Counter Defense Research Articles

Strong Gene Flow Undermines Local Adaptations in a Host Parasite System.

Simple SummaryThe co-evolution of hosts and parasites depends on their ability to adapt to each other’s defense and counter-defense mechanisms. The strength of selection on those mechanisms may vary among populations, resulting in a geographical mosaic of co-evolution. The boreo-montane paper wasp Polistes biglumis and its parasite Polistes atrimandibularis exemplify this type of co-evolutionary system. Here, we used genetic markers to examine the genetic population structures of these wasps in the western Alps. We found that both host and parasite populations displayed similar levels of genetic variation. In the host species, populations located near to each other were genetically similar; in both the host and the parasite species populations farther apart were significantly different. Thus, apparent dispersal barriers (i.e., high mountains) did not seem to restrict gene flow across populations as expected. Furthermore, there were no major differences in gene flow between the two species, perhaps because P. atrimandibularis parasitizes both alpine and lowland host species and annually migrates between alpine and lowland populations. The presence of strong gene flow in a system where local populations experience variable levels of selection pressure challenges the classical hypothesis that restricted gene flow is required for local adaptations to evolve.The co-evolutionary pathways followed by hosts and parasites strongly depend on the adaptive potential of antagonists and its underlying genetic architecture. Geographically structured populations of interacting species often experience local differences in the strength of reciprocal selection pressures, which can result in a geographic mosaic of co-evolution. One example of such a system is the boreo-montane social wasp Polistes biglumis and its social parasite Polistes atrimandibularis, which have evolved local defense and counter-defense mechanisms to match their antagonist. In this work, we study spatial genetic structure of P. biglumis and P. atrimandibularis populations at local and regional scales in the Alps, by using nuclear markers (DNA microsatellites, AFLP) and mitochondrial sequences. Both the host and the parasite populations harbored similar amounts of genetic variation. Host populations were not genetically structured at the local scale, but geographic regions were significantly differentiated from each other in both the host and the parasite in all markers. The net dispersal inferred from genetic differentiation was similar in the host and the parasite, which may be due to the annual migration pattern of the parasites between alpine and lowland populations. Thus, the apparent dispersal barriers (i.e., high mountains) do not restrict gene flow as expected and there are no important gene flow differences between the species, which contradict the hypothesis that restricted gene flow is required for local adaptations to evolve.

Identification of the RNA silencing suppressor activity of sugarcane streak mosaic virus P1 gene.

Sugarcane streak mosaic virus (SCSMV) belonging to Poacevirus, is a causative virus of mosaic disease in sugarcane in many Asian countries with substantial genomic variation. Although the virus infects the crop with Sugarcane mosaic virus (SCMV) a Potyvirus, it predominates over SCMV in spread as well as titre. We have taken up detailed studies to identify the functional activity of viral suppressors of SCSMV genome. Transient expression assay was performed with SCSMV-P1 and HC-Pro genes in the model plant Nicotiana tabacum to establish suppressor role of these genes. The plasmid constructs of both the genes were co-infiltrated with the reporter green fluorescent protein (GFP) and the suppressor activity was measured as enhancement in the GFP fluorescence. Further, the phenotypic expressions were validated by respective gene expression through semi quantitative and qRT-PCR. In the P1 co-infiltrated GFP leaves, suppression in the PTGS mechanism took place that allowed a long term expression of GFP. However, GFP co-infiltrated with HC-Pro did not sustain the GFP expression level for a prolonged period and the expression level was close to GFP control. The study concluded that unlike in other Potyviridae genera, P1 gene of SCSMV is playing the role of RNA silencing suppressor. This study helps in unveiling a new and promising way to understand the regulatory pathway in the host at the time of viral infection. Targeting the P1 gene of SCSMV through RNA silencing approach will be a viable strategy to develop mosaic resistant transgenic sugarcane varieties as they are directly involved in counter defence against the host.

Abortive Infection: Bacterial Suicide as an Antiviral Immune Strategy.

Facing frequent phage challenges, bacteria have evolved numerous mechanisms to resist phage infection. A commonly used phage resistance strategy is abortive infection (Abi), in which the infected cell commits suicide before the phage can complete its replication cycle. Abi prevents the phage epidemic from spreading to nearby cells, thus protecting the bacterial colony. The Abi strategy is manifested by a plethora of mechanistically diverse defense systems that are abundant in bacterial genomes. In turn, phages have developed equally diverse mechanisms to overcome bacterial Abi. This review summarizes the current knowledge on bacterial defense via cell suicide. It describes the principles of Abi, details how these principles are implemented in a variety of natural defense systems, and discusses phage counter-defense mechanisms.

