Geminivirus Rep Protein Interferes with the Plant DNA Methylation Machinery and Modifies the Host Epigenome

The apparent simplicity of viruses hides the complexity of their interactions with their hosts. 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 and Arabidopsis mutants affected in cytosine methylation are hypersusceptible to geminivirus infection. This implies that plants might use methylation as a defense against geminiviruses and that the viral genome is a target for plant DNA methyltransferases. We have found a novel counter-defense strategy used by geminiviruses, that reduces 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. Additionally, we 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 Arabidopsis transgene and of host loci whose expression is strongly controlled by CG methylation. 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

Characterization of the translational landscape of the plant-virus interaction

Tomato yellow leaf curl virus (TYLCV) is responsible for a disease that causes massive damage to tomato crops around the world (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 machinery that is 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 the emerging technique Ribo-Seq. And to deepen our knowledge on 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, and using RIP+MS, we are attempting to uncover the translational machinery associated to viral transcripts to determine if certain riboproteins or translation factors are preferred for the translation of viral transcripts. We will present the advances we have made regarding these objectives.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, a RYC-2017-1218 to CM and the “Plan Propio” from the Universidad de Málaga. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

ttl mutants are impaired in cellulose biosynthesis under osmotic stress

As sessile organisms, plants require mechanisms to sense and respond to the challenging environment, that encompass both biotic and abiotic factors that results in differential development. In these conditions is essential to balance growth and stress responses. As cell walls shape plant growth, this differential growth response cause alterations to the plant cell wall and cellulose is a major component. Therefore, understanding the mechanisms that regulate cellulose biosynthesis is essential to develop strategies to improve plant production. Previous studies have shown that the GSK3 kinase BIN2 modulate cellulose biosynthesis through phosphorylating cellulose synthases and that the expression of cellulose synthases are regulated by Brassinosteroids. Our previous work reveals that the tetratricopeptide-repeat thioreoxin-like (TTL) TTL1, TTL3, and TTL4 genes, in addition to their reported role in abiotic stress tolerance, are positive regulators of BR signaling. We observe association of TTL3 with most core components in traducing BR signalling, such as LRR-RLK BRI1, BIN2 and the transcription factor BES1 that positively regulate cellulose biosynthesis. We show that ttl mutants are affected in cellulose biosynthesis, particularly in osmotic stress conditions. Furthermore, TTL3 associates with LRR-RLKs that have been shown to be important for cellulose biosynthesis such as FEI1 in the FEI1/FEI2/SOS5 pathway. We aim to investigate the mechanisms by which TTL proteins regulate cellulose biosynthesis using a combination of genetics, biochemical, and molecular and cell biology approaches. This work was supported by grants from: (1) Ministerio de Ciencia e Innovación BIO2014-55380-R, BIO2014-56153-REDT; (2) Ministerio de Economía, Industria y Competitividad (BES-2015-071256); (3) Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.This work was supported by grants from: (1) Ministerio de Ciencia e Innovación BIO2014-55380-R, BIO2014-56153-REDT; (2) Ministerio de Economía, Industria y Competitividad (BES-2015-071256); (3) 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

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

Adaptative mechanisms of cellulose synthesis under stress conditions

Cellulose is the main structural compound of the plant cell wall and the most abundant biopolymer on Earth (Bar‐On et al 2018). The essential role of cellulose in plant development and defence highlights the importance of understanding how its synthesis is regulated and will provide new tools to improve crop tolerance to biotic and abiotic stresses. We identified that Tetratricopeptide Thioredoxin‐Like (TTL) proteins function as scaffold components of brassinosteroid signalling components (Amorim‐Silva et al 2019) and as new components of the Cellulose Synthase Complex (CSC) and describe its unique dynamic association with the CSC under cellulose‐deficient conditions (Kesten, García‐Moreno, Amorim‐Silva et al 2022). The TTL‐CESA interaction at the plasma membrane significantly increased under conditions that cause reduced cellulose content, such as salt stress and structural alterations of the CSCs. The relocalization of cytosolic TTLs to the active CSCs allows cellulose synthesis, mediated by a stress‐resilient cortical microtubule array and the stabilization of the CSCs at the plasma membrane. TTLs carry this out by interacting with Cellulose Synthase 1 and promoting the polymerization of microtubules, thus maintaining the stability and integrity of the complex. We propose that TTLs act as bridges connecting stress‐mediated cell wall modification with the regulation of cellulose biosynthesis. We are currently investigating novel components involved in TTL function and how this protein family is regulated. Recently, we have identified the 14‐3‐3 proteins as interactors of TTL3. The 14‐3‐3s are a family of proteins conserved in eukaryotes that target a wide number of proteins (Huang et al 2022). An Arabidopsis line overexpressing 14‐3‐3λ present phenotypes under stress consistent with defects in cellulose biosynthesis. This study will elucidate a possible role of 14‐3‐3 proteins in TTL regulation and cellulose biosynthesis.This work was funded by the Spanish Ministry for Science and Innovation (MCIN/AEI/10.1 0 9/ 011000110 ) and the Andalusian Research Plan cofinanced by the European Union(PAI I 2020-PY20_0008 and UMA20- E ERJA-02 ) to M.A.B.; V.A.-S. was supported by an Emerging Investigator research project (UMA20- E ERJA-007) and cofinanced by the “Programa Operativo E ER 201 -2020” and by the “Consejeria de Economia y Conocimiento de la Junta de Andalucia”. R.P.M. was supported by the Andalusian PRE OC_01 fellowship. A.M. was supported by Shanghai Center for Plant Stress Biology. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Unraveling the mechanism of TTL genes in cellulose biosynthesis

