Discerning the relationship between geminiviral infection and vesicle trafficking using virus induced gene silencing

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 machineries 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 defence mechanisms. The identification of the host proteins involved in viral infection will be an important step towards the understanding of the mechanisms underlying this process. In our laboratory, transgenic Nicotiana benthamiana plants containing a green fluorescent protein (GFP) expression cassette flanked by two direct repeats of the intergenic region of TYLCSV have been constructed (2IR plants). When these plants are infected with TYLCSV, an overexpression of the reporter gene is observed in those cells where the virus is actively replicating. These plants have been used together with virus induced gene silencing (VIGS) in an effort to identify host genes involved in the infection process using a reverse genetics approach. Using this combined technique our group has identified two genes δ-COP and ARF 1, involved in retrograde vesicle trafficking, which are essential for the infectious process. We are currently assaying genes codifying proteins involved in different pathways of the vesicle trafficking system: Sar1b, γ subunit of AP1, Sec24, SYT1 and two that encode the heavy chain of triskelion proteins. Their effect over viral infection will be presented and discussedUniversidad de Málaga. Campues de Excelencia Internacional Andalucía Tech

Geminiviral protein Rep interferes in PCNA sumoylation

Rep is a multifunctional protein essential for replication of geminivirus that interferes with the sumoylation of a key protein in the DNA replication, PCNA (Proliferating Cell Nuclear Antigen). It is known that Rep is capable of interacting with a plethora of plant proteins, including PCNA. Despite the biological significance remains unknown, it’s thought that this interaction should play a key role for generating new copies of the virus genome. Therefore, in order to characterize this interaction, we study which lysines are sumoylated in tomato PCNA (SlPCNA). Considering conservation, location and presence of sumoylation domain criteria, we have identified some candidate lysines and studied how its mutation affects this protein sumoylation in Escherichia coli assays. Finally, we plan to confirm and characterize the Rep interference on SlPCNA sumoylation and determine if this interference occurs in planta.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Identifying the function of vesicle trafficking in geminiviral infection using virus induced gene silencing

Tomato yellow leaf curl Sardinian virus (TYLCSV) is one of the causal agent of the tomato yel-low leaf curl disease, one of the most important threats to tomato crops worldwide. TYLCSV is a monopartite member of the genus Begomovirus from the family Geminiviridae. To carry out a full infection, geminiviruses need to move inside the infected cell and from one cell to an-other for which they depend on diverse cellular factors. While cell-to-cell movement has been described to occur through plasmodesmata, the way in which geminiviruses move inside the host cells is yet unknown. The identification of the host proteins involved in viral infection will be an important step to-wards the understanding of the mechanisms underlying this process. In our laboratory, trans-genic Nicotianabenthamiana plants containing a green fluorescent protein (GFP) expression cassette flanked by two direct repeats of the intergenic region of TYLCSV have been construct-ed (2IR plants). When these plants are infected with TYLCSV, an overexpression of the reporter gene is observed in those cells where the virus replicates. These plants have been used to-gether with virus induced gene silencing (VIGS) in an effort to identify host genes involved in the infection process using a reverse genetics approach. Using this combined technique our group has identified two genes δ-COP and ARF 1, involved in retrograde vesicle trafficking, which are essential for the infectious process. We are current-ly assaying genes codifying proteins involved in different pathways of the vesicle trafficking system: Sar1b, γ subunit of AP1, Sec24, SYT1 and two that encode the heavy chain of triskelion proteins. Their effect over virus infection will be presented and discussed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Geminivirus Rep Protein Interferes with the Plant DNA Methylation Machinery and Suppresses Transcriptional Gene Silencing

Viruses are masters at circumventing host defenses and manipulating the cellular environment for their own benefit. The replication of the largest known family of single-stranded DNA viruses, Geminiviridae, is impaired by DNA methylation but the fact that plants might use methylation as a defense against geminiviruses and the impact that viral genome methylation may have during the infection, remain controversial. We have found that geminiviruses reduce the expression of the plant maintenance DNA methyltransferases, MET1 and CMT3, in both, locally and systemically infected tissues. Furthermore, we demonstrated that the virus-mediated repression of these two maintenance DNA methyltransferases is widely spread among different geminivirus species and we have identified Rep as the geminiviral protein responsible for the repression of MET1 and CMT3. The presence of Rep, suppresses transcriptional gene silencing (TGS) of an Arabidopsis transgene and of host loci whose expression is strongly controlled by MET1. Bisulfite sequencing analyses showed that the expression of Rep caused a substantial reduction in the levels of DNA methylation at certain loci at CG sites. The biological relevance of these findings and the role of Rep as a TGS suppressor will be discussed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Importance of the temperature in the interaction plant-geminivirus

Introduction. Geminivirusare plant viruses that have circular single-stranded DNA (ssDNA) genome encapsidated in twinned quasi-icosahedral (geminate) virionsand are a serious threat to diverse economically important crops such as tomato, pepper and cassava. TYLCV (Tomato yellow leaf curl virus) belongs to the genus begomovirus which is by far, the largest of the seven genera in the family Geminiviridae and it is a pathogen that induces a devastating disease in tomato in the Mediterranean region. Cultured tomatoes are often exposed to a combination of extreme heat and infection with TYLCV and this combination leads to intense disease symptoms and yield losses.The impact of temperature over the interaction tomato- TYLCVhas been characterized (Ghandi et al., 2016) but these results seem to be contradictory to our findings. Objective. The main objective of this work is to improve our understanding of the impact that high temperature has in the interaction plant-geminivirus.Material and methods. Nicotianabenthamiana plants were infected with TYLCV and growth in a chamber at 21 ºC or at 35 ºC. The amount of virus, symptoms, plant height and number of leaves were monitored at 14, 21 and 28 dpi (days post-infection). Three biological replicates were performed, each containing 15 infected plants and 10 non-infected plants (mock). Results. No differences were observed between non-infected and infected plants at both temperatures, in plant height or in the number of leaves, but the amount of virus and the severity of symptoms were significantly reduced in plants at high temperature. Conclusions. We conclude that TYLCV yield decreases in N. benthamiana due to high temperature and ongoing experiments in tomato TYLCV-infected plants will let us determine the importance of this finding in an agronomical important crop and to compare our results with the published data.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Geminivirus C2 protein represses genes involved in sulphur assimilation and this effect can be counteracted by jasmonate treatment

Geminiviruses are plant viruses that infect a broad range of crops and cause extensive losses worldwide, having an important economic impact. C2, a multifunctional pathogenicity factor encoded by geminiviruses, has been recently shown to suppress the responses to jasmonates in the host plant, which might at least partially explain its well-established role in pathogenicity. Sulphur is one of the essential macro-elements for plant life, and is considered to have a role in plant defence, in a phenomenon named sulphur-induced resistance (SIR) or sulphur-enhanced defence (SED). In this work, we show that geminivirus C2 protein represses the expression of genes involved in the sulphur assimilation pathway in Arabidopsis, but, interestingly, this effect can be neutralized by exogenous jasmonate treatment. These preliminary results may raise the idea that geminiviruses might be affecting sulphur metabolism, and maybe counteracting SIR/SED, through the manipulation of the jasmonate signalling pathway, which would define a novel strategy in plant-virus interactions and may unveil SIR/SED as an important player in the plant defence against viruses.Ministerio de Ciencia y Innovación/FEDER AGL2007-66062-C02-02/AGR AGL2010-22287-CO2European Regional Development Fund (ERDF) BIO2010-15201Junta de Andalucía BIO­27

PAMP-triggered immunity against Pseudomonas syringae involves microRNA-mediated regulation of several uncharacterized R genes

