- Email: [email protected]
Enhancing Plant Disease Resistance without R Genes
of GM with non-GM varieties of the same crop or a different crop. Hence, if African countries exporting to the EU adopt productivity-enhancing GM crops, they risk losing their markets in the EU. Given that absolute segregation of GM and non-GM crops is both difficult and expensive to achieve on a commercial basis, African countries eschew the adoption of GM crops, thus forgoing improvements in their agricultural productivity (even developed countries are stressed: the EU closed the border to Canadian exports of flax and American exports of corn, rice, and soybeans recently after detecting trace amounts of traits not approved in the EU). The failure to adopt GM crops in the EU also feeds back into global investment decisions: one result is lower investment into the adaptation of GM crops suited to agronomic conditions in developing countries, especially tropical crops.
cross-species manipulations, there is a movement afoot in the EU, especially in many of the MS that have exercised the opportunity to renationalize decision about GM crops, to outright reject these technologies. This knee-jerk reaction to a real opportunity to accelerate crop productivity, lower the ecological footprint of food production, and improve the lot of many food-insecure farmers and families around the world is a shame. The burden of increasing global food security cannot be in the hands of the few GM crop-adopting countries, led by Argentina, Australia, Brazil, Canada and the USA, but must be shared equally between all industrialized countries. The commercialization of new, higher yielding crops needs all leading food producers to engage. The EU cannot abrogate its duty to use its wealth and resources on behalf of humanity.
This latest move away from science- Resources based regulation in the EU will have i www.fao.org/fileadmin/templates/wsfs/docs/ wide-ranging effects beyond the EU itself, expert_paper/How_to_Feed_the_World_in_2050.pdf effects that will complicate international ii www.ictsd.org/bridges-news/bridges/news/ efforts to achieve global food security. eu-confirms-provisional-agreement-on-nationalWhile numerous GM-adopting nations gmo-bans iii http://ec.europa.eu/research/biosociety/pdf/ have positively responded to the chala_decade_of_eu-funded_gmo_research.pdf lenge of the FAO, the EU continues to iv http://giannini.ucop.edu/media/are-update/files/ ignore the impact of their domestic articles/v12n2_5.pdf choices on this crucial global issue. The EU has secure food supplies and is ignor- 1Department of Agriculture and Resource Economics, ing the cost its politically motivated tech- University of Saskatchewan, 51 Campus Drive, Saskatoon, S7N 5A8, Canada nology choice has on food insecure 2Johnson-Shoyama Graduate School of Public Policy, consumers and countries. The EU has University of Saskatchewan, 101 Diefenbaker Place, failed in its responsibility to its own citizens Saskatoon, S7N 5B8, Canada and to those in developing countries by *Correspondence: [email protected] (S.J. Smyth). ignoring science and evidence. http://dx.doi.org/10.1016/j.tibtech.2016.04.003
Concluding Remarks While the opportunity to use GM technologies in the EU seems to be lost, there is a bigger opportunity that the world cannot afford to waste. A host of new plant breeding techniques and technologies are poised to enter the crop variety development sector. They offer an exciting opportunity to reverse the trend to weaker yield growth. While many of these new technologies and techniques do not involve
References 1. Klümper, W. and Qaim, M. (2014) A meta analysis of the impact of genetically modified crops. PLoS ONE 9, 1–7 2. Kathage, J. and Qaim, M. (2012) Economic impacts and impact dynamics of Bt (Bacillus thuringiensis) cotton in India. PNAS 109, 11652–11656 3. Vitale, J. et al. (2014) Cotton. In Handbook on Agriculture, Biotechnology and Development (Smyth, S.J. et al., eds), pp. 604–620, Edward Elgar 4. Karplus, V.J. (2014) China. In Handbook on Agriculture, Biotechnology and Development (Smyth, S.J. et al., eds), pp. 112–125, Edward Elgar 5. Bennet, R. et al. (2006) The economic impact of genetically modified cotton on South African smallholders: yields, profits and health effects. J. Dev. Studies 42, 662–677
6. Yorobe, J.M. and Smale, M. (2012) Impacts of Bt maize on smallholder income in the Philippines. Agbioforum 15, 152–162 7. Alston, J.M. et al. (2014) The size and distribution of the benefits from the adoption of biotech soybean varieties. In Handbook on Agriculture, Biotechnology and Development (Smyth, S.J. et al., eds), pp. 728–751, Edward Elgar 8. Huang, J. et al. (2010) A decade of Bt cotton in Chinese fields: assessing the direct effects and indirect externalities of Bt cotton adoption in China. Sci. China Life Sci. 53, 981–991 9. Smyth, S.J. et al. (2011) Changes in herbicide use after the adoption of HR canola in Western Canada. Weed Tech. 25, 492–500 10. Kouser, S. and Qaim, M. (2011) Impact of Bt cotton on pesticide poisoning in smallholder agriculture: a panel data analysis. Ecol. Econ. 70, 2105–2113 11. Hobbs, J.E. et al. (2014) The perils of zero tolerance: technology management, supply chains and thwarted globalization. Int. J. Tech. Globalization 7, 203–216
Letter
Enhancing Plant Disease Resistance without R Genes Birinchi Kumar Sarma,1 Harikesh Bahadur Singh,1 Dilantha Fernando,2 Roberto Nascimento Silva,3 and Vijai Kumar Gupta4,* Crop plants encounter constant biotic challenges, and these challenges have historically been best managed with resistance (R) genes. However, the rapid evolution of new pathogenic strains along with the nonavailability or nonidentification of R genes in cultivated crop species against a large number of plant pathogens have led researchers to think beyond R genes. Biotechnological tools have shown promise in dealing with such challenges. Technologies such as transgenerational plant immunity, interspecies transfer of pattern recognition receptors (PRRs), pathogen-derived resistance (PDR), gene regulation, and expression of antimicrobial peptides (AMPs) in host plants from other
Trends in Biotechnology, July 2016, Vol. 34, No. 7
523