Visual acuity and egg spatial chromatic contrast predict egg rejection behavior of American robins.

Color and spatial vision is critical for recognition and discrimination tasks affecting fitness, including finding food and mates, and recognizing offspring. For example, as a counter defense to avoid the cost of raising the unrelated offspring of obligate interspecific avian brood parasites, many host species routinely view, recognize and remove the foreign egg(s) from their nests. Recent research has shown that host species visually attend to both chromatic and spatial pattern features of eggs; yet how hosts simultaneously integrate these features together when recognizing eggs remains an open question. Here, we tested egg rejection responses of American robins (Turdus migratorius) using a range of 3D-printed model eggs covered with blue and yellow checkered patterns differing in relative square sizes. We predicted that robins would reject a model egg if they could visually resolve the blue and yellow squares as separate features, or accept it if the squares blended together and appeared similar in color to the natural blue-green color of robin eggs as perceived by the avian visual system. As predicted, the probability of robins rejecting a model egg increased with greater sizes of its blue and yellow squares. Our results suggest that chromatic visual acuity and viewing distance have the potential to limit the ability of a bird to recognize a foreign egg in its nest, thus providing a limitation to host egg recognition that obligate interspecific avian brood parasites may exploit.

The counter defence system of antioxidants in Coelomycetous emerging human and plant pathogenic fungus Macrophomina phaseolina against copper toxicity.

Macrophomina phaseolina (Tassi) Goid. is primarily a phytopathogen but also exhibits potential to cause infection in human being due to its aggressive and robust nature. Adaptation potential in M. phaseolina was assessed after exposing to heavy metal copper through analysing macro- and microscopic as well as physiological attributes. Considerable modifications were observed in morphology with an increase in concentrations (25, 75, and 100ppm) of copper as compared with control. Total content of protein and activities of the antioxidant enzymes were affected differently at different copper concentrations. The fungal biomass, metal accumulation, metal uptake efficiency, and bioaccumulation factors were decreased due to the increase in copper concentrations. Fourier transformed infrared spectroscopic assessment showed hydroxyl, carboxyl, and amine groups as major metal accumulation sites. It is concluded that Cu-based fungicides can manage this pathogen but excessive application may accumulate in the plant that will transfer along the food chain, reaching ultimately the human body.

Ty-1, a universal resistance gene against geminiviruses that is compromised by co-replication of a betasatellite.

SummaryTomato yellow leaf curl virus (TYLCV), a begomovirus, causes large yield losses and breeding for resistance is an effective way to combat this viral disease. The resistance gene Ty‐1 codes for an RNA‐dependent RNA polymerase and has recently been shown to enhance transcriptional gene silencing of TYLCV. Whereas Ty‐1 was earlier shown to also confer resistance to a bipartite begomovirus, here it is shown that Ty‐1 is probably generic to all geminiviruses. A tomato Ty‐1 introgression line, but also stable transformants of susceptible tomato cv. Moneymaker and Nicotiana benthamiana (N. benthamiana) expressing the Ty‐1 gene, exhibited resistance to begomoviruses as well as to the distinct, leafhopper‐transmitted beet curly top virus, a curtovirus. Stable Ty‐1 transformants of N. benthamiana and tomato showed fewer symptoms and reduced viral titres on infection compared to wild‐type plants. TYLCV infections in wild‐type N. benthamiana plants in the additional presence of a betasatellite led to increased symptom severity and a consistent, slightly lowered virus titre relative to the high averaged levels seen in the absence of the betasatellite. On the contrary, in Ty‐1 transformed N. benthamiana viral titres increased in the presence of the betasatellite. The same was observed when these Ty‐1‐encoding plants were challenged with TYLCV and a potato virus X construct expressing the RNA interference suppressor protein βC1 encoded by the betasatellite. The resistance spectrum of Ty‐1 and the durability of the resistance are discussed in light of antiviral RNA interference and viral counter defence strategies.

Glycosidase and glycan polymorphism control hydrolytic release of immunogenic flagellin peptides.

