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Abstract Fungal pathogens that impact perennial plants or natural ecosystems require management strategies beyond fungicides and breeding for resistance. Rust fungi, some of the most economically and environmentally important plant pathogens, have shown amenability to double-stranded RNA (dsRNA) mediated control. To date, dsRNA treatments have been applied prior to infection or together with the inoculum. Here we show that a dsRNA spray can effectively prevent and cure infection by Austropuccinia psidii (cause of myrtle rust) at different stages of the disease cycle. Significant reductions in disease coverage were observed in plants treated with dsRNA targeting essential fungal genes 48 h pre-infection through to 14 days post-infection. For curative treatments, improvements in plant health and photosynthetic capacity were seen 2–6 weeks post-infection. Two-photon microscopy suggests inhibitory activity of dsRNA on intercellular hyphae or haustoria. Our results show that dsRNA acts both preventively and curatively against myrtle rust disease, with treated plants recovering from severe infection. These findings have immediate potential in the management of the more than 10-year epidemic of myrtle rust in Australia.

The orthotospovirus capsicum chlorosis virus (CaCV) is an important pathogen affecting capsicum plants. Elevated temperatures may affect disease progression and pose a potential challenge to capsicum production. To date, CaCV-resistant capsicum breeding lines have been established; however, the impact of an elevated temperature of 35 °C on this genetic resistance remains unexplored. Thus, this study aimed to investigate how high temperature (HT) influences the response of CaCV-resistant capsicum to the virus. Phenotypic analysis revealed a compromised resistance in capsicum plants grown at HT, with systemic necrotic spots appearing in 8 out of 14 CaCV-infected plants. Molecular analysis through next-generation sequencing identified 105 known and 83 novel microRNAs (miRNAs) in CaCV-resistant capsicum plants. Gene ontology revealed that phenylpropanoid and lignin metabolic processes, regulated by Can-miR408a and Can- miR397, are likely involved in elevated-temperature-mediated resistance-breaking responses. Additionally, real-time PCR validated an upregulation of Can-miR408a and Can-miR397 by CaCV infection at HT; however, only the Laccase 4 transcript, targeted by Can-miR397, showed a tendency of negative correlation with this miRNA. Overall, this study provides the first molecular insights into how elevated temperature affects CaCV resistance in capsicum plants and reveals the potential role of miRNA in temperature-sensitive tospovirus resistance.

Myrteae is the most species-rich tribe in the Myrtaceae family, represented by a range of socioeconomically and ecologically significant species. Many of these species, including commercially relevant ones, have become increasingly threatened in the wild, and now require conservation actions. Tissue culture presents an appropriate in vitro tool to facilitate medium-term and long-term wild germplasm conservation, as well as for commercial propagation to maintain desirable traits of commercial cultivars. So far, tissue culture has not been extensively achieved for Myrteae. Here, tissue culture for Eugenia, one of the most species-rich genera in Myrteae, is reviewed, giving directions for other related Myrteae. This review also focuses on ex situ conservation of Australian Myrteae, including using seed banking and field banking. Despite some progress, challenges to conserve these species remain, mostly due to the increasing threats in the wild and limited research. Research into in vitro methods (tissue culture and cryopreservation) is paramount given that at least some of the species are ‘non-orthodox’. There is an urgent need to develop long-term in vitro conservation for capturing the remaining germplasm of threatened Myrteae.

Bovicola ovis, commonly known as the sheep-biting louse, is an ectoparasite that adversely affects the sheep industry. Sheep louse infestation lowers the quality of products, including wool and leather, causing a loss of approximately AUD 123M per annum in Australia alone. The lack of a high-quality genome assembly for the sheep-biting louse, as well as any closely related livestock lice, has hindered the development of louse research and management control tools. In this study, we present the assembly of B. ovis with a genome size of ~123 Mbp based on a nanopore long-read sequencing library and Illumina RNA sequencing, complemented with a chromosome-level scaffolding using the Pore-C multiway chromatin contact dataset. Combining multiple alignment and gene prediction tools, a comprehensive annotation on the assembled B. ovis genome was conducted and recalled 11,810 genes as well as other genomic features including orf, ssr, rRNA and tRNA. A manual curation using alignment with the available closely related louse species, Pediculus humanus, increased the number of annotated genes to 16,024. Overall, this study reported critical genetic resources and biological insights for the advancement of sheep louse research and the development of sustainable control strategies in the sheep industry.

Improving clonal rootstock propagation of avocado has been a major industry/research challenge globally for many decades. Tissue culture has been a focus for clonal propagation with substantial advancements in recent years. In the process of tissue culture, avocado rootstocks display differences in rooting and acclimation capacity. Such differences may relate to the specific structural characteristics of the rootstock. This study aimed to investigate the structural difference during tissue culture in two rootstocks ‘Reed’ and ‘Velvick’, with differing rooting and acclimation capacity. Histological investigations were carried out of stem vasculature, leaves and roots of tissue cultured plantlets. Quantitative parameters; stomatal index, stomatal density, trichome density, vein-islet density and vein termination density were also analysed. Prominent fascicular cambium and fewer phloem fibres in stems positively correlated with rooting capacity. Acclimation success positively correlated to the presence of fully differentiated secondary xylem in root. Presence of smaller epidermal cells, high stomatal density and reduced vein termination density was associated with reduced acclimation success. These findings will support optimisation strategies for micropropagation of not only difficult-to-root and difficult-to-acclimate avocado rootstocks, but also, other woody perennials experiencing similar problems.

Insects and ectoparasites are causes for major concern throughout the world due to their economic and welfare impacts on livestock agriculture. Current control measures involve chemicals such as acaricides which pose challenges like chemical resistance and longer withholding periods. To enable more sustainable agriculture practices, it is important to develop technologies that combine targeted effectiveness with minimal environmental footprint. RNA interference (RNAi) is a eukaryotic process in which transcript expression is reduced in a sequence-specific manner. This makes it a perfect tool for developing efficient and effective biological control against pests and pathogens. Double-stranded RNA (dsRNA) is the key trigger molecule for inducing RNAi; this concept is widely studied for development of RNA-based biopesticides as an alternative to chemical controls in crop protection for targeting pests and pathogens with accuracy and specificity. In this review, we discuss key advances made using RNAi technology and how they can be applied to improve health in livestock industries. This includes research focused on different delivery mechanisms of dsRNA, important developments in regulatory frameworks, and risk identification, that will enable the future adoption of RNAi technologies to improve animal health.

Abstract Global food production is at risk from many abiotic and biotic stresses and can be affected by multiple stresses simultaneously. Virus diseases damage cultivated plants and decrease the marketable quality of produce. Importantly, the progression of virus diseases is strongly affected by changing climate conditions. Among climate-changing variables, temperature increase is viewed as an important factor that affects virus epidemics, which may in turn require more efficient disease management. In this review, we discuss the effect of elevated temperature on virus epidemics at both macro- and micro-climatic levels. This includes the temperature effects on virus spread both within and between host plants. Furthermore, we focus on the involvement of molecular mechanisms associated with temperature effects on plant defence to viruses in both susceptible and resistant plants. Considering various mechanisms proposed in different pathosystems, we also offer a view of the possible opportunities provided by RNA -based technologies for virus control at elevated temperatures. Recently, the potential of these technologies for topical field applications has been strengthened through a combination of genetically modified (GM)-free delivery nanoplatforms. This approach represents a promising and important climate-resilient substitute to conventional strategies for managing plant virus diseases under global warming scenarios. In this context, we discuss the knowledge gaps in the research of temperature effects on plant-virus interactions and limitations of RNA-based emerging technologies, which should be addressed in future studies.

