Deficiency of multiple RNA silencing-associated genes may contribute to the increased susceptibility of Nicotiana benthamiana to viruses.

KEY MESSAGE: Recently published high-quality reference genome assemblies indicate that, in addition to RDR1-deficiency, the loss of several key RNA silencing-associated genes may contribute to the hypersusceptibility of Nicotiana benthamiana to viruses.

Hierarchical contribution of Argonaute proteins to antiviral protection.

Antiviral RNAi is the main protective measure employed by plants in the fight against viruses. The main steps of this process have been clarified in recent years, primarily relying on the extensive genetic resources of Arabidopsis thaliana. Our knowledge of viral diseases of crops, however, is still limited, mainly due to the fact that A. thaliana is a non-host for many agriculturally important viruses. In contrast, Nicotiana benthamiana has an unparalleled susceptibility to viruses and, since it belongs to the Solanaceae family, it is considered an adequate system for modeling infectious diseases of crops such as tomatoes. We used a series of N. benthamiana mutants created by genome editing to analyze the RNAi response elicited by the emerging tomato pathogen, pepino mosaic virus (PepMV). We uncovered hierarchical roles of several Argonaute proteins (AGOs) in anti-PepMV defense, with the predominant contribution of AGO2. Interestingly, the anti-PepMV activities of AGO1A, AGO5, and AGO10 only become apparent when AGO2 is mutated. Taken together, our results prove that hierarchical actions of several AGOs are needed for the plant to build effective anti-PepMV resistance. The genetic resources created here will be valuable assets for analyzing RNAi responses triggered by other agriculturally important pathogenic viruses.

CRISPR-Cas9 Targeting of the eIF4E1 Gene Extends the Potato Virus Y Resistance Spectrum of the Solanum tuberosum L. cv. Desirée.

Translation initiation factors and, in particular, the eIF4E family are the primary source of recessive resistance to potyviruses in many plant species. However, no eIF4E-mediated resistance to this virus genus has been identified in potato (Solanum tuberosum L.) germplasm. As in tomato, the potato eIF4E gene family consists of eIF4E1, its paralog eIF4E2, eIF(iso)4E, and nCBP. In tomato, eIF4E1 knockout (KO) confers resistance to a subset of potyviruses, while the eIF4E1/2 double KO, although conferring a broader spectrum of resistance, leads to plant developmental defects. Here, the tetraploid potato cv. Desirée owning the dominant Ny gene conferring resistance to potato virus Y (PVY) strain O but not NTN was used to evaluate the possibility to expand its PVY resistance spectrum by CRISPR-Cas9-mediated KO of the eIF4E1 susceptibility gene. After a double process of plant protoplast transfection-regeneration, eIF4E1 KO potatoes were obtained. The knockout was specific for the eIF4E1, and no mutations were identified in its eIF4E2 paralog. Expression analysis of the eIF4E family shows that the disruption of the eIF4E1 does not alter the RNA steady-state level of the other family members. The eIF4E1 KO lines challenged with a PVYNTN isolate showed a reduced viral accumulation and amelioration of virus-induced symptoms suggesting that the eIF4E1 gene was required but not essential for its multiplication. Our data show that eIF4E1 editing can be usefully exploited to broaden the PVY resistance spectrum of elite potato cultivars, such as Desirée, by pyramiding eIF4E-mediated recessive resistance.

A calmodulin-binding transcription factor links calcium signaling to antiviral RNAi defense in plants.

RNA interference (RNAi) is an across-kingdom gene regulatory and defense mechanism. However, little is known about how organisms sense initial cues to mobilize RNAi. Here, we show that wounding to Nicotiana benthamiana cells during virus intrusion activates RNAi-related gene expression through calcium signaling. A rapid wound-induced elevation in calcium fluxes triggers calmodulin-dependent activation of calmodulin-binding transcription activator-3 (CAMTA3), which activates RNA-dependent RNA polymerase-6 and Bifunctional nuclease-2 (BN2) transcription. BN2 stabilizes mRNAs encoding key components of RNAi machinery, notably AGONAUTE1/2 and DICER-LIKE1, by degrading their cognate microRNAs. Consequently, multiple RNAi genes are primed for combating virus invasion. Calmodulin-, CAMTA3-, or BN2-knockdown/knockout plants show increased susceptibility to geminivirus, cucumovirus, and potyvirus. Notably, Geminivirus V2 protein can disrupt the calmodulin-CAMTA3 interaction to counteract RNAi defense. These findings link Ca2+ signaling to RNAi and reveal versatility of host antiviral defense and viral counter-defense.

