From Metagenomics to Ecogenomics: NGS-Based Approaches for Discovery of New Circular DNA Single-Stranded Viral Species.

Viruses comprise the most abundant genetic material in the biosphere; however, global viral genomic population (virome) has been largely underestimated. Recently, high-throughput sequencing (HTS) has provided a powerful tool for the detection of known viruses and the discovery of novel viral species from environmental and individual samples using metagenomics and ecogenomics approaches, respectively. Viruses with circular DNA single-stranded (ssDNA) genomes belonging to the begomovirus genera (family Geminiviridae) constitute the largest group of emerging plant viruses worldwide. The knowledge of begomoviruses viromes is mostly restricted to crop plant systems; nevertheless, it has been described that noncultivated plants specifically at the interface between wild and cultivated plants are important reservoirs leading to viral evolution and the emergence of new diseases. Here we present a protocol that allows the identification and isolation of known and novel begomoviruses species infecting cultivated and noncultivated plant species. The method consists of circular viral molecules enrichment by rolling circle amplification (RCA) from begomovirus-positive total plant DNA, followed by NGS-based metagenomic sequencing. Subsequently, metagenomic reads are processed for taxonomic classification using Viromescan software and a customized Geminiviridae family database, and begomovirus-related reads are used for contigs assembly and annotation using Spades software and Blastn algorithm, respectively. Then, the obtained begomovirus-related signatures are used as templates for specific primers design and implemented for PCR-based ecogenomic identification of individual samples harboring the corresponding viral species. Lastly, full-length begomovirus genomes are obtained by RCA-based amplification from total plant DNA of selected individual samples, cloning, and viral molecular identity corroborated by Sanger sequencing. Conclusively, the identification and isolation of a novel monopartite begomovirus species native to the New World (NW) named Gallium leaf deformation virus (GLDV) is shown.

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.

Regulation of plant immunity via small RNA-mediated control of NLR expression.

Plants use different receptors to detect potential pathogens: membrane-anchored pattern recognition receptors (PRRs) activated upon perception of pathogen-associated molecular patterns (PAMPs) that elicit pattern-triggered immunity (PTI); and intracellular nucleotide-binding leucine-rich repeat proteins (NLRs) activated by detection of pathogen-derived effectors, activating effector-triggered immunity (ETI). The interconnections between PTI and ETI responses have been increasingly reported. Elevated NLR levels may cause autoimmunity, with symptoms ranging from fitness cost to developmental arrest, sometimes combined with run-away cell death, making accurate control of NLR dosage key for plant survival. Small RNA-mediated gene regulation has emerged as a major mechanism of control of NLR dosage. Twenty-two nucleotide miRNAs with the unique ability to trigger secondary siRNA production from target transcripts are particularly prevalent in NLR regulation. They enhance repression of the primary NLR target, but also bring about repression of NLRs only complementary to secondary siRNAs. We summarize current knowledge on miRNAs and siRNAs in the regulation of NLR expression with an emphasis on 22 nt miRNAs and propose that miRNA and siRNA regulation of NLR levels provides additional links between PTI and NLR defense pathways to increase plant responsiveness against a broad spectrum of pathogens and control an efficient deployment of defenses.

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.

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.

SUMO E3 ligase SIZ1 connects sumoylation and reactive oxygen species homeostasis processes in Arabidopsis.

The ubiquitin-like modifying peptide SMALL UBIQUITIN-LIKE MODIFIER (SUMO) has become a known modulator of the plant response to multiple environmental stimuli. A common feature of many of these external stresses is the production of reactive oxygen species (ROS). Taking into account that SUMO conjugates rapidly accumulate in response to an external oxidative stimulus, it is likely that ROS and sumoylation converge at the molecular and regulatory levels. In this study, we explored the SUMO-ROS relationship, using as a model the Arabidopsis (Arabidopsis thaliana) null mutant of the major SUMO-conjugation enhancer, the E3 ligase SAP AND MIZ 1 (SIZ1). We showed that SIZ1 is involved in SUMO conjugate increase when primed with both exogenous and endogenous ROS. In siz1, seedlings were sensitive to oxidative stress imposition, and mutants accumulated different ROS throughout development. We demonstrated that the deregulation in hydrogen peroxide and superoxide homeostasis, but not of singlet O2 (1O2), was partially due to SA accumulation in siz1. Furthermore, transcriptomic analysis highlighted a transcriptional signature that implicated siz1 with 1O2 homeostasis. Subsequently, we observed that siz1 displayed chloroplast morphological defects and altered energy dissipation activity and established a link between the chlorophyll precursor protochlorophyllide and deregulation of PROTOCHLOROPHYLLIDE OXIDOREDUCTASE A (PORA), which is known to drive overproduction of 1O2. Ultimately, network analysis uncovered known and additional associations between transcriptional control of PORA and SIZ1-dependent sumoylation. Our study connects sumoylation, and specifically SIZ1, to the control of chloroplast functions and places sumoylation as a molecular mechanism involved in ROS homeostatic and signaling events.

miR825-5p targets the TIR-NBS-LRR gene MIST1 and down-regulates basal immunity against Pseudomonas syringae in Arabidopsis.

