The molecular arms race: mechanisms of fungal antiviral defense and viral counterstrategies.

Combinatorial RNAi: A Winning Strategy for the Race Against Evolving Targets?

Summary The coat protein (CP) of Melon necrotic spot virus (MNSV) is structurally composed of three major domains. The middle S‐domain builds a robust protein shell around the viral genome, whereas the C‐terminal protruding domain, or P‐domain, is involved in the attachment of virions to the transmission vector. Here, we have shown that the N‐terminal domain, or R‐domain, and the arm region, which connects the R‐domain and S‐domain, are involved in different key steps of the viral cycle, such as cell‐to‐cell movement and the suppression of RNA silencing and pathogenesis through their RNA‐binding capabilities. Deletion mutants revealed that the CP RNA‐binding ability was abolished only after complete, but not partial, deletion of the R‐domain and the arm region. However, a comparison of the apparent dissociation constants for the CP RNA‐binding reaction of several partial deletion mutants showed that the arm region played a more relevant role than the R‐domain in in vitro RNA binding. Similar results were obtained in in vivo assays, although, in this case, full‐length CPs were required to encapsidate full‐length genomes. We also found that the R‐domain carboxyl portion and the arm region were essential for efficient cell‐to‐cell movement, for enhancement of Potato virus X pathogenicity, for suppression of systemic RNA silencing and for binding of small RNAs. Therefore, unlike other carmovirus CPs, the R‐domain and the arm region of MNSV CP have acquired, in addition to other essential functions such as genome binding and encapsidation functions, the ability to suppress RNA silencing by preventing systemic small RNA transport.

Viral RNA silencing suppressors (RSS): Novel strategy of viruses to ablate the host RNA interference (RNAi) defense system

The RNA interference (RNAi) pathway has evolved numerous functionalities in eukaryotes, with many on display in Kingdom Fungi. RNAi can regulate gene expression, facilitate drug resistance, or even be altogether lost to improve growth potential in some fungal pathogens. In the WHO fungal priority pathogen, Aspergillus fumigatus, the RNAi system is known to be intact and functional. To extend our limited understanding of A. fumigatus RNAi, we first investigated the genetic variation in RNAi-associated genes in a collection of 217 environmental and 83 clinical genomes, where we found that RNAi components are conserved even in clinical strains. Using endogenously expressed inverted-repeat transgenes complementary to a conditionally essential gene (pabA) or a nonessential gene (pksP), we determined that a subset of the RNAi componentry is active in inverted-repeat transgene silencing in conidia and mycelium. Analysis of mRNA-seq data from RNAi double-knockout strains linked the A. fumigatus dicer-like enzymes (DclA/B) and RNA-dependent RNA polymerases (RrpA/B) to regulation of conidial ribosome biogenesis genes; however, surprisingly few endogenous small RNAs were identified in conidia that could explain this broad change. Although RNAi was not clearly linked to growth or stress response defects in the RNAi knockouts, serial passaging of RNAi knockout strains for six generations resulted in lineages with diminished spore production over time, indicating that loss of RNAi can exert a fitness cost on the fungus. Cumulatively, A. fumigatus RNAi appears to play an active role in defense against double-stranded RNA species alongside a previously unappreciated housekeeping function in regulation of conidial ribosomal biogenesis genes.

RNA interference (RNAi) has emerged as a promising tool for controlling fungal plant pathogens, offering a targeted and environmentally friendly alternative to traditional chemical fungicides. Botrytis cinerea, the causative agent of gray mold disease, serves as a model and plant pathogen for investigating RNAi-based strategies due to its wide host range and economic impact. This review synthesizes current knowledge on RNAi mechanisms in B. cinerea, and that several factors influence the efficacy of RNAi in B. cinerea, including the stability and uptake of double-stranded RNAs (dsRNAs), the efficiency of RNA processing machinery, and environmental conditions. Furthermore, RNAi responses can vary significantly across strains, developmental stages, and infection modes, underscoring the complexity of fungal responses. With this review, I also aim to present the field trials reported so far, underscoring the practicality of RNAi. This review identifies current hotspots and outlines future directions for deploying RNAi as a sustainable control strategy against fungal pathogens.

