Publications by authors named "Neena Mitter"

56 Publications

Current Status and Potential of RNA Interference for the Management of Tomato Spotted Wilt Virus and Thrips Vectors.

Pathogens 2021 Mar 9;10(3). Epub 2021 Mar 9.

Centre for Horticultural Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, QLD 4067, Australia.

Tomato spotted wilt virus (TSWV) is the type member of the genus in the family 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.
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http://dx.doi.org/10.3390/pathogens10030320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001667PMC
March 2021

Characterization of the Biodistribution of a Silica Vesicle Nanovaccine Carrying a Protective Antigen With Live Animal Imaging.

Front Bioeng Biotechnol 2020 18;8:606652. Epub 2021 Jan 18.

Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, QLD, Australia.

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. (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 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-C (Rho-SV-140-C) vesicles were used to adsorb the Cy5-labeled Bm86 antigen (Cy5-Bm86) to enable detection and characterization of the biodistribution of SV as well as antigen in a small animal model for up to 28 days using optical fluorescence imaging. We tracked the 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.
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http://dx.doi.org/10.3389/fbioe.2020.606652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848120PMC
January 2021

Synergistic Effect of Two Nanotechnologies Enhances the Protective Capacity of the Sporozoite p67C Antigen in Cattle.

J Immunol 2021 Feb 8;206(4):686-699. Epub 2021 Jan 8.

Animal and Human Health Program, International Livestock Research Institute, Nairobi 00100, Kenya.

East Coast fever (ECF), caused by , is the most important tick-borne disease of cattle in sub-Saharan Africa. Practical disadvantages associated with the currently used live-parasite vaccine could be overcome by subunit vaccines. An 80-aa polypeptide derived from the C-terminal portion of p67, a sporozoite surface Ag and target of neutralizing Abs, was the focus of the efforts on subunit vaccines against ECF and subjected to several vaccine trials with very promising results. However, the vaccination regimen was far from optimized, involving three inoculations of 450 μg of soluble p67C (s-p67C) Ag formulated in the Seppic adjuvant Montanide ISA 206 VG. Hence, an improved formulation of this polypeptide Ag is needed. In this study, we report on two nanotechnologies that enhance the bovine immune responses to p67C. Individually, HBcAg-p67C (chimeric hepatitis B core Ag virus-like particles displaying p67C) and silica vesicle (SV)-p67C (s-p67C adsorbed to SV-140-C, octadecyl-modified SVs) adjuvanted with ISA 206 VG primed strong Ab and T cell responses to p67C in cattle, respectively. Coimmunization of cattle () with HBcAg-p67C and SV-p67C resulted in stimulation of both high Ab titers and CD4 T cell response to p67C, leading to the highest subunit vaccine efficacy we have achieved to date with the p67C immunogen. These results offer the much-needed research depth on the innovative platforms for developing effective novel protein-based bovine vaccines to further the advancement.
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http://dx.doi.org/10.4049/jimmunol.2000442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851744PMC
February 2021

Current scenario of RNAi-based hemipteran control.

Pest Manag Sci 2021 May 6;77(5):2188-2196. Epub 2020 Nov 6.

Queensland Alliance for Agriculture and Food Innovation, Centre for Horticultural Sciences, The University of Queensland, Brisbane, Australia.

RNA interference (RNAi) is an homology-dependent gene silencing mechanism that is a feasible and sustainable avenue for the management of hemipteran pests. Commercial implementation of RNAi-based control strategies is impeded by limited knowledge about the mechanism of double-stranded RNA (dsRNA) uptake, the function of core RNAi genes and systemic RNAi mechanisms in hemipteran insects. This review briefly summarizes recent progress in RNAi-based studies aimed to reduce insect populations, viral transmission and insecticide resistance focusing on hemipteran pests. This review explores RNAi-mediated management of hemipteran insects and offers potential solutions, including in silico approaches coupled with laboratory-based toxicity assays to circumvent potential off-target effects against beneficial organisms. We further explore ways to mitigate degradation of dsRNA in the environment and the insect such as stacking and formulation of dsRNA effectors. Finally, we conclude by considering nontransformative RNAi approaches, concatomerization of RNAi sequences and pyramiding RNAi with active constituents to reduce dsRNA production and application cost, and to improve broad-spectrum hemipteran pest control. © 2020 Society of Chemical Industry.
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http://dx.doi.org/10.1002/ps.6153DOI Listing
May 2021

RNAi-Based Functional Genomics in Hemiptera.