Two main types of noncoding small RNA molecules have been found in plants: microRNAs (miRNAs) and small interfering RNAs (siRNAs). They differ in their biogenesis and mode of action, but share similar sizes (20-24 nt). Their precursors are processed by Dicer-Like RNase III (dcl) proteins present in Arabidopsis thaliana, and in their mature form can act as negative regulators of gene expression, being involved in a vast array of plant processes, including plant development, genomic integrity or response to stress. Small-RNA mediated regulation can occurs at transcriptional level (TGS) or at post-transcriptional level (PTGS). In recent years, the role of gene silencing in the regulation of expression of genes related to plant defence responses against bacterial pathogens is becoming clearer. Comparisons carried out in our lab between the expression profiles of different mutants affected in gene silencing, and plants challenged with Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, differentially expressed in these conditions. Through the use of bioinformatics tools, we found a miRNA* of 22 nt putatively responsible for down-regulating expression of these R genes through the generation of siRNAs. We have also found that the corresponding pri-miRNA is down-regulated after PAMP-perception in a SA-dependent manner. We also demonstrate that plants with altered levels of miRNA* (knockdown lines or overexpression lines) exhibit altered PTI-associated phenotypes, suggesting a role for this miRNA* in this defence response against bacteria. In addition we identify one of the target genes as a negative regulator of defence response against Pseudomonas syringae.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. MINECO, FEDE

Importance of vesicle trafficking in the establichsment of a geminiviral infection

Importance of vesicle trafficking in the establishment of a geminiviral infection P. CANA-QUIJADA1, T. ROSAS-DÍAZ2, LOZANO-DURÁN R. 2 AND E.R. BEJARANO1 1Dpto. Biología Celular, Genética y Fisiología. Área de Genética Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” (IHSM-UMA-CSIC), Universidad de Málaga, Málaga, Spain E-mail: [email protected] 2. Shanghai Center for Plant Stress Biology (PSC), Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai 201602, China. INTRODUCTION Geminiviruses produces some of the most devastating diseases for agriculture worldwide. Geminiviral genomes encode only 5 to 7 proteins, forcing them to rely heavily on host cellular machineries and to interact with a high amount of host proteins in order to complete a full infection. The identification of the host proteins involved in viral infection will be an important step towards the understanding of the mechanisms underlying this process and develop new strategies to generate new sources of resistance. Previous efforts from our group have identified several genes involved in vesicle trafficking. OBJECTIVES The main aim of this work is to elucidate the role of the plant cell’s vesicle trafficking in a geminiviral infection. MATHERIALS AND METHODS Transgenic Nicotiana benthamiana plants containing a green fluorescent protein (GFP) expression cassette flanked by two direct repeats of the intergenic region of TYLCSV have been constructed (2IR plants). When these plants are infected with TYLCSV, an overexpression of the reporter gene is observed in those cells where the virus is actively replicating. 2IR plants were used in combination with virus induced gene silencing (VIGS), to identify vesicle trafficking genes involved in the infectious process. Viral replication was monitorized by GFP expression. Viral accumulation was determined using qPCR. RESULTS When silenced, four of the assessed genes reduced dramatically the viral amounts or completely abolished the infection. On the other hand, three of them had no significant effect over the infection and one of them seems to cause a slight increase in viral accumulation. CONCLUSIONS The vesicle trafficking machinery plays an essential role in geminivirus infection. Assays to determine whether the described effect is due to a lack of replication or movement of the virus inside the plant cells are in progress.Universidad de Málaga. Campues de Excelencia Internacional Andalucía Tech