Duboisia is an Australian native, commercially valuable for tropane alkaloid extraction. Clonal propagation of elite selections is essential to establish highly productive plantations. The current propagation system using stem cuttings is proven to be inefficient, prompting the industry to seek a more efficient and effective propagation tool. Tissue culture is a cost-effective alternative for mass propagation of true-to-type plants, particularly ideal for propagating elite Duboisia selections. In this context, attempts were made to develop a commercially viable high throughput micropropagation system for three Duboisia species: Duboisia myoporoides, Duboisia leichhradtii and Duboisia hopwoodii. Various nutrient media, hormone combinations and incubating conditions were tested to optimise each stage of the micropropagation pipeline. The findings revealed that the tissue culture media composition and hormone requirements are species-specific. With the optimised conditions, an efficient tissue culture system was developed, achieving successful meristem induction and multiplication. Species-specific rooting protocol optimisation resulted in 100% rooting for D. myoporoides and D. leichhardtii, and 70% rooting for D. hopwoodii. Furthermore, an optimised acclimatisation protocol supported 100% survival of D. myoporoides and D. leichhardtii and 80% of D. hopwoodii plantlets. This study, for the first time, demonstrated the capacity of successful meristem culture of three Duboisia species, establishing the foundation for high throughput micropropagation of Duboisia species.

The Duboisia species, a group of plants native to Australia, have been historically valued for their pharmacological properties and have played a significant role in traditional medicine and pharmaceutical research. Persistent efforts are underway to enhance the efficacy of the active ingredient scopolamine, employing both conventional breeding methods and advanced biotechnology tools. The primary objective of this research was to establish a highly efficient method for isolating mesophyll protoplasts and facilitating their regeneration, thereby laying a robust foundation for the application of various advanced plant biotechnology tools in the pursuit of genetic enhancement. The mesophyll protoplast isolation process was developed for hybrid D. myoporoides × D. hopwoodii with careful optimisation of the following parameters: leaf strip size; incubation conditions; physical treatment; and enzyme concentration. The optimal parameters were combined in each individual step; the best enzyme concentration was determined to be 2% (w/v) cellulysin and 0.5% (w/v) macerase. Protoplast yield was found to be greatly affected by the enzyme concentrations. The isolated protoplasts were cultured at a density of 0.5 × 105 to best sustain the highest cell division (33.2%) and a microcalli induction frequency of 17.9%. After 40 days of culture in a modified KM8P medium at 25 °C in darkness, visible microcalli were transferred to a solidified Murashige and Skoog (MS) medium with 1 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction under a 16 h photoperiod. After 30 days of culture, compact organogenic calli were transferred into a solid MS medium with 6-benzylaminopurine (BA) alone or thidiazuron (TDZ) alone or in combination with BA or naphthalene acetic acid (NAA) for shoot regeneration. The maximum shoot regeneration frequency (63.3%) was observed in the medium with 1.5 mg L−1 TDZ alone. For the first time, a reliable protoplast isolation and regeneration system from mesophyll cells was established for Duboisia with high protoplast viability, successful microcalli formation, and intact plant regeneration. This innovation will significantly contribute towards the genetic enhancement of the Duboisia species.

Abstract Neptunia amplexicaulis is an herbaceous legume endemic to the Richmond area in central Queensland, Australia and is one of the strongest known Selenium hyperaccumulators on earth, showing significant potential to be utilised in Se phytoextraction applications. Here a protocol was established for in vitro micropropagation of Se hyperaccumulator N. amplexicaulis using nodal segments from in vitro-germinated seedlings. Shoot multiplication was achieved on Murashige and Skoog (MS) basal media supplemented with various concentrations of 6-Benzylaminopurine (BA) (1.0, 2.0, 3.0 mg L−1) alone or in combination with low levels of Naphthaleneacetic acid (NAA) (0.1, 0.2, 0.3 mg L−1), with 2.0 mg L−1 BA + 0.2 mg L−1 NAA found to be most effective. Elongated shoots were rooted in vitro using NAA, with highest root induction rate of 30% observed at 0.2 mg L−1 NAA. About 95% of the in vitro rooted shoots survived acclimatization. Clonally propagated plantlets were dosed with selenate/selenite solution and assessed for Se tissue concentrations using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) and found to retain their ability to hyperaccumulate. The protocol developed for this study has potential to be optimised for generating clonal plants of N. amplexicaulis for use in research and phytoextraction industry applications.

Our duty to conserve global natural ecosystems is increasingly in conflict with our need to feed an expanding population. The use of conventional pesticides not only damages the environment and vulnerable biodiversity but can also still fail to prevent crop losses of 20–40% due to pests and pathogens. There is a growing call for more ecologically sustainable pathogen control measures. RNA-based biopesticides offer an eco-friendly alternative to the use of conventional fungicides for crop protection. The genetic modification (GM) of crops remains controversial in many countries, though expression of transgenes inducing pathogen-specific RNA interference (RNAi) has been proven effective against many agronomically important fungal pathogens. The topical application of pathogen-specific RNAi-inducing sprays is a more responsive, GM-free approach to conventional RNAi transgene-based crop protection. The specific targeting of essential pathogen genes, the development of RNAi-nanoparticle carrier spray formulations, and the possible structural modifications to the RNA molecules themselves are crucial to the success of this novel technology. Here, we outline the current understanding of gene silencing pathways in plants and fungi and summarize the pioneering and recent work exploring RNA-based biopesticides for crop protection against fungal pathogens, with a focus on spray-induced gene silencing (SIGS). Further, we discuss factors that could affect the success of RNA-based control strategies, including RNA uptake, stability, amplification, and movement within and between the plant host and pathogen, as well as the cost and design of RNA pesticides.

Reproductively mature horticultural trees undergo an annual flowering cycle that repeats each year of their reproductive life. This annual flowering cycle is critical for horticultural tree productivity. However, the molecular events underlying the regulation of flowering in tropical tree crops such as avocado are not fully understood or documented. In this study, we investigated the potential molecular cues regulating the yearly flowering cycle in avocado for two consecutive crop cycles. Homologues of flowering-related genes were identified and assessed for their expression profiles in various tissues throughout the year. Avocado homologues of known floral genes FT, AP1, LFY, FUL, SPL9, CO and SEP2/AGL4 were upregulated at the typical time of floral induction for avocado trees growing in Queensland, Australia. We suggest these are potential candidate markers for floral initiation in these crops. In addition, DAM and DRM1, which are associated with endodormancy, were downregulated at the time of floral bud break. In this study, a positive correlation between CO activation and FT in avocado leaves to regulate flowering was not seen. Furthermore, the SOC1-SPL4 model described in annual plants appears to be conserved in avocado. Lastly, no correlation of juvenility-related miRNAs miR156, miR172 with any phenological event was observed.