Plants encode numerous intracellular receptors known as nucleotide-binding leucine-rich repeat receptors (NLRs) that recognize pathogen-derived effectors or their activity to activate defenses. miRNAs regulate NLR genes in many species, often triggering the production of phased siRNAs (phasiRNAs). Most such examples involve genes encoding NLRs carrying coiled-coil domains, although a few include genes encoding NLRs carrying a Toll/interleukin-1 domain (TNL). Here, we characterize the role of miR825-5p in Arabidopsis, using a combination of bioinformatics, transgenic plants with altered miRNA levels and/or reporters, small RNAs, and virulence assays. We demonstrate that miR825-5p down-regulates the TNL MIST1 by targeting for endonucleolytic cleavage the sequence coding for TIR2, a highly conserved amino acid motif, linked to a catalytic residue essential for immune function. miR825-5p acts as a negative regulator of basal resistance against Pseudomonas syringae. miR825-5p triggers the production from MIST1 of a large number of phasiRNAs that can mediate cleavage of both MIST1 and additional TNL gene transcripts, potentially acting as a regulatory hub. miR825-5p is expressed in unchallenged leaves and transcriptionally down-regulated in response to pathogen-associated molecular patterns (PAMPs). Our results show that miR825-5p, which is required for full expression of PAMP-triggered immunity, establishes a link between PAMP perception and expression of uncharacterized TNL genes.

ER Bodies Are Induced by Pseudomonas syringae and Negatively Regulate Immunity.

ER bodies are endoplasmic reticulum-derived organelles present in plants belonging to the Brassicales order. In Arabidopsis thaliana, ER bodies are ubiquitous in cotyledons and roots and are present only in certain cell types in rosette leaves. However, both wounding and jasmonic acid treatment induce the formation of ER bodies in leaves. Formation of this structure is dependent on the transcription factor NAI1. The main components of the ER bodies are ß-glucosidases (BGLUs), enzymes that hydrolyze specialized compounds. In Arabidopsis, PYK10 (BGLU23) and BGLU18 are the most abundant ER body proteins. In this work, we found that ER bodies are downregulated as a consequence of the immune responses induced by bacterial flagellin perception. Arabidopsis mutants defective in ER body formation show enhanced responses upon flagellin perception and enhanced resistance to bacterial infections. Furthermore, the bacterial toxin coronatine induces the formation of de novo ER bodies in leaves and its virulence function is partially dependent on this structure. Finally, we show that performance of the polyphagous beet armyworm herbivore Spodoptera exigua increases in plants lacking ER bodies. Altogether, we provide new evidence for the role of the ER bodies in plant immune responses.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Plant DNA polymerases α and δ mediate replication of geminiviruses.

Geminiviruses are causal agents of devastating diseases in crops. Geminiviruses have circular single-stranded (ss) DNA genomes that are replicated in the nucleus of the infected plant cell through double-stranded (ds) DNA intermediates by the plant DNA replication machinery. Which host DNA polymerase mediates geminiviral multiplication, however, has so far remained elusive. Here, we show that subunits of the nuclear replicative DNA polymerases α and δ physically interact with the geminivirus-encoded replication enhancer protein, C3, and that these polymerases are required for viral replication. Our results suggest that, while DNA polymerase α is essential to generate the viral dsDNA intermediate, DNA polymerase δ mediates the synthesis of new copies of the geminiviral ssDNA genome, and that the virus-encoded C3 may act selectively, recruiting DNA polymerase δ over ε to favour productive replication.

Ménage à Trois: Unraveling the Mechanisms Regulating Plant-Microbe-Arthropod Interactions.

Plant-microbe-arthropod (PMA) three-way interactions have important implications for plant health. However, our poor understanding of the underlying regulatory mechanisms hampers their biotechnological applications. To this end, we searched for potential common patterns in plant responses regarding taxonomic groups or lifestyles. We found that most signaling modules regulating two-way interactions also operate in three-way interactions. Furthermore, the relative contribution of signaling modules to the final plant response cannot be directly inferred from two-way interactions. Moreover, our analyses show that three-way interactions often result in the activation of additional pathways, as well as in changes in the speed or intensity of defense activation. Thus, detailed, basic knowledge of plant-microbe-arthropod regulation will be essential for the design of environmentally friendly crop management strategies.

