Identification and Expression Analysis of GRAS Transcription Factor Genes Involved in the Control of Arbuscular Mycorrhizal Development in Tomato

The formation and functioning of arbuscular mycorrhizal (AM) symbiosis are complex and tightly regulated processes. Transcriptional regulation mechanisms have been reported to mediate gene expression changes closely associated with arbuscule formation, where nutrients move between the plant and fungus. Numerous genes encoding transcription factors (TFs), with those belonging to the GRAS family being of particular importance, are induced upon mycorrhization. In this study, a screening for candidate transcription factor genes differentially regulated in AM tomato roots showed that more than 30% of known GRAS tomato genes are upregulated upon mycorrhization. Some AM-upregulated GRAS genes were identified as encoding for transcription factors which are putative orthologs of previously identified regulators of mycorrhization in other plant species. The symbiotic role played by other newly identified AM-upregulated GRAS genes remains unknown. Preliminary results on the involvement of tomato SlGRAS18, SlGRAS38, and SlGRAS43 from the SCL3, SCL32, and SCR clades, respectively, in mycorrhization are presented. All three showed high transcript levels in the late stages of mycorrhization, and the analysis of promoter activity demonstrated that SlGRAS18 and SlGRAS43 are significantly induced in cells containing arbuscules. When SlGRAS18 and SlGRAS38 genes were silenced using RNA interference in hairy root composite tomato plants, a delay in mycorrhizal infection was observed, while an increase in mycorrhizal colonization was observed in SlGRAS43 RNAi roots. The possible mode of action of these TFs during mycorrhization is discussed, with a particular emphasis on the potential involvement of the SHR/SCR/SCL3 module of GRAS TFs in the regulation of gibberellin signaling during mycorrhization, which is analogous to other plant developmental processes

Gibberellin–Abscisic Acid Balances during Arbuscular Mycorrhiza Formation in Tomato

Plant hormones have become appropriate candidates for driving functional plant mycorrhization programs, including the processes that regulate the formation of arbuscules in arbuscular mycorrhizal (AM) symbiosis. Here, we examine the role played by ABA/GA interactions regulating the formation of AM in tomato. We report differences in ABA and GA metabolism between control and mycorrhizal roots. Active synthesis and catabolism of ABA occur in AM roots. GAs level increases as a consequence of a symbiosis-induced mechanism that requires functional arbuscules which in turn is dependent on a functional ABA pathway. A negative interaction in their metabolism has been demonstrated. ABA attenuates GA-biosynthetic and increases GA-catabolic gene expression leading to a reduction in bioactive GAs. Vice versa, GA activated ABA catabolism mainly in mycorrhizal roots. The negative impact of GA3 on arbuscule abundance in wild-type plants is partially offset by treatment with ABA and the application of a GA biosynthesis inhibitor rescued the arbuscule abundance in the ABA-deficient sitiens mutant. These findings, coupled with the evidence that ABA application leads to reduce bioactive GA1, support the hypothesis that ABA could act modifying bioactive GA level to regulate AM. Taken together, our results suggest that these hormones perform essential functions and antagonize each other by oppositely regulating AM formation in tomato roots.This study was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (CICYT) and Fondos Europeos de Desarrollo Regional (FEDER) through the Ministerio de Economía y Competitividad in Spain (AGL2008-00742; AGL2011-25930) as well as the Junta de Andalucía (Research Group BIO 260). JM-R was supported by a research fellowship from the FPU-MICINN program. The work on hormone measurements was funded by a Grant Agency of the Czech Republic (grant no. 14-34792S) and by the Ministry of Education, Youth and Sports of the Czech Republic through the National Program for Sustainability I (Nr. LO1204).Peer reviewedPeer Reviewe

Arbuscular Mycorrhizal Fungi. Methods and Protocols.