Comparative analysis identifies amino acids critical for citrus tristeza virus (T36CA) encoded proteins involved in suppression of RNA silencing and differential systemic infection in two plant species.

Complementary (c)DNA clones corresponding to the full-length genome of T36CA (a Californian isolate of Citrus tristeza virus with the T36 genotype), which shares 99.1% identity with that of T36FL (a T36 isolate from Florida), were made into a vector system to express the green fluorescent protein (GFP). Agroinfiltration of two prototype T36CA-based vectors (pT36CA) to Nicotiana benthamiana plants resulted in local but not systemic GFP expression/viral infection. This contrasted with agroinfiltration of the T36FL-based vector (pT36FL), which resulted in both local and systemic GFP expression/viral infection. A prototype T36CA systemically infected RNA silencing-defective N. benthamiana lines, demonstrating that a genetic basis for its defective systemic infection was RNA silencing. We evaluated the in planta bioactivity of chimeric pT36CA-pT36FL constructs and the results suggested that nucleotide variants in several open reading frames of the prototype T36CA could be responsible for its defective systemic infection. A single amino acid substitution in each of two silencing suppressors, p20 (S107G) and p25 (G36D), of prototype T36CA facilitated its systemic infectivity in N. benthamiana (albeit with reduced titre relative to that of T36FL) but not in Citrus macrophylla plants. Enhanced virus accumulation and, remarkably, robust systemic infection of T36CA in N. benthamiana and C. macrophylla plants, respectively, required two additional amino acid substitutions engineered in p65 (N118S and S158L), a putative closterovirus movement protein. The availability of pT36CA provides a unique opportunity for comparative analysis to identify viral coding and noncoding nucleotides or sequences involved in functions that are vital for in planta infection.

Targeted inactivation of the AGO1 homeologues of Nicotiana benthamiana reveals their distinct roles in development and antiviral defence.

The Solanaceae family includes numerous highly valuable crops. Understanding the viral diseases that affect them is of great importance. Nicotiana benthamiana has contributed greatly to unravelling antiviral RNA interference, and can also be regarded as an adequate model for studying viral diseases of solanaceous crops. This species, however, as with many of its relatives, possesses an allopolyploid genome, in which homeologous gene pairs frequently occur. AGO1 is a pivotal component of most plant RNA silencing pathways. The Nicotiana benthamiana genome encodes two highly similar AGO1 homeologues: AGO1A and AGO1B. To understand their roles in planta, their genes were selectively inactivated. Given the inherent limitations of RNA interference-based techniques, we used genome editing to achieve this goal. We found that AGO1A was not required for normal development, while AGO1B was indispensable for that. By contrast, the two homeologues both contributed to antiviral defence. Additionally, we observed that AGO1B utilised miR168 poorly, which may help to retain a significant level of antiviral RNA interference during viral infection. Our results have important implications for the better understanding of viral diseases of economically important solanaceous crops.

Abscisic Acid Connects Phytohormone Signaling with RNA Metabolic Pathways and Promotes an Antiviral Response that Is Evaded by a Self-Controlled RNA Virus.

A complex network of cellular receptors, RNA targeting pathways, and small-molecule signaling provides robust plant immunity and tolerance to viruses. To maximize their fitness, viruses must evolve control mechanisms to balance host immune evasion and plant-damaging effects. The genus Potyvirus comprises plant viruses characterized by RNA genomes that encode large polyproteins led by the P1 protease. A P1 autoinhibitory domain controls polyprotein processing, the release of a downstream functional RNA-silencing suppressor, and viral replication. Here, we show that P1Pro, a plum pox virus clone that lacks the P1 autoinhibitory domain, triggers complex reprogramming of the host transcriptome and high levels of abscisic acid (ABA) accumulation. A meta-analysis highlighted ABA connections with host pathways known to control RNA stability, turnover, maturation, and translation. Transcriptomic changes triggered by P1Pro infection or ABA showed similarities in host RNA abundance and diversity. Genetic and hormone treatment assays showed that ABA promotes plant resistance to potyviral infection. Finally, quantitative mathematical modeling of viral replication in the presence of defense pathways supported self-control of polyprotein processing kinetics as a viral mechanism that attenuates the magnitude of the host antiviral response. Overall, our findings indicate that ABA is an active player in plant antiviral immunity, which is nonetheless evaded by a self-controlled RNA virus.

In Vivo Reporter System for miRNA-Mediated RNA Silencing.

Argonaute proteins play a central role in the evolutionarily conserved mechanisms of RNA silencing. Programmed by a variety of small RNAs, including miRNAs, they recognize their target nucleic acids and modulate gene expression by various means. Argonaute proteins are large complex molecules. Therefore, to better understand the mechanisms they use to regulate gene expression, it is necessary to identify regions of them bearing functional importance (protein-protein interaction surfaces, acceptor sites of posttranslational modifications, etc.). Identification of these regions can be performed using a variety of mutant screens. Here we describe a transient reporter assay system, which is suitable to carry out rapid functional assessment of mutant Argonaute molecules before proceeding to their more detailed biochemical characterization.

Double-Stranded-RNA-Binding Protein 2 Participates in Antiviral Defense.

Double-stranded RNA (dsRNA) is a common pattern formed during the replication of both RNA and DNA viruses. Perception of virus-derived dsRNAs by specialized receptor molecules leads to the activation of various antiviral measures. In plants, these defensive processes include the adaptive RNA interference (RNAi) pathway and innate pattern-triggered immune (PTI) responses. While details of the former process have been well established in recent years, the latter are still only partially understood at the molecular level. Nonetheless, emerging data suggest extensive cross talk between the different antiviral mechanisms. Here, we demonstrate that dsRNA-binding protein 2 (DRB2) of Nicotiana benthamiana plays a direct role in potato virus X (PVX)-elicited systemic necrosis. These results establish that DRB2, a known component of RNAi, is also involved in a virus-induced PTI response. In addition, our findings suggest that RNA-dependent polymerase 6 (RDR6)-dependent dsRNAs play an important role in the triggering of PVX-induced systemic necrosis. Based on our data, a model is formulated whereby competition between different DRB proteins for virus-derived dsRNAs helps establish the dominant antiviral pathways that are activated in response to virus infection.IMPORTANCE Plants employ multiple defense mechanisms to restrict viral infections, among which RNA interference is the best understood. The activation of innate immunity often leads to both local and systemic necrotic responses, which confine the virus to the infected cells and can also provide resistance to distal, noninfected parts of the organism. Systemic necrosis, which is regarded as a special form of the local hypersensitive response, results in necrosis of the apical stem region, usually causing the death of the plant. Here, we provide evidence that the dsRNA-binding protein 2 of Nicotiana benthamiana plays an important role in virus-induced systemic necrosis. Our findings are not only compatible with the recent hypothesis that DRB proteins act as viral invasion sensors but also extends it by proposing that DRBs play a critical role in establishing the dominant antiviral measures that are triggered during virus infection.

The virological model plant, Nicotiana benthamiana expresses a single functional RDR6 homeolog.

The RNA dependent RNA polymerase, RDR6 is involved in a variety of processes including the biogenesis of endogenous regulatory small RNAs, maintaining post-transcriptional gene silencing of transgenes and establishing efficient antiviral RNA silencing. In the virological model plant, Nicotiana benthamiana, functional studies of RDR6 has so far only been depended on RNAi based methodologies. These techniques however have inherent limitations, especially in the context of antiviral RNA silencing. To overcome this issue, we created rdr6 mutant N. benthamiana by the CRISPR/Cas9 genome editing system. Using the mutant, most of the proposed functions of RDR6 was confirmed. Additionally, the rdr6 N. benthamiana plant recapitulated closely the phenotype of the equivalent Arabidopsis mutant. In summary, the rdr6 N. benthamiana described here may be employed as a model system not only for the better understanding of the role of RDR6 in pathogen elicited immune responses but in various developmental processes as well.

Expression and antiviral function of ARGONAUTE 2 in Nicotiana benthamiana plants infected with two isolates of tomato ringspot virus with varying degrees of virulence.

ARGONAUTEs (notably AGO1 and AGO2) are effectors of plant antiviral RNA silencing. AGO1 was shown to be required for the temperature-dependent symptom recovery of Nicotiana benthamiana plants infected with tomato ringspot virus (isolate ToRSV-Rasp1) at 27 °C. In this study, we show that symptom recovery from isolate ToRSV-GYV shares similar hallmarks of antiviral RNA silencing but occurs at a wider range of temperatures (21-27 °C). At 21 °C, an early spike in AGO2 mRNAs accumulation was observed in plants infected with either ToRSV-Rasp1 or ToRSV-GYV but the AGO2 protein was only consistently detected in ToRSV-GYV infected plants. Symptom recovery from ToRSV-GYV at 21 °C was not prevented in an ago2 mutant or by silencing of AGO1 or AGO2. We conclude that other factors (possibly other AGOs) contribute to symptom recovery under these conditions. The results also highlight distinct expression patterns of AGO2 in response to ToRSV isolates and environmental conditions.

Establishment of an In Vivo ARGONAUTE Reporter System in Plants.

RNA silencing is not only an evolutionarily conserved gene regulatory mechanism, but in plants also serves as the basis for robust adaptive antiviral immune responses. ARGONAUTE (AGO) proteins form the catalytic cores of the RNA-guided ribonuclease complexes, which play a central role in RNA silencing. Here we describe an in vivo assay system for analyzing the activities of AGO proteins in the virological model plant Nicotiana benthamiana .

Crispr/Cas9 Mediated Inactivation of Argonaute 2 Reveals its Differential Involvement in Antiviral Responses.

RNA silencing constitutes an important antiviral mechanism in plants. Small RNA guided Argonaute proteins fulfill essential role in this process by acting as executors of viral restriction. Plants encode multiple Argonaute proteins of which several exhibit antiviral activities. A recent addition to this group is AGO2. Its involvement in antiviral responses is established predominantly by studies employing mutants of Arabidopsis thaliana. In the virological model plant, Nicotiana benthamiana, the contribution of AGO2 to antiviral immunity is much less certain due to the lack of appropriate genetic mutants. Previous studies employed various RNAi based tools to down-regulate AGO2 expression. However, these techniques have several disadvantages, especially in the context of antiviral RNA silencing. Here, we have utilized the CRISPR/Cas9 technology to inactivate the AGO2 gene of N. benthamiana. The ago2 plants exhibit differential sensitivities towards various viruses. AGO2 is a critical component of the plants' immune responses against PVX, TuMV and TCV. In contrast, AGO2 deficiency does not significantly influence the progression of tombusvirus and CMV infections. In summary, our work provides unequivocal proof for the virus-specific antiviral role of AGO2 in a plant species other than A. thaliana for the first time.

Functional dissection of a plant Argonaute.

RNA guided ribonuclease complexes play central role in RNA interference. Members of the evolutionarily conserved Argonaute protein family form the catalytic cores of these complexes. Unlike a number of other plant Argonautes, the role of AGO2 has been obscure until recently. Newer data, however, have indicated its involvement in various biotic and abiotic stress responses. Despite its suggested importance, there is no detailed characterization of this protein to date. Here we report cloning and molecular characterization of the AGO2 protein of the virological model plant Nicotiana benthamiana. We show that AGO2 can directly repress translation via various miRNA target site constellations (ORF, 3' UTR). Interestingly, although AGO2 seems to be able to silence gene expression in a slicing independent fashion, its catalytic activity is still a prerequisite for efficient translational repression. Additionally, mismatches between the 3' end of the miRNA guide strand and the 5' end of the target site enhance gene silencing by AGO2. Several functionally important amino acid residues of AGO2 have been identified that affect its small RNA loading, cleavage activity, translational repression potential and antiviral activity. The data presented here help us to understand how AGO2 aids plants to deal with stress.

The fate of the primary cilium during myofibroblast transition.

Myofibroblasts, the culprit of organ fibrosis, can originate from mesenchymal and epithelial precursors through fibroblast-myofibroblast and epithelial-myofibroblast transition (EMyT). Because certain ciliopathies are associated with fibrogenesis, we sought to explore the fate and potential role of the primary cilium during myofibroblast formation. Here we show that myofibroblast transition from either precursor results in the loss of the primary cilium. During EMyT, initial cilium growth is followed by complete deciliation. Both EMyT and cilium loss require two-hit conditions: disassembly/absence of intercellular contacts and transforming growth factor-ß1 (TGFß) exposure. Loss of E-cadherin-dependent junctions induces cilium elongation, whereas both stimuli are needed for deciliation. Accordingly, in a scratch-wounded epithelium, TGFß provokes cilium loss exclusively along the wound edge. Increased contractility, a key myofibroblast feature, is necessary and sufficient for deciliation, since constitutively active RhoA, Rac1, or myosin triggers, and down-regulation of myosin or myocardin-related transcription factor prevents, this process. Sustained myosin phosphorylation and consequent deciliation are mediated by a Smad3-, Rac1-, and reactive oxygen species-dependent process. Transitioned myofibroblasts exhibit impaired responsiveness to platelet-derived growth factor-AA and sonic hedgehog, two cilium-associated stimuli. Although the cilium is lost during EMyT, its initial presence contributes to the transition. Thus myofibroblasts represent a unique cilium-less entity with profoundly reprogrammed cilium-related signaling.

Role of SUMO modification of human PCNA at stalled replication fork.