Insects 2020 Aug 20;11(9). Epub 2020 Aug 20.

Queensland Alliance for Agriculture and Food Innovation, Centre for Horticultural Sciences, The University of Queensland, Brisbane 4072, Queensland, Australia.

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.
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http://dx.doi.org/10.3390/insects11090557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564473PMC
August 2020

The (TSWV) Genome is Differentially Targeted in TSWV-Infected Tomato () with or without Gene.

Viruses 2020 03 26;12(4). Epub 2020 Mar 26.

Department of Plant Pathology, Washington State University, Pullman, WA 99164, USA.

Tospoviruses cause significant losses to a wide range of agronomic and horticultural crops worldwide. The type member, (TSWV), causes systemic infection in susceptible tomato cultivars, whereas its infection is localized in cultivars carrying the 5 resistance gene. The response to TSWV infection in tomato cultivars with or without -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 -5(+) compared to -5(-) genotypes. In the -5(+) cultivar, the L RNA had the highest number of viral small-interfering RNAs (vsiRNAs), whereas in the -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 -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.
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http://dx.doi.org/10.3390/v12040363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232525PMC
March 2020

A Perspective on RNAi-Based Biopesticides.

Front Plant Sci 2020 12;11:51. Epub 2020 Feb 12.

Centre for Horticultural Science, Queensland Alliance for Agriculture and Food Innovation, University of Queensland, Saint Lucia, QLD, Australia.

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.
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http://dx.doi.org/10.3389/fpls.2020.00051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7028687PMC
February 2020

Can-Seq: a PCR and DNA sequencing strategy for identifying new alleles of known and candidate genes.

Plant Methods 2020 13;16:16. Epub 2020 Feb 13.

1School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072 Australia.

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 mutants with defects in systemic spreading of RNA interference (RNAi), or more specifically in () 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 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 defect. Importantly, by exclusion, the Can-Seq pipeline also identified 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.
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http://dx.doi.org/10.1186/s13007-020-0555-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017465PMC
February 2020

Scion control of miRNA abundance and tree maturity in grafted avocado.

BMC Plant Biol 2019 Sep 3;19(1):382. Epub 2019 Sep 3.

Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia, Brisbane, Queensland, 4072, Australia.

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.
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http://dx.doi.org/10.1186/s12870-019-1994-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6724330PMC
September 2019

The avocado genome informs deep angiosperm phylogeny, highlights introgressive hybridization, and reveals pathogen-influenced gene space adaptation.

Proc Natl Acad Sci U S A 2019 08 6;116(34):17081-17089. Epub 2019 Aug 6.

Unidad de Genomica Avanzada/Langebio, Centro de Investigación y de Estudios Avanzados, Irapuato 36821, México;

The avocado, , is a fruit crop of immense importance to Mexican agriculture with an increasing demand worldwide. Avocado lies in the anciently diverged magnoliid clade of angiosperms, which has a controversial phylogenetic position relative to eudicots and monocots. We sequenced the nuclear genomes of the Mexican avocado race, var. , and the most commercially popular hybrid cultivar, Hass, and anchored the latter to chromosomes using a genetic map. Resequencing of Guatemalan and West Indian varieties revealed that ∼39% of the Hass genome represents Guatemalan source regions introgressed into a Mexican race background. Some introgressed blocks are extremely large, consistent with the recent origin of the cultivar. The avocado lineage experienced 2 lineage-specific polyploidy events during its evolutionary history. Although gene-tree/species-tree phylogenomic results are inconclusive, syntenic ortholog distances to other species place avocado as sister to the enormous monocot and eudicot lineages combined. Duplicate genes descending from polyploidy augmented the transcription factor diversity of avocado, while tandem duplicates enhanced the secondary metabolism of the species. Phenylpropanoid biosynthesis, known to be elicited by (anthracnose) pathogen infection in avocado, is one enriched function among tandems. Furthermore, transcriptome data show that tandem duplicates are significantly up- and down-regulated in response to anthracnose infection, whereas polyploid duplicates are not, supporting the general view that collections of tandem duplicates contribute evolutionarily recent "tuning knobs" in the genome adaptive landscapes of given species.
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http://dx.doi.org/10.1073/pnas.1822129116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6708331PMC
August 2019

