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

High-throughput mapping of cell wall glycans to unveil cell wall disassembly, a key process determining strawberry fruit softening

The short shelf life of strawberry fruit is a major limitation that produces important economic losses related to postharvest spoiling. Fruit texture of fleshy fruits is a complex trait but mainly rely on mechanical properties of parenchyma cell walls. Several studies support the relevance of cell wall modifying enzymes on cell wall deconstruction, decreasing cell wall strength and cell to cell adhesion, and ultimately producing the softening of the fruit at macroscopic level. Previous studies on our group showed that transgenic silencing of ripening-specific genes encoding some of these enzymes reduced softening and increased postharvest shelf life in strawberry (Fragaria × ananassa, cv. ‘Chandler’) fruits. In this research, to further investigate the cell wall remodelling process associated to strawberry softening a high-throughput analysis of cell wall composition based on monoclonal antibodies against different polysaccharide epitopes has been performed. To this purpose, cell walls were isolated from non-transgenic fruits at different developmental stages as well as from ripe fruits of selected transgenic lines with genes involved in metabolism of pectins (pectate lyase, polygalacturonase, β-galactosidase, pectin acetil esterase), hemicellulose/cellulose (endo-β-glucanase) or lignin (cinnamyl alcohol dehydrogenase) down-regulated. These transgenic lines showed a large variability in fruit firmness at ripening. Cell walls were fractionated and subjected to a carbohydrate microarray. The results obtained unveiled a common pattern of cell wall composition on those transgenic lines with firmer phenotypes, specially defined by the higher content of pectins on those cell wall fractions more imbricated in the matrix, which can be interpreted as a less degraded cell wall structure.This research was supported by FEDER EU Funds and the Ministerio de Economía y Competitividad of Spain (grant reference AGL2017-86531-C2-1-R). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Obtención de plantas compuestas de olivo mediante transformación con Agrobacterium rhizogenes

La transformación con Agrobacterium rhizogenes ha sido utilizada como herramienta para estudios de genómica funcional en raíces (Collier et al. 2005 Plant Journal 43:449-457; Baranski et al. 2006 Plant Cell Reports 25:190-197). La obtención de plantas compuestas de olivo (sistema radicular transgénico y parte aérea no transgénica), mediante esta técnica, sería de gran ayuda para estudiar la interacción de esta especie con los patógenos de suelo Verticillium dahliae y Rosellinia necatrix. En este trabajo, se presentan las primeras aproximaciones para la transformación de brotes micropropagados de olivo mediante A. rhizogenes. Se han utilizado 2 genotipos procedentes de semilla del cv. Picual, uno con baja capacidad de enraizamiento, P1, y otro, con alta capacidad, P138, y dos cepas de A. rhizogenes: A4, que contiene el plásmido silvestre Ri, y K599, con el plásmido binario pKGWFS7.0-35SP, que incluye el gen marcador gfp. En el caso del genotipo P1, en la fase de co-cultivo con la bacteria, se añadieron al medio 3 mg/l AIB, para facilitar la formación de raíces. En el genotipo P138, se obtuvo un 100% de enraizamiento tanto en el tratamiento control como en el de brotes infectados con la cepa A4; sin embargo, aquéllos inoculados con la cepa K599 sólo alcanzaron un 70% de enraizamiento. Asimismo, se observó que el 61% de las raíces obtenidas tras la infección con K599 mostraron fluorescencia verde bajo el microscopio confocal. En el genotipo P1, el 60% de las plantas control formaron raíces, frente al 10% de plantas infectadas con A4 y ninguna con la cepa K599. La naturaleza transgénica de las raíces obtenidas tras la infección con A4, en ambos genotipos, se evaluará mediante amplificación por PCR del gen RolB.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Transformación de olivo con el gen AtNPR1 para inducir tolerancia a patógenos fúngicos.