DNA double-strand breaks (DSBs) can be generated not only by reactive agents but also as a result of replication fork collapse at unrepaired DNA lesions. Whereas ubiquitylation of proliferating cell nuclear antigen (PCNA) facilitates damage bypass, modification of yeast PCNA by small ubiquitin-like modifier (SUMO) controls recombination by providing access for the Srs2 helicase to disrupt Rad51 nucleoprotein filaments. However, in human cells, the roles of PCNA SUMOylation have not been explored. Here, we characterize the modification of human PCNA by SUMO in vivo as well as in vitro. We establish that human PCNA can be SUMOylated at multiple sites including its highly conserved K164 residue and that SUMO modification is facilitated by replication factor C (RFC). We also show that expression of SUMOylation site PCNA mutants leads to increased DSB formation in the Rad18(-/-) cell line where the effect of Rad18-dependent K164 PCNA ubiquitylation can be ruled out. Moreover, expression of PCNA-SUMO1 fusion prevents DSB formation as well as inhibits recombination if replication stalls at DNA lesions. These findings suggest the importance of SUMO modification of human PCNA in preventing replication fork collapse to DSB and providing genome stability.

TTRAP is a novel component of the non-canonical TRAF6-TAK1 TGF-ß signaling pathway.

Transforming growth factor-ß (TGF-ß) principally relays its effects through the Smad pathway however, accumulating evidence indicate that alternative signaling routes are also employed by this pleiotropic cytokine. For instance recently, we have demonstrated that ligand occupied TGF-ß receptors can directly trigger the TRAF6-TAK1 signaling module, resulting in MAP kinase activation. Here we report identification of the adaptor molecule TTRAP as a novel component of this non-canonical TGF-ß pathway. We show that the protein associates with TGF-ß receptors and components of the TRAF6-TAK1 signaling module, resulting in differential regulation of TGF-ß activated p38 and NF-κB responses. Modulation of cellular TTRAP level affects cell viability in the presence of TGF-ß, suggesting that the protein is an important component of the TGF-ß induced apoptotic process.

TRAF6 mediates Smad-independent activation of JNK and p38 by TGF-beta.

In many physiological and disease processes, TGF-beta usurps branches of MAP kinase pathways in conjunction with Smads to induce apoptosis and epithelial-to-mesenchymal transition, but the detailed mechanism of how a MAP kinase cascade is activated by TGF-beta receptors is not clear. We report here that TRAF6 is specifically required for the Smad-independent activation of JNK and p38, and its carboxyl TRAF homology domain physically interacts with TGF-beta receptors. TGF-beta induces K63-linked ubiquitination of TRAF6 and promotes association between TRAF6 and TAK1. Our results indicate that TGF-beta activates JNK and p38 through a mechanism similar to that operating in the interleukin-1beta/Toll-like receptor pathway.

Proteasomal ATPases are associated with rDNA: the ubiquitin proteasome system plays a direct role in RNA polymerase I transcription.

Significant amount of data have accumulated in the last several years pointing to the essential role of the ubiquitin proteasome system in the regulation of RNA polymerase II transcription; however, its involvement in RNA polymerase I transcription has remained largely unexplored. In this study, we demonstrate that proteasome activity is required for pre-rRNA synthesis. We can detect the association of proteasomal ATPases with both the rDNA promoter and coding region. Additionally, we show that the RNA polymerase I associated transcription factor, TIF-IA interacts with proteasomal ATPases, representing a potential link via which proteasomes and/or proteasome related complexes are recruited to rRNA genes. In summary, our findings suggest that the ubiquitin proteasome system is directly involved in RNA polymerase I transcription in analogy to the RNA polymerase II system.

Human HLTF functions as a ubiquitin ligase for proliferating cell nuclear antigen polyubiquitination.

Human helicase-like transcription factor (HLTF) is frequently inactivated in colorectal and gastric cancers. Here, we show that HLTF is a functional homologue of yeast Rad5 that promotes error-free replication through DNA lesions. HLTF and Rad5 share the same unique structural features, including a RING domain embedded within a SWI/SNF helicase domain and an HIRAN domain. We find that inactivation of HLTF renders human cells sensitive to UV and other DNA-damaging agents and that HLTF complements the UV sensitivity of a rad5Delta yeast strain. Also, similar to Rad5, HLTF physically interacts with the Rad6-Rad18 and Mms2-Ubc13 ubiquitin-conjugating enzyme complexes and promotes the Lys-63-linked polyubiquitination of proliferating cell nuclear antigen at its Lys-164 residue. A requirement of HLTF for error-free postreplication repair of damaged DNA is in keeping with its cancer-suppression role.