Juvenility and Vegetative Phase Transition in Tropical/Subtropical Tree Crops.

Front Plant Sci 2019 4;10:729. Epub 2019 Jun 4.

Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, QLD, Australia.

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 (), mango (), and macadamia (). 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- 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 genes as well as the accumulation level of 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 and 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.
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http://dx.doi.org/10.3389/fpls.2019.00729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6558100PMC
June 2019

Moving policy and regulation forward for nanotechnology applications in agriculture.

Nat Nanotechnol 2019 Jun;14(6):508-510

Centre for Policy Futures, Faculty of Humanities and Social Sciences, The University of Queensland, Brisbane, Queensland, Australia.

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http://dx.doi.org/10.1038/s41565-019-0464-4DOI Listing
June 2019

How nanocarriers delivering cargos in plants can change the GMO landscape.

Nat Nanotechnol 2019 Jun;14(6):512-514

Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, Queensland, Australia.

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http://dx.doi.org/10.1038/s41565-019-0463-5DOI Listing
June 2019

Exogenous Application of RNAi-Inducing Double-Stranded RNA Inhibits Aphid-Mediated Transmission of a Plant Virus.

Front Plant Sci 2019 15;10:265. Epub 2019 Mar 15.

Centre of Horticultural Science, Queensland Alliance of Agriculture and Food Innovation, The University of Queensland, Brisbane, QLD, Australia.

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 and cowpea () 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 . Spraying detached leaves with BCMVCP-BioClay 5 days prior to exposure to viruliferous aphids protected the leaves from infection by BCMV. Importantly, spraying of intact 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.
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http://dx.doi.org/10.3389/fpls.2019.00265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6429036PMC
March 2019

Molecular characterization and analysis of conserved potyviral motifs in bean common mosaic virus (BCMV) for RNAi-mediated protection.

Arch Virol 2019 Jan 9;164(1):181-194. Epub 2018 Oct 9.

Centre of Horticultural Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, Brisbane, QLD, 4072, Australia.

Australian bean common mosaic virus (BCMV) isolates were sequenced, and the sequences were compared to global BCMV and bean common mosaic necrosis virus (BCMNV) sequences and analysed for conserved potyviral motifs to generate in planta RNA-interference (RNAi) resistance. Thirty-nine out of 40 previously reported potyvirus motifs were conserved among all 77 BCMV/BCMNV sequences. Two RNAi target regions were selected for dsRNA construct design, covering 920 bp of the nuclease inclusion b (NIb) protein and 461 bp of the coat protein (CP). In silico prediction of the effectiveness of these constructs for broad-spectrum defence against the 77 BCMV and BCMNV sequences was done via analysis of putative 21-nucleotide (nt) and 22-nt small-interfering RNAs (siRNAs) generated from the target regions. The effectiveness of both constructs for siRNA generation and BCMV RNAi-mediated resistance was validated in Nicotiana benthamiana transient assays.
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http://dx.doi.org/10.1007/s00705-018-4065-6DOI Listing
January 2019

Detection and profiling of circular RNAs in uninfected and maize Iranian mosaic virus-infected maize.

Plant Sci 2018 Sep 20;274:402-409. Epub 2018 Jun 20.

Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia 4072, QLD, Australia.

Circular RNAs (circRNAs) are covalently closed non-coding RNAs that are usually derived from exonic regions of genes, but can also arise from intronic and intergenic regions. Studies of circRNAs in humans, animals and several plant species have shown an altered population of circRNAs in response to abiotic and biotic stress. Recently it was shown that circRNAs also occur in maize, but it is unknown if maize circRNAs are responsive to stress. Maize Iranian mosaic virus (MIMV, genus Nucleorhabdovirus, family Rhabdoviridae) causes an economically important disease in maize and other gramineous crops in Iran. In this study, we used data from RNA-Seq of MIMV-infected maize and uninfected controls to identify differentially expressed circRNAs. Such circRNAs were confirmed by two-dimensional polyacrylamide gel electrophoresis, northern blot, RT-qPCR and sequencing. A total of 1443 circRNAs were identified in MIMV-infected maize and 1165 circRNAs in uninfected maize. Two hundred and one circRNAs were in common between MIMV-infected and uninfected samples. Of these, 155 circRNAs were up-regulated and 5 down-regulated in MIMV infected plants, compared to the uninfected control. This study for the first time identified and profiled circRNA expression in maize in response to virus infection. Moreover, we predict that 33 circRNAs may bind 23 maize miRNAs, possibly affecting plant metabolism and development. Our data suggest a role for circRNAs in plant cell regulation and response to biotic stress such as virus infection, and give new insights into the complexity of plant-microbe interactions.
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http://dx.doi.org/10.1016/j.plantsci.2018.06.016DOI Listing
September 2018

SCRAM: a pipeline for fast index-free small RNA read alignment and visualization.

Bioinformatics 2018 08;34(15):2670-2672

School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.

Summary: Small RNAs play key roles in gene regulation, defense against viral pathogens and maintenance of genome stability, though many aspects of their biogenesis and function remain to be elucidated. SCRAM (Small Complementary RNA Mapper) is a novel, simple-to-use short read aligner and visualization suite that enhances exploration of small RNA datasets.

Availability And Implementation: The SCRAM pipeline is implemented in Go and Python, and is freely available under MIT license. Source code, multiplatform binaries and a Docker image can be accessed via https://sfletc.github.io/scram/.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty161DOI Listing
August 2018

A Genetic Screen for Impaired Systemic RNAi Highlights the Crucial Role of DICER-LIKE 2.

Plant Physiol 2017 Nov 19;175(3):1424-1437. Epub 2017 Sep 19.

School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia

Posttranscriptional gene silencing (PTGS) of transgenes involves abundant 21-nucleotide small interfering RNAs (siRNAs) and low-abundance 22-nucleotide siRNAs produced from double-stranded RNA (dsRNA) by DCL4 and DCL2, respectively. However, DCL2 facilitates the recruitment of RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) to ARGONAUTE 1-derived cleavage products, resulting in more efficient amplification of secondary and transitive dsRNA and siRNAs. Here, we describe a reporter system where RDR6-dependent PTGS is initiated by restricted expression of an inverted-repeat dsRNA specifically in the Arabidopsis () root tip, allowing a genetic screen to identify mutants impaired in RDR6-dependent systemic PTGS. Our screen identified but not mutants. Moreover, grafting experiments showed that DCL2, but not DCL4, is required in both the source rootstock and the recipient shoot tissue for efficient RDR6-dependent systemic PTGS. Furthermore, rootstocks produced more DCL2-dependent 22-nucleotide siRNAs than the wild type and showed enhanced systemic movement of PTGS to grafted shoots. Thus, along with its role in recruiting RDR6 for further amplification of PTGS, DCL2 is crucial for RDR6-dependent systemic PTGS.
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http://dx.doi.org/10.1104/pp.17.01181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664484PMC
November 2017

Induction of virus resistance by exogenous application of double-stranded RNA.

Curr Opin Virol 2017 10 3;26:49-55. Epub 2017 Aug 3.

School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia.

