Heterologous expression of AtNPR1 gene in olive for increasing fungal tolerance

The NPR1 gene encodes a key component of SAR signaling mediated by salicylic acid (SA). After a pathogen infection, the accumulation of SA releases NPR1 monomers in the cytosol that are translocated to the nucleus, activating the expression of pathogenesis-related (PR) genes. Overexpression of NPR1 has conferred resistance to fungal, viral and bacterial pathogens in several plant species. The aim of this research was to generate transgenic olive plants expressing the gene AtNPR1 from Arabidopsis thaliana to obtain material resistant to fungal pathogens. Three transgenic lines expressing AtNPR1 gene under the control of the constitutive promoter CaMV35S were obtained following the protocol of Torreblanca et al. (2010), using an embryogenic line derived from a seed of cv. Picual. Level of AtNPR1 expression in transgenic calli varied greatly among the different lines, being higher in the line NPR1-780. The elicitation of embryogenic calli in liquid medium with AS did not increase endochitinase activity, a PR protein. However, jasmonic acid induced a transient increase in chitinase activity after 24 h of treatment in all the lines, being the increment higher in transgenic NPR1 than in control. After maturation and germination of transgenic somatic embryos, plants were micropropagated and acclimated to ex vitro conditions. The expression of AtNPR1 did not alter the growth of transgenic plants neither in vitro nor in the greenhouse. Experiments are in progress to determine the resistance of transgenic AtNPR1 plants to V. dalihae and R. necatrix.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Research projects: Plan Nacional AGL2014-52518-C2-1-R; AGL2017-83368-C2-1-R and Junta de Andalucía P11-AGR799

Screening for candidate bacterial biocontrol agents against soilborne fungal plant pathogens

Over the years, many bacterial isolates have been evaluated as potential biocontrol agents against soilborne fungal phytopathogens. However, few of them were ultimately successful after evaluation in field trials. One of the major reasons for this failure is the lack of appropriate screening procedures to select the most suitable microorganisms for disease control in diverse soil environments. For this reason, the study of bacterial screening has a future that is characterised by many technical and conceptual challenges. In this review, we summarise and discuss the convenience of use of the main screening methods currently applied to select bacterial candidates for biocontrol of fungal and oomycete soilborne phytopathogens. Also, a comparative case study of the application of different screening methods applied to an experimental pathosystem is shown, revealing the success of bacterial candidates selected by different strategies for biocontrol of the phytopathogenic fungus Rosellinia necatrix in avocado plants. Screening for antagonism against this fungal pathogen, one of the more straightforward methods used for the selection of bacterial biocontrol agents, was proven to be a valid strategy for this experimental system

Agrobacterium-mediated transformation of olive (Olea europaea L.) with an antifungal protein from Aspergillus giganteus.

Broad-spectrum resistance to pests and diseases is difficult to obtain through classical breeding programs, hence, this is a targeted trait for accelerating the development of major olive cultivars using plant trans- formation technologies. Olive Verticillium wilt, caused by Verticillium dahliae, is considered to be an important constraint for cultivation of olive trees (López-Escudero and Mercado-Blanco 2010). Different transgenic approaches have been proposed to engineer plants for resistance to fungal diseases, including production of antifungal proteins (Gurr and Rushton 2005). Regarding this approach, among different anti- fungal compounds, the antifungal protein (AFP) from Aspergillus giganteus can be considered a promising candidate for practical applications in crop protection (Meyer 2007). AFP is a defensin-like protein that belongs to a group of small-sized secretory proteins rich in cysteine residues. The protein possesses in vitro antifungal activity inhibiting the growth of several fungal pathogens. Previous work has already shown that afp gene can be expressed in transgenic rice plants inducing resistance to the fungus Magnaporthe grisea and indicating the usefulness of such approach for protection against rice blast. (Coca et al. 2004). In this work, transgenic olive plants were generated by Agrobacterium-mediated transformation as des- cribed by Torreblanca et al. (2010). The AGL-1 strain containing the pBIN61-afp binary vector was used. This plasmid contains the nptII gene for paromomycin selection and a chemically synthesized codon-op- timized afp gene under the control of the 35S CaMV promoter. Globular somatic embryos derived from a mature seed of cultivar `Picual ́ were transformed obtaining an average success rate around 2%. Plants were regenerated from six independent lines and transgenic nature was confirmed by PCR studying nptII and afp insertion. With the aim of studying whether the afp gene can be used to induce resistance against fungal diseases in olive, susceptibility to the fungal pathogens Rosellinia necatrix and Verticilium dahliae will be evaluated. In addition, the inhibitory effect of proteins extracts from transgenic leaves on the in vitro growth of these fungal pathogens will also be examined.Universidad de Málaga. Campus de Excelencia Internacional Andalucia Tec

Response to fungal exudates of the rhizosphere isolate Pseudomonas sp. UMAF110 involves a GGDEF/EAL domain-containing protein