As sessile organisms, plants require mechanisms to sense and respond to the challenging environment, that encompass both biotic and abiotic factors that results in differential development. In these conditions is essential to balance growth and stress responses. As cell walls shape plant growth, this differential growth response cause alterations to the plant cell wall where cellulose is the major component. Therefore, understanding the mechanisms that regulate cellulose biosynthesis is essential to develop strategies to improve plant production. In Arabidopsis, the TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) gene family is composed by four members (TTL1 to TTL4) and mutations in TTL1, TTL3, and TTL4 genes cause reduced growth under salt and osmotic stress due to defects in plant cell wall integrity. We observe association of TTL3 with most core components in traducing BR signalling, such as LRR-RLK BRI1 or GSK3 BIN2 that modulate cellulose biosynthesis through phosphorylating cellulose synthases. Here, we show that ttl mutants present defects in the plant cell wall, particularly in Isoxaben, salt or sucrose stress. Spinning disk microscopy in etiolated hypocotyls reveals that, TTL proteins are responsible for the cellulose synthase complex (CSC) stability in plasma membrane (PM) upon sucrose stress. Moreover, TTL3 associates with LRR-RLKs that have been shown to be important for cellulose biosynthesis such as FEI1 in the FEI1/FEI2/SOS5 pathway. We aim to investigate the mechanisms by which TTL proteins regulate CesA stability in PM under stress, using a combination of genetics, biochemical, and molecular and cell biology approaches.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This work was supported by grants from: (1) Ministerio de Ciencia e Innovación BIO2014-55380-R, BIO2014-56153-REDT; (2) Ministerio de Economía, Industria y Competitividad (BES-2015-071256

Overcoming stress: new insights in the regulation of cell wall biosynthesis

In addition to being crucial in plant development and defence, cellulose is the most abundant organic compound of all biomass on Earth1. Therefore, it is essential to elucidate the regulation of its biosynthesis to improve crop's tolerance to biotic and abiotic stresses. We have found the Tetratricopeptide Thioredoxin-Like (TTL)2 proteins as new players in the regulation of the cellulose synthase complex (CSC), identifying its dynamic association with the CSC under cellulose-deficient conditions3. We have found that TTLs are essential to maintain cellulose synthesis under salt stress, mediated by a stress-resilient cortical microtubule array and the stabilization of the CSCs at the plasma membrane. TTLs carry this out by interacting with Cellulose Synthase 1 and promoting the polymerization of microtubules. This dynamic behaviour of TTLs is not specific to salinity stress, and other modifications that cause reduced cellulose content also lead to the re-localization from the cytosol to the CSC. We conclude that TTLs act as intermediates between stress perception and regulation of cellulose biosynthesis to overcome adverse environmental conditions. All TTL proteins contain an Intrinsic Disordered Region at the end terminus, and we are now investigating how changes in phosphorylation regulate the activity and dynamic localization of these proteins.This work was funded by the Spanish Ministry for Science and Innovation (MCIN/AEI/ 10.13039/501100011033) (PGC2018-098789-B-I00) and (PID2019-107657RB-C22) to MAB, NRL and AC respectively. The Andalusian Research Plan co-financed by the European Union (PAIDI 2020-PY20_00084) to MAB and Junta de Andalucía UMA-FEDER project (grant UMA18-FEDERJA-154) to NRL, and the Swiss National foundation to CSR (SNF 31003A_163065/1 to AM). CK was supported by a Peter und Traudl Engelhorn-Stiftung fellowship, an ETH Career Seed Grant (SEED-05 19-2) of the ETH Foundation, an Emerging Investigator grant (NNF20OC0060564) of the Novo Nordisk Foundation, and an Experiment grant (R346-2020-1546) of the Lundbeck foundation. AGM and FP were supported by BES-2015-071256 and FPU19/02219 fellowships respectively, and meeting attendance was supported by Plan Propio de Investigación, Transfe-rencia y Divulgación Científica de la Universidad de Málaga (UMA) Campus de Excelencia Internacional Andalucía Tech. VAS was supported by an Emerging Investigator research project (UMA20-FEDERJA-007) and co-financed by the “Programa Operativo FEDER 2014-2020” and by the “Consejería de Economía y Conocimiento de la Junta de Andalucía”. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Characterization of ripe fruit epidermis-specific transcription factors in strawberry