Plants and animals recognize conserved flagellin fragments as a signature of bacterial invasion. These immunogenic elicitor peptides are embedded in the flagellin polymer and require hydrolytic release before they can activate cell surface receptors. Although much of flagellin signaling is understood, little is known about the release of immunogenic fragments. We discovered that plant-secreted β-galactosidase 1 (BGAL1) of Nicotiana benthamiana promotes hydrolytic elicitor release and acts in immunity against pathogenic Pseudomonas syringae strains only when they carry a terminal modified viosamine (mVio) in the flagellin O-glycan. In counter defense, P. syringae pathovars evade host immunity by using BGAL1-resistant O-glycans or by producing a BGAL1 inhibitor. Polymorphic glycans on flagella are common to plant and animal pathogenic bacteria and represent an important determinant of host immunity to bacterial pathogens.

Paving the Way to Tospovirus Infection: Multilined Interplays with Plant Innate Immunity.

Tospoviruses are among the most important plant pathogens and cause serious crop losses worldwide. Tospoviruses have evolved to smartly utilize the host cellular machinery to accomplish their life cycle. Plants mount two layers of defense to combat their invasion. The first one involves the activation of an antiviral RNA interference (RNAi) defense response. However, tospoviruses encode an RNA silencing suppressor that enables them to counteract antiviral RNAi. To further combat viral invasion, plants also employ intracellular innate immune receptors (e.g., Sw-5b and Tsw) to recognize different viral effectors (e.g., NSm and NSs). This leads to the triggering of a much more robust defense against tospoviruses called effector-triggered immunity (ETI). Tospoviruses have further evolved their effectors and can break Sw-5b-/Tsw-mediated resistance. The arms race between tospoviruses and both layers of innate immunity drives the coevolution of host defense and viral genes involved in counter defense. In this review, a state-of-the-art overview is presented on the tospoviral life cycle and the multilined interplays between tospoviruses and the distinct layers of defense.

Defence and counter defence.

This study shows that a plant pathogen uses extracellular DNases as a defence mechanism against host-secreted extracellular DNA.

A DNase from a Fungal Phytopathogen Is a Virulence Factor Likely Deployed as Counter Defense against Host-Secreted Extracellular DNA.

Histone-linked extracellular DNA (exDNA) is a component of neutrophil extracellular traps (NETs). NETs have been shown to play a role in immune response to bacteria, fungi, viruses, and protozoan parasites. Mutation of genes encoding group A Streptococcus extracellular DNases (exDNases) results in reduced virulence in animals, a finding that implies that exDNases are deployed as counter defense against host DNA-containing NETs. Is the exDNA/exDNase mechanism also relevant to plants and their pathogens? It has been demonstrated previously that exDNA is a component of a matrix secreted from plant root caps and that plants also carry out an extracellular trapping process. Treatment with DNase I destroys root tip resistance to infection by fungi, the most abundant plant pathogens. We show that the absence of a single gene encoding a candidate exDNase results in significantly reduced virulence of a fungal plant pathogen to its host on leaves, the known infection site, and on roots. Mg2+-dependent exDNase activity was demonstrated in fungal culture filtrates and induced when host leaf material was present. It is speculated that the enzyme functions to degrade plant-secreted DNA, a component of a complex matrix akin to neutrophil extracellular traps of animals.IMPORTANCE We document that the absence of a single gene encoding a DNase in a fungal plant pathogen results in significantly reduced virulence to a plant host. We compared a wild-type strain of the maize pathogen Cochliobolus heterostrophus and an isogenic mutant lacking a candidate secreted DNase-encoding gene and demonstrated that the mutant is reduced in virulence on leaves and on roots. There are no previous reports of deletion of such a gene from either an animal or plant fungal pathogen accompanied by comparative assays of mutants and wild type for alterations in virulence. We observed DNase activity, in fungal culture filtrates, that is Mg2+ dependent and induced when plant host leaf material is present. Our findings demonstrate not only that fungi use extracellular DNases (exDNases) for virulence, but also that the relevant molecules are deployed in above-ground leaves as well as below-ground plant tissues. Overall, these data provide support for a common defense/counter defense virulence mechanism used by animals, plants, and their fungal and bacterial pathogens and suggest that components of the mechanism might be novel targets for the control of plant disease.

RNA-Seq Analyses of Midgut and Fat Body Tissues Reveal the Molecular Mechanism Underlying Spodoptera litura Resistance to Tomatine.

