Transmission Of dsRNA Research Articles

New insights into transmission pathways and possible off-target effects of insecticidal dsRNA released by treated plants

RNAi has shown great potential in controlling pests and pathogens, and dsRNA-based pesticides have been used in different ways. Due to off-target effects, the transmission pathways and possible impacts of dsRNA on non-target organisms after release should be researched. Here, we tested pathways of dsRNA transmission through the rice-hopper-spider food chain and their efficiency for triggering RNAi.The results revealed five new pathways by which plants transfer dsRNA into the environment through the food chain. We found that ingestion of the tissues or guttation droplets of treated plant could trigger both targeted and off-target RNAi both in consumers and predators. Ingestion of consumer hoppers could also result in localized RNAi in the midguts of the predator spiders. Trace amounts of dsRNA were detected in plant root excretions and in hopper honeydew. Cutting the root tips dramatically increased the levels of dsRNA in root excretions. Host shifting experiments proved that hoppers could transfer a trace amount of dsRNA via vomit. With specially designed dsRNAs, we showed that dsRNA sharing matching sequences of 29 bp or 32 bp in length with non-target genes could trigger off-target RNAi, but that dsRNA sharing 13 bp matching sequences could not.We conclude that field-released pesticidal dsRNA could be transmitted via the hydrophilic transport system in plants, and that this may pose a safety risk to non-target animal consumers that are closely related to target pests. Rational use of pesticidal dsRNAs should involve careful consideration of dsRNA design to manage the biosafety risk.

Persistent Parental RNAi in the Beetle Tribolium castaneum Involves Maternal Transmission of Long Double-Stranded RNA.

Parental RNA interference (pRNAi) is a powerful and widely used method for gene-specific knockdown. Yet in insects its efficacy varies between species, and how the systemic response is transmitted from mother to offspring remains elusive. Using the beetle Tribolium castaneum, an RT-qPCR strategy to distinguish the presence of double-stranded RNA (dsRNA) from endogenous mRNA is reported. It is found that injected dsRNA is directly transmitted into the egg and persists throughout embryogenesis. Despite this depletion of dsRNA from the mother, it is shown that strong pRNAi can persist for months before waning at strain-specific rates. In seeking the receptor proteins for cellular uptake of long dsRNA into the egg, a phylogenomics profiling approach of candidate proteins is also presented. A visualization strategy based on taxonomically hierarchical assessment of orthology clustering data to rapidly assess gene age and copy number changes, refined by sequence-based evidence, is demonstrated. Repeated losses of SID-1-like channel proteins in the arthropods, including wholesale loss in the Heteroptera (true bugs), which are nonetheless highly sensitive to pRNAi, are thereby documented. Overall, practical considerations for insect pRNAi against a backdrop of outstanding questions on the molecular mechanism of dsRNA transmission for long-term, systemic knockdown are elucidated.

Cysteine protease enhances plant-mediated bollworm RNA interference.

Oral ingestion of plant-expressed double stranded RNA (dsRNA) triggers target gene suppression in insect. An important step of this process is the transmission of dsRNA from plant to midgut cells. Insect peritrophic matrix (PM) presents a barrier that prevents large molecules from entering midgut cells. Here, we show that uptake of plant cysteine proteases, such as GhCP1 from cotton (Gossypium hirsutum) and AtCP2 from Arabidopsis, by cotton bollworm (Helicoverpa armigera) larvae resulted in attenuating the PM. When GhCP1 or AtCP2 pre-fed larvae were transferred to gossypol-containing diet, the bollworm accumulated higher content of gossypol in midgut. Larvae previously ingested GhCP1 or AtCP2 were more susceptible to infection by Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV), a dsRNA virus. Furthermore, the pre-fed larvae exhibited enhanced RNAi effects after ingestion of the dsRNA-expressing plant. The bollworm P450 gene CYP6AE14 is involved in the larval tolerance to gossypol; cotton plants producing dsRNA of CYP6AE14 (dsCYP6AE14) were more resistant to bollworm feeding (Mao et al. in Transgenic Res 20:665–673, 2011). We found that cotton plants harboring both 35S:dsCYP6AE14 and 35S:GhCP1 were better protected from bollworm than either of the single-transgene lines. Our results demonstrate that plant cysteine proteases, which have the activity of increasing PM permeability, can be used to improve the plant-mediated RNAi against herbivorous insects.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-013-0030-7) contains supplementary material, which is available to authorized users.