Duboisia is an Australian native woody species of the Solanaceae family, a crucial source of alkaloids, and is naturally extracted for pharmaceuticals. The alkaloid content of the four naturally occurring species of Duboisia, i.e., Duboisia myoporoides R. Br., Duboisia leichhardtii F. Muell., Duboisia hopwoodii F. Muell. and Duboisia arenitensis, is not conducive for large-scale commercial extraction. High-value hybrids between D. myoporoides R. Br. and D. leichhardtii F. Muell. have become the commercial crop for the industry. Propagation of these hybrids is key for progression of this industry, especially for the establishment and expansion of plantations and to replenish old plantations. Commercial propagation of Duboisia completely depends on cutting propagation to ensure true-to-type propagules. Cutting propagation of this species is associated with several challenges and has been a hurdle for industry expansion for many years. Micropropagation can be an efficient and sustainable alternative for Duboisia clonal propagation and is a faster and cleaner propagation avenue for elite propagules. This review compiles the research attempts made in the space of Duboisia micropropagation and provides an update on recent advancements to understand the technical capacity, progress and challenges towards a commercial micropropagation platform.

Abstract Background Forward genetic screens are a powerful approach for identifying the genes contributing to a trait of interest. However, mutants arising in genes already known can obscure the identification of new genes contributing to the trait. Here, we describe a strategy called Candidate gene-Sequencing (Can-Seq) for rapidly identifying and filtering out mutants carrying new alleles of known and candidate genes. Results We carried out a forward genetic screen and identified 40 independent Arabidopsis mutants with defects in systemic spreading of RNA interference (RNAi), or more specifically in root-to-shoot transmission of post-transcriptional gene silencing (rtp). To classify the mutants as either representing a new allele of a known or candidate gene versus carrying a mutation in an undiscovered gene, bulk genomic DNA from up to 23 independent mutants was used as template to amplify a collection of 47 known or candidate genes. These amplified sequences were combined into Can-Seq libraries and deep sequenced. Subsequently, mutations in the known and candidate genes were identified using a custom Snakemake script ( https://github.com/Carroll-Lab/can_seq ), and PCR zygosity tests were then designed and used to identify the individual mutants carrying each mutation. Using this approach, we showed that 28 of the 40 rtp mutants carried homozygous nonsense, missense or splice site mutations in one or more of the 47 known or candidate genes. We conducted complementation tests to demonstrate that several of the candidate mutations were responsible for the rtp defect. Importantly, by exclusion, the Can-Seq pipeline also identified rtp mutants that did not carry a causative mutation in any of the 47 known and candidate genes, and these mutants represent an undiscovered gene(s) required for systemic RNAi. Conclusions Can-Seq offers an accurate, cost-effective method for classifying new mutants into known versus unknown genes. It has several advantages over existing genetic and DNA sequencing approaches that are currently being used in forward genetic screens for gene discovery. Using Can-Seq in conjunction with map-based gene cloning is a cost-effective approach towards identifying the full complement of genes contributing to a trait of interest.

Abstract The agrochemical is often applied to enhance the agricultural production. Nanotechnology has advanced agricultural biotechnology in the new “agri‐tech revolution.” Eco‐friendly nanoparticles as carriers can essentially actualize the application of biomolecules as sustainable agrochemicals to increase their biological performance and reduce deterioration of the ecosystem. Particularly, these nanocarrier‐biomolecule hybrids will be ideal for topical applications to enhance the crop yield and quality. This review summarizes the current efforts in developing nanoparticles as carriers of biomolecules for topical delivery. First, the applicable biomolecules for plant growth regulation and the pathogen control are briefly introduced. The criteria and strategies for research and industrial development of nanocarrier‐biomolecule hybrids are then proposed, which better involves both researchers and manufacturers in the beginning for an efficient R&D process. The review further discusses several typical nanoparticle‐biomolecule hybrids as the candidates of next‐generation agrochemicals with advanced scientific merits and high perspectives of industrialization as the conclusion.

Advances in DNA sequencing have made it easier to sequence and assemble plant genomes. Here, we extend an earlier study, and compare recent methods for long read sequencing and assembly. Updated Oxford Nanopore Technology software improved assemblies. Using more accurate sequences produced by repeated sequencing of the same molecule (Pacific Biosciences HiFi) resulted in less fragmented assembly of sequencing reads. Using data for increased genome coverage resulted in longer contigs, but reduced total assembly length and improved genome completeness. The original model species, Macadamia jansenii, was also compared with three other Macadamia species, as well as avocado (Persea americana) and jojoba (Simmondsia chinensis). In these angiosperms, increasing sequence data volumes caused a linear increase in contig size, decreased assembly length and further improved already high completeness. Differences in genome size and sequence complexity influenced the success of assembly. Advances in long read sequencing technology continue to improve plant genome sequencing and assembly. However, results were improved by greater genome coverage, with the amount needed to achieve a particular level of assembly being species dependent.

Abstract Background Grafting is the common propagation method for avocado and primarily benefits orchard production by reducing the time to tree productivity. It also allows use of scions and rootstocks specifically selected for improved productivity and commercial acceptance. Rootstocks in avocado may be propagated from mature tree cuttings (‘mature’), or from seed (‘juvenile’). While the use of mature scion material hastens early bearing/maturity and economic return, the molecular factors involved in the role of the scion and/or rootstock in early bearing/reduced juvenility of the grafted tree are still unknown. Results Here, we utilized juvenility and flowering associated miRNAs; miR156 and miR172 and their putative target genes to screen pre-graft and post-graft material in different combinations from avocado. The abundance of mature miR156, miR172 and the miR156 target gene SPL4, showed a strong correlation to the maturity of the scion and rootstock material in avocado. Graft transmissibility of miR156 and miR172 has been explored in annual plants. Here, we show that the scion may be responsible for grafted tree maturity involving these factors, while the rootstock maturity does not significantly influence miRNA abundance in the scion. We also demonstrate that the presence of leaves on cutting rootstocks supports graft success and contributes towards intergraft signalling involving the carbohydrate-marker TPS1. Conclusion Here, we suggest that the scion largely controls the molecular ‘maturity’ of grafted avocado trees, however, leaves on the rootstock not only promote graft success, but can influence miRNA and mRNA abundance in the scion. This constitutes the first study on scion and rootstock contribution towards grafted tree maturity using the miR156-SPL4-miR172 regulatory module as a marker for juvenility and reproductive competence.

Abstract Fusarium wilt, caused by the soil-borne fungal pathogen, Fusarium oxysporum f. sp. cubense (Foc), is considered as one of the most threatening diseases of banana. The Cavendish variety, resistant to Foc race 1 (R1), is susceptible to tropical race 4 (TR4), an aggressive race of the pathogen which is of increasing concern worldwide. Previous studies have revealed that plant small RNAs (sRNAs) play crucial roles in the host response to pathogen infection. To investigate the roles of sRNAs involved in the interaction of the banana-Foc pathosystem, small RNA profiles of Cavendish banana roots inoculated with Foc TR4 and Foc R1 were obtained and analyzed in the present study using Next-Generation Sequencing (NGS) technology. A total of 112 discrete mature microRNAs (miRNAs) belonging to 22 known miRNA families were found across all constructed sRNA libraries. The expression of miR166, miR159 and miR156 was up-regulated in TR4-inoculated samples as compared to mock-inoculated samples, while the expression of these miRNAs was approximately the same in R1-inoculated and mock-inoculated samples. Consistent with the sequencing data, qRT-PCR results demonstrated up-regulation of these miRNAs and down-regulation of their target genes in TR4-inoculated samples, but not in R1-inoculated samples. Considering Cavendish banana is resistant to R1 and susceptible to TR4, it is possible that these sRNAs and their target genes are involved in particular plant defence pathways such as salicylic acid-based defence. The findings will pave way for future investigations of the defence mechanism and potential approaches of resistance improvement.

