Deciphering molecular events behind Systemin-induced resistance against Botrytis cinerea in tomato plants.

Plant defense peptides are paramount endogenous danger signals secreted after a challenge intensifying the plant immune response. The peptidic hormone Systemin (Sys) was shown to participate in resistance in several plant-pathosystems, although the mechanisms behind Sys-IR when exogenously applied remain elusive. We performed proteomic, metabolomic and enzymatic studies to decipher the Sys-induced changes in tomato plants either in the absence or the presence of Botrytis cinerea infection. Sys-treatments triggered direct proteomic rearrangement mostly involved in carbon metabolism and photosynthesis. However, the final induction of defense proteins required concurrent challenge, triggering priming of pathogen-targeted proteins. Conversely, at the metabolomic level, Sys-treated plants showed an alternative behaviour following a general priming profile. Out of the primed metabolites, the flavonoids rutin and isorhamnetin and two alkaloids correlated with the proteins 4-coumarate-CoA-ligase and chalcone-flavanone-isomerase triggered by Sys treatment. In addition, the proteomic and enzymatic analyses revealed that Sys conditioned the primary metabolism towards the production of available sugars that could be fuelling the priming of callose deposition in Sys-treated plants, furthermore PR1 appeared as as key element in Sys-induced resistance. Collectively, the direct induction of proteins and priming of specific secondary metabolites in Sys-treated plants indicated that posttranslational protein regulation is an additional component of priming against necrotrophic fungi.

Transcriptional and epigenetic changes during tomato yellow leaf curl virus infection in tomato.

BACKGROUND: Geminiviruses are DNA plant viruses that cause highly damaging diseases affecting crops worldwide. During the infection, geminiviruses hijack cellular processes, suppress plant defenses, and cause a massive reprogramming of the infected cells leading to major changes in the whole plant homeostasis. The advances in sequencing technologies allow the simultaneous analysis of multiple aspects of viral infection at a large scale, generating new insights into the molecular mechanisms underlying plant-virus interactions. However, an integrative study of the changes in the host transcriptome, small RNA profile and methylome during a geminivirus infection has not been performed yet. Using a time-scale approach, we aim to decipher the gene regulation in tomato in response to the infection with the geminivirus, tomato yellow leaf curl virus (TYLCV). RESULTS: We showed that tomato undergoes substantial transcriptional and post-transcriptional changes upon TYLCV infection and identified the main altered regulatory pathways. Interestingly, although the principal plant defense-related processes, gene silencing and the immune response were induced, this cannot prevent the establishment of the infection. Moreover, we identified extra- and intracellular immune receptors as targets for the deregulated microRNAs (miRNAs) and established a network for those that also produced phased secondary small interfering RNAs (phasiRNAs). On the other hand, there were no significant genome-wide changes in tomato methylome at 14 days post infection, the time point at which the symptoms were general, and the amount of viral DNA had reached its maximum level, but we were able to identify differentially methylated regions that could be involved in the transcriptional regulation of some of the differentially expressed genes. CONCLUSION: We have conducted a comprehensive and reliable study on the changes at transcriptional, post-transcriptional and epigenetic levels in tomato throughout TYLCV infection. The generated genomic information is substantial for understanding the genetic, molecular and physiological changes caused by TYLCV infection in tomato.

Differential Expression of miRNAs Involved in Response to Candidatus Liberibacter asiaticus Infection in Mexican Lime at Early and Late Stages of Huanglongbing Disease.

