A viral effector suppresses cell-to-cell spread of silencing by targeting two plasmodesmal receptor-like kinases

RNA interference (RNAi) in plants can move from cell to cell, allowing for systemic spread of an anti-viral immune response. How this cell-to-cell spread of silencing is regulated is currently unknown. Here, we describe that the C4 protein from Tomato yellow leaf curl virus can inhibit the intercellular spread of RNAi. Using this viral protein as a probe, we have identified the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1) as a positive regulator of the cell-to-cell movement of RNAi, and determined that BAM1 and its closest homologue, BAM2, play a redundant role in this process. C4 interacts with the intracellular domain of BAM1 and BAM2 at the plasma membrane and plasmodesmata, the cytoplasmic connections between plant cells, interfering with the function of these RLKs in the cell-to-cell spread of RNAi. Our results identify BAM1 as an element required for the cell-to-cell spread of RNAi and highlight that signalling components have been co-opted to play multiple functions in plants.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Exploring the functional interactions between geminivirus and host

Los geminivirus son virus de plantas con genomas circulares de DNA de cadena sencilla que infectan numerosas especies de interés agronómico en todo el mundo, provocando cuantiosas pérdidas que pueden llegar hasta el 100% de la cosecha. Los genomas de estos virus están muy reducidos, y codifican sólo 6 u 8 proteínas, dependiendo de la especie. Esta reducción genómica hace que el virus dependa de factores celulares para el desarrollo de la infección y la compleción de su ciclo vital, incluyendo las fases de replicación y tráfico dentro de la célula o en la planta. Dado que los geminivirus precisan proteínas de la planta hospedadora, la identificación de dichas proteínas supondría un importante paso hacia la comprensión del proceso de infección, lo que en último término podría suponer un importante aporte en la lucha contra la enfermedad. El objetivo de éste trabajo consiste en explorar las interacciones funcionales entre los geminivirus y sus hospedadores. En el primer capítulo se estudió la importancia de la proteína C2 de los geminivirus durante la señalización de jasmonatos (JA) y la respuesta de defensa en planta. Trabajos previos en nuestro grupo mostraron que la expresión de las proteínas C2 es capaz de interferir con la ruta de ubiquitinación en plantas de Arabidopsis. Con la finalidad de estudiar más a detalle el efecto que tiene la expresión de C2 en Arabidopsis, se hizo un análisis de microarray. El análisis transcriptómico de las plantas transgénicas expresan C2 de Tomato yellow curl Sardinia virus (TYLCSV) reveló que C2 altera múltiples procesos celulares. Entre los procesos más destacados están la represión de la respuesta a JA y al metabolismo secundario. Además, el análisis transcriptómico de las plantas transgénicas de Arabidopsis que expresan C2 tratadas con JA exógeno, puntualizó que la represión causada por C2 es a través la respuesta específica de genes inducidos por JA; por lo tanto es dudoso que ésta inhibición sea a través de la inhibición de la E3-ligasa SCFCOI1. Por otro lado, observamos que la proteína C2 interacciona en levaduras e in planta con la proteína represora de la respuesta a JA llamada JAZ8. Por lo tanto, hemos propuesto que la proteína C2 de los geminivirus podría estar interfiriendo con la respuesta a JA en varios niveles. El segundo capítulo tuvo como objetivo llevar a cabo la identificación de genes del hospedador involucrados en la infección por geminivirus usando una aproximación de genética reversa. Para llevar a cabo esto, usamos plantas transgénicas de Nicotiana benthamiana denominadas 2IRGFP. Estas plantas presentan una sobreexpresión de la GFP dependiente de la actividad de la proteína viral Rep de TYLCSV, que dispara la formación de replicones mGFP. La acumulación de GFP actúa como marcador de la replicación de TYLCSV, permitiendo la detección y seguimiento de este proceso de manera rápida, sencilla, semi-cuantitativa y a tiempo real. Además, en combinación con una técnica de silenciamiento como el silenciamiento génico inducido por virus (VIGS), las plantas 2IRGFP son una poderosa herramienta en estudios de genética reversa dirigidos a la identificación de genes de la planta necesarios para la infección viral. Siguiendo este concepto, se silenciaron 37 genes candidatos en las plantas 2IRGFP a las que paralelamente se infectaron con TYLCSV. De acuerdo con el efecto de su silenciamiento sobre la infección TYLCSV, medida como tiempo de aparición y la intensidad de la expresión de GFP, se agruparon los genes del huésped en tres clases: aquellos cuyo silenciamiento no causó cambios en la expresión (grupo A), o aquellos cuya silenciamiento adelantó (grupo B) o por el contrario retrasó o llegó a ser nula (grupo C) la expresión de GFP. En total hemos identificado 18 genes implicados en varios procesos celulares cuyos silenciamiento altera infección TYLCSV. En particular, 15 de estos genes son descritos por primera vez como factores implicados en infecciones virales. Por lo tanto, nuestros resultados proporcionan nuevos conocimientos sobre los posibles mecanismos moleculares que subyacen a las infecciones por geminivirus, y al mismo tiempo revelan el sistema 2IRGFP/ VIGS como una poderosa herramienta para los estudios de genética funcional reversa. Como tercera meta nos propusimos analizar el papel que cumple el tráfico vesícula retrogrado durante la infección por geminivirus. Éste capítulo se inició por el sorprendente hallazgo que se obtuvo en el segundo capítulo del presente trabajo, donde observamos que silenciamiento del gen que codifica para subunidad delta del complejo de coatomero COPI (-COP) imposibilita por completo la infección de TYLCSV en plantas de N. benthamiana. Para obtener mayor información sobre el rol del tráfico retrógrado sobre la infección por geminivirus, decidimos silenciar otro gen implicado en ésta ruta. Este gen fué ADP-ribosilación 1 (ARF1), la GTPasa específica que impulsa la formación de las vesículas de COPI. Observamos que los geminivirus TYLCSV y Beet curly top virus no infectan plantas de N. benthamiana donde se silenciaron los genes -COP como ARF1. Además pudimos concluir que es un efecto específico de los geminivirus, porque al infectar con otros patógenos como virus de RNA y Pseudomonas syringae pv tomato no se observó ningún efecto. En resumen, los geminivirus requieren un sistema de transporte retrógrado activo. El silenciamiento génico de dos de los principales componentes de ésta ruta afecta negativamente la infección de TYLCSV y BCTV, pero no altera la interacción con otros patógenos de planta