RNA interference (RNAi) is a powerful tool that is being increasingly utilized for crop protection against viruses, fungal pathogens, and insect pests. The non-transgenic approach of spray-induced gene silencing (SIGS), which relies on spray application of double-stranded RNA (dsRNA) to induce RNAi, has come to prominence due to its safety and environmental benefits in addition to its wide host range and high target specificity. However, along with promising results in recent studies, several factors limiting SIGS RNAi efficiency have been recognized in insects and plants. While sprayed dsRNA on the plant surface can produce a robust RNAi response in some chewing insects, plant uptake and systemic movement of dsRNA is required for delivery to many other target organisms. For example, pests such as sucking insects require the presence of dsRNA in vascular tissues, while many fungal pathogens are predominately located in internal plant tissues. Investigating the mechanisms by which sprayed dsRNA enters and moves through plant tissues and understanding the barriers that may hinder this process are essential for developing efficient ways to deliver dsRNA into plant systems. In this review, we assess current knowledge of the plant foliar and cellular uptake of dsRNA molecules. We will also identify major barriers to uptake, including leaf morphological features as well as environmental factors, and address methods to overcome these barriers.

Tospoviruses infect numerous crop species worldwide, causing significant losses throughout the supply chain. As a defence mechanism, plants use RNA interference (RNAi) to generate virus-derived small-interfering RNAs (vsiRNAs), which target viral transcripts for degradation. Small RNA sequencing and in silico analysis of capsicum and N. benthamiana infected by tomato spotted wilt virus (TSWV) or capsicum chlorosis virus (CaCV) demonstrated the presence of abundant vsiRNAs, with host-specific differences evident for each pathosystem. Despite the biogenesis of vsiRNAs in capsicum and N. benthamiana, TSWV and CaCV viral loads were readily detectable. In response to tospovirus infection, the solanaceous host species also generated highly abundant virus-activated small interfering RNAs (vasiRNAs) against many endogenous transcripts, except for an N. benthamiana accession lacking a functional RDR1 gene. Strong enrichment for ribosomal protein-encoding genes and for many genes involved in protein processing in the endoplasmic reticulum suggested co-localisation of viral and endogenous transcripts as a basis for initiating vasiRNA biogenesis. RNA-seq and RT-qPCR-based analyses of target transcript expression revealed an inconsistent role for vasiRNAs in modulating gene expression in N. benthamiana, which may be characteristic of this tospovirus-host pathosystem.

Development of veterinary subunit vaccines comes with a spectrum of challenges, such as the choice of adjuvant, antigen delivery vehicle, and optimization of dosing strategy. Over the years, our laboratory has largely focused on investigating silica vesicles (SVs) for developing effective veterinary vaccines for multiple targets. Rhipicephalus microplus (cattle tick) are known to have a high impact on cattle health and the livestock industry in the tropical and subtropical regions. Development of vaccine using Bm86 antigen against R. microplus has emerged as an attractive alternative to control ticks. In this study, we have investigated the biodistribution of SV in a live animal model, as well as further explored the SV ability for vaccine development. Rhodamine-labeled SV-140-C18 (Rho-SV-140-C18) vesicles were used to adsorb the Cy5-labeled R. microplus Bm86 antigen (Cy5-Bm86) to enable detection and characterization of the biodistribution of SV as well as antigen in vivo in a small animal model for up to 28 days using optical fluorescence imaging. We tracked the in vivo biodistribution of SVs and Bm86 antigen at different timepoints (days 3, 8, 13, and 28) in BALB/c mice. The biodistribution analysis by live imaging as well as by measuring the fluorescent intensity of harvested organs over the duration of the experiment (28 days) showed greater accumulation of SVs at the site of injection. The Bm86 antigen biodistribution was traced in lymph nodes, kidney, and liver, contributing to our understanding how this delivery platform successfully elicits antibody responses in the groups administered antigen in combination with SV. Selected tissues (skin, lymph nodes, spleen, kidney, liver, and lungs) were examined for any cellular abnormalities by histological analysis. No adverse effect or any other abnormalities were observed in the tissues.

Sustainable agriculture relies on practices and technologies that combine effectiveness with a minimal environmental footprint. RNA interference (RNAi), a eukaryotic process in which transcript expression is reduced in a sequence-specific manner, can be co-opted for the control of plant pests and pathogens in a topical application system. Double-stranded RNA (dsRNA), the key trigger molecule of RNAi, has been shown to provide protection without the need for integration of dsRNA-expressing constructs as transgenes. Consequently, development of RNA-based biopesticides is gaining momentum as a narrow-spectrum alternative to chemical-based control measures, with pests and pathogens targeted with accuracy and specificity. Limitations for a commercially viable product to overcome include stable delivery of the topically applied dsRNA and extension of the duration of protection. In addition to the research focus on delivery of dsRNA, development of regulatory frameworks, risk identification, and establishing avoidance and mitigation strategies is key to widespread deployment of topical RNAi technologies. Once in place, these measures will provide the crop protection industry with the certainty necessary to expend resources on the development of innovative dsRNA-based products. Readily evident risks to human health appear minimal, with multiple barriers to uptake and a long history of consumption of dsRNA from plant material. Unintended impacts to the environment are expected to be most apparent in species closely related to the target. Holistic design practices, which incorporate bioinformatics-based dsRNA selection along with experimental testing, represent important techniques for elimination of adverse impacts.

Capsicum, an important vegetable crop in Queensland, Australia, is vulnerable to both elevated temperatures and capsicum chlorosis virus (CaCV). Thus, it is imperative to understand the genetic responses of capsicum plants (Capsicum annuum) to CaCV under elevated temperature conditions. Here, we challenged susceptible plants (cv. Yolo Wonder) with CaCV and investigated the effects of elevated temperature on symptom expression, the accumulation of virus-derived short interfering RNA (vsiRNA) and viral RNA, and the expression of plant defense-associated genes. CaCV-inoculated plants initially showed more severe symptoms and higher viral concentrations at a higher temperature (HT, 35 °C) than at ambient temperature (AT, 25 °C). However, symptom recovery and reduced viral RNA accumulation were seen in the CaCV-infected plants grown at HT at later stages of infection. We also observed that HT enhanced the accumulation of vsiRNAs and that, concurrently, RNA interference (RNAi)-related genes, including Dicer-like2 (DCL2), DCL4, RNA-dependent RNA polymerase 1 (RdRp1), RdRp6, and Argonaute2 (AGO2), were upregulated early during infection. Moreover, continuous high levels of vsiRNAs were observed during later stages of CaCV infection at HT. Overall, our investigation suggests that HT facilitates CaCV replication during early infection stages. However, this appears to lead to an early onset of antiviral RNA silencing, resulting in a subsequent recovery from CaCV in systemic leaves.