Huanglongbing (HLB) is one of the most destructive diseases threatening citriculture worldwide. This disease has been associated with α-proteobacteria species, namely Candidatus Liberibacter. Due to the unculturable nature of the causal agent, it has been difficult to mitigate the disease, and nowadays a cure is not available. MicroRNAs (miRNAs) are key regulators of gene expression, playing an essential role in abiotic and biotic stress in plants including antibacterial responses. However, knowledge derived from non-model systems including Candidatus Liberibacter asiaticus (CLas)-citrus pathosystem remains largely unknown. In this study, small RNA profiles from Mexican lime (Citrus aurantifolia) plants infected with CLas at asymptomatic and symptomatic stages were generated by sRNA-Seq, and miRNAs were obtained with ShortStack software. A total of 46 miRNAs, including 29 known miRNAs and 17 novel miRNAs, were identified in Mexican lime. Among them, six miRNAs were deregulated in the asymptomatic stage, highlighting the up regulation of two new miRNAs. Meanwhile, eight miRNAs were differentially expressed in the symptomatic stage of the disease. The target genes of miRNAs were related to protein modification, transcription factors, and enzyme-coding genes. Our results provide new insights into miRNA-mediated regulation in C. aurantifolia in response to CLas infection. This information will be useful to understand molecular mechanisms behind the defense and pathogenesis of HLB.

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 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.

Begomovirus Tomato Leaf Curl New Delhi Virus Is Seedborne but Not Seed Transmitted in Melon.

Seed transmission can be of considerable relevance to the dissemination of plant viruses in nature and for their prevalence and perpetuation. Long-distance spread of isolates of the begomovirus species Tomato leaf curl New Delhi virus (genus Begomovirus, family Geminiviridae) has recently occurred from Asia to the Middle East and the Mediterranean Basin. Here, we investigated the possible transmission by melon (Cucumis melo L.) seeds of a tomato leaf curl New Delhi virus (ToLCNDV) isolate of the "Spain" strain widely distributed in the Mediterranean area as an alternative mechanism for long-distance spread. PCR amplification detection of ToLCNDV in floral parts and mature seeds of melon plants reveals that this virus is seedborne. "Seedborne" is defined as the ability of a virus to be carried through seeds, which does not necessarily lead to transmission to the next generation. Treatment with a chemical disinfectant significantly reduced the detectable virus associated with melon seeds, suggesting ToLCNDV contamination of the external portion of the seed coat. Also, when the internal fraction of the mature seed (seed cotyledons + embryo) was analyzed by quantitative PCR amplification, ToLCNDV was detectable at low levels, suggesting the potential for viral contamination or infection of the internal portions of seed. However, grow-out studies conducted with melon progeny plants germinated from mature seeds collected from ToLCNDV-infected plants and evaluated at early (1-leaf) or at late (20-leaf) growth stages did not support the transmission of ToLCNDV from seeds to offspring.

Functional Analysis of V2 Protein of Beet Curly Top Iran Virus.

Geminivirus beet curly top Iran virus (BCTIV) is one of the main causal agents of the beet curly top disease in Iran and the newly established Becurtovirus genus type species. Although the biological features of known becurtoviruses are similar to those of curtoviruses, they only share a limited sequence identity, and no information is available on the function of their viral genes. In this work, we demonstrate that BCTIV V2, as the curtoviral V2, is also a local silencing suppressor in Nicotiana benthamiana and can delay the systemic silencing spreading, although it cannot block the cell-to-cell movement of the silencing signal to adjacent cells. BCTIV V2 shows the same subcellular localization as curtoviral V2, being detected in the nucleus and perinuclear region, and its ectopic expression from a PVX-derived vector also causes the induction of necrotic lesions in N. benthamiana, such as the ones produced during the HR, both at the local and systemic levels. The results from the infection of N. benthamiana with a V2 BCTIV mutant showed that V2 is required for systemic infection, but not for viral replication, in a local infection. Considering all these results, we can conclude that BCTIV V2 is a functional homologue of curtoviral V2 and plays a crucial role in viral pathogenicity and systemic movement.

Peripheral membrane proteins modulate stress tolerance by safeguarding cellulose synthases.

