénicos germinados.Este trabajo se ha realizado en el marco del proyecto AGL2008 - 05453 - C02 - 01/AGR.Peer Reviewe

Use of ChIP-Seq data for the design of a multiple promoter-alignment method

We address the challenge of regulatory sequence alignment with a new method, Pro-Coffee, a multiple aligner specifically designed for homologous promoter regions. Pro-Coffee uses a dinucleotide substitution matrix estimated on alignments of functional binding sites from TRANSFAC. We designed a validation framework using several thousand families of orthologous promoters. This dataset was used to evaluate the accuracy for predicting true human orthologs among their paralogs. We found that whereas other methods achieve on average 73.5% accuracy, and 77.6% when trained on that same dataset, the figure goes up to 80.4% for Pro-Coffee. We then applied a novel validation procedure based on multi-species ChIP-seq data. Trained and untrained methods were tested for their capacity to correctly align experimentally detected binding sites. Whereas the average number of correctly aligned sites for two transcription factors is 284 for default methods and 316 for trained methods, Pro-Coffee achieves 331, 16.5% above the default average. We find a high correlation between a method's performance when classifying orthologs and its ability to correctly align proven binding sites. Not only has this interesting biological consequences, it also allows us to conclude that any method that is trained on the ortholog data set will result in functionally more informative alignments

ORegAnno: an open-access community-driven resource for regulatory annotation

ORegAnno is an open-source, open-access database and literature curation system for community-based annotation of experimentally identified DNA regulatory regions, transcription factor binding sites and regulatory variants. The current release comprises 30 145 records curated from 922 publications and describing regulatory sequences for over 3853 genes and 465 transcription factors from 19 species. A new feature called the ‘publication queue’ allows users to input relevant papers from scientific literature as targets for annotation. The queue contains 4438 gene regulation papers entered by experts and another 54 351 identified by text-mining methods. Users can enter or ‘check out’ papers from the queue for manual curation using a series of user-friendly annotation pages. A typical record entry consists of species, sequence type, sequence, target gene, binding factor, experimental outcome and one or more lines of experimental evidence. An evidence ontology was developed to describe and categorize these experiments. Records are cross-referenced to Ensembl or Entrez gene identifiers, PubMed and dbSNP and can be visualized in the Ensembl or UCSC genome browsers. All data are freely available through search pages, XML data dumps or web services at: http://www.oreganno.org

A genome-wide association study follow-up suggests a possible role for PPARG in systemic sclerosis susceptibility

Introduction: A recent genome-wide association study (GWAS) comprising a French cohort of systemic sclerosis (SSc) reported several non-HLA single-nucleotide polymorphisms (SNPs) showing a nominal association in the discovery phase. We aimed to identify previously overlooked susceptibility variants by using a follow-up strategy.<p></p> Methods: Sixty-six non-HLA SNPs showing a P value <10-4 in the discovery phase of the French SSc GWAS were analyzed in the first step of this study, performing a meta-analysis that combined data from the two published SSc GWASs. A total of 2,921 SSc patients and 6,963 healthy controls were included in this first phase. Two SNPs, PPARG rs310746 and CHRNA9 rs6832151, were selected for genotyping in the replication cohort (1,068 SSc patients and 6,762 healthy controls) based on the results of the first step. Genotyping was performed by using TaqMan SNP genotyping assays. Results: We observed nominal associations for both PPARG rs310746 (PMH = 1.90 × 10-6, OR, 1.28) and CHRNA9 rs6832151 (PMH = 4.30 × 10-6, OR, 1.17) genetic variants with SSc in the first step of our study. In the replication phase, we observed a trend of association for PPARG rs310746 (P value = 0.066; OR, 1.17). The combined overall Mantel-Haenszel meta-analysis of all the cohorts included in the present study revealed that PPARG rs310746 remained associated with SSc with a nominal non-genome-wide significant P value (PMH = 5.00 × 10-7; OR, 1.25). No evidence of association was observed for CHRNA9 rs6832151 either in the replication phase or in the overall pooled analysis.<p></p> Conclusion: Our results suggest a role of PPARG gene in the development of SSc

