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

Towards defining the role of translation regulation during viral infection in plants.

Tomato yellow leaf curl virus (TYLCV) is responsible for massive damage to tomato crops worldwide (Prasad et al., 2020). Due to its viral nature, it requires the host’s cellular machinery to be able to infect, which implies complex interactions between the virus and the plant. Most studies about this association are based on transcriptomics and interactomics, while translatomics analyses have, so far, been scarce. Understanding the translational mechanisms responsible for the production of viral proteins and, consequently, its propagation will allow to shed some light on these interactions and gain knowledge about the changes at the translational level that tomato plants experience upon infection. To that end, we are characterizing the translational landscape of the plant-virus interaction using RNA-seq of polysomal RNA. To deepen our knowledge of the regulatory mechanisms involved in the translational response, two isogenic tomato lines, one resistant (the ty-5 mutant) and one susceptible (Santa Clara) to TYLCV are being employed. Ty-5 is a recessive mutation located on the Pelota gene, which is involved in the recycling phase of the translation cycle (Lapidot et al., 2015), so the study of this mutant will inform about the role of the translational machinery in the viral infection. In addition, using RIP+MS, we are attempting to uncover the translational machinery associated with viral transcripts to determine if certain riboproteins or translation factors are preferred for translating viral transcripts. We will present the advances we have made regarding these objectives. References Lapidot et al. PLoS Genet. 11:e1005538. Prasad et al. Trends Plant Sci 2020 Sep;25(9):897-911. Acknowledgments: The authors are grateful to Rafael Fernández-Muñoz (IHSM) for sharing the ty-5 and Santa Clara tomato seeds. This work is funded by Grant P18-RT-1218 from the Junta de Andalucía to CM and ERB, and grants RYC2017-22323 and PID2021-12324NB-100 to CM.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

Transcriptomic analysis of the interaction geminivirus-tomato

Geminiviridae family is one of the main families of plant pathogenic viruses with large relevance as they cause great losses worldwide in commercial crops and crops destined to food production. Geminiviruses present a little single-stranded DNA genome and a capsid composed of two twin icosahedral parts. Tomato Yellow Leaf Curl Virus (TYLCV) belongs to the Begomovirus genus and is transmitted by the whitefly Bemisia tabaci. With only 6 viral proteins, this geminivirus must create a proper environment for viral replication, transcription and propagation. Behind the apparent simplicity of geminiviruses lies a complex network of molecular interactions with their host and even their natural vector, which induces a wide variety of transcriptional, post-transcriptional and chromatinic changes in both the plant and the geminivirus. In order to study these changes and decipher the effects of the transmission vector on the infection, we carried out a global approximation of the TYLCV-tomato interaction to generate integrated single-base resolution maps by NGS (next-generation sequencing) of the transcriptome, smallRNAome and methylome of the pathogen and the host. Tomato plants (Moneymaker) were infected with TYLCV under controlled conditions of light and temperature using Agrobacterium tumefaciens or its natural vector. Apical tissue from these plants was collected at different time points (2, 7, 14 and 21 days after inoculation), and three biological replicas were generated for each treatment and time. Total RNA and DNA was extracted and analysed by RNA-Seq, smallRNA-Seq and Bisulfite-Seq. The transcriptome of the tomato-TYLCV interaction will be presented and discussed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Complex interaction among virus-plant-vector in the Tomato yellow leaf curl disease (TYLCD)

Geminiviruses constitute the largest plant family of DNA viruses that cause diseases in crops worldwide. Among them, Tomato yellow leaf curl disease (TYLCD) is one of the most devastating viral diseases affecting tomato crops in tropical, subtropical and temperate areas worldwide. Tomato yellow leaf curl virus (TYLCV), the first known causal agent of TYLCD is a monopartite Begomovirus transmitted by the whitefly Bemisia tabaci. In the recent years, several works have revealed a complex scenario in the interaction among the players that participate in the disease, the virus, the whitefly and the host plant, that may produce a different outcome depending on the interaction of each of the organism involved. To get insight to these three-way interaction, we have analysed the changes in the transcriptome, the smallRNA profile and the methylome of tomato plants infected with TYLCV either by agroinoculation of by B. tabaci. DNA and RNA samples were extracted from infected apical tomato leaves at 7, 14, 14 and 21 dpi (days post infection) and RNAseq, smallRNAseq and WGBS (Whole Genome Bisulfite Sequencing) was performed. Comparative analysis of the infection mediated by Agrobacterium and the whitefly lighted-up genes and DNA methylated regions, that were deregulated and in plants TYLCV-infected by B. tabaci and not by agroinculation, suggesting a partially different plants response to TYLCV depending on the infection method used.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Study of the functional domains of the PTGS suppressor V2 from geminivirus Beet curly top virus (BCTV)

Geminiviruses constitute a group of plant viruses that infect vegetable crops all over the world. Among the Geminiviridae family, the genera Mastrevirus, Begomovirus and Curtovirus are the most abundant. Suppression of gene silencing is a key mechanism for viral infection in plants. In begomovirus, V2 is a strong posttranscriptional gene silencing suppressor. We recently showed that V2 from curtovirus Beet curly top virus (BCTV) is a PTGS suppressor by impairing the RDR6/SGS3 pathway, as V2 from begomovirus. In order to identify the domains involved in the suppression activity and viral pathogenicity, we performed an alignment of several begomovirus and curtovirus V2 proteins. A protein kinase C (PKC) phosphorylation motif essential for suppression activity in begomovirus (P1) was found in all analysed sequences. We also found similar hydrophobic profiles, with two hydrophobic domains (H1 and H2) followed by a long hydrophilic domain. Then we generated BCTV V2 mutant proteins and performed transient assays in Nicotiana benthamiana plants to test their suppression activity. We also expressed them from a Potato virus X-derived vector to check the symptoms produced. Additionally, their subcellular localization was determined. Finally, we produced BCTV viruses mutated in the different domains and N. benthamiana plants were infected, analysing virus levels and symptoms produced. The results showed that P1, H1 and H2 are involved in the suppression activity and viral pathogenicity.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Characterization of Curtoviral V2 Protein

Geminiviruses are single-stranded DNA plant viruses with circular genomes packaged within geminate particles. Among the Geminiviridae family, Begomovirus and Curtovirus comprise the two best characterized genera. Curtovirus and Old World begomovirus possess similar genome structures with six to seven open-reading frames (ORF). Among them, begomovirus and curtovirus V2 ORFs share the same location in the viral genome, encode proteins of similar size, but show extremely poor sequence homology between the genera. V2 from Beet curly top virus (BCTV), the model species for the Curtovirus genus, as it begomoviral counterpart, suppresses post-transcriptional gene silencing (PTGS) by impairing the RDR6/SGS3 pathway and localizes in the nucleus spanning from the perinuclear region to the cell periphery. By aminoacid sequence comparison we have identified that curtoviral and begomoviral V2 proteins shared two hydrophobic domains and a putative phosphorylation motif. These three domains are essential for BCTV V2 silencing suppression activity, for the proper nuclear localization of the protein and for systemic infection. The lack of suppression activity in the mutated versions of V2 is complemented by the impaired function of RDR6 in Nicotiana benthamiana but the ability of the viral mutants to produce a systemic infection is not recovered in gene silencing mutant backgrounds. We have also demonstrated that, as its begomoviral homolog, V2 from BCTV is able to induce systemic symptoms and necrosis associated with a hypersensitive response-like (HR-like) when expressed from Potato virus X vector in N. benthamiana, and that this pathogenicity activity does not dependent of its ability to supress PTG

V2 from a curtovirus is a suppressor of post-transcriptional gene silencing

The suppression of gene silencing is a key mechanism for the success of viral infection in plants. DNA viruses from the Geminiviridae family encode several proteins that suppress transcriptional and post-transcriptional gene silencing (TGS/PTGS). In Begomovirus, the most abundant genus of this family, three out of six genome-encoded proteins, namely C2, C4 and V2, have been shown to suppress PTGS, with V2 being the strongest PTGS suppressor in transient assays. Beet curly top virus (BCTV), the model species for the Curtovirus genus, is able to infect the widest range of plants among geminiviruses. In this genus, only one protein, C2/L2, has been described as inhibiting PTGS. We show here that, despite the lack of sequence homology with its begomoviral counterpart, BCTV V2 acts as a potent PTGS suppressor, possibly by impairing the RDR6 (RNA-dependent RNA polymerase 6)/suppressor of gene silencing 3 (SGS3) pathway

Study of the PTGS suppressor activity of V2 protein from geminivirus Beet curly top virus

Suppression of gene silencing is a key mechanism for the success of viral infection in plants. DNA viruses from the Geminiviridae family encode several proteins that suppress post- and transcriptional gene silencing (PTGS/TGS). In Begomovirus V2 has been shown to be the strongest PTGS suppressor in transient assays. Beet curly top virus (BCTV), the model species for the Curtovirus genus, is able to infect the widest range of plants among geminiviruses. In this genus, only C2 protein has been described to inhibit PTGS and TGS. Objective: Our main goal is to test the PTGS suppressor activity of BCTV V2 and to study further its gene-silencing suppression mechanism. Material and methods: To determine whether BCTV V2 is also a gene silencing suppressor we carried out transient expression assays in Nicotiana benthamiana wild-type or the 16c GFP-expressing line: plant leaves were agroinfiltrated with binary constructs to express GFP (35S:GFP) and V2. Visual detection of GFP fluorescence was confirmed by western blot. Relative levels of the GFP-specific siRNAs were determined by northern blot. We also expressed the V2 ORF from a Potato virus X-derived vector in N. benthamiana plants. As an approach to identify a genetic target of V2 in the antiviral silencing pathway, we carried out a complementation analysis of BCTV-∆V2 in a series of Arabidopsis thaliana mutants deficient in specific components of this pathway involved in DNA virus siRNA production and amplification (DCL2, DCL3, DCL4, RDR6 and RDR2). Arabidopsis plants were infected by agroinoculation and the amount of viral DNA was measured by real-time qPCR. To gain more insight into the gene silencing suppression mechanism of V2, we generated Arabidopsis plants overexpressing the viral protein. Wild-type (Col-0) plants, as well as, plants containing the SUC-SUL hairpin or the AMPLICON (AMP) constructs were transformed with the same V2 expression cassette used for the gene silencing assays. Results: Like its begomoviral counterpart, BCTV V2 is a potent PTGS suppressor and produces an HR-like response in N. benthamiana plants when expressed from PVX. The molecular and genetic analysis of transgenic plants expressing V2 indicates that, as the begomoviral V2, BCTV V2 inhibits the RDR6/SGS3-dependent silencing pathway. Finally, infection assays in Arabidopsis mutants confirm the importance of the RDR6/SGS3 pathway in defence against curtoviruses, and reveal an additional RDR6/SGS3-independent gene-silencing suppression mechanism of V2. Conclusions: BCTV V2, as begomovirus V2 protein, suppresses PTGS by impairing the RDR6/SGS3 pathway. Keywords: Geminivirus, BCTV V2, RNA-silencing suppressor.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech