Optical microscopy on agrobacterium-mediated transient transformated arabidopsis nahg plants

Agrobacterium tumefaciens-mediated transient transformation has demonstrated to be an invaluable tool in plant cell biology. However, low efficiency and inconsistency of this method in Arabidopsis has forced the implementation of Nicotiana benthamiana as a surrogate system, limiting applicability. Previous results suggested that hormone-mediated defence responses against bacteria might be responsible for the low efficiency of Agrobacterium-mediated transient transformation in Arabidopsis. In this work, we evaluate the efficiency of Agrobacterium-mediated transient transformation in Arabidopsis genotypes affected in JA perception or signalling (coi1, jin1), or with low SA or JA content (sid2, NahG, aos). We demonstrate that expression of the NahG transgene dramatically improves this process. Arabidopsis NahG plants can be efficiently used for transient expression-based optical microscopy assays routinely performed in N. benthamiana, such as determination of subcellular localization of GFP-fused proteins or analysis of protein-protein interactions by Bimolecular Fluorescent Complementation. Considering the wide-spread use of Agrobacterium-mediated transient transformation, this system can enormously facilitate research in the model plant Arabidopsis, allowing for an efficient use of the full potential of the numerous tools and resources currently available to the community.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Arabidopsis NahG Plants as a Suitable and Efficient System for Transient Expression using Agrobacterium tumefaciens

aken together, our results show that expression of the NahG transgene in Arabidopsis dramatically enhances the efficiency of Agrobacterium-mediated transformation in rosette leaves, enabling the routine use of this technique in transient assays. This method can be successfully applied in SA-depleted Arabidopsis plants for transient expression-based functional assays routinely done in N. benthamiana, which would facilitate the use of the plethora of tools and knowledge generated in Arabidopsis. The use of this assay does not require complex inoculation media, supplements, or specific growth conditions, and can be used with different Agrobacterium strains. A high level of expression has also been previously achieved in Arabidopsis transgenic plants containing an inducible cassette to express the bacterial effector AvrPto from a DEX-inducible promoter (Tsuda et al., 2012). However, the assay reported here overcomes the use of DEX treatment and the putative multi-side effects potentially derived from the expression of a bacterial effector that interferes with multiple receptor-like kinases and whose constitutive expression is most likely detrimental for the plant.This work was supported by the Spanish Ministerio de Ciencia y Tecnología ( AGL2013-48913-C2 and BIO2014-55380R ); and the Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, and the 100 Talent Program from the Chinese Academy of Sciences (RL-D). T.R.-D. is funded by a postdoctoral President's International Fellowship Initiative (PIFI) number 2016PB042 from the Chinese Academy of Sciences

The transcriptional regulator JAZ8 interacts with the C2 protein from geminiviruses and limits the geminiviral infection in Arabidopsis

Jasmonates (JAs) are phytohormones that finely regulate critical biological processes, including plant development and defense. JASMONATE ZIM-DOMAIN (JAZ) proteins are crucial transcriptional regulators that keep JA-responsive genes in a repressed state. In the presence of JA-Ile, JAZ repressors are ubiquitinated and targeted for degradation by the ubiquitin/proteasome system, allowing the activation of downstream transcription factors and, consequently, the induction of JA-responsive genes. A growing body of evidence has shown that JA signalling is crucial in defending against plant viruses and their insect vectors. Here, we describe the interaction of C2 proteins from two tomato-infecting geminiviruses from the genus Begomovirus, tomato yellow leaf curl virus (TYLCV) and tomato yellow curl Sardinia virus (TYLCSaV), with the transcriptional repressor JAZ8 from Arabidopsis thaliana and its closest orthologue in tomato, SlJAZ9. Both JAZ and C2 proteins colocalize in the nucleus, forming discrete nuclear speckles. Overexpression of JAZ8 did not lead to altered responses to TYLCV infection in Arabidopsis; however, knock-down of JAZ8 favours geminiviral infection. Low levels of JAZ8 likely affect the viral infection specifically, since JAZ8-silenced plants do not display obvious developmental phenotypes nor present differences in their interaction with the viral insect vector. In summary, our results show that the geminivirus-encoded C2 interacts with JAZ8 in the nucleus, and suggest that this plant protein exerts an anti-geminiviral effect.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The transcriptional regulator JAZ8 interactswith the C2 protein from geminivirusesand limits the geminiviral infection in Arabidopsis