Transcriptome changes during strawberry fruit ripening have been previously reported using either complete fruits or achenes (actual fruits) and receptacles (fleshy part) separately. In order to perform a more detailed study, we have performed a tissue- and stage-specific transcriptome analysis in receptacles of Fragaria vesca fruits, allowing us to infer Gene Regulatory Networks (GRN) in each tissue and stage. In the study, we have focused on the epidermis at the ripe stage, since it plays an important role in defense, as it is the external cell layer in direct contact with the environment, and, in contrast to receptacles of the commercial species, it is the only part of the fruit that accumulates anthocyanins. MapMan analysis of the GRN in ripe epidermis showed that wax and flavonoid biosynthesis were significantly overrepresented functions. Three out of the several TFs found among the main hubs in this GRN were selected to study their biological role, one of them belonging to the MYB family, and two bHLH genes. Protein interaction assays revealed that the MYB protein physically interacts with the two bHLHs, leading to the subcellular relocalization from the cytoplasm to the nucleus in one of them. DAP-seq analyses showed that the bHLH TFs do not bind DNA by themselves, but that genes involved in cuticle formation and flavonoid biosynthesis are among the MYB targets, which were validated by a transactivation assay using the Luciferase/Renilla system. Consistently, MYB-overexpressing stable lines exhibited an upregulation of genes related to cuticle and wax biosynthesis in ripe fruits, and an accumulation of higher amounts of epicuticular waxes in young leaves compared to the WT. We are currently establishing RNAi and CRISPR lines for these three ripe-epidermis specific TFs to further investigate their biological role and performing analyses to understand the effect on gene expression of the interaction between them.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Calling for reinforcements: the role of TTL proteins in the regulación of cell wall biosynthesis

Cellulose is the most abundant organic compound of all biomass on Earth1, with highly relevant roles in plant development and defence. Hence, it is essential to understand the regulation of its biosynthesis to improve the crop's tolerance to biotic and abiotic stresses. Tetratricopeptide Thioredoxin-Like (TTL)2 proteins have been identified as new players in regulating the cellulose synthase complex (CSC), uncovering their dynamic association with the CSC under cellulose-deficient conditions3. We show that TTLs are essential to maintain cellulose synthesis under salt stress, mediated by a reinforced cortical microtubule array and the stabilization of the CSCs at the plasma membrane. To perform this, TTLs interact with Cellulose Synthase 1 and promote microtubules polymerization. This dynamic behaviour of TTLs is not specific to salinity stress, and other factors that cause defects in cellulose also cause the re-localization from the cytosol to the CSC. We conclude that TTLs act as intermediates between stress sensing and the regulation of cellulose biosynthesis to overcome adverse environmental conditions. We are now investigating how changes in phosphorylation of the Intrinsic Disordered Region at the end terminus of TTLs regulate their activity and dynamic localization.This work was funded by the Spanish Ministry for Science and Innovation (MCIN/AEI/ 10.13039/501100011033) (PGC2018-098789-B-I00) and (PID2019-107657RB-C22) to MAB, NRL and AC respectively. The Andalusian Research Plan co-financed by the European Union (PAIDI 2020-PY20_00084) to MAB and Junta de Andalucía UMA-FEDER project (grant UMA18-FEDERJA-154, UMA20-FEDERJA-023) to NRL and MAB respectively, and the Swiss National foundation to CSR (SNF 31003A_163065/1 to AM). CK was supported by a Peter und Traudl Engelhorn-Stiftung fellowship, an ETH Career Seed Grant (SEED-05 19-2) of the ETH Foundation, an Emerging Investigator grant (NNF20OC0060564) of the Novo Nordisk Foundation, and an Experiment grant (R346-2020-1546) of the Lundbeck foundation. AGM and FP were supported by BES-2015-071256 and FPU19/02219 fellowships respectively, and meeting attendance was supported by Plan Propio de Investigación, Transferencia y Divulgación Científica de la Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. VAS was supported by an Emerging Investigator research project (UMA20-FEDERJA-007) and co-financed by the “Programa Operativo FEDER 2014-2020” and by the “Consejería de Economía y Conocimiento de la Junta de Andalucía”