Controlled primary cell wall remodeling allows plant growth under stressful conditions, but how these changes are conveyed to adjust cellulose synthesis is not understood. Here, we identify the TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins as new members of the cellulose synthase complex (CSC) and describe their unique and hitherto unknown dynamic association with the CSC under cellulose-deficient conditions. We find that TTLs are essential for maintaining cellulose synthesis under high-salinity conditions, establishing a stress-resilient cortical microtubule array, and stabilizing CSCs at the plasma membrane. To fulfill these functions, TTLs interact with CELLULOSE SYNTHASE 1 (CESA1) and engage with cortical microtubules to promote their polymerization. We propose that TTLs function as bridges connecting stress perception with dynamic regulation of cellulose biosynthesis at the plasma membrane.

Glucose-6-P/phosphate translocator2 mediates the phosphoglucose-isomerase1-independent response to microbial volatiles.

In Arabidopsis (Arabidopsis thaliana), the plastidial isoform of phosphoglucose isomerase (PGI1) mediates photosynthesis, metabolism, and development, probably due to its involvement in the synthesis of isoprenoid-derived signals in vascular tissues. Microbial volatile compounds (VCs) with molecular masses of <45 Da promote photosynthesis, growth, and starch overaccumulation in leaves through PGI1-independent mechanisms. Exposure to these compounds in leaves enhances the levels of GLUCOSE-6-PHOSPHATE/PHOSPHATE TRANSLOCATOR2 (GPT2) transcripts. We hypothesized that the PGI1-independent response to microbial volatile emissions involves GPT2 action. To test this hypothesis, we characterized the responses of wild-type (WT), GPT2-null gpt2-1, PGI1-null pgi1-2, and pgi1-2gpt2-1 plants to small fungal VCs. In addition, we characterized the responses of pgi1-2gpt2-1 plants expressing GPT2 under the control of a vascular tissue- and root tip-specific promoter to small fungal VCs. Fungal VCs promoted increases in growth, starch content, and photosynthesis in WT and gpt2-1 plants. These changes were substantially weaker in VC-exposed pgi1-2gpt2-1 plants but reverted to WT levels with vascular and root tip-specific GPT2 expression. Proteomic analyses did not detect enhanced levels of GPT2 protein in VC-exposed leaves and showed that knocking out GPT2 reduced the expression of photosynthesis-related proteins in pgi1-2 plants. Histochemical analyses of GUS activity in plants expressing GPT2-GUS under the control of the GPT2 promoter showed that GPT2 is mainly expressed in root tips and vascular tissues around hydathodes. Overall, the data indicated that the PGI1-independent response to microbial VCs involves resetting of the photosynthesis-related proteome in leaves through long-distance GPT2 action.

The role of GDP-l-galactose phosphorylase in the control of ascorbate biosynthesis.

The enzymes involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler [SW] pathway) are well established. Here, we analyzed their subcellular localizations and potential physical interactions and assessed their role in the control of ascorbate synthesis. Transient expression of C terminal-tagged fusions of SW genes in Nicotiana benthamiana and Arabidopsis thaliana mutants complemented with genomic constructs showed that while GDP-d-mannose epimerase is cytosolic, all the enzymes from GDP-d-mannose pyrophosphorylase (GMP) to l-galactose dehydrogenase (l-GalDH) show a dual cytosolic/nuclear localization. All transgenic lines expressing functional SW protein green fluorescent protein fusions driven by their endogenous promoters showed a high accumulation of the fusion proteins, with the exception of those lines expressing GDP-l-galactose phosphorylase (GGP) protein, which had very low abundance. Transient expression of individual or combinations of SW pathway enzymes in N. benthamiana only increased ascorbate concentration if GGP was included. Although we did not detect direct interaction between the different enzymes of the pathway using yeast-two hybrid analysis, consecutive SW enzymes, as well as the first and last enzymes (GMP and l-GalDH) associated in coimmunoprecipitation studies. This association was supported by gel filtration chromatography, showing the presence of SW proteins in high-molecular weight fractions. Finally, metabolic control analysis incorporating known kinetic characteristics showed that previously reported feedback repression at the GGP step, combined with its relatively low abundance, confers a high-flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.