Plants produce secondary metabolites to provide chemical defense against herbivorous insects, whereas insects can induce the expression of detoxification metabolism-related unigenes in counter defense to plant xenobiotics. Tomatine is an important secondary metabolite in tomato (Lycopersicon esculentum L.) that can protect the plant from bacteria and insects. However, the mechanism underlying the adaptation of Spodoptera litura, a major tomato pest, to tomatine in tomato is largely unclear. In this study, we first found that the levels of tomatine in tomatoes subjected to S. litura treatment were significantly increased. Second, we confirmed the inhibitory effect of tomatine on S. litura by adding moderate amounts of commercial tomatine to an artificial diet. Then, we utilized RNA-Seq to compare the differentially expressed genes (DEGs) in the midgut and fat body tissues of S. litura exposed to an artificial diet supplemented with tomatine. In total, upon exposure to tomatine, 134 and 666 genes were upregulated in the S. litura midgut and fat body, respectively. These DEGs comprise a significant number of detoxification-related genes, including 7 P450 family genes, 8 glutathione S-transferases (GSTs) genes, 6 ABC transport enzyme genes, 9 UDP-glucosyltransferases genes and 3 carboxylesterases genes. Moreover, KEGG analysis demonstrated that the upregulated genes were enriched in xenobiotic metabolism by cytochrome P450s, ABC transporters and drug metabolism by other enzymes. Furthermore, as numerous GSTs were induced by tomatine in S. litura, we chose one gene, namely GSTS1, to confirm the detoxification function on tomatine. Expression profiling revealed that GSTS1 transcripts were mainly expressed in larvae, and the levels were the highest in the midgut. Finally, when larvae were injected with double-stranded RNA specific to GSTS1, the transcript levels in the midgut and fat body decreased, and the negative effect of the plant xenobiotic tomatine on larval growth was magnified. These results preliminarily clarified the molecular mechanism underlying the resistance of S. litura to tomatine, establishing a foundation for subsequent pest control.

Chemical-mediated counter defense: attraction of two parasitoid species to the defensive secretion of host larvae

Carnivorous animals put selection pressure on their herbivorous preys, leading to the development of defensive traits in prey species and counter defensive traits in carnivores—an evolutionary arms race for survival. For instance, larvae of the moth Heliothis virescens use defensive oral secretions to deter attacking parasitic wasps (parasitoids). However, it is not clear whether parasitoids also use the same secretion for host location. If carnivores follow the herbivore model, evolution should favor specialist over generalist parasitoid species in their ability to manipulate host defensive chemicals. To test this prediction, Microplitis croceipes and Cotesia marginiventris, both larval endoparasitoids of H. virescens, were used as a study system. Microplitis croceipes is relatively specialized on Heliothis/Helicoverpa species while C. marginiventris is a generalist parasitoid on several noctuid host species. Innate behavioral responses of parasitoids to host defensive secretion were tested in Petri dish arena and Y-tube olfactometer bioassays. Heliothis virescens defensive secretion was manually applied to a spot in a sector (secretion zone) of the arena. Tracking data suggests that both parasitoid species are likely to locate the secretion spot once they enter the secretion zone. Comparing the two species, however, M. croceipes (specialist) spent significantly more time in the secretion zone and spot than C. marginiventris (generalist). In Y-tube olfactometer bioassays, the secretion elicited a strong attraction in M. croceipes but not in C. marginiventris, supporting our prediction. We discussed chemical-mediated counter defense in natural enemies of pest insects and the significance of the results to parasitoid-host coevolution.

Phage Therapy Faces Evolutionary Challenges.

Antibiotic resistance evolution in bacteria indicates that one of the challenges faced by phage therapy is that, sooner or later, bacteria will evolve resistance to phages. Evidently, this is the case of every known antimicrobial therapy, but here this is also part of a ubiquitous natural process of co-evolution between phages and bacteria. Fundamental evolutionary studies hold some clues that are crucial to limit the problematic process of bacterial resistance during phage applications. First, I discuss here the importance of defining evolutionary and ecological factors influencing bacterial resistance and phage counter-defense mechanisms. Then, I comment on the interest of determining the co-evolutionary dynamics between phages and bacteria that may allow for selecting the conditions that will increase the probability of therapeutic success. I go on to suggest the varied strategies that may ensure the long-term success of phage therapy, including analysis of internal phage parameters and personalized treatments. In practical terms, these types of approaches will define evolutionary criteria regarding how to develop, and when to apply, therapeutic phage cocktails. Integrating this perspective in antimicrobial treatments, such as phage therapy, is among the necessary steps to expand its use in the near future, and to ensure its durability and success.