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.

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.

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.

Protocol: low cost fast and efficient generation of molecular tools for small RNA analysis.

BACKGROUND: Small RNAs are sequence-dependent negative regulators of gene expression involved in many relevant plant processes such as development, genome stability, or stress response. Functional characterization of sRNAs in plants typically relies on the modification of the steady state levels of these molecules. State-of-the-art strategies to reduce plant sRNA levels include molecular tools such as Target Mimics (MIMs or TMs), Short Tandem Target Mimic (STTMs), or molecular SPONGES (SPs). Construction of these tools routinely involve many different molecular biology techniques, steps, and reagents rendering such processes expensive, time consuming, and difficult to implement, particularly high-throughput approaches. RESULTS: We have developed a vector and a cloning strategy that significantly reduces the number of steps required for the generation of MIMs against any given small RNA (sRNA). Our pGREEN-based binary expression vector (pGREEN-DLM100) contains the IPS1 gene from A. thaliana bisected by a ccdB cassette that is itself flanked by restriction sites for a type IIS endonuclease. Using a single digestion plus a sticky-end ligation step, the ccdB cassette that functions as a negative (counter) selection system is replaced by a pair of 28 nt self-annealing primers that provide specificity against the selected target miRNA/siRNA. The method considerably reduces the number of steps and the time required to generate the construct, minimizes the errors derived from long-range PCRs, bypasses bottlenecks derived from subcloning steps, and eliminates the need for any additional cloning technics and reagents, overall saving time and reagents. CONCLUSIONS: Our streamlined system guarantees a low cost, fast and efficient cloning process that it can be easily implemented into high-throughput strategies, since the same digested plasmid can be used for any given sRNA. We believe this method represents a significant technical improvement on state-of-the-art methods to facilitate the characterization of functional aspects of sRNA biology.

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.

High-Throughput Sequencing Reveals Differential Begomovirus Species Diversity in Non-Cultivated Plants in Northern-Pacific Mexico.

Plant DNA viruses of the genus Begomovirus have been documented as the most genetically diverse in the family Geminiviridae and present a serious threat for global horticultural production, especially considering climate change. It is important to characterize naturally existing begomoviruses, since viral genetic diversity in non-cultivated plants could lead to future disease epidemics in crops. In this study, high-throughput sequencing (HTS) was employed to determine viral diversity of samples collected in a survey performed during 2012-2016 in seven states of Northern-Pacific Mexico, areas of diverse climatic conditions where different vegetable crops are subject to intensive farming. In total, 132 plant species, belonging to 34 families, were identified and sampled in the natural ecosystems surrounding cultivated areas (agro-ecological interface). HTS analysis and subsequent de novo assembly revealed a number of geminivirus-related DNA signatures with 80 to 100% DNA similarity with begomoviral sequences present in the genome databank. The analysis revealed DNA signatures corresponding to 52 crop-infecting and 35 non-cultivated-infecting geminiviruses that, interestingly, were present in different plant species. Such an analysis deepens our knowledge of geminiviral diversity and could help detecting emerging viruses affecting crops in different agro-climatic regions.

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.

A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein.

Geminiviruses are single-stranded DNA (ssDNA) viruses that infect a wide range of plants. To promote viral replication, geminiviruses manipulate the host cell cycle. The viral protein Rep is essential to reprogram the cell cycle and then initiate viral DNA replication by interacting with a plethora of nuclear host factors. Even though many protein domains of Rep have been characterized, little is known about its nuclear targeting. Here, we show that one conserved lysine in the N-terminal part of Rep is pivotal for nuclear localization of the Rep protein from Tomato yellow leaf curl virus (TYLCV), with two other lysines also contributing to its nuclear import. Previous work had identified that these residues are essential for Rep from Tomato golden mosaic virus (TGMV) to interact with the E2 SUMO-conjugating enzyme (SCE1). We here show that mutating these lysines leads to nuclear exclusion of TYLCV Rep without compromising its interaction with SCE1. Moreover, the ability of TYLCV Rep to promote viral DNA replication also depends on this highly conserved lysine independently of its role in nuclear import of Rep. Our data thus reveal that this lysine potentially has a broad role in geminivirus replication, but its role in nuclear import and SCE1 binding differs depending on the Rep protein examined.IMPORTANCE Nuclear activity of the replication initiator protein (Rep) of geminiviruses is essential for viral replication. We now define that one highly conserved lysine is important for nuclear import of Rep from three different begomoviruses. To our knowledge, this is the first time that nuclear localization has been mapped for any geminiviral Rep protein. Our data add another key function to this lysine residue, besides its roles in viral DNA replication and interaction with host factors, such as the SUMO E2-conjugating enzyme.