High-quality DNA and RNA forms the basis of genomic and genetic investigations. The extraction of DNA and RNA from woody trees, like avocado (Persea americana Mill.), is challenging due to compounds which interact with nucleic acids and influence separation. Previously reported methods of DNA and RNA extraction from avocado have issues of low yield, quality and applicability across different cultivars and tissue types. In the current study, methods have been optimised for high-quality DNA extraction from 40 avocado cultivars and RNA extraction from multiple tissue types, including roots, stem, leaves, flowers and fruits. The method is based on the modification of the cetyltrimethylammonium bromide buffer, centred around the specific optimisation of chemicals, such as sodium dodecyl sulphate, polyvinylpyrrolidone, sodium sulphite, polyethylene glycol and β-mercaptoethanol. The DNA extraction method yielded high-molecular weight DNA from the leaf tissue of 40 avocado cultivars belonging to Mexican, Guatemalan and West Indian avocado horticultural groups. The method was further optimised for RNA extraction from different avocado plant parts, enabling extraction using amounts as low as ~10 mg of starting material. The DNA and RNA extracted was successfully used for long- and short-read sequencing and gene expression analysis. The methods developed may also be applicable to other recalcitrant plant species.

Abstract Background Potato virus Y (PVY) is one of the most economically important pathogen of potato that is present as biologically distinct strains. The virus-derived small interfering RNAs (vsiRNAs) from potato cv. Russet Burbank individually infected with PVY-N, PVY-NTN and PVY-O strains were recently characterized. Plant defense RNA-silencing mechanisms deployed against viruses produce vsiRNAs to degrade homologous viral transcripts. Based on sequence complementarity, the vsiRNAs can potentially degrade host RNA transcripts raising the prospect of vsiRNAs as pathogenicity determinants in virus-host interactions. This study investigated the global effects of PVY vsiRNAs on the host potato transcriptome. Methods The strain-specific vsiRNAs of PVY, expressed in high copy number, were analyzed in silico for their proclivity to target potato coding and non-coding RNAs using psRobot and psRNATarget algorithms. Functional annotation of target coding transcripts was carried out to predict physiological effects of the vsiRNAs on the potato cv. Russet Burbank. The downregulation of selected target coding transcripts was further validated using qRT-PCR. Results The vsiRNAs derived from biologically distinct strains of PVY displayed diversity in terms of absolute number, copy number and hotspots for siRNAs on their respective genomes. The vsiRNAs populations were derived with a high frequency from 6 K1, P1 and Hc-Pro for PVY-N, P1, Hc-Pro and P3 for PVY-NTN, and P1, 3′ UTR and NIa for PVY-O genomic regions. The number of vsiRNAs that displayed interaction with potato coding transcripts and number of putative coding target transcripts were comparable between PVY-N and PVY-O, and were relatively higher for PVY-NTN. The most abundant target non-coding RNA transcripts for the strain specific PVY-derived vsiRNAs were found to be MIR821, 28S rRNA,18S rRNA, snoR71, tRNA-Met and U5. Functional annotation and qRT-PCR validation suggested that the vsiRNAs target genes involved in plant hormone signaling, genetic information processing, plant-pathogen interactions, plant defense and stress response processes in potato. Conclusions The findings suggested that the PVY-derived vsiRNAs could act as a pathogenicity determinant and as a counter-defense strategy to host RNA silencing in PVY-potato interactions. The broad range of host genes targeted by PVY vsiRNAs in infected potato suggests a diverse role for vsiRNAs that includes suppression of host stress responses and developmental processes. The interactome scenario is the first report on the interaction between one of the most important Potyvirus genome-derived siRNAs and the potato transcripts.

In plants, juvenile to adult phase transition is regulated by the sequential activity of two microRNAs: miR156 and miR172. A decline in miR156 and increase in miR172 abundance is associated with phase transition. There is very limited information on phase transition in economically important horticultural tree crops, which have a significantly long vegetative phase affecting fruit bearing. Here, we profiled various molecular cues known to be involved in phase transition and flowering, including the microRNAs miR156 and miR172, in three horticultural tree crops: avocado (Persea americana), mango (Mangifera indica), and macadamia (Macadamia integrifolia). We observed that miR156 expression decreases as these trees age and can potentially be used as a juvenility marker. Consistent with findings in annual plants, we also observed conserved regulation of the miR156-SPL3/4/5 regulatory module in these genetically distant tree crops, suggesting that this pathway may play a highly conserved role in vegetative identity. Meanwhile, the abundance of miR172 and its target AP2-like genes as well as the accumulation level of SPL9 transcripts were not related with plant age in these crops except in avocado where miR172 expression increased steadily. Finally, we demonstrate that various floral genes, including AP1 and SOC1 were upregulated in the reproductive phase and can be used as potential markers for the reproductive phase transition. Overall, this study provides an insight into the molecular associations of juvenility and phase transition in horticultural trees where crop breeding and improvement are encumbered by long juvenile phases.

Plant viruses are difficult to control, and they decrease both the quality and yield of crops, thus threatening global food security. A new approach that uses topical application of double-stranded RNA (dsRNA) to induce antiviral RNA-interference has been shown to be effective at preventing virus infection in a range of plants following mechanical inoculation. In this study, topical application of dsRNA was effective against mechanical inoculation and aphid-mediated inoculation with the potyvirus bean common mosaic virus (BCMV). Topical application of dsRNAs targeting either the coding region of the potyviral nuclear inclusion b (NIb) protein (BCMVNIb-dsRNA) or the coat protein (CP) coding region (BCMVCP-dsRNA) protected Nicotiana benthamiana and cowpea (Vigna unguiculata) plants against mechanical inoculation with BCMV. BCMVCP-dsRNA was selected for subsequent aphid transmission experiments. BCMVCP-dsRNA was loaded onto layered double hydroxide nanoparticles to form BCMVCP-BioClay which is a more stable formulation for delivering dsRNA than naked dsRNA. BCMVCP-BioClay was shown to be successful in protecting plants against BCMV transmission by the aphid Myzus persicae. Spraying detached N. benthamiana leaves with BCMVCP-BioClay 5 days prior to exposure to viruliferous aphids protected the leaves from infection by BCMV. Importantly, spraying of intact N. benthamiana and cowpea plants with BCMVCP-BioClay 5 days prior to exposure to viruliferous aphids protected plants of both species from BCMV infection. This study demonstrates that topical application of dsRNA using BioClay protects plants from aphid-mediated virus transmission, which is an important first step toward developing practical application of this approach in crop protection.