Towards resolving the transcription factor network controlling myelin gene expression

In the central nervous system (CNS), myelin is produced from spirally-wrapped oligodendrocyte plasma membrane and, as exemplified by the debilitating effects of inherited or acquired myelin abnormalities in diseases such as multiple sclerosis, it plays a critical role in nervous system function. Myelin sheath production coincides with rapid up-regulation of numerous genes. The complexity of their subsequent expression patterns, along with recently recognized heterogeneity within the oligodendrocyte lineage, suggest that the regulatory networks controlling such genes drive multiple context-specific transcriptional programs. Conferring this nuanced level of control likely involves a large repertoire of interacting transcription factors (TFs). Here, we combined novel strategies of computational sequence analyses with in vivo functional analysis to establish a TF network model of coordinate myelin-associated gene transcription. Notably, the network model captures regulatory DNA elements and TFs known to regulate oligodendrocyte myelin gene transcription and/or oligodendrocyte development, thereby validating our approach. Further, it links to numerous TFs with previously unsuspected roles in CNS myelination and suggests collaborative relationships amongst both known and novel TFs, thus providing deeper insight into the myelin gene transcriptional network

Analysis of the association between CD40 and CD40 ligand polymorphisms and systemic sclerosis.

Introduction: The aim of the present study was to investigate the possible role of CD40 and CD40 ligand (CD40LG) genes in the susceptibility and phenotype expression of systemic sclerosis (SSc). Methods: In total, 2,670 SSc patients and 3,245 healthy individuals from four European populations (Spain, Germany, The Netherlands, and Italy) were included in the study. Five single-nucleotide polymorphisms (SNPs) of CD40 (rs1883832, rs4810485, rs1535045) and CD40LG (rs3092952, rs3092920) were genotyped by using a predesigned TaqMan allele-discrimination assay technology. Meta-analysis was assessed to determine whether an association exists between the genetic variants and SSc or its main clinical subtypes. Results: No evidence of association between CD40 and CD40LG genes variants and susceptibility to SSc was observed. Similarly, no significant statistical differences were observed when SSc patients were stratified by the clinical subtypes, the serologic features, and pulmonary fibrosis. Conclusions: Our results do not suggest an important role of CD40 and CD40LG gene polymorphisms in the susceptibility to or clinical expression of SSc

Rheumatoid arthritis response to treatment across IgG1 allotype - anti-TNF incompatibility: a case-only study.

INTRODUCTION: We have hypothesized that incompatibility between the G1m genotype of the patient and the G1m1 and G1m17 allotypes carried by infliximab (INX) and adalimumab (ADM) could decrease the efficacy of these anti-tumor necrosis factor (anti-TNF) antibodies in the treatment of rheumatoid arthritis (RA). METHODS: The G1m genotypes were analyzed in three collections of patients with RA totaling 1037 subjects. The first, used for discovery, comprised 215 Spanish patients. The second and third were successively used for replication. They included 429 British and Greek patients and 393 Spanish and British patients, respectively. Two outcomes were considered: change in the Disease Activity Score in 28 joint (ΔDAS28) and the European League Against Rheumatism (EULAR) response criteria. RESULTS: An association between less response to INX and incompatibility of the G1m1,17 allotype was found in the discovery collection at 6 months of treatment (P = 0.03). This association was confirmed in the replications (P = 0.02 and 0.08, respectively) leading to a global association (P = 0.001) that involved a mean difference in ΔDAS28 of 0.4 units between compatible and incompatible patients (2.3 ± 1.5 in compatible patients vs. 1.9 ± 1.5 in incompatible patients) and an increase in responders and decrease in non-responders according to the EULAR criteria (P = 0.03). A similar association was suggested for patients treated with ADM in the discovery collection, but it was not supported by replication. CONCLUSIONS: Our results suggest that G1m1,17 allotypes are associated with response to INX and could aid improved therapeutic targeting in RA