Mycovirus inPseudocercospora griseola, the causal agent of angular leaf spot in common bean

Pseudocercospora griseola (Sacc.) Crous & Braun is a widespread fungal phytopathogen that is responsible for angular leaf spot in the common bean (Phaseolus vulgaris L.). A number of fungal phytopathogens have been shown to harbour mycoviruses, and this possibility was investigated in populations of Pseudocercospora griseola. The total nucleic acid extracts of 61 fungal isolates were subjected to agarose gel electrophoresis. Small fragments (800-4800 bp) could be identified in 42 of the samples. The presence of dsRNA in isolate Ig838 was confirmed by treatment of total nucleic acid with DNase, RNase A, and nuclease S1. Transmission electron microscopy revealed the presence of viral-like particles 40 nm in diameter in the mycelia of 2 fungal isolates, namely 29-3 and Ig838. The transmission of dsRNA by means of conidia was 100% for isolate 29-3, but there was loss of 1-6 fragments of dsRNA in monosporic colonies of isolate Ig848. Cycloheximide treatment failed to inhibit the mycovirus in isolate 29-3, but proved efficient in the elimination of the 2.2, 2.0, 1.8, 1.2 and 1.0 kb fragments in 2 colonies of isolate Ig848. The occurrence of a mycovirus in Pseudocercospora griseola was demonstrated for the first time in the present study.

Transmission of the M2 double-stranded RNA in Rhizoctonia solani anastomosis group 3 (AG-3)

Horizontal transmission of the 3.57 kb M2 double-stranded RNA (dsRNA) between mycelia of somatically incompatible isolates of Rhizoctonia solani anastomosis group 3 (AG-3), an economically important pathogen of cultivated plants in the family Solanaceae, was investigated. Nine donor isolates of R. solani AG-3 containing the M2 dsRNA were paired on potato-dextrose agar with each of three different recipient isolates where the M2 dsRNA was absent. Reverse-transcription PCR (RT-PCR) was used to detect horizontal transmission of the M2 dsRNA via hyphal anastomosis from donor to recipient isolates by examining hyphal explants taken 3 cm from the hyphal interaction zone. PCR-RFLP genetic-based markers of two nuclear loci and one mitochondrial locus were used to confirm identity and transmission between donor and recipient isolates of R. solani AG-3. The frequency of transmission observed between 72 pairings of the eight donor and three recipient isolates was approximately 4% of the total pairings, and differences in the phenotype of the recipient isolates after acquisition of the M2 dsRNA via horizontal transmission were observed. To our knowledge this represents the first demonstration of transmission of dsRNA between genetically different individuals of R. solani confirmed with nuclear and mitochondrial markers. These results suggest that transmission can occur between somatically incompatible isolates of R. solani AG-3 but that maintenance of the dsRNA in the recipient isolates was not stable after repeated subculturing on nutrient medium.

Transmission of the M2 double-stranded RNA in Rhizoctonia solani anastomosis group 3 (AG-3)

Horizontal transmission of the 3.57 kb M2 double-stranded RNA (dsRNA) between mycelia of somatically incompatible isolates of Rhizoctonia solani anastomosis group 3 (AG-3), an economically important pathogen of cultivated plants in the family Solanaceae, was investigated. Nine donor isolates of R. solani AG-3 containing the M2 dsRNA were paired on potato-dextrose agar with each of three different recipient isolates where the M2 dsRNA was absent. Reverse-transcription PCR (RT-PCR) was used to detect horizontal transmission of the M2 dsRNA via hyphal anastomosis from donor to recipient isolates by examining hyphal explants taken 3 cm from the hyphal interaction zone. PCR-RFLP genetic-based markers of two nuclear loci and one mitochondrial locus were used to confirm identity and transmission between donor and recipient isolates of R. solani AG-3. The frequency of transmission observed between 72 pairings of the eight donor and three recipient isolates was approximately 4% of the total pairings, and differences in the phenotype of the recipient isolates after acquisition of the M2 dsRNA via horizontal transmission were observed. To our knowledge this represents the first demonstration of transmission of dsRNA between genetically different individuals of R. solani confirmed with nuclear and mitochondrial markers. These results suggest that transmission can occur between somatically incompatible isolates of R. solani AG-3 but that maintenance of the dsRNA in the recipient isolates was not stable after repeated subculturing on nutrient medium.