Exogenous application of double-stranded RNA (dsRNA) for virus resistance in plants represents a very attractive alternative to virus resistant transgenic crops or pesticides targeting virus vectors. However, the instability of dsRNA sprayed onto plants is a major challenge as spraying naked dsRNA onto plants provides protection against homologous viruses for only 5 days. Innovative approaches, such as the use of nanoparticles as carriers of dsRNA for improved stability and sustained release, are emerging as key disruptive technologies. Knowledge is still limited about the mechanism of entry, transport and processing of exogenously applied dsRNA in plants. Cost of dsRNA and regulatory framework will be key influencers towards practical adoption of this technology.
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http://dx.doi.org/10.1016/j.coviro.2017.07.009DOI Listing
October 2017

The effects of potato virus Y-derived virus small interfering RNAs of three biologically distinct strains on potato (Solanum tuberosum) transcriptome.

Virol J 2017 07 17;14(1):129. Epub 2017 Jul 17.

Department of Plant Pathology, Washington State University, Pullman, WA, 99164, USA.

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.
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http://dx.doi.org/10.1186/s12985-017-0803-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513076PMC
July 2017

Transcriptome-wide identification of host genes targeted by tomato spotted wilt virus-derived small interfering RNAs.

Virus Res 2017 06 22;238:13-23. Epub 2017 May 22.

Department of Plant Pathology, Washington State University, Pullman, WA 99164, USA. Electronic address:

RNA silencing mechanism functions as a major defense against invading viruses. The caveat in the RNA silencing mechanism is that the effector small interfering RNAs (siRNAs) act on any RNA transcripts with sequence complementarity irrespective of target's origin. A subset of highly expressed viral small interfering RNAs (vsiRNAs) derived from the tomato spotted wilt virus (TSWV; Tospovirus: Bunyaviridae) genome was analyzed for their propensity to downregulate the tomato transcriptome. A total of 11898 putative target sites on tomato transcripts were found to exhibit a propensity for down regulation by TSWV-derived vsiRNAs. In total, 2450 unique vsiRNAs were found to have potential cross-reacting capability with the tomato transcriptome. VsiRNAs were found to potentially target a gamut of host genes involved in basal cellular activities including enzymes, transcription factors, membrane transporters, and cytoskeletal proteins. KEGG pathway annotation of targets revealed that the vsiRNAs were mapped to secondary metabolite biosynthesis, amino acids, starch and sucrose metabolism, and carbon and purine metabolism. Transcripts for protein processing, hormone signalling, and plant-pathogen interactions were the most likely targets from the genetic, environmental information processing, and organismal systems, respectively. qRT-PCR validation of target gene expression showed that none of the selected transcripts from tomato cv. Marglobe showed up regulation, and all were down regulated even upto 20 folds (high affinity glucose transporter). However, the expression levels of transcripts from cv. Red Defender revealed differential regulation as three among the target transcripts showed up regulation (Cc-nbs-lrr, resistance protein, AP2-like ethylene-responsive transcription factor, and heat stress transcription factor A3). Accumulation of tomato target mRNAs of corresponding length was proved in both tomato cultivars using 5' RACE analysis. The TSWV-tomato interaction at the sRNA interface points to the ability of tomato cultivars to overcome vsiRNA-mediated targeting of NBS-LRR class R genes. These results suggest the prevalence of vsiRNA-induced RNA silencing of host transcriptome, and the interactome scenario is the first report on the interaction between tospovirus genome-derived siRNAs and tomato transcripts, and provide a deeper understanding of the role of vsiRNAs in pathogenicity and in perturbing host machinery.
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http://dx.doi.org/10.1016/j.virusres.2017.05.014DOI Listing
June 2017

Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses.

Nat Plants 2017 01 9;3:16207. Epub 2017 Jan 9.

Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia.