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

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

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

NAHG increases transient transformation of arabidopsis leaves by agrobacterium

Agrobacterium tumefaciens-mediated transient transformation has demonstrated to be an invaluable tool in plant molecular biology studies. However, low efficiency and inconsistency of this method in Arabidopsis has forced the implementation of Nicotiana benthamiana as a surrogate system, limiting applicability. One of the main reasons to underlie the recalcitrance of Arabidopsis to Agrobacterium-mediated transformation is the activation of plant immune responses upon perception of the bacteria. Perception of bacterial pathogen-associated molecular patterns (PAMPs), including EF-Tu, leads to activation of PAMP-triggered immunity (PTI). Activation of PTI ultimately induces salicylic acid (SA) accumulation, which in turn shuts down expression of the vir genes, potentially interfering with the transfer of the T-DNA, and therefore T-DNA gene expression (Yuan et al., 2007; Anand et al., 2008). However, previous results suggested that besides depletion of SA other hormone-mediated defence responses, including jasmonic acid (JA), might be responsible for the low efficiency of transient transformation in Arabidopsis (Tsuda et al., 2012). 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). The results show that impairment of JA signalling reduces or does not affect transient expression in mature leaves, but expression of the NahG transgene dramatically improves this process. We demonstrate that Arabidopsis NahG plants can be efficiently used for transient expression-based functional 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.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Geminivirus replication protein decreases PCNA sumoylation at two acceptor sites

Geminiviruses are plant viruses with circular, single-stranded DNA (ssDNA) genomes that infect a broad range of plants causing substantial crop diseases worldwide. They replicate in nuclei of infected cells by using host DNA replication machinery and an essential protein encoded in their genome designated Rep (replication-associated protein). This multifunctional protein induces the accumulation of the host factors involved in replication and it is capable of interacting with a lot of plant proteins including PCNA (Proliferating Cell Nuclear Antigen), a processivity factor that coordinates a wide range of processes involved in maintenance, duplication and transmission of the genome, and the sumoylation enzyme that conjugates SUMO to target proteins (SUMO-conjugating enzyme- SCE). PCNA modification by SUMO, and also ubiquitin, has long been known to be of key importance for determining how DNA damage is processed by the replisome and for maintenance of overall genome integrity. In yeast, PCNA sumoylation has been associated to DNA repair involving homologous recombination (HR). Previously, we reported that Rep ectopic expression does not result in broad changes in the sumoylation pattern of plant cells, but it modifies the sumoylation state of selected host proteins. In this work, we show, using a reconstituted sumoylation system in Escherichia coli, that tomato PCNA is sumoylated at two residues, K254 and K164, and that co-expression of the Rep protein suppresses PCNA sumoylation at these lysines. Finally, we confirm that PCNA is sumoylated and that Rep also interferes with PCNA sumoylation in planta.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

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

Identification of Host Genes Involved in Geminivirus Infection Using a Reverse Genetics Approach

Geminiviruses, like all viruses, rely on the host cell machinery to establish a successful infection, but the identity and function of these required host proteins remain largely unknown. Tomato yellow leaf curl Sardinia virus (TYLCSV), a monopartite geminivirus, is one of the causal agents of the devastating Tomato yellow leaf curl disease (TYLCD). The transgenic 2IRGFP N. benthamiana plants, used in combination with Virus Induced Gene Silencing (VIGS), entail an important potential as a tool in reverse genetics studies to identify host factors involved in TYLCSV infection. Using these transgenic plants, we have made an accurate description of the evolution of TYLCSV replication in the host in both space and time. Moreover, we have determined that TYLCSV and Tobacco rattle virus (TRV) do not dramatically influence each other when co-infected in N. benthamiana, what makes the use of TRV-induced gene silencing in combination with TYLCSV for reverse genetic studies feasible. Finally, we have tested the effect of silencing candidate host genes on TYLCSV infection, identifying eighteen genes potentially involved in this process, fifteen of which had never been implicated in geminiviral infections before. Seven of the analyzed genes have a potential anti-viral effect, whereas the expression of the other eleven is required for a full infection. Interestingly, almost half of the genes altering TYLCSV infection play a role in postranslational modifications. Therefore, our results provide new insights into the molecular mechanisms underlying geminivirus infections, and at the same time reveal the 2IRGFP/VIGS system as a powerful tool for functional reverse genetics studies