The genome of the fungal wheat pathogen Zymoseptoria tritici consists of thirteen essential chromosomes and several so-called dispensable chromosomes. These dispensable chromosomes encode only 6% of the protein coding genes of Z. tritici. To date no genes involved in pathogenicity are described on the dispensable chromosomes which can be lost after meiosis or mitotic cell division without any apparent effect on fitness. To investigate the underlying molecular basis of instability of the dispensable chromosomes, the epigenetic components of both the essential and dispensable chromosomes were characterized here. Chromatin immunoprecipitation and sequencing of DNA associated with the centromere specific histone (CenH3) was conducted to identify the centromeres of Z. tritici. It was shown that the centromeres of Z. tritici are small, sequence independent and lack any conserved motif. The centromeres are AT-rich, but not located in the most abundant AT-rich region of the chromosomes, and the centromeric organization is similar for both essential and dispensable chromosomes. To study centromere dynamic, parental and progeny strains derived from a meiotic cross were included in the study. The centromeres of these strains were shown to be conserved among Z. tritici strains. The deletion of the centromere of the dispensable chromosome 14 resulted in several strains were chromosome 14 was completely lost, while only a single strain was identified with a neocentromere on chromosome 14. The chromatin content of both types of chromosomes was also investigated. Three histone modifications specific for either euchromatin or heterochromatin were characterized. The essential chromosomes are enriched with euchromatin while the dispensable chromosomes are mainly heterochromatic. Several repeat rich regions with low gene density were also enriched with heterochromatin on the essential chromosomes. One particularly large region of 780 kb of the essential chromosome 7 was in addition found to be enriched with facultative heterochromatin. Genes in this region are silenced both during axenic and infectious growth. Based on the obtained results, it can be concluded that the difference between the essential and dispensable chromosomes cannot be associated with the centromeres. However, differences in the chromatin states is a main difference between the two types of chromosomes. To investigate the hemibiotrophic lifestyle switch in Z. tritici the epigenetic component of infectious growth was studied with a focus on RNA interference (RNAi). Five mutant strains of several proteins involved in the RNAi pathway were created. It could be demonstrated that Dicer and Argonaute genes play a role during the formation of asexual fruiting bodies called pycnidia. In contrast to the Dicer gene, the Argonaute genes show an unusual degree of sequence variation among Z. tritici strains. Collectively, the work presented here underlines the importance of epigenetics in both genome stability as well as pathogenicity in the fungal pathogen Z. tritici.%%%%Das…

RNA interference (RNAi) is an ancient, natural process conserved among species from different kingdoms. RNAi is a transcriptional and post-transcriptional gene silencing mechanism in which, double-stranded RNA or hairpin RNA is cleaved by an RNase III-type enzyme called Dicer into small interfering RNA duplex. This subsequently directs sequence-specific, homology dependent, Watson-Crick base-pairing post-transcriptional gene silencing by binding to its complementary RNA and initiating its elimination through degradation or by persuading translational inhibition. In plants, worms, and insects, RNAi is the main and strong antiviral defense mechanism. It is clear that RNAi silencing, contributes in restriction of viral infection in vertebrates. In a short period, RNAi has progressed to become a significant experimental tool for the analysis of gene function and target validation in mammalian systems. In addition, RNA silencing has then been found to be involved in translational repression, transcriptional inhibition, and DNA degradation. RNAi machinery required for robust RNAi-mediated antiviral response are conserved throughout evolution in mammals and plays a crucial role in antiviral defense of invertebrates, but despite these important functions RNAi contribution to mammalian antiviral innate immune defense has been underestimated and disputed. In this article, we review the literature concerning the roles of RNAi as components of innate immune system in mammals and how, the RNAi is currently one of the most hopeful new advances toward disease therapy. This review highlights the potential of RNAi as a therapeutic strategy for viral infection and gene regulation to modulate host immune response to viral infection.

The 2b proteins encoded by cucumovirus act as post-transcriptional gene silencing suppressors to counter host defence during infection. Here we report the crystal structure of Tomato aspermy virus 2b (TAV2b) protein bound to a 19 bp small interfering RNA (siRNA) duplex. TAV2b adopts an all alpha-helix structure and forms a homodimer to measure siRNA duplex in a length-preference mode. TAV2b has a pair of hook-like structures to recognize simultaneously two alpha-helical turns of A-form RNA duplex by fitting its alpha-helix backbone into two adjacent major grooves of siRNA duplex. The conserved pi-stackings between tryptophan and the 5'-terminal base of siRNA duplex from both ends enhance the recognition. TAV2b further oligomerizes to form a dimer of dimers through the conserved leucine-zipper-like motif at its amino-terminal alpha-helix. Biochemical experiments suggest that TAV2b might interfere with the post-transcriptional gene silencing pathway by directly binding to siRNA duplex.

Plants, fungi, and many other eukaryotes have evolved an RNA interference (RNAi) mechanism that is key for regulating gene expression and the control of pathogens. RNAi inhibits gene expression, in a sequence-specific manner, by recognizing and deploying cognate double-stranded RNA (dsRNA) either from endogenous sources (e.g. pre-micro RNAs) or exogenous origin (e.g. viruses, dsRNA, or small interfering RNAs, siRNAs). Recent studies have demonstrated that fungal pathogens can transfer siRNAs into plant cells to suppress host immunity and aid infection, in a mechanism termed cross-kingdom RNAi. New technologies, based on RNAi are being developed for crop protection against insect pests, viruses, and more recently against fungal pathogens. One example, is host-induced gene silencing (HIGS), which is a mechanism whereby transgenic plants are modified to produce siRNAs or dsRNAs targeting key transcripts of plants, or their pathogens or pests. An alternative gene regulation strategy that also co-opts the silencing machinery is spray-induced gene silencing (SIGS), in which dsRNAs or single-stranded RNAs (ssRNAs) are applied to target genes within a pathogen or pest. Fungi also use their RNA silencing machinery against mycoviruses (fungal viruses) and mycoviruses can deploy virus-encoded suppressors of RNAi (myco-VSRs) as a counter-defence. We propose that myco-VSRs may impact new dsRNA-based management methods, resulting in unintended outcomes, including suppression of management by HIGS or SIGS. Despite a large diversity of mycoviruses being discovered using high throughput sequencing, their biology is poorly understood. In particular, the prevalence of mycoviruses and the cellular effect of their encoded VSRs are under-appreciated when considering the deployment of HIGS and SIGS strategies. This review focuses on mycoviruses, their VSR activities in fungi, and the implications for control of pathogenic fungi using RNAi.