Diversity, complexity and transmission of double-stranded RNA elements in Chalara elegans (synanam. Thielaviopsis basicola)

Double-stranded (ds) RNA banding patterns were determined in 21 wild-type strains of the soilborne plant pathogen Chalara elegans originating from different geographic regions worldwide. Five strains, each with a unique dsRNA pattern, were selected for cDNA cloning, northern blot analysis and dsRNA transmission experiments. Four strains contained multiple (up to 6) dsRNA elements (2.0 kbp to 12 kbp in size) and one strain contained a single 2.8 kbp fragment. These five strains were distinguished from one another by their unique RAPD-PCR patterns. Seven partial cDNA clones were derived from the predominant 2.8, 5.3, and 12 kbp dsRNA elements. Nucleotide sequence analysis and northern blot hybridizations revealed a high degree of genetic dissimilarity among the different molecular-size dsRNA elements, even those found within a single strain. Four clones from the 5.3 kbp dsRNA fragment showed a 23-43 % amino acid identity to either the coat protein or RNA-dependent RNA polymerase regions of viruses in the Totiviridae. One clone from the 2.8 kbp dsRNA fragment had a 55-57 % amino acid identity to the RdRp region of viruses in the Narnaviridae. Two clones from the 12 kbp dsRNA fragment showed no significant homology to any known virus group. Colonies derived from 100 single-conidia isolates of C. elegans strains with the 2.8, 5.3 and 12 kbp elements all contained the corresponding dsRNA element, indicating that dsRNA transmission through conidia was highly efficient, regardless of molecular size. However, transmission of dsRNA between the mycelium of strains of C. elegans could not be achieved in this study. Genetically unique strains carrying diverse dsRNA elements appear to have evolved within populations of C. elegans. Based on our findings, there are at least 3 groups of viruses present in C. elegans.

Double-stranded RNA transmission through basidiospores of Heterobasidion annosum

A search for double-stranded RNA (dsRNA) was conducted among 106 isolates of the pathogenic basidiomycete Heterobasidion annosum. Of these isolates, 47 were tissue isolates from fruit bodies and 59 were isolated from decayed wood. Nucleic acids were extracted from freeze-dried mycelia and dsRNA was separated by cellulose CF-11 chromatography and confirmed by digestions with specific nucleases. dsRNA was present in 19 and 14% of the tissue and wood isolates, respectively. From five of the fruit bodies containing dsRNA basidiospores were investigated and 10-84% of the germinated basidiospores contained dsRNA. On high nutrient media, the germination frequency of basidiospores was reduced by presence of dsRNA in the fruit body (P < 0.05); germination frequencies were 34 and 78% for spores from fruit bodies with and without dsRNA, respectively. The same trend was present also on low nutrient media, although not statistically significant; germination was 3 and 10% for spores from infected and dsRNA free fruit bodies, respectively. Transmission of dsRNA in H. annosum from mycelia into basidiospores together with the lowered germination frequency are likely to play a significant role in the life cycle of the fungus. The relative importance of different transmission routes for dsRNA in H. annosum is discussed.

Transmissibility of viral double-stranded RNA between strains of the violet root rot fungus Helicobasidium mompa and the potential for viral dsRNA infection to this fungus using monokaryotic strains

To examine the potential of a method of doublestranded (ds) RNA infection to Helicobasidium mompa, the transmissibility of dsRNA between strains of this fungus was investigated. Strain V70 was used as a dsRNA donor. The dsRNA recipients were six strains that were mycelially incompatible with V70, plus two monokaryotic strains. Random amplified polymorphic DNA analysis suggested that the mycelially incompatible strains were genetically different mutually; however, the analysis also suggested that V70 was genetically homogeneous with the two monokaryotic strains. When V70 was paired with either of the mycelially incompatible strains, the dsRNAs did not transmit to the recipients at all. When V70 was paired with the two monokaryotic strains, the dsRNAs were transmitted to the monokaryotic strains. The two monokaryotic strains, which had acquired dsRNAs from V70 in the previous experiment, were used as new dsRNA donors in a next experiment so that we could investigate dsRNA transmission from these monokaryotic strains to the six strains used in the previous experiment. One of the monokaryotic strains permitted dsRNA transmission to two of the six recipients. We conclude that we can infect genetically different strains of H. mompa with dsRNA using the monokaryotic strains.