Jasmonates (JAs) are phytohormones that finely regulate critical biological processes, including plant development and defense. JASMONATE ZIM-DOMAIN (JAZ) proteins are crucial transcriptional regulators that keep JA-responsive genes in a repressed state. In the presence of JA-Ile, JAZ repressors are ubiquitinated and targeted for degradation by the ubiquitin/proteasome system, allowing the activation of downstream transcription factors and, consequently, the induction of JA-responsive genes. A growing body of evidence has shown that JA signaling is crucial in defending against plant viruses and their insect vectors. Here, we describe the interaction of C2 proteins from two tomato-infecting geminiviruses from the genus Begomovirus, tomato yellow leaf curl virus (TYLCV) and tomato yellow curl Sardinia virus (TYLCSaV), with the transcriptional repressor JAZ8 from Arabidopsis thaliana and its closest orthologue in tomato, SlJAZ9. Both JAZ and C2 proteins colocalize in the nucleus, forming discrete nuclear speckles. Overexpression of JAZ8 did not lead to altered responses to TYLCV infection in Arabidopsis; however, knock-down of JAZ8 favors geminiviral infection. Low levels of JAZ8 likely affect the viral infection specifically, since JAZ8-silenced plants neither display obvious developmental phenotypes nor present differences in their interaction with the viral insect vector. In summary, our results show that the geminivirus-encoded C2 interacts with JAZ8 in the nucleus, and suggest that this plant protein exerts an anti-geminiviral effect.This work was supported by the Spanish Ministerio de Ciencia y Tecnología (PID2019-107657RB-C22) (ER-B), FEDER program (UMA20-FEDERJA-021) (AG-C), the Shanghai Center for Plant Stress Biology, the Chinese Academy of Sciences, and the Federal Ministry of Education and Research (BMBF) and the Baden-Württemberg Ministry of Science as part of the Excellence Strategy of the German Federal and State Governments (RL-D). TR-D was supported by a President's International Fellowship Initiative (PIFI) postdoctoral fellowship (No. 2016PB042) from the Chinese Academy of Sciences, the “Programa Juan de la Cierva” (IJCI-2017-33367) from the MCIN and FEDER program UMA20-FEDERJA-132 by AEI and by “ERDF A way of making Europe,” by the “European Union.” Funding for Open Access charge: Universidad de Málaga / CBUA

Deciphering the role of vesicle trafficking in geminiviral infection

Esta tesis está dividida en dos partes claramente diferenciadas. En la primera parte se ha investigado el papel que juega el tráfico vesicular en las infecciones causadas por geminivirus, mientras que la segunda se ha centrado en el desarrollo de un método para la realización de expresión transitoria de proteínas por medio de Agrobacterium tumefaciens en plantas de la especie Arabidopsis thaliana. Durante el desarrollo de la primera parte se han encontrado una serie de genes implicados en tráfico vesicular que tiene una gran relevancia en las infecciones con geminivirus. En ella se comprueba y discute acerca de las rutas de tráfico vesisuclar en que intervienen estos genes y que son de importancia para la infección. También se han descubierto nuevos procesos implicados en la formación de vesículas en las que intervienen los geminivirus. Como conclusión se llega al razonamiento de que los geminivirus se están desplazando por el interior celular y exterior mediante vesículas. En la segunda parte se ha puesto a punto un sistema de expresión transitoria en arabidopsis con el que se han podido publicar dos artículos

Unraveling the importance of vesicle trafficking for the movement of geminiviruses

Tomato yellow leaf curl disease is one of the most important threats to tomato crops worldwide. One of its causal agents, Tomato yellow leaf curl Sardinian virus (TYLCSV), is a monopartite member of the genus Begomovirus from the family Geminiviridae. Due to the few proteins encoded by their viral genome, geminiviruses rely heavily on host cellular machinery and interact with a wide range of plant proteins to complete all processes required for infection, such as viral replication, movement and suppression or evasion of plant defense mechanisms. Therefore, identifying the host proteins involved in viral infection will be an essential step towards understanding the mechanisms underlying this process. Using a reverse genetic approach our group identified a series of genes involved in vesicle trafficking, which affect geminivirus infection. Four of them are essential as their silencing produce a complete abolishment of TYLCSV infection (-COP, ARF1, CHC1, and CHC2). However, these genes do not affect TYLCSV replication. A series of experiments using confocal microscopy and viral proteins bound to fluorescent markers have been carried out to determine the effect of inhibiting vesicle trafficking over the subcellular localization of TYLCSV’s movement proteins. The biological relevance of vesicle trafficking during geminivirus infection will be presented and discussed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Dissecting the relevance of vesicle trafficking for the movement of geminiviruses

Tomato yellow leaf curl disease is one of the most important threats to tomato crops worldwide. One of its causal agents, Tomato yellow leaf curl Sardinian virus (TYLCSV), is a monopartite member of the genus Begomovirus from the family Geminiviridae. Due to the few proteins encoded by their viral genome, geminiviruses rely heavily on host cellular machinery and interact with a wide range of plant proteins to complete all processes required for infection, such as viral replication, movement and suppression or evasion of plant defense mechanisms. Therefore, identifying the host proteins involved in viral infection will be an essential step towards understanding the mechanisms underlying this process. Using a reverse genetic approach our group identified a series of genes involved in vesicle trafficking, which affect geminivirus infection. Four of them are essential as their silencing produce a complete abolishment of TYLCSV infection ( δ-COP, ARF1, CHC1, and CHC2). However, these genes do not affect TYLCSV replication. A series of experiments using confocal microscopy and viral proteins bound to fluorescent markers have been carried out to determine the effect of inhibiting vesicle trafficking over the subcellular localization of TYLCSV’s movement proteins. The biological relevance of vesicle trafficking during geminivirus infection will be presented and discussed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech