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

Cytosine methylation is an epigenetic mark that promotes gene silencing and plays an important role in genome defense against transposons and invading DNA viruses. So far just one geminiviral protein, C2 from Tomato golden mosaic virus (TGMV), Beet curly top virus(BCTV) and Beet severe curly top virus (BSCTV) and a betasatellite protein from Tomato yellow leaf curl China virus (TYLCCNV), have been shown to act as TGS suppressors by interfering with the proper functioning of the plant methylation cycle. We have found that geminiviral infection reduces the expression of the plant maintenance DNA methyltransferases, MET1 and CMT3, in both, locally and systemically infected tissues of Arabidopsis and Nicotianabenthamiana and we demonstrated that the virus-mediated repression of these two maintenance DNA methyltransferases is widely spread among different geminivirus species (TGMV, African cassava mosaic virus (ACMV), Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV-Mld).Furthermore, we have identified Rep as the geminiviral protein responsible for the repression of MET1 and CMT3, and another viral protein, C4, as an ancillary player in MET1 downregulation. The presence of Rep, suppresses TGS of an Arabidopsistransgene and of host loci whose expression is strongly controlled by CG methylation. Additionally, bisulfite sequencing analyses showed that the expression of Rep caused a substantial reduction in the levels of DNA methylation at CG sites. Our findings suggest that Rep, the only viral protein essential for geminiviral replication, displays TGS suppressor activity through a mechanism distinct from the one thus far described for geminiviruses.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Studying the role of the strawberry Fra protein family in the flavonoid metabolism during fruit ripening

Strawberry fruits are highly appreciated worldwide due to their pleasant flavor and aroma and to the health benefits associated to their consumption. An important part of these properties is due to their content in secondary metabolites, especially phenolic compounds, of which flavonoids are the most abundant in the strawberry fruit. Although the flavonoid biosynthesis pathway is uncovered, little is known about its regulation. The strawberry Fra a (Fra) genes constitute a large family of homologs of the major birch pollen allergen Bet v 1 and for which no equivalents exist in Arabidopsis. Our group has shown that Fra proteins are involved in the formation of colored compounds in strawberries (Muñoz et al., 2010), which mainly depends on the production of certain flavonoids; that they are structurally homologs to the PYR/PYL/RCAR Arabidopsis ABA receptor, and that they are able to bind flavonoids (Casañal et al., 2013). With these previous results, our working hypothesis is that the Fra proteins are involved in the regulation of the flavonoids pathway. They would mechanistically act as the ABA receptor, binding a protein interactor and a ligand to regulate a signaling cascade and/or act as molecular carriers. The main objective of this research is to characterize the Fra family in strawberry and gain insight into their role in the flavonoid metabolism. By RNAseq expression analysis in ripening fruits we have identified transcripts for 10 members of the Fra family. Although expressed in all tissues analyzed, each family member presents a unique pattern of expression, which suggests functional specialization for each Fra protein. Then, our next approach was to identify the proteins that interact with Fras and their ligands to gain knowledge on the role that these proteins play in the flavonoids pathway. To identify the interacting partners of Fras we have performed a yeast two hybrid (Y2H) screening against cDNA libraries of strawberry fruits at the green and red stages. A protein that shares a 95% homology to the Heat stress transcription factor A-4-C like of Fragaria vesca (HSA4C) interacts specifically with Fra1 and not with other family members, which suggests functional diversification of Fra proteins in specific signaling pathways. The Y2H screening is not yet saturated, so characterization of other interacting proteins with other members of the Fra family will shed light on the functional diversity within this gene family. This research will contribute to gain knowledge on how the flavonoid pathway, and hence, the fruit ripening, is regulated in strawberry; an economically important crop but for which basic research is still very limited. References: Muñoz, C, et al. (2010). The Strawberry Fruit Fra a Allergen Functions in Flavonoid Biosynthesis. Molecular Plant, 3(1): 113–124. Casañal, A, et al (2013). The Strawberry Pathogenesis-related 10 (PR-10) Fra a Proteins Control Flavonoid Biosynthesis by Binding Metabolic Intermediates. Journal of Biological Chemistry, 288(49): 35322–35332.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 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

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

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

Plasticity of Fission Yeast CENP-A Chromatin Driven by Relative Levels of Histone H3 and H4

The histone H3 variant CENP-A assembles into chromatin exclusively at centromeres. The process of CENP-A chromatin assembly is epigenetically regulated. Fission yeast centromeres are composed of a central kinetochore domain on which CENP-A chromatin is assembled, and this is flanked by heterochromatin. Marker genes are silenced when placed within kinetochore or heterochromatin domains. It is not known if fission yeast CENP-ACnp1 chromatin is confined to specific sequences or whether histone H3 is actively excluded. Here, we show that fission yeast CENP-ACnp1 can assemble on noncentromeric DNA when it is inserted within the central kinetochore domain, suggesting that in fission yeast CENP-ACnp1 chromatin assembly is driven by the context of a sequence rather than the underlying DNA sequence itself. Silencing in the central domain is correlated with the amount of CENP-ACnp1 associated with the marker gene and is also affected by the relative level of histone H3. Our analyses indicate that kinetochore integrity is dependent on maintaining the normal ratio of H3 and H4. Excess H3 competes with CENP-ACnp1 for assembly into central domain chromatin, resulting in less CENP-ACnp1 and other kinetochore proteins at centromeres causing defective kinetochore function, which is manifest as aberrant mitotic chromosome segregation. Alterations in the levels of H3 relative to H4 and CENP-ACnp1 influence the extent of DNA at centromeres that is packaged in CENP-ACnp1 chromatin and the composition of this chromatin. Thus, CENP-ACnp1 chromatin assembly in fission yeast exhibits plasticity with respect to the underlying sequences and is sensitive to the levels of CENP-ACnp1 and other core histones

Factors That Promote H3 Chromatin Integrity during Transcription Prevent Promiscuous Deposition of CENP-A(Cnp1) in Fission Yeast

Specialized chromatin containing CENP-A nucleosomes instead of H3 nucleosomes is found at all centromeres. However, the mechanisms that specify the locations at which CENP-A chromatin is assembled remain elusive in organisms with regional, epigenetically regulated centromeres. It is known that normal centromeric DNA is transcribed in several systems including the fission yeast, Schizosaccharomyces pombe. Here, we show that factors which preserve stable histone H3 chromatin during transcription also play a role in preventing promiscuous CENP-A(Cnp1) deposition in fission yeast. Mutations in the histone chaperone FACT impair the maintenance of H3 chromatin on transcribed regions and promote widespread CENP-A(Cnp1) incorporation at non-centromeric sites. FACT has little or no effect on CENP-A(Cnp1) assembly at endogenous centromeres where CENP-A(Cnp1) is normally assembled. In contrast, Clr6 complex II (Clr6-CII; equivalent to Rpd3S) histone deacetylase function has a more subtle impact on the stability of transcribed H3 chromatin and acts to prevent the ectopic accumulation of CENP-A(Cnp1) at specific loci, including subtelomeric regions, where CENP-A(Cnp1) is preferentially assembled. Moreover, defective Clr6-CII function allows the de novo assembly of CENP-A(Cnp1) chromatin on centromeric DNA, bypassing the normal requirement for heterochromatin. Thus, our analyses show that alterations in the process of chromatin assembly during transcription can destabilize H3 nucleosomes and thereby allow CENP-A(Cnp1) to assemble in its place. We propose that normal centromeres provide a specific chromatin context that limits reassembly of H3 chromatin during transcription and thereby promotes the establishment of CENP-A(Cnp1) chromatin and associated kinetochores. These findings have important implications for genetic and epigenetic processes involved in centromere specification

Characterization of ripe fruit epidermis-specific transcription factors in strawberry

The epidermis is the external cell layer in direct contact with the environment, and it plays essential biological roles. Transcriptome analysis (RNA-seq) of Fragaria vesca fruit receptacles at four ripening stages (green, white, turning and red) and of different tissue types of receptacles (pith, vascular bundles, cortex and epidermis) at two ripening stages (green and red) allowed us to infer tissue- and stage-specific Gene Regulatory Networks (GRN). Due to the potential role of the epidermis in defense and in the differential anthocyanin accumulation pattern that shows at the ripe stage of F. vesca fruits (the skin is red, while the inner part is white), we have focused on the GRN of the ripe epidermis. In this study, we aim at the functional characterization of two transcription factors (TFs) that constituted the main hubs of this GRN: a MYB-like gene, and a member of the NAC family of TFs. A MapMan analysis of the genes constituting the GRN in ripe epidermis showed that wax and flavonoid biosynthesis were significantly overrepresented functions in this tissue at the ripe stage. Using the Luciferase/Renilla (Luc/Ren) system, the interaction of the MYB and NAC TFs with their wax-related putative targets was validated. To gain insight into the target genes of these two TFs, we mapped the genome-wide binding sites using DAP-seq analyses. Consistently, MYB bound to a set of genes involved in cuticle formation and flavonoid biosynthesis, while a number of genes involved in solute transport were enriched among the NAC targets. Currently, we are generating CRISPR/Cas9 mutant lines to functionally characterize these two TFs. Furthermore, we are performing protein interaction assays to decipher whether the MYB and NAC TFs interact with each other and with other TFs from the red epidermis GRN.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Lifetime mental health problems in Adult Lower Secondary Education: a student survey

Background/Objective: Adult Lower Secondary Education is an education program for basic qualifications for the labor market. Our study aimed to compare lifetime mental health problems between current Adult Lower Secondary Education students and higher Vocational Education students, as the former constitutes a highly distinct and understudied group. Methods: Findings were based on a cross-sectional self-report survey. Lifetime relative odds of occurrence of mental disorders [i.e., psychiatric disorders typically diagnosed in adults, learning difficulties or deficit hyperactivity disorder (ADHD)] were compared between Adult Lower Secondary Education students (n = 134) and Vocational Education students (n = 149). Results: While the frequency of mental health problems was high in both groups, psychiatric disorders typically diagnosed in adults were more common in Adult Lower Secondary Education students than among other students. Vocational Education students reported higher rates of ADHD. Conclusion: There is a need for additional psychological resources for Adult Lower Secondary Education students, an educational level that is the last path for many to acquire a basic degree