Characterization of Curtovirus V2 Protein, a Functional Homolog of Begomovirus V2.

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 PTGS.

Cutting-edge technology to generate plant immunity against geminiviruses.

Geminiviruses (viruses with circular, single-stranded DNA genomes) are one of the major groups of plant viruses causing severe economic problems for agriculture worldwide. The control of these pathogens has become a priority to maintain the production of important crops, including cotton, maize, cassava, and other vegetables. Obtaining resistant plants is the most powerful strategy and a key factor to stablish an effective integrated pest management for a robust control. In the last few decades, numerous studies have successfully approached that goal using diverse strategies based on plant variability or on the engineered expression of proteins/RNAs. The increasing knowledge of the mechanisms involved in the geminivirus-plant-vector interactions, in combination with the development of gene editing technology and nanoparticles, draw new and promising strategies for a durable control of these emerging pathogens.

Gene Expression Profile of Mexican Lime (Citrus aurantifolia) Trees in Response to Huanglongbing Disease caused by Candidatus Liberibacter asiaticus.

Nowadays, Huanglongbing (HLB) disease, associated with Candidatus Liberibacter asiaticus (CLas), seriously affects citriculture worldwide, and no cure is currently available. Transcriptomic analysis of host-pathogen interaction is the first step to understand the molecular landscape of a disease. Previous works have reported the transcriptome profiling in response to HLB in different susceptible citrus species; however, similar studies in tolerant citrus species, including Mexican lime, are limited. In this work, we have obtained an RNA-seq-based differential expression profile of Mexican lime plants challenged against CLas infection, at both asymptomatic and symptomatic stages. Typical HLB-responsive differentially expressed genes (DEGs) are involved in photosynthesis, secondary metabolism, and phytohormone homeostasis. Enrichment of DEGs associated with biotic response showed that genes related to cell wall, secondary metabolism, transcription factors, signaling, and redox reactions could play a role in the tolerance of Mexican lime against CLas infection. Interestingly, despite some concordance observed between transcriptional responses of different tolerant citrus species, a subset of DEGs appeared to be species-specific. Our data highlights the importance of studying the host response during HLB disease using as model tolerant citrus species, in order to design new and opportune diagnostic and management methods.

De novo assembly and functional annotation of Citrus aurantifolia transcriptome from Candidatus Liberibacter asiaticus infected and non-infected trees.

Mexican lime (Citrus aurantifolia) belongs to the Rutaceae family and nowadays is one of the major commercial citrus crops in different countries. In Mexico, Mexican lime production is impaired by Huanglongbing (HLB) disease associated to Candidatus Liberibacter asiaticus (CLas) bacteria. To date, transcriptomic studies of CLas-Citrus interaction, have been performed mainly in sweet citrus models at symptomatic (early) stage where pleiotropic responses could mask important, pathogen-driven host modulation as well as, host antibacterial responses. Additionally, well-assembled reference transcriptomes for acid limes including C. aurantifolia are not available. The development of improved transcriptomic resources for CLas-citrus pathosystem, including both asymptomatic (early) and symptomatic (late) stages, could accelerate the understanding of the disease. Here, we provide the first transcriptomic analysis from healthy and HLB-infected C. aurantifolia leaves at both asymptomatic and symptomatic stages, using a RNA-seq approach in the Illumina NexSeq500 platform. The construction of the assembled transcriptome was conducted using the predesigned workflow Transflow and a total of 41,522 tentative transcripts (TTs) obtained. These C. aurantifolia TTs were functionally annotated using TAIR10 and UniProtKB databases. All raw reads were deposited in the NCBI SRA with accession numbers SRR10353556, SRR10353558, SRR10353560 and SRR10353562. Overall, this dataset adds new transcriptomic valuable tools for future breeding programs, will allow the design of novel diagnostic molecular markers, and will be an essential tool for studying the HLB disease.

Revisiting Seed Transmission of the Type Strain of Tomato yellow leaf curl virus in Tomato Plants.

Isolates of the Tomato yellow leaf curl virus (TYLCV) species (genus Begomovirus, family Geminiviridae) infect tomato crops worldwide, causing severe economic damage. Members of the whitefly Bemisia tabaci sibling species group are the vector of begomoviruses, including TYLCV. However, transmission of isolates of the type strain (Israel [IL]) of TYLCV (TYLCV-IL) by tomato seed has recently been reported based on infections occurring in Korea. Because of the consequences of this finding on the epidemiology and control of the disease caused by TYLCV and on the seed market, it was considered essential to revisit and expand those results to other tomato-growing areas. TYLCV DNA content was detected in tomato and Nicotiana benthamiana seed collected from plants naturally or experimentally infected with TYLCV-IL, supporting its seedborne nature. The TYLCV-IL replication detected in tomato and N. benthamiana flower reproductive organs demonstrated close association of this virus with the seed during maturation. However, the significant reduction of TYLCV DNA load after surface disinfections of tomato seed suggests that most of the virus is located externally, as contaminant of the seed coat. Transmission assays, carried out with seven tomato genotypes and more than 3,000 tomato plants, revealed no evidence of seed transmission from "surface-disinfected" or untreated seed for two Mediterranean isolates of TYLCV-IL. Similar results were also obtained for seed collected from TYLCV-IL-infected N. benthamiana plants. The results support the conclusion that TYLCV-IL is seedborne but is not seed transmitted in tomato or N. benthamiana, suggesting that transmission through seed is not a general property of TYLCV.

TTL Proteins Scaffold Brassinosteroid Signaling Components at the Plasma Membrane to Optimize Signal Transduction in Arabidopsis.

Brassinosteroids (BRs) form a group of steroidal hormones essential for plant growth, development, and stress responses. BRs are perceived extracellularly by plasma membrane receptor-like kinases that activate an interconnected signal transduction cascade, leading to the transcriptional regulation of BR-responsive genes. TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) genes are specific for land plants, and their encoded proteins are defined by the presence of protein-protein interaction motives, that is, an intrinsic disordered region at the N terminus, six tetratricopeptide repeat domains, and a C terminus with homology to thioredoxins. TTL proteins thus likely mediate the assembly of multiprotein complexes. Phenotypic, molecular, and genetic analyses show that TTL proteins are positive regulators of BR signaling in Arabidopsis (Arabidopsis thaliana). TTL3 directly interacts with a constitutively active BRASSINOSTEROID INSENSITIVE1 (BRI1) receptor kinase, BRI1-SUPPRESSOR1 phosphatase, and the BRASSINAZOLE RESISTANT1 transcription factor and associates with BR-SIGNALING KINASE1, BRASSINOSTEROID INSENSITIVE2 kinases, but not with BRI1-ASSOCIATED KINASE1. A functional TTL3-green fluorescent protein (GFP) shows dual cytoplasmic plasma membrane localization. Depleting the endogenous BR content reduces plasma membrane localization of TTL3-GFP, while increasing BR content causes its plasma membrane relocalization, where it strengthens the association of BR signaling components. Our results reveal that TTL proteins promote BR responses and suggest that TTL proteins may function as scaffold proteins by bringing together cytoplasmic and plasma membrane BR signaling components.

Integrated single-base resolution maps of transcriptome, sRNAome and methylome of Tomato yellow leaf curl virus (TYLCV) in tomato.