ABSTRACTRapid evolution of fungal pathogens poses a serious threat to medicine and agriculture. The mutation rate determines the pace of evolution of a fungal pathogen. Hypermutator fungal strains have an elevated mutation rate owing to certain defects such as those in the DNA mismatch repair system. Studies in Saccharomyces cerevisiae show that hypermutators expedite evolution by generating beneficial alleles at a faster pace than the wild-type strains. However, an accumulation of deleterious alleles in a hypermutator may reduce its fitness. The balance between fitness cost and mutation benefit determines the prevalence of hypermutators in a population. This balance is affected by a complex interaction of ploidy, mode of reproduction, population size, and recent population history. Studies in human fungal pathogens like Aspergillus fumigatus, Candida albicans, Candida glabrata, Cryptococcus deuterogattii, and Cryptococcus neoformans have highlighted the importance of hypermutators in host adaptation and development of antifungal resistance. However, a critical examination of hypermutator biology, experimental evolution studies, and epidemiological studies suggests that hypermutators may impact evolutionary investigations. This review aims to integrate the knowledge about biology, experimental evolution, and dynamics of fungal hypermutators to critically examine the evolutionary role of hypermutators in fungal pathogen populations and project implications of hypermutators in the evolution of fungal plant pathogen populations. Understanding the factors determining the emergence and evolution of fungal hypermutators can open a novel avenue of managing rapidly evolving fungal pathogens in medicine and agriculture.

The question of whether RNA interference (RNAi) acts as an antiviral mechanism in mammalian cells remains controversial. The antiviral interferon (IFN) response cannot easily be distinguished from a possible antiviral RNAi pathway owing to the involvement of double-stranded RNA (dsRNA) as a common inducer molecule. The non-structural protein 3 (NS3) protein of rice hoja blanca virus (RHBV) is an RNA silencing suppressor (RSS) that exclusively binds to small dsRNA molecules. Here, we show that this plant viral RSS lacks IFN antagonistic activity, yet it is able to substitute the RSS function of the Tat protein of human immunodeficiency virus type 1. An NS3 mutant that is deficient in RNA binding and its associated RSS activity is inactive in this complementation assay. This cross-kingdom suppression of RNAi in mammalian cells by a plant viral RSS indicates the significance of the antiviral RNAi response in mammalian cells and the usefulness of well-defined RSS proteins.

Several proteins and RNAs expressed by mammalian viruses have been reported to interfere with RNA interference (RNAi) activity. We investigated the ability of the HIV-1-encoded RNA elements Trans-Activation Response (TAR) and Rev-Response Element (RRE) to alter RNAi. MicroRNA let7-based assays showed that RRE is a potent suppressor of RNAi activity, while TAR displayed moderate RNAi suppression. We demonstrate that RRE binds to TAR-RNA Binding Protein (TRBP), an essential component of the RNA Induced Silencing Complex (RISC). The binding of TAR and RRE to TRBP displaces small interfering (si)RNAs from binding to TRBP. Several stem-deleted RRE mutants lost their ability to suppress RNAi activity, which correlated with a reduced ability to compete with siRNA-TRBP binding. A lentiviral vector expressing TAR and RRE restricted RNAi, but RNAi was restored when Rev or GagPol were coexpressed. Adenoviruses are restricted by RNAi and encode their own suppressors of RNAi, the Virus-Associated (VA) RNA elements. RRE enhanced the replication of wild-type and VA-deficient adenovirus. Our work describes RRE as a novel suppressor of RNAi that acts by competing with siRNAs rather than by disrupting the RISC. This function is masked in lentiviral vectors co-expressed with viral proteins and thus will not affect their use in gene therapy. The potent RNAi suppressive effects of RRE identified in this study could be used to enhance the expression of RNAi restricted viruses used in oncolysis such as adenoviruses.

West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteins to block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in mosquitoes depleted for RNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whether or not flaviviruses counteract RNAi via expression of an RSS. First, we established that flaviviral RNA replication suppressed siRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encoded proteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast, we found that the 3'-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressed siRNA- and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibits in vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as a nucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses.

RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation.

Polymorphism of het-genes prevents resource plundering in Neurospora crassa