Recent development and implementation of crop cryopreservation protocols has increased the capacity to maintain recalcitrant seeded germplasm collections via cryopreserved in vitro material. To preserve the greatest possible plant genetic resources globally for future food security and breeding programs, it is essential to integrate in situ and ex situ conservation methods into a cohesive conservation plan. In vitro storage using tissue culture and cryopreservation techniques offers promising complementary tools that can be used to promote this approach. These techniques can be employed for crops difficult or impossible to maintain in seed banks for long-term conservation. This includes woody perennial plants, recalcitrant seed crops or crops with no seeds at all and vegetatively or clonally propagated crops where seeds are not true-to-type. Many of the world’s most important crops for food, nutrition and livelihoods, are vegetatively propagated or have recalcitrant seeds. This review will look at ex situ conservation, namely field repositories and in vitro storage for some of these economically important crops, focusing on conservation strategies for avocado. To date, cultivar-specific multiplication protocols have been established for maintaining multiple avocado cultivars in tissue culture. Cryopreservation of avocado somatic embryos and somatic embryogenesis have been successful. In addition, a shoot-tip cryopreservation protocol has been developed for cryo-storage and regeneration of true-to-type clonal avocado plants.

Tomato spotted wilt virus (TSWV) is the type member of the genus Orthotospovirus in the family Tospoviridae and order Bunyavirales. TSWV, transmitted by several species of thrips, causes significant disease losses to agronomic and horticultural crops worldwide, impacting both the yield and quality of the produce. Management strategies include growing virus-resistant cultivars, cultural practices, and managing thrips vectors through pesticide application. However, numerous studies have reported that TSWV isolates can overcome host-plant resistance, while thrips are developing resistance to pesticides that were once effective. RNA interference (RNAi) offers a means of host defence by using double-stranded (ds) RNA to initiate gene silencing against invading viruses. However, adoption of this approach requires production and use of transgenic plants and thus limits the practical application of RNAi against TSWV and other viruses. To fully utilize the potential of RNAi for virus management at the field level, new and novel approaches are needed. In this review, we summarize RNAi and highlight the potential of topical or exogenous application of RNAi triggers for managing TSWV and thrips vectors.

RNA interference (RNAi) is a powerful approach for sequence-specific gene silencing, displaying tremendous potential for functional genomics studies in hemipteran insects. Exploiting RNAi allows the biological roles of critical genes to be defined and aids the development of RNAi-based biopesticides. In this review, we provide context to the rapidly expanding field of RNAi-based functional genomics studies in hemipteran insects. We highlight the most widely used RNAi delivery strategies, including microinjection, oral ingestion and topical application. Additionally, we discuss the key variables affecting RNAi efficacy in hemipteran insects, including insect life-stage, gene selection, the presence of nucleases, and the role of core RNAi machinery. In conclusion, we summarise the application of RNAi in functional genomics studies in Hemiptera, focusing on genes involved in reproduction, behaviour, metabolism, immunity and chemical resistance across 33 species belonging to 14 families.

Animal health measures mainly rely on vaccination or chemical control for major pests and pathogens, causing issues of residue, toxicity and development of resistance. For example, control of Sheep flystrike and lice-infestation affecting the Australia’s sheep/wool industry (>3.5 B) have developed resistance to nearly all control chemicals used in the past. Topicals RNAi provides an innovative clean-green, non-toxic, environmentally sustainable biological control solution. Biodegradable clay particles as carriers can be used to deliver double stranded RNA (dsRNA), the key trigger molecule of RNA interference pathway. As an early proof of concept, we investigated the stability dsRNA loaded on two types of Clay particles: Clay 1 (releases dsRNA under acidic conditions) and Clay 2 (releases dsRNA under alkaline conditions) on cattle hide. Cattle skin was treated with Cy3 labelled dsRNA alone and Cy3 labelled dsRNA loaded on Clay1 or Clay2. The skin samples treated with the Cy3 formulations were imaged using confocal microscopy. Once imaged, the skin samples were washed and stored at room temperature for 5 days, later the samples were re-imaged to detect the fluorescent signal (Figure 1). The dsRNA loaded on clay particles was stable unlike naked Cy3-dsRNA which degraded and was not visible after washing. This increased inherent stability of the dsRNA molecules, combined with the environmental stability afforded by the Clay particles, offers promise to provide a sustainable solution for animal health. Topical RNAi can reduce reliance on trade withholding periods of meat/wool without chemical residues, enhance animal welfare and increase production of premium quality meat/wool, improve export potential, competitiveness and long-term profitability of livestock industry.

Tospoviruses cause significant losses to a wide range of agronomic and horticultural crops worldwide. The type member, Tomato spotted wilt tospovirus (TSWV), causes systemic infection in susceptible tomato cultivars, whereas its infection is localized in cultivars carrying the Sw-5 resistance gene. The response to TSWV infection in tomato cultivars with or without Sw-5 was determined at the virus small RNA level in the locally infected leaf. Predicted reads were aligned to TSWV reference sequences. The TSWV genome was found to be differentially processed among each of the three-viral genomic RNAs—Large (L), Medium (M) and Small (S)—in the Sw-5(+) compared to Sw-5(−) genotypes. In the Sw-5(+) cultivar, the L RNA had the highest number of viral small-interfering RNAs (vsiRNAs), whereas in the Sw-5(−) cultivar the number was higher in the S RNA. Among the three-viral genomic RNAs, the distribution of hotspots showed a higher number of reads per million reads of vsiRNAs of 21 and 22 nt class at the 5′ and 3′ ends of the L and the S RNAs, with less coverage in the M RNA. In the Sw-5(−) cultivar, the nature of the 5′ nucleotide-end in the siRNAs varied significantly; reads with 5′-adenine-end were most abundant in the mock control, whereas cytosine and uracil were more abundant in the infected plants. No such differences were seen in case of the resistant genotype. Findings provided insights into the response of tomato cultivars to TSWV infection.

Modern agricultural improvements rely on the planting of elite crop lines to maximise the [...]

The whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) is a significant global pest of economically important vegetable, fibre, and ornamental crops. Whiteflies directly damage the plants by piercing and sucking essential nutrients, indirectly through honeydew secretion and by transmitting more than 200 plant viruses that cause millions of dollars in produce losses per year. Whitefly management is mostly reliant on the heavy use of chemical insecticides. However, this ultimately leads to increasing resistance development, detrimental effects on beneficial insects and biomagnification of ecologically harmful chemicals in the environment. Responding to consumer demands for more selective, less toxic, non-GM insect control strategies, RNA interference (RNAi) has emerged as a potential game-changing solution. The RNA interference (RNAi) is a homology-dependent mechanism of gene silencing that represents a feasible and sustainable technology for the management of insect pests. In the present study, twenty-two whitefly genes were selected based on their essential function in the insect and tested in artificial diet bioassays for mortality and gene silencing efficacy. The nine most effective dsRNA constructs showed moderate-to-high whitefly mortality as compared to negative controls six days post-feeding. qPCR analysis further demonstrated significant knockdown of target gene mRNA expression. Additionally, uptake and spread of fluorescently labelled dsRNA was evident beyond the midgut of the whitefly supporting the systemic spreading of RNAi effectors. Taken together, the oral delivery of dsRNA shows effective RNAi mediated gene silencing of target genes and offers a viable approach for the development of dsRNA biopesticides against hemipteran pest.