Interspecific Transmission of Double-Stranded RNA and Hypovirulence fromSclerotinia sclerotiorumtoS. minor

ABSTRACT Interspecific transmission of a hypovirulence-associated double-stranded RNA (dsRNA) and hypovirulent phenotype was attempted from hypovirulent isolate Ss275 of Sclerotinia sclerotiorum to five virulent isolates of S. minor. dsRNA and the hypovirulent phenotype were successfully transmitted to one of the five isolates, Sm10. Three putative converted isolates of Sm10 were slow growing and developed atypical colony morphologies characteristic of the hypovirulent phenotype. These isolates were assayed for virulence and produced significantly smaller lesions than isolate Sm10 on detached leaves of Romaine lettuce. One of these putative converted isolates, designated Sm10T, was tested to confirm interspecific transmission of dsRNA. In northern hybridizations, dsRNA isolated from Sm10T hybridized with a digoxigenin-labeled cDNA probe prepared from dsRNA isolated from Ss275. Random amplified polymorphic DNA analysis confirmed that isolate Sm10T was derived from Sm10 and not from Ss275 or a hybrid of the two species. The dsRNA and hypovirulent phenotype were subsequently transmitted intraspecifically from Sm10T to Sm8. To our knowledge, this is the first report of interspecific transmission of dsRNA and an associated hypovirulent phenotype between fungal plant pathogens by hyphal anastomosis.

Transmission of double-stranded RNA in Heterobasidion annosum

Transmission of dsRNA viruses between homo- and heterokaryotic mycelia paired on agar plates and into conidia has been studied in Heterobasidion annosum. Horizontal transmission of dsRNA occurred between both homo- and heterokaryotic isolates, as well as between isolates belonging to different intersterility groups. The proportions of vertical transmission into conidia were 3% and 55%, respectively, for the two isolates included in the study. RT-PCR of dsRNA and PCR–RFLP of mitochondrial markers were used to confirm transmission of dsRNA between the cytoplasms of different mycelia. The identity of nuclei and nuclear migration during experiments were verified using PCR–RFLP of several nuclear markers.

Double-stranded RNA elements in Rhizoctonia solani AG 3

All isolates of Rhizoctonia solani (teleomorph Thanatephorus cucumeris ) anastomosis group (AG) 3 from potato tubers in commercial fields in Scotland were found to contain more than one double-stranded RNA (dsRNA) element ranging in size from 0.3 to 15 kb. Strains of other AGs (2–1, 3, 4, 5) from culture collections also contained dsRNA elements. Isolates of AG 3 from different black scurf lesions on single tubers showed similar dsRNA profiles and, when opposed on agar plates, underwent compatible hyphal fusion where the colonies met. Isolates from different tubers in the same commercial field often showed somatic incompatibility (mean 44%) leading to cytoplasmic death when colonies fused. Isolates from different fields always showed somatic incompatibility after hyphal fusion, suggesting that the complex population structure of AG 3 could significantly limit the transmission of dsRNA between strains. The incidence of different dsRNA elements in AG 3 isolates could not be related to pathogenicity in seedling tests on tomato, carrot, lettuce, radish, cress or onion. Double-stranded RNA elements could not be eliminated from AG 3 isolates by repeated hyphal-tip subculture or extended (8 week) incubation on agar containing cycloheximide. Some isolates lost the smaller dsRNA elements (1.0 to 1.6 kb) during these procedures, and one isolate (of AG 4) lost a 6.5 kb dsRNA element, but the larger elements (up to 9.5 or 12 kb) were always retained. When assessed by low stringency hybridisation, the dsRNA elements of R. solani AG 3 and of strains from AGs 2–1, 4 and 5 showed no homology with a plasmid containing cDNA of the complete hypovirus dsRNA genome of Cryphonectria parasitica .

Molecular Characterization of Two Endogenous Double-stranded RNAs in Rice and Their Inheritance by Interspecific Hybrids

We completely sequenced 13,936 nucleotides (nt) of a double-stranded RNA (dsRNA) of wild rice (W-dsRNA). A single long open reading frame (13,719 nt) containing the conserved motifs of RNA-dependent RNA polymerase and RNA helicase was located in the coding strand. The identity between entire nucleotide sequence of W-dsRNA and that of the dsRNA of temperate japonica rice (J-dsRNA, 13,952 nt) was 75.5%. A site-specific discontinuity (nick) was identified at nt 1,197 from the 5' end of the coding strand of W-dsRNA. This nick is also located at nt 1,211 from the 5' end in the coding strand of J-dsRNA. The dsRNA copy number was increased more than 10-fold in pollen grains of both rice plants. This remarkable increase may be responsible for the highly efficient transmission of J-dsRNA via pollen that we already reported. J-dsRNA and W-dsRNA were also efficiently transmitted to interspecific F1 hybrids. Seed-mediated dsRNA transmission to F2 plants was also highly efficient when the maternal parent was wild rice. The efficiency of dsRNA transmission to F2 plants was reduced when the maternal parent was temperate japonica rice; however, the reduced rates in F2 plants were returned to high levels in F3 plants.