Pseudomonas sp. UMAF110, isolated from rhizosphere soil in Spain, display in vitro antagonism towards the pythopathogenic fungus Rosellinia necatrix and is able grow in fungal exudates (BM-RE medium). A transposon mutant library of this strain was constructed and several mutants were selected by their reduced competitiveness in BM-RE medium. Pseudomonas sp. UMAF110-G3, which contains the transposon into a gene encoding a putative REC/PAS/GGDEF/EAL protein, was selected for further characterization. Blastn searches using the sequence of the gene interrupted by the transposon in UMAF110-G3, here called cmpA (c-di-GMP Metabolizing Protein), yielded a single positive hit (98% cover, 78% identity) with a gene from a terpene-degrading Pseudomonas sp. strain isolated from soil. Context analysis of the cmpA gene in Pseudomonas sp. UMAF110 showed that this gene is located downstream from several genes involved in flagellar motility/chemotaxis. RT-PCR experiments further confirmed that cmpA form a transcriptional unit with the che gene cluster. Expression analysis of cmpA by qRT-PCR clearly showed upregulation of this gene after transfer of Pseudomonas sp. UMAF110 cells to BM-RE medium, suggesting a role for this operon in response to fungal exudates. Deletion of cmpA in Pseudomonas sp. UMAF110 did not affect the ability of the strain to form biofilms under the conditions tested. However, overexpression of wild type CmpA in Pseudomonas putida KT2440 negatively regulated biofilm formation in this strain. Together, these results suggest that CmpA could be involved in signal transduction pathways regulating flagellar motility/chemotaxis in response to fungal exudates.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Caracterización de variantes somaclonales de olivo obtenidos tras la exposición al filtrado crudo del hongo Rosellinia necatrix.

Líneas embriogénicas de olivo tolerantes al filtrado crudo (FC) del hongo Rosellinia necatrix, un patógeno muy extendido por el sur de España y que afecta al aguacate y al olivo, fueron previamente obtenidas (Palomo-Ríos et al. 2017). Dos de estas líneas fueron seleccionadas para la regeneración de plantas, R2-L3 y R2-L4, ambas tolerantes al 60% (v/v) del FC del hongo. En este trabajo se ha llevado a cabo una caracterización del crecimiento de estas plantas y de su comportamiento tras la inoculación con R. necatrix.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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

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

Deciphering molecular mechanisms underlaying biological control of the fungal pathogen Rosellinia necatrix by Pseudomonas pseudoalcaligenes AVO110

Comunicación en póster.Pseudomonas pseudoalcaligenes AVO110 was selected as an efficient avocado root tip colonizer, displaying antagonism towards Rosellinia necatrix, the causal agent of avocado white root rot. The most likely biocontrol mechanisms used by this strain is competition for niches and nutrients. In fact, AVO110 is able to colonise competitively the surface of fungal hyphae, using fungal exudates. In a previous study, we used signature-tagged mutagenesis (STM) to screen a library of AVO110 transposon mutants for their ability to grow in Rosellinia exudates (BM-RE medium). A pool of 26 growth-attenuated mutants (GAM) was identified. In this work, we used the recently obtained draft genome sequence of AVO110 to analyse the genetic context of the genes affected by the transposon in seven GAM mutants. Among the traits affected, we have identified homologs of an exodeoxyribonuclease, a GGDEF/EAL/PAS protein, a transcriptional regulator, a histidine kinase, a putative enterotoxin, a peptidase and a putative small RNA. Phenotypic characteristics such as biofilm formation, persistence and colonisation of avocado roots and colonisation of fungal hyphae were studied. Mutants affected in the transcriptional regulator and the GGDEF/EAL/PAS proteins were altered in all features analysed when compared to the wild type strain. The remaining mutants presented variations in persistence and colonisation of fungal and root surfaces, except for that affected in a putative enterotoxin, which was only altered in fungal colonisation. Finally, quantitative RT-PCR experiments were performed in BM-RE to evaluate the expression dynamics of these genes in the presence of the fungal metabolites along the time. Results showed no evident correlation between gene expression and phenotypic behaviour.Universidad de Málaga, Campus de Excelencia Internacional Andalucía Tech. AGL2011-30354-CO20

Transcriptome analysis of Neofusicoccum luteum during avocado branch and fruit infection

The most important aerial diseases affecting avocado orchards on the Andalusian coast (Spain) are caused by species of fungi belonging to the Botryosphaeriaceae family, one of the most common being Neofusicoccum luteum. The symptoms produced by this fungus are branch dieback and fruit rot. Currently, it is essential to develop strategies to control this disease. In this line, knowledge of the infection mechanisms of the pathogen is considered an essential objective. Therefore, a transcriptomic study was carried out by RNAseq of N. luteum growing on avocado branch and fruit in comparison with its in vitro growth in PDA medium. Transcriptome analysis revealed a total of 903 and 1271 genes significantly downregulated (- 2 > fold change > 2) during growth on branch and fruit compared to growth on PDA, respectively. Among the genes overexpressed in the N. luteum/branch/fruit interaction, genes related to mycotoxin production, wall degradation, detoxification of harmful compounds, protein degradation and candidate effector proteins were identified, three of which showed 100% probability (Effector P3) and apoplastic localization. The analysis of N. luteum transcriptome during the infection process will be a very useful tool to better understand the biology and virulence of this emerging pathogen and will help in the development of strategies for its controlUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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