Geminiviruses are plant ssDNA viruses that replicate through dsDNA intermediates and form minichromosomes which carry the same epigenetic marks as the host chromatin. During the infection, geminiviruses are targets of the post-transcriptional and transcriptional gene silencing machinery. To obtain insights into the connection between virus-derived small RNAs (vsRNAs), viral genome methylation and gene expression, we obtained the transcriptome, sRNAome and methylome from the geminivirus Tomato yellow leaf curl virus-infected tomato plants. The results showed accumulation of transcripts just at the viral ORFs, while vsRNAs spanned the entire genome, showing a prevalent accumulation at regions where the viral ORFs overlapped. The viral genome was not homogenously methylated showing two highly methylated regions located in the C1 ORF and around the intergenic region (IR). The compilation of those results showed a partial correlation between vsRNA accumulation, gene expression and DNA methylation. We could distinguish different epigenetic scenarios along the viral genome, suggesting that in addition to its function as a plant defence mechanism, DNA methylation could have a role in viral gene regulation. To our knowledge, this is the first report that shows integrative single-nucleotide maps of DNA methylation, vsRNA accumulation and gene expression from a plant virus.

Arabidopsis thaliana SPF1 and SPF2 are nuclear-located ULP2-like SUMO proteases that act downstream of SIZ1 in plant development.

Post-translational modifiers such as the small ubiquitin-like modifier (SUMO) peptide act as fast and reversible protein regulators. Functional characterization of the sumoylation machinery has determined the key regulatory role that SUMO plays in plant development. Unlike components of the SUMO conjugation pathway, SUMO proteases (ULPs) are encoded by a relatively large gene family and are potential sources of specificity within the pathway. This study reports a thorough comparative genomics and phylogenetic characterization of plant ULPs, revealing the presence of one ULP1-like and three ULP2-like SUMO protease subgroups within plant genomes. As representatives of an under-studied subgroup, Arabidopsis SPF1 and SPF2 were subjected to functional characterization. Loss-of-function mutants implicated both proteins with vegetative growth, flowering time, and seed size and yield. Mutants constitutively accumulated SUMO conjugates, and yeast complementation assays associated these proteins with the function of ScUlp2 but not ScUlp1. Fluorescence imaging placed both proteins in the plant cell nucleoplasm. Transcriptomics analysis indicated strong regulatory involvement in secondary metabolism, cell wall remodelling, and nitrate assimilation. Furthermore, developmental defects of the spf1-1 spf2-2 (spf1/2) double-mutant opposed those of the major E3 ligase siz1 mutant and, most significantly, developmental and transcriptomic characterization of the siz1 spf1/2 triple-mutant placed SIZ1 as epistatic to SPF1 and SPF2.

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.

Panspecies small-molecule disruptors of heterochromatin-mediated transcriptional gene silencing.

Heterochromatin underpins gene repression, genome integrity, and chromosome segregation. In the fission yeast Schizosaccharomyces pombe, conserved protein complexes effect heterochromatin formation via RNA interference-mediated recruitment of a histone H3 lysine 9 methyltransferase to cognate chromatin regions. To identify small molecules that inhibit heterochromatin formation, we performed an in vivo screen for loss of silencing of a dominant selectable kanMX reporter gene embedded within fission yeast centromeric heterochromatin. Two structurally unrelated compounds, HMS-I1 and HMS-I2, alleviated kanMX silencing and decreased repressive H3K9 methylation levels at the transgene. The decrease in methylation caused by HMS-I1 and HMS-I2 was observed at all loci regulated by histone methylation, including centromeric repeats, telomeric regions, and the mating-type locus, consistent with inhibition of the histone deacetylases (HDACs) Clr3 and/or Sir2. Chemical-genetic epistasis and expression profiles revealed that both compounds affect the activity of the Clr3-containing Snf2/HDAC repressor complex (SHREC). In vitro HDAC assays revealed that HMS-I1 and HMS-I2 inhibit Clr3 HDAC activity. HMS-I1 also alleviated transgene reporter silencing by heterochromatin in Arabidopsis and a mouse cell line, suggesting a conserved mechanism of action. HMS-I1 and HMS-I2 bear no resemblance to known inhibitors of chromatin-based activities and thus represent novel chemical probes for heterochromatin formation and function.