El gen NPR1 codifica un componente esencial de la respuesta SAR mediada por ácido salicílico (AS). Tras la infección por el patógeno, la acumulación de AS libera los monómeros NPR1 en el citoplasma, los cuáles son translocados al núcleo activando la expresión de genes relacionados con la patogénesis (PR). La sobreexpresión del gen NPR1 de Arabidopsis thaliana ha incrementado la resistencia a hongos, bacterias y virus, en distintas especies. El objetivo de esta investigación fue sobreexpresar este gen en olivo con objeto de evaluar su efecto en la tolerancia a dos hongos de suelo, el hemibiotrofo Verticillium dahliae (Vd), una de las mayores amenazas del cultivo y el necrotrofo Rosellinia necatrix, un patógeno emergente en nuevas plantaciones. Se obtuvieron 3 líneas transgénicas, a partir de una línea embriogénica derivada de semilla del cv. Picual. Las líneas mostraron diferencias en el nivel de expresión del transgen en hoja, aunque estas diferencias no afectaron a los niveles de actividad endoquitinasa basal, similar a la de plantas control. La respuesta a Vd varió con el patotipo; así, todas las plantas murieron 50 días tras su inoculación con la cepa defoliante (D) V-138. Por otra parte, la respuesta a patotipos no defoliantes (ND) también fue variable, en función de la raza; tras la inoculación con la cepa V1242 (ND, raza 2), los síntomas aparecieron transcurridos 44-55 días, siendo la línea NPR1-780, con mayor expresión del transgen, la que mostró menor índice de severidad de la enfermedad. Esta línea también mostró un comportamiento superior al control tras la inoculación con la cepa V1558 (ND, raza 1), aunque las diferencias no fueron tan acusadas. En la respuesta a R. necatrix, las líneas transgénicas mostraron un ligero retraso en el desarrollo de la enfermedad con valores AUDPC entre 7-15% inferiores al control.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Global economic burden of unmet surgical need for appendicitis

Background There is a substantial gap in provision of adequate surgical care in many low- and middle-income countries. This study aimed to identify the economic burden of unmet surgical need for the common condition of appendicitis. Methods Data on the incidence of appendicitis from 170 countries and two different approaches were used to estimate numbers of patients who do not receive surgery: as a fixed proportion of the total unmet surgical need per country (approach 1); and based on country income status (approach 2). Indirect costs with current levels of access and local quality, and those if quality were at the standards of high-income countries, were estimated. A human capital approach was applied, focusing on the economic burden resulting from premature death and absenteeism. Results Excess mortality was 4185 per 100 000 cases of appendicitis using approach 1 and 3448 per 100 000 using approach 2. The economic burden of continuing current levels of access and local quality was US $92 492 million using approach 1 and$73 141 million using approach 2. The economic burden of not providing surgical care to the standards of high-income countries was $95 004 million using approach 1 and$75 666 million using approach 2. The largest share of these costs resulted from premature death (97.7 per cent) and lack of access (97.0 per cent) in contrast to lack of quality. Conclusion For a comparatively non-complex emergency condition such as appendicitis, increasing access to care should be prioritized. Although improving quality of care should not be neglected, increasing provision of care at current standards could reduce societal costs substantially

Global economic burden of unmet surgical need for appendicitis

Background There is a substantial gap in provision of adequate surgical care in many low- and middle-income countries. This study aimed to identify the economic burden of unmet surgical need for the common condition of appendicitis. Methods Data on the incidence of appendicitis from 170 countries and two different approaches were used to estimate numbers of patients who do not receive surgery: as a fixed proportion of the total unmet surgical need per country (approach 1); and based on country income status (approach 2). Indirect costs with current levels of access and local quality, and those if quality were at the standards of high-income countries, were estimated. A human capital approach was applied, focusing on the economic burden resulting from premature death and absenteeism. Results Excess mortality was 4185 per 100 000 cases of appendicitis using approach 1 and 3448 per 100 000 using approach 2. The economic burden of continuing current levels of access and local quality was US $92 492 million using approach 1 and$73 141 million using approach 2. The economic burden of not providing surgical care to the standards of high-income countries was $95 004 million using approach 1 and$75 666 million using approach 2. The largest share of these costs resulted from premature death (97.7 per cent) and lack of access (97.0 per cent) in contrast to lack of quality. Conclusion For a comparatively non-complex emergency condition such as appendicitis, increasing access to care should be prioritized. Although improving quality of care should not be neglected, increasing provision of care at current standards could reduce societal costs substantially