Thrips-transmitted tospoviruses are economically important viruses affecting a wide range of field and horticultural crops worldwide. Tomato spotted wilt virus is the type member of the Tospovirus genus with a broad host range of more than 900 plant species. Interactions between these viruses and their plant hosts and insect vectors via RNA interference pathways are likely a key determinant of pathogenicity. The current investigation, for the first time, compares biogenesis of small RNAs between the plant host and insect vector in the presence or absence of TSWV. Unique viral small interfering RNA (vsiRNA) profiles are evident for Arachis hypogaea (peanut) and Frankliniella fusca (thrips vector) following infection with TSWV. Differences between vsiRNA profiles for these plant and insect species, such as the relative abundance of 21 nt and 22 nt vsiRNAs and locations of alignment hotspots, reflect the diverse siRNA biosynthesis pathways of their respective kingdoms. The presence of unique vsiRNAs in F. fusca samples indicates that vsiRNA generation takes place within the thrips, and not solely through uptake via feeding on vsiRNAs produced in infected A. hypogaea. The study also shows key vsiRNA profile differences for TSWV among plant families, which are evident in the case of A. hypogaea, a legume, and members of Solanaceae (S. lycopersicum and N. benthamiana). Distinctively, overall small RNA biogenesis in A. hypogaea is markedly affected with an absence of the 24 nt small RNAs in TSWV-infected plants, possibly leading to wide-spread molecular and phenotypic perturbations specific to this species. These findings add significant information on the host-virus-vector interaction in terms of RNAi pathways and may lead to better crop and vector specific control strategies.

Anaplasma marginale is the most prevalent tick-borne livestock pathogen and poses a significant threat to cattle industry. In contrast to currently available live blood-derived vaccines against A. marginale, alternative safer and better-defined subunit vaccines will be of great significance. Two proteins (VirB9-1 and VirB9-2) from the Type IV secretion system of A. marginale have been shown to induce humoral and cellular immunity. In this study, Escherichia coli were used to express VirB9-1 and VirB9-2 proteins. Silica vesicles having a thin wall of 6 nm and pore size of 5.8 nm were used as the carrier and adjuvant to deliver these two antigens both as individual or mixed nano-formulations. High loading capacity was achieved for both proteins, and the mouse immunisation trial with individual as well as mixed nano-formulations showed high levels of antibody titres over 107 and strong T-cell responses. The mixed nano-formulation also stimulated high-level recall responses in bovine T-cell proliferation assays. These results open a promising path towards the development of efficient A. marginale vaccines and provide better understanding on the role of silica vesicles to deliver multivalent vaccines as mixed nano-formulations able to activate both B-cell and T-cell immunity, for improved animal health.

Each year, 20%⁻40% of crops are lost due to plant pests and pathogens. Existing plant disease management relies predominantly on toxic pesticides that are potentially harmful to humans and the environment. Nanotechnology can offer advantages to pesticides, like reducing toxicity, improving the shelf-life, and increasing the solubility of poorly water-soluble pesticides, all of which could have positive environmental impacts. This review explores the two directions in which nanoparticles can be utilized for plant disease management: either as nanoparticles alone, acting as protectants; or as nanocarriers for insecticides, fungicides, herbicides, and RNA-interference molecules. Despite the several potential advantages associated with the use of nanoparticles, not many nanoparticle-based products have been commercialized for agricultural application. The scarcity of commercial applications could be explained by several factors, such as an insufficient number of field trials and underutilization of pest⁻crop host systems. In other industries, nanotechnology has progressed rapidly, and the only way to keep up with this advancement for agricultural applications is by understanding the fundamental questions of the research and addressing the scientific gaps to provide a rational and facilitate the development of commercial nanoproducts.

Bovine Viral Diarrhoea Virus (BVDV) is one of the most serious pathogen, which causes tremendous economic loss to the cattle industry worldwide, meriting the development of improved subunit vaccines. Structural glycoprotein E2 is reported to be a major immunogenic determinant of BVDV virion. We have developed a novel hollow silica vesicles (SV) based platform to administer BVDV-1 Escherichia coli-expressed optimised E2 (oE2) antigen as a nanovaccine formulation. The SV-140 vesicles (diameter 50 nm, wall thickness 6 nm, perforated by pores of entrance size 16 nm and total pore volume of 0.934 cm3 g(-1)) have proven to be ideal candidates to load oE2 antigen and generate immune response. The current study for the first time demonstrates the ability of freeze-dried (FD) as well as non-FD oE2/SV140 nanovaccine formulation to induce long-term balanced antibody and cell mediated memory responses for at least 6 months with a shortened dosing regimen of two doses in small animal model. The in vivo ability of oE2 (100 μg)/SV-140 (500 μg) and FD oE2 (100 μg)/SV-140 (500 μg) to induce long-term immunity was compared to immunisation with oE2 (100 μg) together with the conventional adjuvant Quil-A from the Quillaja saponira (10 μg) in mice. The oE2/SV-140 as well as the FD oE2/SV-140 nanovaccine generated oE2-specific antibody and cell mediated responses for up to six months post the final second immunisation. Significantly, the cell-mediated responses were consistently high in mice immunised with oE2/SV-140 (1,500 SFU/million cells) at the six-month time point. Histopathology studies showed no morphological changes at the site of injection or in the different organs harvested from the mice immunised with 500 μg SV-140 nanovaccine compared to the unimmunised control. The platform has the potential for developing single dose vaccines without the requirement of cold chain storage for veterinary and human applications.

Bovine viral diarrhoea virus 1 (BVDV-1) is arguably the most important viral disease of cattle. It is associated with reproductive, respiratory and chronic diseases in cattle across the world. In this study we have investigated the capacity of the major immunological determinant of BVDV-1, the E2 protein combined with hollow type mesoporous silica nanoparticles with surface amino functionalisation (HMSA), to stimulate immune responses in sheep. The current work also investigated the immunogenicity of the E2 nanoformulation before and after freeze-drying processes. The optimal excipient formulation for freeze-drying of the E2 nanoformulation was determined to be 5% trehalose and 1% glycine. This excipient formulation preserved both the E2 protein integrity and HMSA particle structure. Sheep were immunised three times at three week intervals by subcutaneous injection with 500 μg E2 adsorbed to 6.2 mg HMSA as either a non-freeze-dried or freeze-dried nanoformulation. The capacity of both nanovaccine formulations to generate humoral (antibody) and cell-mediated responses in sheep were compared to the responses in sheep immunisation with Opti-E2 (500 μg) together with the conventional adjuvant Quil-A (1 mg), a saponin from the Molina tree (Quillaja saponira). The level of the antibody responses detected to both the non-freeze-dried and freeze-dried Opti-E2/HMSA nanoformulations were similar to those obtained for Opti-E2 plus Quil-A, demonstrating the E2 nanoformulations were immunogenic in a large animal, and freeze-drying did not affect the immunogenicity of the E2 antigen. Importantly, it was demonstrated that the long term cell-mediated immune responses were detectable up to four months after immunisation. The cell-mediated immune responses were consistently high in all sheep immunised with the freeze-dried Opti-E2/HMSA nanovaccine formulation (>2,290 SFU/million cells) compared to the non-freeze-dried nanovaccine formulation (213-500 SFU/million cells). This study is the first to demonstrate that a freeze-dried silica mesoporous nanovaccine formulation gives balanced immune responses in a production animal.