Topical application of pathogen-specific double-stranded RNA (dsRNA) for virus resistance in plants represents an attractive alternative to transgenic RNA interference (RNAi). However, the instability of naked dsRNA sprayed on plants has been a major challenge towards its practical application. We demonstrate that dsRNA can be loaded on designer, non-toxic, degradable, layered double hydroxide (LDH) clay nanosheets. Once loaded on LDH, the dsRNA does not wash off, shows sustained release and can be detected on sprayed leaves even 30 days after application. We provide evidence for the degradation of LDH, dsRNA uptake in plant cells and silencing of homologous RNA on topical application. Significantly, a single spray of dsRNA loaded on LDH (BioClay) afforded virus protection for at least 20 days when challenged on sprayed and newly emerged unsprayed leaves. This innovation translates nanotechnology developed for delivery of RNAi for human therapeutics to use in crop protection as an environmentally sustainable and easy to adopt topical spray.
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http://dx.doi.org/10.1038/nplants.2016.207DOI Listing
January 2017

A partially purified outer membrane protein VirB9-1 for low-cost nanovaccines against Anaplasma marginale.

Vaccine 2017 01 24;35(1):77-83. Epub 2016 Nov 24.

Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD 4072, Australia. Electronic address:

Anaplasma marginale is a devastating tick-borne pathogen causing anaplasmosis in cattle and results in significant economic loss to the cattle industry worldwide. Currently, there is no widely accepted vaccine against A. marginale. New generation subunit vaccines against A. marginale, which are much safer, more efficient and cost-effective, are in great need. The A. marginale outer membrane protein VirB9-1 is a promising antigen for vaccination. We previously have shown that soluble recombinant VirB9-1 protein can be expressed and purified from Escherichia coli and induce a high level of humoral and cellular immunity in mice. In this study, we re-formulated the nanovaccines using the partially-purified VirB9-1 protein as the antigen and hollow nano-size silica vesicles (SV-100) as the adjuvant. We simplified the purification method to obtain the partially-purified antigen VirB9-1 with a six-fold higher yield. The new formulations using the partially-purified VirB9-1 protein achieved higher antibody and cell-mediated immune responses compared to the purified ones. This finding suggests that the partially-purified VirB9-1 protein performs better than the purified ones in the vaccination against A. marginale, and a certain level of contaminants in the protein antigen can be self-adjuvant and boost immunogenicity together with the nanoparticle adjuvant. This may lead to finding a "Goldilocks" level of contaminants. The new nanovaccine formulation using partially-purified antigens along with nanoparticle adjuvants offers an alternative strategy for making cheaper veterinary vaccines.
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http://dx.doi.org/10.1016/j.vaccine.2016.11.037DOI Listing
January 2017

Hollow mesoporous carbon nanocarriers for vancomycin delivery: understanding the structure-release relationship for prolonged antibacterial performance.

J Mater Chem B 2016 Nov 18;4(43):7014-7021. Epub 2016 Oct 18.

Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia.

Mono-dispersed mesoporous hollow carbon (MHC) nanospheres with comparable structures have been designed as nanocarriers for the delivery of vancomycin (Van) to inhibit bacterial growth. It is demonstrated that MHC materials possess a Van loading capacity of 861 mg g, much higher than that of any Van nanocarrier in previous reports. By comparing the drug loading, release and antibacterial performance of MHC nanospheres with controllable structures, it is shown that MHC with a pore size of 5.8 nm and a wall thickness of 25 nm exhibits compromising storage-release behaviour and achieves extended bactericidal activity of Van towards E. coli and S. epidermidis compared to free Van and other MHC nanocarriers. This study provides new knowledge about the rational design of carbon based nanocarriers to enhance the therapeutic efficacy of antibiotics.
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http://dx.doi.org/10.1039/c6tb01778aDOI Listing
November 2016

The Tomato Spotted Wilt Virus Genome Is Processed Differentially in its Plant Host Arachis hypogaea and its Thrips Vector Frankliniella fusca.

Front Plant Sci 2016 7;7:1349. Epub 2016 Sep 7.

Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia QLD, Australia.

Thrips-transmitted tospoviruses are economically important viruses affecting a wide range of field and horticultural crops worldwide. Tomato spotted wilt virus (TSWV) 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 RNAi 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 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 Nicotiana benthamiana). Distinctively, overall small RNA (sRNA) biogenesis in A. hypogaea is markedly affected with an absence of the 24 nt sRNAs 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.
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http://dx.doi.org/10.3389/fpls.2016.01349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5013717PMC
September 2016

Shaping Nanoparticles with Hydrophilic Compositions and Hydrophobic Properties as Nanocarriers for Antibiotic Delivery.

ACS Cent Sci 2015 Sep 9;1(6):328-34. Epub 2015 Sep 9.

Australian Institute for Bioengineering and Nanotechnology, The University of Queensland , Brisbane, Queensland 4072, Australia.

Inspired by the lotus effect in nature, surface roughness engineering has led to novel materials and applications in many fields. Despite the rapid progress in superhydrophobic and superoleophobic materials, this concept of Mother Nature's choice is yet to be applied in the design of advanced nanocarriers for drug delivery. Pioneering work has emerged in the development of nanoparticles with rough surfaces for gene delivery; however, the preparation of nanoparticles with hydrophilic compositions but with enhanced hydrophobic property at the nanoscale level employing surface topology engineering remains a challenge. Herein we report for the first time the unique properties of mesoporous hollow silica (MHS) nanospheres with controlled surface roughness. Compared to MHS with a smooth surface, rough mesoporous hollow silica (RMHS) nanoparticles with the same hydrophilic composition show unusual hydrophobicity, leading to higher adsorption of a range of hydrophobic molecules and controlled release of hydrophilic molecules. RMHS loaded with vancomycin exhibits an enhanced antibacterial effect. Our strategy provides a new pathway in the design of novel nanocarriers for diverse bioapplications.
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http://dx.doi.org/10.1021/acscentsci.5b00199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4827501PMC
September 2015

Silica Nanopollens Enhance Adhesion for Long-Term Bacterial Inhibition.

J Am Chem Soc 2016 05 13;138(20):6455-62. Epub 2016 May 13.

Australian Institute for Bioengineering and Nanotechnology, The University of Queensland , Brisbane 4072, Australia.

Nature's creations with spiky topological features typically exhibit intriguing surface adhesive properties. From micrometer-sized pollen grains that can easily stick to hairy insects for pollination to nanoscale virus particles that are highly infectious toward host cells, multivalent interactions are formed taking advantage of rough surfaces. Herein, this nature-inspired concept is employed to develop novel drug delivery nanocarriers for antimicrobial applications. A facile new approach is developed to fabricate silica nanopollens (mesoporous silica nanospheres with rough surfaces), which show enhanced adhesion toward bacteria surfaces compared to their counterparts with smooth surfaces. Lysozyme, a natural antimicrobial enzyme, is loaded into silica nanopollens and shows sustained release behavior, potent antimicrobial activity, and long-term total bacterial inhibition up to 3 days toward Escherichia coli. The potent antibacterial activity of lysozyme-loaded silica nanopollens is further demonstrated ex vivo by using a small-intestine infection model. Our strategy provides a novel pathway in the rational design of nanocarriers for efficient drug delivery.
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http://dx.doi.org/10.1021/jacs.6b00243DOI Listing
May 2016

Immunogenicity of Outer Membrane Proteins VirB9-1 and VirB9-2, a Novel Nanovaccine against Anaplasma marginale.

PLoS One 2016 26;11(4):e0154295. Epub 2016 Apr 26.

Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD, 4072, Australia.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0154295PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846087PMC
March 2017

Nanoparticle-Based Delivery of Anaplasma marginale Membrane Proteins; VirB9-1 and VirB10 Produced in the Pichia pastoris Expression System.

Nanomaterials (Basel) 2016 Nov 5;6(11). Epub 2016 Nov 5.

Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, QLD 4072, Australia.

Bovine anaplasmosis or cattle-tick fever is a tick-borne haemolytic disease caused by the rickettsial haemoparasite 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 . Soluble VirB9-1 and VirB10 were successfully expressed using . 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 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.
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http://dx.doi.org/10.3390/nano6110201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5245741PMC
November 2016