Immunity and resistance to the KP6 toxin of Ustilago maydis.

The KP6 toxin of Ustilago maydis, encoded by segmented double-stranded (ds) RNA viruses, is lethal to sensitive strains of the same species and related species. The toxin consists of two polypeptides, alpha and beta, synthesized as a single preprotoxin, which are not covalently linked. Neither polypeptide alone is toxic, but killer activity can be restored by in vitro and in vivo complementation. Killer-secreting strains are resistant to the toxin they produce. Resistance is conferred by a single recessive nuclear gene. This study describes a search for cytoplasmic factors that may confer resistance, also referred to as immunity. The approaches used to detect cytoplasmic immunity included transmission of dsRNA and transmission of virus particles to sensitive cells by cytoduction, cytoplasmic mixing in diploids and infection with viruses. An alternative approach was also used to express cloned cDNAs of the KP6 toxin-encoding dsRNA and of the alpha and beta polypeptides. The results indicated that no immunity to KP6 can be detected. While KP6, alpha and beta polypeptides were expressed by resistant cells, neither KP6 nor beta were expressed in sensitive strains. The alpha polypeptide was expressed in sensitive cells, but it did not confer immunity. These results suggest that neither the preprotoxin nor the alpha or beta polypeptides confer immunity and thus beta may be the toxic component of the binary toxin.

Control of plant pathogens with viruses and related agents

Cytoplasmically transmissible agents causing diseases of plant pathogenic fungi characterized by reductions in pathogenicity, ability to form sexual and asexual spores, spore viability and growth rate, are often associated with the presence of one or more specific segments of virus-like double-stranded RNA (dsRNA). In Italy, hypovirulent dsRNA-containing strains of the chestnut blight fungus,Endothia(Cryphonectria)parasitica, have become predominant in many areas where blight is no longer a serious problem. dsRNA-containing strains of other pathogens, with various degrees of debilitation, survive in natural populations but have not become predominant or resulted in any great reduction in disease. Examples include the Dutch elm disease fungus,Ophiostoma(Ceratocystis)ulmi, and the wheat take-all fungus,Gaeumannomyces graminisvar.tritici. Successful biological control of such pathogens could probably be achieved, however, if methods could be developed to suppress the loss of dsRNA that occurs during the sexual and other stages of their life cycles, and to suppress the vegetative incompatibility reactions that reduce the cytoplasmic transmission of dsRNA. Systemic infection with attenuated strains of plant viruses can protect plants from later infection by virulent strains of the same or closely related viruses. Despite some notable successes, e.g. control of citrus tristeza and tomato mosaic viruses, such ‘cross-protection’ has not been widely applied because of the cost and difficulty of application, and caution about the widespread distribution of infectious agents in the environment. These problems could be overcome if cross-protection could be achieved by the expression of a single viral gene rather than infection with intact virus, and consideration of possible mechanisms of cross protection suggests novel ways of producing virus-resistant plants.

Isolation of a Virus from Virulent Strains of Rhizoctonia solani

SUMMARY Double-stranded RNA viruses were detected in virulent strains of Rhizoctonia solani. The virus particles were 33 nm in diameter, contained two or three major segments of dsRNA (mol. wt. 1.6 × 106, 1.45 × 106 and 1.25 × 106), had a density of 1.34 g/ml and had an estimated sedimentation coefficient of 161S. The major coat protein had a mol. wt. of 46000. An RNA-dependent RNA polymerase activity was associated with the viral capsids. Native hypovirulent strains of R. solani were devoid of these viruses and attenuation of virulent strains to hypovirulence appeared to be correlated with the loss of some or all of the segments of dsRNA. Transmission of dsRNA from a virulent to a hypovirulent strain by heterokaryon formation was associated with the transmission of virulence.