Post-transcriptional gene silencing (PTGS), an intrinsic plant defense mechanism, can be efficiently triggered by double stranded (ds)RNA-producing transgenes and can provide high level virus resistance by specific targeting of cognate viral RNA. The discovery of virus-encoded suppressors of PTGS led to concerns about the stability of such resistance. Here, we show that Cucumber mosaic virus (CMV) is able to suppress dsRNA-induced PTGS and the associated Potato virus Y (PVY) immunity in tobacco. CMV suppression supported only a transient PVY accumulation and did not prevent recovery of the transgenic plants from PVY infection. CMV inoculation resulted in strongly increased transgene mRNA levels due to suppression of PTGS, but accumulation of PVY-specific small interfering (si)RNA was unaffected. However, PVY accumulation in previously immune plants resulted in increased PVY siRNA levels and transgene mRNA was no longer detected, despite the presence of CMV. Transgene mRNA returned to high levels once PVY was no longer detected in CMV-infected plants. Recovered and chronically CMV-infected tissues were immune to further PVY infection.

BACKGROUND:Viral small RNAs (vsiRNAs) in the infected host can be generated from viral double-stranded RNA replicative intermediates, self-complementary regions of the viral genome or from the action of host RNA-dependent RNA polymerases on viral templates. The vsiRNA abundance and profile as well as the endogenous small RNA population can vary between different hosts infected by the same virus influencing viral pathogenicity and host response. There are no reports on the analysis of vsiRNAs of Tomato spotted wilt virus (TSWV), a segmented negative stranded RNA virus in the family Bunyaviridae, with two of its gene segments showing ambisense gene arrangement. The virus causes significant economic losses to numerous field and horticultural crops worldwide. PRINCIPAL FINDINGS:Tomato spotted wilt virus (TSWV)-specific vsiRNAs were characterized by deep sequencing in virus-infected experimental host Nicotiana benthamiana and a commercial, susceptible host tomato. The total small (s) RNA reads in TSWV-infected tomato sample showed relatively equal distribution of 21, 22 and 24 nt, whereas N. benthamiana sample was dominated by 24 nt total sRNAs. The number of vsiRNA reads detected in tomato was many a magnitude (~350:1) higher than those found in N. benthamiana, however the profile of vsiRNAs in terms of relative abundance 21, 22 and 24 nt class size was similar in both the hosts. Maximum vsiRNA reads were obtained for the M RNA segment of TSWV while the largest L RNA segment had the least number of vsiRNAs in both tomato and N. benthamiana. Only the silencing suppressor, NSs, of TSWV recorded higher antisense vsiRNA with respect to the coding frame among all the genes of TSWV. SIGNIFICANCE:Details of the origin, distribution and abundance of TSWV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. It also has major implications toward our understanding of the differential processing of vsiRNAs in antiviral defense and viral pathogenicity.

Bovine anaplasmosis or cattle-tick fever is a tick-borne haemolytic disease caused by the rickettsial haemoparasite Anaplasma marginale in tropical and subtropical areas of the world. While difficult to express, the proteins VirB9-1 and VirB10 are immunogenic components of the outer membrane type IV secretion system that have been identified as candidate antigens for vaccines targeting of A. marginale. Soluble VirB9-1 and VirB10 were successfully expressed using Pichia pastoris. When formulated with the self-adjuvanting silica vesicles, SV-100 (diameter: 50 nm, and pore entrance size: 6 nm), 200 µg of VirB9-1 and VirB10 were adsorbed per milligram of nanoparticle. The VirB9-1 and VirB10, SV-100 formulations were shown to induce higher antibody responses in mice compared to the QuilA formulations. Moreover, intracellular staining of selected cytokines demonstrated that both VirB9-1 and VirB10 formulations induced cell-mediated immune responses in mice. Importantly, the SV-100 VirB9-1 and VirB10 complexes were shown to specifically stimulate bovine T-cell linages derived from calves immunised with A. marginale outer membrane fractions, suggesting formulations will be useful for bovine immunisation and protection studies. Overall this study demonstrates the potential of self-adjuvanting silica vesicle formulations to address current deficiencies in vaccine delivery applications.

BACKGROUND: New viruses pathogenic to plants continue to emerge due to mutation, recombination, or reassortment among genomic segments among individual viruses. Tospoviruses cause significant economic damage to a wide range of crops in many parts of the world. The genetic or molecular basis of the continued emergence of new tospoviruses and new hosts is not well understood though it is generally accepted that reassortment and/or genetic complementation among the three genomic segments of individual viruses could be contributing to this variability since plants infected with more than one tospovirus are not uncommon in nature. METHODOLOGY/PRINCIPAL FINDINGS: Two distinct and economically important tospoviruses, Iris yellow spot virus (IYSV) and Tomato spotted wilt virus (TSWV), were investigated for inter-virus interactions at the molecular level in dually-infected plants. Datura (Datura stramonium) is a permissive host for TSWV, while it restricts the movement of IYSV to inoculated leaves. In plants infected with both viruses, however, TSWV facilitated the selective movement of the viral gene silencing suppressor (NSs) gene of IYSV to the younger, uninoculated leaves. The small RNA expression profiles of IYSV and TSWV in single- and dually-infected datura plants showed that systemic leaves of dually-infected plants had reduced levels of TSWV N gene-specific small interfering RNAs (siRNAs). No TSWV NSs-specific siRNAs were detected either in the inoculated or systemic leaves of dually-infected datura plants indicating a more efficient suppression of host silencing machinery in the presence of NSs from both viruses as compared to the presence of only TSWV NSs. CONCLUSION/SIGNIFICANCE: Our study identifies a new role for the viral gene silencing suppressor in potentially modulating the biology and host range of viruses and underscores the importance of virally-coded suppressors of gene silencing in virus infection of plants. This is the first experimental evidence of functional complementation between two distinct tospoviruses in the Bunyaviridae family.

MicroRNA (miRNA) and other types of small regulatory RNAs play a crucial role in the regulation of gene expression in eukaryotes. Several distinct classes of small regulatory RNAs have been discovered in recent years. To extend the repertoire of small RNAs characterized in mammals and to examine relationship between host miRNA expression and viral infection we used Illumina's ultrahigh throughput sequencing approach. We sequenced three small RNA libraries prepared from cell line derived from the adult bovine kidney under normal conditions and upon infection of the cell line with Bovine herpesvirus 1. We used a bioinformatics approach to distinguish authentic mature miRNA sequences from other classes of small RNAs and short RNA fragments represented in the sequencing data. Using this approach we detected 219 out of 356 known bovine miRNAs and 115 respective miRNA* sequences. In addition we identified five new bovine orthologs of known mammalian miRNAs and discovered 268 new cow miRNAs many of which are not identifiable in other mammalian genomes and thus might be specific to the ruminant lineage. In addition we found seven new bovine mirtron candidates. We also discovered 10 small nucleolar RNA (snoRNA) loci that give rise to small RNA with possible miRNA-like function. Results presented in this study extend our knowledge of the biology and evolution of small regulatory RNAs in mammals and illuminate mechanisms of small RNA biogenesis and function. New miRNA sequences and the original sequencing data have been submitted to miRNA repository (miRBase) and NCBI GEO archive respectively. We envisage that these resources will facilitate functional annotation of the bovine genome and promote further functional and comparative genomics studies of small regulatory RNA in mammals.