Ferrol, N.; Lanfranco, L. (Eds.) 2020 Arbuscular Mycorrhizal Fungi. Methods and Protocols. Vol 2146. pp. 257. ISBN 978 1 0716-0602-

Análisis y carcterización funcional de dos genes de tomate implicados en el proceso de micorrización

Los hongos micorrícico arbusculares (MA) son hongos microscópicos que viven en simbiosis con las raíces de la mayoría de las plantas. Entre los beneficios que aporta la formación de MA para las plantas caben destacar desde una mejor nutrición, hasta una mayor defensa ante estreses tanto bióticos como abióticos. Como consecuencia de la larga coevolución en simbiosis, dado que posiblemente esta asociación se formó desde el inicio de la adaptación de las plantas al medio terrestre, los mecanismos de regulación y control del desarrollo de la simbiosis han ido evolucionando a la par que la propia evolución de las plantas. Así, el desarrollo y establecimiento de la simbiosis MA están regulados muy finamente, tanto por condicionantes medioambientales incluyendo aspectos nutricionales, como por mecanismos de diálogo y señalización molecular, así como por una compleja red de factores y cofactores transcripcionales. La comprensión del proceso simbiótico y de los elementos clave en su regulación es esencial a la hora de averiguar los mecanismos por los que la planta es beneficiada en su interacción con los hongos MA, y desarrollar estrategias que permitan una mejor gestión de dicha asociación y de su aprovechamiento como alternativa al uso de fertilizantes químicos y plaguicidas. Nuestro equipo de trabajo está focalizado en desgranar los procesos moleculares que ocurren en la simbiosis MA, tomando como modelo la planta de tomate, ya que, además de suponer uno de los cultivos más importantes a escala mundial, es una planta muy adecuada para el estudio de la micorrización, entre otras razones por ser una planta modelo de estudios fisiológicos y genéticos. En este trabajo se ha realizado la caracterización y el análisis funcional de dos genes de tomate inducidos por micorrización, y denominados tsb y SlDLK2, candidatos a jugar un papel relevante en la simbiosis MA. Concretamente, uno de ellos, el tsb, se eligió por ser posiblemente importante para la reorganización del citosqueleto de las células de la raíz durante la micorrización, y el segundo de ellos, SlDLK2, por codificar para una proteína de la familia α/β-hidrolasa que podría ser un posible receptor hormonal relevante en la señalización del proceso micorrícico. Como primer paso y para evaluar de manera rápida y eficaz la funcionalidad de estos genes en la simbiosis MA, se ha implementado un método para la obtención de plantas “compuestas” de tomate, es decir, plantas con un sistema radical transformado pero con la parte aérea silvestre, generadas mediante transformación por Agrobacterium rhizogenes. Se ha conseguido un protocolo optimizado para la transformación y generación de plántulas compuestas para experimentos de micorrización con una gran tasa de éxito, y que además permite la identificación inequívoca de aquellas raíces transformadas desde el inicio hasta el momento de su cosecha mediante una selección visual, usando el fluoróforo DsRed, y sin necesidad de antibióticos. Se dispone de una colección de vectores binarios de transformación, tanto para experimentos de silenciamiento, sobrexpresión, o análisis de la actividad de promotores, que nos ha permitido realizar un análisis funcional de los genes objeto de estudio.Arbuscular mycorrhizal (AM) fungi are microscopic fungi that live in symbiosis with the roots of most plants. Among the benefits provided to the plants by this interaction we must highlight a better nutrition and a higher resistance to biotic and abiotic stresses. As both symbionts have coevolved for a long time, probably from the beginning of plant adaptation to the land environment, it is expected that the mechanisms for regulation of mycorrhizal development have also coevolved with plants. In this manner, the development and establishment of the AM symbiosis are fine-tuned regulated by environmental factors including nutritional conditions, as well as by molecular dialog and signaling mechanisms, and by a complex network of transcription factors and cofactors. The understanding of the symbiotic process and the key components for its regulation is essential in order to elucidate by what mechanisms the plant is benefited from the interaction with the AM fungi, and to develop strategies to improve the management of the mycorrhizal associations in order to use them as an alternative to the chemical fertilizers and pesticides. Our team work is focused in the analysis of the molecular processes underlying the AM symbiosis using the tomato plant, which constitutes a model plant for physiological and genetic studies and, in addition, it is a worldwide important crop. In this work, two AM-induced genes from tomato, tsb and SlDLK2, are subjected to their functional characterization and analysis, due to their possible role in the mycorrhization process. Particularly, tsb was chosen because of its possible function in cytoskeleton rearrangements during mycorrhization; while SlDLK2, encoding for a protein from the α,β-hydrolase family, because of its possible role as a relevant hormonal receptor involved in signaling during the mycorrhizal process. First of all, in order to quickly and easily screen the functionality of these genes during mycorrhization, a method for obtaining composite tomato plants using Agrobacterium rhizogenes-mediated transformation was implemented. The resulting plants were composed of a transformed root system and a wild type aerial part. An optimized protocol was successfully set up for the transformation and generation of composite seedlings for mycorrhizal studies, with high success rates and that allows to undoubtedly identifying and selecting tomato cotransformed roots from the beginning until the harvesting time through visual selection using the fluorophore marker DsRed, without the requirement of antibiotics. Three different binary vectors were tested for silencing, overexpressing and promoter-GUS expression studies, that have allowed us to successfully perform a functional analysis of the candidate genes.Tesis Univ. Granada.Programa Oficial de Doctorado en Biología Fundamental y de Sistemasfinanciada por el proyecto de investigación AGL2011-25930 concedido por el Ministerio de Economía y Competitividad, así como con las siguientes ayudas asociadas al mismo: Beca de Formación de Personal Investigador (Ref. ayuda FPI: : BES-2012-052057) desde el 1 de marzo de 2013 al 28 de febrero del 2017 (4 años). - Estancia Breve en el Structural Biology Laboratory, Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST) en Nara, Japón, bajo la supervisión del Dr. Toshio Hakoshima (Ref. EEBB-I-14-07870) del 21 de agosto de 2014 hasta 18 de diciembre de 2014 (4 meses). - Estancia Breve la Plant Biology Division de la Samuel Roberts Noble Foundation en Ardmore, Oklahoma, EEUU, bajo la supervisión de Michael Udvardi (Ref. EEBB-I-16- 11379), desde el día 31 de marzo de 2016 hasta el día 28 de julio de 2016 (4 meses)

Tolerancia a toxicidad por cesio inducida por microorganismos beneficiosos del suelo

Máster Oficial Biología Agraria y Acuicultura.Peer reviewe

Analysis and functional characterization of two tomato genes involved in the mycorrhization process

Arbuscular mycorrhizal (AM) fungi are microscopic fungi that live in symbiosis with the roots of most plants. Among the benefits provided to the plants by this interaction we must highlight a better nutrition and a higher resistance to biotic and abiotic stresses. As both symbionts have coevolved for a long time, probably from the beginning of plant adaptation to the land environment, it is expected that the mechanisms for regulation of mycorrhizal development have also coevolved with plants. In this manner, the development and establishment of the AM symbiosis are fine-tuned regulated by environmental factors including nutritional conditions, as well as by molecular dialog and signalling mechanisms, and by a complex network of transcription factors and cofactors. The understanding of the symbiotic process and the key components for its regulation is essential in order to elucidate by what mechanisms the plant is benefited from the interaction with the AM fungi, and to develop strategies to improve the management of the mycorrhizal associations in order to use them as an alternative to the chemical fertilizers and pesticides. Our team work is focused in the analysis of the molecular processes underlying the AM symbiosis using the tomato plant, which constitutes a model plant for physiological and genetic studies and, in addition, it is a worldwide important crop. In this work, two AM-induced genes from tomato, tsb and SlDLK2, are subjected to their functional characterization and analysis, due to their possible role in the mycorrhization process. Particularly, tsb was chosen because of its possible function in cytoskeleton rearrangements during mycorrhization; while SlDLK2, encoding for a protein from the α,β-hydrolase family, because of its possible role as a relevant hormonal receptor involved in signalling during the mycorrhizal processFirst of all, in order to quickly and easily screen the functionality of these genes during mycorrhization, a method for obtaining composite tomato plants using Agrobacterium rhizogenes-mediated transformation was implemented. The resulting plants were composed of a transformed root system and a wild type aerial part. An optimized protocol was successfully set up for the transformation and generation of composite seedlings for mycorrhizal studies, with high success rates and that allows to undoubtedly identifying and selecting tomato cotransformed roots from the beginning until the harvesting time through visual selection using the fluorophore marker DsRed, without the requirement of antibiotics. Three different binary vectors were tested for silencing, overexpressing and promoter-GUS expression studies, that have allowed us to successfully perform a functional analysis of the candidate genes. This method, which have been recently published (Ho-Plágaro et al. 2018), is presented in Chapter 1 of this doctoral dissertation. The research carried out regarding the tsb gene is shown in Chapter 2. Tsb encodes a putative MAP (Microtubule Associated Protein) that belongs to a family of MAPs unique from Solanaceae plants. Some of the members of this MAP family have been previously described as pollen specific and with a function in cytoskeleton rearrangements and the formation of the pollen tube (Zhao et al. 2006; Huang et al. 2007; Liu et al. 2013). Here, through the analysis of tsb-silenced and tsb-overexpressing composite plants, a decreased expression was observed for genes related to arbuscule functionality in the AM tsb-silenced roots compared to the wildtype AM roots. In agreement with this result, tsb-overexpressing roots showed an induction all genes used as markers of arbuscule and fungal activities. Moreover, the cortical microtubule array seems to be perturbed in tsb-overexpressing roots. Microtubules and microfilaments are not only known to determine the structure of the cell and its organelles, but also to be involved in the intracellular transportation mechanisms, because they act as “tracks” for the clathrin vesicle trafficking, themain means of transport of the cell, consequently determining the endocytosis and exocytotic pathways. The results obtained in this work point to a role of tsb in restructuring the microtubule cytoskeleton in the cortical cells of the roots during mycorrhization and indicate that the action of tsb is related to the specific exocytosis processes for the delivery of proteins and compounds to the periarbuscular membrane and the symbiotic interface to allow arbuscule functionality and activity. The second gene studied in this work is a gene that encodes for a protein belonging to the α.β-hydrolase family, particularly to the DLK2 group. For this reason, it was named as SlDLK2. Curiously, the DLK2 protein group is phylogenetically very close to the strigolactone receptors (D14) and the karrikin receptors (KAI2). Strigolactones are plant hormones that play an important role in presymbiotic signalling during mycorrhization. In addition, the karrikin receptor KAI2 has been recently shown to be essential for the AM symbiosis (Gutjahr et al. 2015). However, DLK2 α.β-hydrolases are little characterized, and no relation has yet been found between DLK2 and the mycorrhization process. In Chapter 3 is presented our research work performed to elucidate if SlDLK2 is a signalling receptor important during MA symbiosis. Composite plants with transformed roots were obtained, and we observed that SlDLK2 silencing showed a significant increase of AM fungal development in the host roots. However, SlDLK2 overexpression gives place to an anomalous arbuscule development, with lack of branching, what suggest that SlDLK2 has a clear role in the development and branching of the arbuscules. Additionally, SlDLK2 has a conserved catalytic triad, responsible of the hydrolytic activity described for the strigolactone and karrikin receptors, and here we have actually probed that SlDLK2 retains some ability to interact and hydrolyse synthetic strigolactones. Although our research have not allowed us to elucidate the chemical structure of the specific ligand for SlDLK2, theresults obtained so far point to the C13 α-ionols derivatives as the possible ligands. Finally, in Chapter 4, in order to clarify the possible signalling role of the SlDLK2 receptor during mycorrhization, transcriptomic alterations and the predicted changes in metabolic pathways in SlDLK2-silenced plants are shown, and the causes and consequences of these changes are discussed. Biggest changes observed were associated to a nutrient starvation and induced defence signature in the SlDLK2-RNAi mycorrhizal roots, probably as a consequence of the higher AM development and/or activity in these plants. However, examination of many other differentially expressed genes supports the idea of SlDLK2 as a negative regulator of mycorrhization, and that SlDLK2 might be important to activate GA signalling, restrict hexose production and carbohydrate supply to the AM fungus, and induce a number of resistance mechanisms, in order to control AM fungal development.(A)N

Multifarious and interactive roles of GRAS transcription factors during arbuscular mycorrhiza development

Arbuscular mycorrhiza (AM) is a mutualistic symbiotic interaction between plant roots and AM fungi (AMF). This interaction is highly beneficial for plant growth, development and fitness, which has made AM symbiosis the focus of basic and applied research aimed at increasing plant productivity through sustainable agricultural practices. The creation of AM requires host root cells to undergo significant structural and functional modifications. Numerous studies of mycorrhizal plants have shown that extensive transcriptional changes are induced in the host during all stages of colonization. Advances have recently been made in identifying several plant transcription factors (TFs) that play a pivotal role in the transcriptional regulation of AM development, particularly those belonging to the GRAS TF family. There is now sufficient experimental evidence to suggest that GRAS TFs are capable to establish intra and interspecific interactions, forming a transcriptional regulatory complex that controls essential processes in the AM symbiosis. In this minireview, we discuss the integrative role of GRAS TFs in the regulation of the complex genetic re-programming determining AM symbiotic interactions. Particularly, research being done shows the relevance of GRAS TFs in the morphological and developmental changes required for the formation and turnover of arbuscules, the fungal structures where the bidirectional nutrient translocation occurs.This study was supported by grant (PID2020-115336GB-I00) funded by Spanish MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe,” by the “European Union”

Molecular regulation of arbuscular mycorrhizal symbiosis

Plant-microorganism interactions at the rhizosphere level have a major impact on plant growth and plant tolerance and/or resistance to biotic and abiotic stresses. Of particular importance for forestry and agricultural systems is the cooperative and mutualistic interaction between plant roots and arbuscular mycorrhizal (AM) fungi from the phylum Glomeromycotina, since about 80% of terrestrial plant species can form AM symbiosis. The interaction is tightly regulated by both partners at the cellular, molecular and genetic levels, and it is highly dependent on environmental and biological variables. Recent studies have shown how fungal signals and their corresponding host plant receptor-mediated signalling regulate AM symbiosis. Host-generated symbiotic responses have been characterized and the molecular mechanisms enabling the regulation of fungal colonization and symbiosis functionality have been investigated. This review summarizes these and other recent relevant findings focusing on the molecular players and the signalling that regulate AM symbiosis. Future progress and knowledge about the underlying mechanisms for AM symbiosis regulation will be useful to facilitate agro-biotechnological procedures to improve AM colonization and/or efficiency.This study was supported by grant (PID2020-115336GB-I00) funded by Spanish MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”, by the “European Union”

Microtubule cytoskeleton and mycorrhizal roots

For the establishment of the Arbuscular Mycorrhiza (AM) symbiosis it is essential that epidermis and cortical cells from plant roots suffer a strong reorganization to allow the penetration of intracellular fungal hyphae. In the same manner, the new formation of a periarbuscular membrane and a symbiotic interface with specific compositions are required for a functional symbiosis. It is believed that the cytoskeleton of the plant host plays an essential role in these processes, particularly the microtubule (MT) cytoskeleton, as huge modifications have been observed in the MT array of root cells accompanying the establishment of the AM symbiosis. Recent research has established a link between microtubule rearrangements and arbuscule functioning. However, further research is required to elucidate the specific functions of MT cytoskeleton along the different stages of the arbuscule life cycle and to unravel the signals triggering these changes.This study was supported by grant [PID2020-115336GB-100] funded by Spanish MCIN/AEI/ 10.13039/501100011033 and by “ERDF A way of making Europe”, by the “European Union

Functional analysis of plant genes related to arbuscular mycorrhiza symbiosis using Agrobacterium rhizogenes-mediated root transformation and hairy root production.

Srivastava V., Mehrotra S., Mishra S. (eds) 2020 Hairy Root Cultures Based Applications. Rhizosphere Biology. pp. 237. ISBN: 978-981-15-4054-7Arbuscular Mycorrhizal symbiosis is a mutualistic endosymbiosis widely distributed in the plant kingdom which has a significant impact on plant growth and health. Agrobacterium rhizogenes-mediated root transformation and composite plant generation have been described as a rapid method to assess gene functions in roots without the need for stable transformation plant production. We describe an optimized protocol for composite tomato plant obtaining achieved through A. rhizogenes-mediated transformation, and we also highlight key differences with other protocols that should be taken into account to adjust this method to the transformation of other plant species. This protocol has been adopted as a useful tool for localizing the promoter expression of genes putatively associated with mycorrhization or for functional analyses in mycorrhizal studies by reverse genetics.This work was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (CICYT) and Fondos Europeos de Desarrollo Regional (FEDER) through the Ministerio de Economía, industria y Competitividad in Spain (AGL2014-52298-P, AGL2017-83871-P)