Structural insights into the inhibition mechanism of bacterial toxin LsoA by bacteriophage antitoxin Dmd.

Bacteria have obtained a variety of resistance mechanisms including toxin-antitoxin (TA) systems against bacteriophages (phages), whereas phages have also evolved to overcome bacterial anti-phage mechanisms. Dmd from T4 phage can suppress the toxicities of homologous toxins LsoA and RnlA from Escherichia coli, representing the first example of a phage antitoxin against multiple bacterial toxins in known TA systems. Here, the crystal structure of LsoA-Dmd complex showed Dmd is inserted into the deep groove between the N-terminal repeated domain (NRD) and the Dmd-binding domain (DBD) of LsoA. The NRD shifts significantly from a 'closed' to an 'open' conformation upon Dmd binding. Site-directed mutagenesis of Dmd revealed the conserved residues (W31 and N40) are necessary for LsoA binding and the toxicity suppression as determined by pull-down and cell toxicity assays. Further mutagenesis identified the conserved Dmd-binding residues (R243, E246 and R305) of LsoA are vital for its toxicity, and suggested Dmd and LsoB may possess different inhibitory mechanisms against LsoA toxicity. Our structure-function studies demonstrate Dmd can recognize LsoA and inhibit its toxicity by occupying the active site possibly via substrate mimicry. These findings have provided unique insights into the defense and counter-defense mechanisms between bacteria and phages in their co-evolution.

Modeling and Analysis of Attacks and Counter Defense Mechanisms for Cyber Physical Systems

In this paper, we develop an analytical model based on stochastic Petri nets to capture the dynamics between adversary behavior and defense for cyber physical systems. We consider several types of failures including attrition failure, pervasion failure, and exfiltration failure which can happen to a cyber physical system. Using a modernized electrical grid as an example, we illustrate the parameterization process. Our results reveal optimal design conditions, including the intrusion detection interval, and the redundancy level, under which the modernized electrical grid's mean time to failure is maximized. Further, there exists a design tradeoff between exfiltration failure, attrition failure, and pervasion failure when using redundancy to improve the overall system reliability.

Heavy Metals and Human Health: Mechanistic Insight into Toxicity and Counter Defense System of Antioxidants.

Heavy metals, which have widespread environmental distribution and originate from natural and anthropogenic sources, are common environmental pollutants. In recent decades, their contamination has increased dramatically because of continuous discharge in sewage and untreated industrial effluents. Because they are non-degradable, they persist in the environment; accordingly, they have received a great deal of attention owing to their potential health and environmental risks. Although the toxic effects of metals depend on the forms and routes of exposure, interruptions of intracellular homeostasis include damage to lipids, proteins, enzymes and DNA via the production of free radicals. Following exposure to heavy metals, their metabolism and subsequent excretion from the body depends on the presence of antioxidants (glutathione, α-tocopherol, ascorbate, etc.) associated with the quenching of free radicals by suspending the activity of enzymes (catalase, peroxidase, and superoxide dismutase). Therefore, this review was written to provide a deep understanding of the mechanisms involved in eliciting their toxicity in order to highlight the necessity for development of strategies to decrease exposure to these metals, as well as to identify substances that contribute significantly to overcome their hazardous effects within the body of living organisms.

Geminivirus-encoded TrAP suppressor inhibits the histone methyltransferase SUVH4/KYP to counter host defense.

Transcriptional gene silencing (TGS) can serve as an innate immunity against invading DNA viruses throughout Eukaryotes. Geminivirus code for TrAP protein to suppress the TGS pathway. Here, we identified an Arabidopsis H3K9me2 histone methyltransferase, Su(var)3-9 homolog 4/Kryptonite (SUVH4/KYP), as a bona fide cellular target of TrAP. TrAP interacts with the catalytic domain of KYP and inhibits its activity in vitro. TrAP elicits developmental anomalies phenocopying several TGS mutants, reduces the repressive H3K9me2 mark and CHH DNA methylation, and reactivates numerous endogenous KYP-repressed loci in vivo. Moreover, KYP binds to the viral chromatin and controls its methylation to combat virus infection. Notably, kyp mutants support systemic infection of TrAP-deficient Geminivirus. We conclude that TrAP attenuates the TGS of the viral chromatin by inhibiting KYP activity to evade host surveillance. These findings provide new insight on the molecular arms race between host antiviral defense and virus counter defense at an epigenetic level.