La proteína Rep/AL1 de geminivirus altera la sumoilación de PCNA

Los geminivirus, llamados así por la forma icosaédrica de su cápside, son una familia de virus patógenos de plantas que causan algunas de las enfermedades con mayor impacto económico a nivel mundial. Estos pequeños virus de ADN se replican en el núcleo de las células vegetales utilizando la maquinaria celular del hospedador, además de requerir la presencia de la proteína viral Rep. Rep es la única proteína del genoma del virus imprescindible para su replicación y es capaz de interaccionar con una gran variedad de proteínas del huésped. Trabajos previos de nuestro grupo demostraron que dos de esas proteínas son PCNA (Proliferating cell nuclear antigen), esencial en el metabolismo del DNA, y SCE (SUMO Conjugating Enzime), enzima que interviene en la sumoilación, uno de los principales mecanismos de modificación postraduccional que está implicado en la respuesta de la planta a estrés abiótico, en el desarrollo, el crecimiento y la respuesta a patógenos. Además, otros resultados que hemos obtenido demuestran que la expresión en Escherichia coli de la proteína Rep del begomovirus TGMV (Tomato golden mosaic virus) reduce la sumoilación de PCNA en plantas y que dicha interferencia no depende de la interacción de la proteína viral con SCE1. Para profundizar en el conocimiento de la interacción Rep-PCNA nos hemos propuesto identificar las lisinas de PCNA que se sumoilan. Teniendo en cuenta criterios de conservación, localización y presencia de dominio de sumoilación se han identificado varias lisinas candidatas y se ha estudiado cómo su mutación afecta a la sumoilación de la proteína. También nos hemos planteado confirmar y caracterizar la interferencia de Rep sobre la sumoilación de PCNA y determinar si dicha interferencia se produce en la planta.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Análisis de la infección por geminivirus en plantas con la maquinaria de metilación del DNA alterada.

Los geminivirus son virus de plantas pertenecientes a la familia Geminiviridae. Poseen un genoma compuesto por una o dos moléculas de DNA circular de cadena simple y una cápside compuesta de 2 partes icosaédricas gemelas, de ahí el nombre de geminivirus. Uno de los geminivirus más estudiados es el virus del rizado amarillo de la hoja de tomate (TYLCV, Tomato yellow leaf curl virus), causante de importantes pérdidas en las cosechas en zonas templadas, subtropicales y tropicales. Este geminivirus codifica 6 proteínas, de las cuales sólo la proteína Rep es esencial para su replicación. La metilación del DNA es una marca epigenética que promueve el silenciamiento génico a nivel transcripcional (TGS) y juega un importante papel en el mantenimiento de la integridad del genoma mediante el silenciamiento de transposones. Diversos estudios sugieren que participa de manera relevante en la defensa de la planta frente a virus de DNA como los geminivirus (Raja et al, 2008; Yang et al, 2011, Zhang et al, 2011). Los geminivirus son capaces de interferir en el mecanismo de TGS propio de la planta; una de las proteínas implicadas en la supresión de dicho mecanismo es Rep, la cual induce una disminución en los niveles de expresión de las metiltransferasas de mantenimiento MET1 y CMT3 de Arabidopsis thaliana y Nicotiana benthamiana, revierte el silenciamiento génico transcripcional de loci endógenos y transgenes e induce la hipometilación de loci cuya metilación es esencialmente dependiente de MET1 (Rodríguez-Negrete et al., 2013). Considerando que los datos previos sugieren que los geminivirus suprimen el mecanismo de TGS como un mecanismo de contra-defensa, nos popusimos evaluar la importancia de la metilación del DNA como mecanismo de defensa frente a los geminivirus. Para ello se midieron los niveles del geminivus TYLCV-Mld (Tomato yellow leaf curl virus, aislado Mld) tras la infección de mutantes de A. thaliana deficientes en la maquinaria de metilación: met1-3 (mutante en MET1), ros1-4 (mutante en la desmetilasa ROS1) y ddc (triple mutante DRM1, DRM2 y CMT3, siendo DRM1 y DRM2 metiltransferasas de novo). Por otro lado se midieron niveles del mismo geminivirus en plantas infectadas de N. benthamiana que presentan reducidos niveles de expresión de MET1, CMT3 o ROS1; la reducción de la expresión de estos genes de N. benthamiana fue generada mediante silenciamiento génico inducido por virus (VIGS). Se presentarán y discutirán dichos resultados.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech