RNAi for Plant Improvement and Protection

Bruno Mezzetti, Jeremy Sweet, Lorenzo Burgos

• Publisher: CAB International
• Year: 2021
• ISBN:
  9781789248913
• Topic: Biological Sciences
• Subtopic: Genetics & Genomics
• Index: Biological Sciences

RNA interference (RNAi) has the potential to make major contributions towards sustainable crop production and protection with minimal environmental impacts compared to other technologies. RNAi is being developed and exploited both within plants (i.e. host-induced gene silencing, HIGS) and/or as topical applications (e.g. spray-induced gene silencing, SIGS) for targeting pest and pathogen genes and for manipulating endogenous gene expression in plants. Chapters by international experts review current knowledge on RNAi, methods for developing RNAi systems in GM plants and applications for crop improvement, crop production and crop protection. Chapters examine both endogenous systems in GM plants and exogenous systems where interfering RNAs are applied to target plants, pests and pathogens. The biosafety of these different systems is examined and methods for risk assessment for food, feed and environmental safety are discussed. Finally, aspects of the regulation of technologies exploiting RNAi and the socio-economic impacts of RNAi technologies are discussed.

1 Introduction to RNAi in Plant Production and Protection

Bruno Mezzetti¹*, Jeremy Sweet² and Lorenzo Burgos³

¹Università Politecnica delle Marche, Ancona, Italy; ²Sweet Environmental Consultants, Cambridge; UK; ³CEBAS-CSIC, Murcia, Spain

*Corresponding author: [email protected]

© CAB International 2021. RNAi for Plant Improvement and Protection (eds B. Mezzetti et al.)

DOI: 10.1079/9781789248890.0001

RNA interference (RNAi) has the potential to have a major impact on agriculture, horticulture and forestry with many different applications for plant improvement in terms of both quality of products and productivity. In addition, crop protection applications are being developed which provide ‘green’ alternatives to conventional pest control methods. RNAi is a naturally occurring process present in plants and animals, in which double-stranded RNA (dsRNA) molecules interfere with homologous RNA. It allows genes to be targeted to remove unwanted products in plants and improve plant productivity and quality of plant products. These RNAi mechanisms were only discovered and described 20 years ago and their discovery led to a Nobel prize in 2006. RNAi is now being developed within plants to silence genes often described as host-induced gene silencing (HIGS). Also, external and topical applications, such as sprays and seed treatments, are being developed to substitute for other types of pesticides or growth regulator treatments. An example is the spray-induced gene silencing (SIGS) approach for targeting pest and pathogen genes and for manipulating endogenous gene expression in plants. Examples of plant improvement applications include: improving fatty acid profiles of soybeans; delayed ripening and improved shelf life of fruits such as apples and tomatoes; or removing unwanted compounds, toxins and allergens from crop products such as decaffeinated coffee, gossypol in cotton seeds and hypoallergenic fruits and cereals.

For pest and disease control applications, dsRNA can be selected for silencing essential genes in pests, pathogens and viruses, expressed either in transformed plants or in exogenous applications. dsRNA can be very specifically targeted at genetic sequences in these targets so that off-target effects are avoided or minimized. Recent advances in genomics and transcriptomics have provided sequence data that enable the design of highly targeted dsRNAs, providing efficient silencing while minimizing the risk of effects on off-target genes or the silencing of gene expression in non-target organisms. Due to the involvement of RNA in virus replication, several virus-resistant plants have been developed (e.g. papaya, plum, squash and tomato) and many more virus control applications are in the pipeline. More recently, plant resistance to a range of other pests and fungal diseases is being developed, including insect pests such as Colorado potato beetle (Leptinotarsa decemlineata) and insect vectors of viruses. The fungal disease targets include a range of diseases such as cereal rusts and Botrytis grey mould on fruit. In the USA, maize transformed to express a dsRNA targeting a gene in corn rootworm (Diabrotica spp.) has been developed and commercialized.

RNAi provides additional options for plant breeders to improve plant varieties compared with other new breeding techniques (NBTs) such as clustered regularly interspaced short palindromic repeats/CRISPR-association protein (CRISPR/Cas) or transcription activator-like effector nucleases (TALENs). For example, RNAi provides a method for reducing gene expression (knockdown) rather than complete blocking of the expression (knockout). This is important for providing reduced levels of gene expression, or when a specific stage in a physiological process is to be targeted. Another important feature of RNAi is that dsRNA molecules can be highly mobile in plants. Therefore, dsRNA produced in part of the plant (e.g. rootstock) can have the potential to spread into the grafted parts of the plant to confer resistance to disease to the whole plant, including fruit. This results in fruits that are not genetically modified but protected by the presence of target-specific degradable small RNA molecules (Limera et al., 2017). In addition, dsRNA molecules can be formulated and applied as a topical treatment to plants to change their physiology or combat pests and pathogens. This approach will avoid genetically modified organism (GMO) regulations if no GMOs are present in the products.

Research on RNAi is being conducted mainly in Europe, the USA and China. However, in Europe and some regions of the world the technology and its applications are being held back by policies and legislation on biotechnologies, by failures in the implementation of GMO regulations and by failure to develop appropriate methods for the regulation and assessment of novel plant protection products. This is inhibiting investment in research and development (R&D) on novel ‘green’ applications of RNAi, as can be seen by the reduction in patent applications in Europe. It has been shown that RNAi has the potential to make major contributions towards sustainable crop production and protection with minimal environmental impacts compared with other technologies. In regions where legislation prevents the use of RNAi technology, farmers will not have access to the technology and important options for improving productivity and economic competitiveness (Taning et al., 2019; Mezzetti et al., 2020). Ironically this will be at a time when governments are trying to introduce more sustainable ‘green’ agricultural practices and when food demand is increasing and food supplies are at risk from climate change, new invasive species and urbanization.

In 1971 a European Cooperation in Science and Technology (COST) programme had been created. In 2016 the iPlanta COST Action CA15223 ‘Modifying plants to produce interfering RNA’ (available at https://iplanta.univpm.it, accessed 1 November 2020) was established with the objective of bringing together experts from a wide range of fields to develop a deeper understanding of the science of RNA, the applications of RNAi, the biosafety of these applications and the socio-economic aspects of these potential applications. This book contains a series of chapters by experts from many countries, who are participating in iPlanta, to review the current scientific knowledge on RNAi, methods for developing RNAi systems in GM plants and a range of applications for crop improvement, crop production and crop protection. Chapters examine both endogenous systems in GM plants and exogenous systems where interfering RNAs are applied to target plants, pests and pathogens. The biosafety of these different systems is examined and methods for risk assessment for food, feed and environmental safety are discussed. Finally, aspects of the regulation of technologies exploiting RNAi and the socio-economic impacts of RNAi technologies are discussed.

References

3 Limera, C., Sabbadini, S., Sweet, J.B. and Mezzetti, B. (2017) New biotechnological tools for the genetic improvement of major woody fruit species. Frontiers in Plant Science 8, 1418. DOI: 10.3389/fpls.2017.01418.

Mezzetti, B., Smagghe, G., Arpaia, S., Christiaens, O., Dietz-Pfeilstetter, A. et al. (2020) RNAi: what is its position in agriculture? Journal of Pest Science 93(4), 1125–1130. DOI: 10.1007/s10340-020-01238-2.

Taning, C.N., Arpaia, S., Christiaens, O., Dietz-Pfeilstetter, A., Jones, H. et al. (2019) RNA-based biocontrol compounds: current status and perspectives to reach the market. Pest Management Science 76(3), 841–845. DOI: 10.1002/ps.5686.

2 Gene Silencing to Induce Pathogen-derived Resistance in Plants

Elena Zuriaga, Ángela Polo-Oltra and Maria Luisa Badenes* Centre of Citriculture and Plant Production, Valencian Institute for Agricultural Research (IVIA), Valencia, Spain

*Corresponding author: [email protected]

© CAB International 2021. RNAi for Plant Improvement and Protection (eds B. Mezzetti et al.)

DOI: 10.1079/9781789248890.0002

2.1 Introduction: Concept and Historical Overview of the Use of Pathogen-derived Resistance in Plants

The discovery and use of RNA interference (RNAi) and pathogen-derived resistance (PDR) in plants has a large history that has been previously reviewed by Gottula and Fuchs (2009), Lindbo (2012) and Rosa et al. (2018), among others. The concept of PDR was introduced by Sanford and Johnston (1985) describing the use of a pathogen’s own genome to confer resistance via genetic engineering as an alternative strategy to avoid problems in identifying and isolating host resistance genes, the polygenic control of the resistance or, directly, the lack of available resistance genes. This approach is based upon the disruption of parasite-encoded cellular functions that are essential to the parasite but not to the host. As a model, Sanford and Johnston (1985) used genes of the bacteriophage Qß to confer resistance in Escherichia coli against this bacteriophage. Before the discovery and description of RNAi, transgenic tobacco plants expressing the coat protein (CP) gene of the tobacco mosaic virus (TMV) were the first demonstration of PDR against a plant virus (Abel et al., 1986). As a result of these experiments, some transgenic lines showed no symptoms, or a delay in the development of the disease. Afterwards, numerous studies were conducted using CP genes and also other viral sequences (reviewed by Gottula and Fuchs, 2009), but the mechanism of the engineered resistance was not well understood at the time. It was suggested that the expression of the viral CP in a transgenic plant interfered with the virus replication, translation or virion assembly. Later, during an experiment to obtain plants resistant to the tobacco etch virus (TEV), transgenic lines expressing the TEV CP were obtained, and also other lines that expressed a non-translatable, sense-stranded mRNA for the TEV CP that were called RNA control (RC) lines (Lindbo and Dougherty, 1992). Surprisingly, during the TEV challenge, several of the RC lines were immune to the infection. In these plants, the accumulation of antisense RNA was responsible for this protection and not the ectopic expression of a viral protein, but, once again, at this time the cellular mechanism was not fully understood.

RNAi was first recognized in plants in the late 1980s and early 1990s. During experiments to increase the pigment content in purple petunia flowers using genetic engineering, some transgenic plant lines had flowers that were totally white or variegated (Napoli et al., 1990; van der Krol et al., 1990). These authors called this phenomenon ‘cosuppression’ or ‘gene silencing’ of both the transgene and the homologous endogenous genes. However, the mechanisms involved were still unknown. Lindbo and collaborators, following their experiments with TEV-resistant transgenic plants, proposed that cytoplasmic activity targeting specific RNA sequences was responsible for the virus resistance in these plants, as transgene mRNA levels were 12- or 22-fold higher in unchallenged transgenic tissues compared with recovered transgenic plants of the same developmental stage (Lindbo et al., 1993). In this publication, the authors proposed a mechanism for post-transcriptional gene silencing (PTGS)/RNA silencing, where the RNA-dependent RNA polymerase (RdRP, also known as RDR) used the overexpressed viral transgene as a template to produce small RNAs that could rebind to new target RNA (viral and transgene) sequences. This model was further expanded by Dougherty and Parks (1995) suggesting that 10–20 nucleotide (nt) RNAs, generated from aberrant or overexpressed transgenes, were part of a cellular sequence-specific RNA targeting and degradation system. In fact, Hamilton and Baulcombe (1999) detected ~25 nt antisense RNAs, complementary to targeted mRNAs, in four types of transgene- or virus-induced PTGS in plants, that were likely synthesized from an RNA template. These authors suggested that these 25 nt antisense RNAs were components of the systemic signal and specificity determinants of PTGS.

Studies with other biological systems contributed to a deeper understanding of the mechanism of PTGS. The discovery of double-stranded RNA (dsRNA) as a potent inducer of PTGS in plants (Waterhouse et al., 1998) and nematodes (Montgomery and Fire, 1998) was a key contribution. Waterhouse et al. (1998) transformed tobacco and rice with gene constructs that produce RNAs capable of duplex formation to confer virus immunity or gene silencing to plants. In parallel, Fire et al. (1998) demonstrated that the direct injection into adult animals of dsRNA molecules was substantially more effective in producing interference effects than either strand was individually, and just a few molecules were required per affected cell. These authors described dsRNA as a potent trigger for RNAi. The use of direct dsRNA injection was suggested as a new tool for gene function studies in Caenorhabditis elegans, but also for other nematodes, other invertebrates and, potentially, in vertebrates and plants. As the genetic screens got easier, the identification of the genes required for RNAi in C. elegans, and their comparison with the ones required for gene silencing in Drosophila, plants and fungi, showed the existence of a common underlying mechanism (Mello and Conte, 2004). In 2006, Andrew Z. Fire and Craig C. Mello were awarded the Nobel Prize in Physiology or Medicine for their discovery of ‘RNA interference – gene silencing by double-stranded RNA’.

RNA silencing, or RNAi, is a conserved regulatory mechanism of gene expression in...

Table of contents

• Cover
• Half-title Page
• Title Page
• Copyright
• Contents
• Contributors
• Acknowledgements
• 1. Introduction to RNAi in Plant Production and Protection
• 2. Gene Silencing to Induce Pathogen-derived Resistance in Plants
• 3. Exogenous Application of RNAs as a Silencing Tool for Discovering Gene Function
• 4. The ‘Trojan Horse’ Approach for Successful RNA Interference in Insects
• 5. Biogenesis and Functional RNAi in Fruit Trees
• 6. Gene Silencing or Gene Editing: the Pros and Cons
• 7. Application of RNAi Technology in Forest Trees
• 8. Host-induced Gene Silencing and Spray-induced Gene Silencing for Crop Protection Against Viruses
• 9. Small Talk and Large Impact: the Importance of Small RNA Molecules in the Fight Against Plant Diseases
• 10. The Stability of dsRNA During External Applications – an Overview
• 11. Boosting dsRNA Delivery in Plant and Insect Cells with Peptide- and Polymer-based Carriers: Case-based Current Status and Future Perspectives
• 12. Environmental Safety Assessment of Plants Expressing RNAi for Pest Control
• 13. Food and Feed Safety Assessment of RNAi Plants and Products
• 14. Regulatory Aspects of RNAi in Plant Production
• 15. The Economics of RNAi-based Innovation: from the Innovation Landscape to Consumer Acceptance
• 16. Future Plant Solutions by Interfering RNA and Key Messages for Communication and Dissemination
• Glossary
• Index
• Backcover