Function and diversity of P0 proteins among cotton leafroll dwarf virus isolates.

BackgroundThe RNA silencing pathway is an important anti-viral defense mechanism in plants. As a counter defense, some members of the viral family Luteoviridae are able to evade host immunity by encoding the P0 RNA silencing suppressor protein. Here we explored the functional diversity of P0 proteins among eight cotton leafroll dwarf virus (CLRDV) isolates, a virus associated with a worldwide cotton disease known as cotton blue disease (CBD).MethodsCLRDV-infected cotton plants of different varieties were collected from five growing fields in Brazil and their P0 sequences compared to three previously obtained isolates. P0’s silencing suppression activities were scored based on transient expression experiments in Nicotiana benthamiana leaves.ResultsHigh sequence diversity was observed among CLRDV P0 proteins, indicating that some isolates found in cotton varieties formerly resistant to CLRDV should be regarded as new genotypes within the species. All tested proteins were able to suppress local and systemic silencing, but with significantly variable degrees. All P0 proteins were able to mediate the decay of ARGONAUTE proteins, a key component of the RNA silencing machinery.ConclusionsThe sequence diversity observed in CLRDV P0s is also reflected in their silencing suppression capabilities. However, the strength of local and systemic silencing suppression was not correlated for some proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0356-7) contains supplementary material, which is available to authorized users.

Overexpression of potato miR482e enhanced plant sensitivity to Verticillium dahliae infection.

Verticillium wilt of potato is caused by the fungus pathogen Verticillium dahliae. Present sRNA sequencing data revealed that miR482 was in response to V. dahliae infection, but the function in potato is elusive. Here, we characterized potato miR482 family and its putative role resistance to Verticillium wilt. Members of the potato miR482 superfamily are variable in sequence, but all variants target a class of disease-resistance proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) motifs. When potato plantlets were infected with V. dahliae, the expression level of miR482e was downregulated, and that of several NBS-LRR targets of miR482e were upregulated. Transgenic potato plantlets overexpressing miR482e showed hypersensitivity to V. dahliae infection. Using sRNA and degradome datasets, we validated that miR482e targets mRNAs of NBS-LRR disease-resistance proteins and triggers the production of trans-acting (ta)-siRNAs, most of which target mRNAs of defense-related proteins. Thus, the hypersensitivity of transgenic potato could be explained by enhanced miR482e and miR482e-derived ta-siRNA-mediated silencing on NBS-LRR-disease-resistance proteins. It is speculated that a miR482-mediated silencing cascade mechanism is involved in regulating potato resistance against V. dahliae infection and could be a counter defense action of plant in response to pathogen infection.

Analysis of pineapple mealybug wilt associated virus -1 and -2 for potential RNA silencing suppressors and pathogenicity factors.

Higher plants use RNA silencing to defend against viral infections. As a counter defense, plant viruses have evolved proteins that suppress RNA silencing. Mealybug wilt of pineapple (MWP), an important disease of pineapple, has been associated with at least three distinct viruses, Pineapple mealybug wilt associated virus -1, -2, and -3 (PMWaV-1, -2, and -3). Selected open reading frames (ORFs) of PMWaV-1 and PMWaV-2 were screened for their local and systemic suppressor activities in Agrobacterium-mediated transient assays using green fluorescent protein (GFP) in Nicotiana benthamiana. Results indicate that PMWaV-2 utilizes a multiple-component RNA silencing suppression mechanism. Two proteins, p20 and CP, target both local and systemic silencing in N. benthamiana, while the p22 and CPd proteins target only systemic silencing. In the related virus PMWaV-1, we found that only one of the encoded proteins, p61, had only systemic suppressor activity. Of all the proteins tested from both viruses, only the PMWaV-2 p20 protein suppressed local silencing induced by double-stranded RNA (dsRNA), but only when low levels of inducing dsRNA were used. None of the proteins analyzed could interfere with the short distance spread of silencing. We examined the mechanism of systemic suppression activity by investigating the effect of PMWaV-2-encoded p20 and CP proteins on secondary siRNAs. Our results suggest that the PMWaV-2 p20 and CP proteins block the systemic silencing signal by repressing production of secondary siRNAs. We also demonstrate that the PMWaV-2 p20 and p22 proteins enhanced the pathogenicity of Potato virus X in N. benthamiana.