plant species have led to enhanced receptor-like proteins (RLPs); signals Similarly, disruption of the main brassinosdisease resistance and increased from potential pests/pathogens received teroid receptor (BRI1, brassinosteroid by the PRR result in a conformational insensitive 1) in both Brachypodium disfood security. Lombardo et al. recently reviewed new approaches to implementing R genes for insect and herbicide resistance in plants [1]. Similar approaches are also relevant for pathogen resistance in plants. Here, we summarize alternative approaches to engineer disease resistance in plants. In addition to genome editing [2] and antisense technologies [3], several other promising technologies have also emerged as potential instruments for countering the limitations of R gene-mediated resistance in plants against pathogens and insect herbivores. Technologies such as transgenerational epigenetics (TE) in plants could be employed to improve some traits in a shorter period of time. TE can be defined as an epigenetic change that persists across one or more subsequent generations. TE can also be utilized to manage plant pathogens, insect herbivores, and other abiotic stresses such as mechanical injuries. The TE blockage in the expression of some particular genes in response to environmental stimuli, due to either DNA methylation or histone modifications, may accelerate the plant's ability to restrict pest/ pathogen development [4]. When outside stimuli induce such blockages in plant gene expression, loss of expression may be permanent in that generation and passed to the next generation in the same state through seeds. Plants thereby pass on the perceived environmental threats of both biotic and abiotic stresses to offspring. Additionally, interspecies transfer of receptors has also shown promise in managing biotic stresses. Broad-spectrum resistance in plants can be developed by transferring pattern recognition receptors (PRRs) between plant species [5]. Engineering broad-spectrum disease resistance through transferring PRRs has shown promise, and the impact is expected to be long-lasting. These PRRs are either receptor kinases (RKs) or
524
Trends in Biotechnology, July 2016, Vol. 34, No. 7
change in the receptor, leading to the activation of downstream defense signaling genes. Transferring PRRs from related species, or wild relatives, that can perceive a pathogen threat from one plant cultivar or species to another and thereby increasing the level of resistance in a susceptible plant cultivar or species against a specific pathogen.
tachyon and barley enhanced disease resistance against some cereal fungal pathogens [9]. Further, when the rice heme activator protein (HAP) gene (OsHAP2E), which is known to regulate plant growth, development, and stress responses, was overexpressed, it not only enhanced disease resistance to a fungal and a bacterial pathogen but also conferred rice plants resistance to abiotic stresses such as Genetic engineering and biotechnology drought and salinity [10]. can open doors for counteracting infection; one strategy is introducing genes In addition, generating antimicrobial pepfrom various sources into plants, which tides (AMPs) in plants to fight pathogens could generate disease resistance with has also become an interesting tool to none of the species boundaries that apply reduce crop losses. AMP chitin-binding to conventional methodologies. The capability plays a crucial role in antifungal expression of structural viral nucleic acid activity. The AMP antiviral effect depends sequences (e.g., coat protein, movement on different factors, such as the direct protein, or replicase protein genes) in interaction with the viral envelope, displants, known as pathogen-derived resis- rupting or destabilizing it; competition tance (PDR), generally offers a broader with viruses for the host membrane, prerange of resistance even to the related venting the viral connection with particuviruses. The technique is effective against lar cell receptors; and prevention of a low level of inoculums but, as with most expression of viral genes in the earlier viral proteins, this strategy can elicit R infection stages, affecting the propagagene-driven effector triggered immunity tion and the viral infection [11]. Interest(ETI), causing hypersensitive response ingly, some endogenous peptides are (HR), a mechanism leading to rapid cell even perceived as danger signals and a death surrounding a pathogen infection stereotypical defense response is that limits the pathogen spread [6]. Bioen- induced in host against the invading gineering against viral pathogens is partic- pathogens [12]. Technological intervenularly important as only around 40 R tions may lead to greater utilization of genes have been identified so far against such peptides in reducing pathogen more than 1000 identified plant viruses. progress in hosts. Further, plantibodies have also been developed to protect plants, which could Recent technological advancements in be constructed to focus on any pathogen, biological sciences have thus created sequestering the antigens that are fre- the future possibility for disease resistance quently required to complete the infection in crop plants to those pathogens against cycle, thus preventing diseases [7]. which neither R genes nor available chemicals have been effective. Moreover, overSimilarly, regulating genes that are not con- reliance on R genes for disease resistance sidered to have direct roles in governing has become counterproductive in several host resistance may also lead to enhanced instances. Therefore, new technologies disease resistance against both pathogens are desirable as they are proving to be and insect herbivores. Downregulating cel- viable, complementary, or supplementary lulose synthase (CesA) increases Arabi- to R genes, environmentally acceptable, dopsis resistance to Botrytis cinerea [8]. and ecologically feasible alternatives to R
to capture thousands of single-cell trangene-mediated resistance. Although, in Spotlight most cases detailed work is necessary scriptomes from an individual experimento make these technologies commercially tal sample. Together with the reduced viable, technologies such as PDR and viral cost of sequencing, these developments antisense are already well established in are paving the way for single-cell RNA-seq certain crops and are in the marketplace. to soon become a laboratory staple. Once the newer technologies are perSamantha A. Morris1,@,* fected, they could be used to increase Enabled by these methodological advanfood production and reduce hunger by ces, it is becoming commonplace to gentackling pests and pathogens with great erate single-cell transcriptome data for Single-cell RNA sequencing (RNA- tens of thousands of cells. How to approspecificity. seq) technology is hitting its stride priately and effectively analyze these data 1 Department of Mycology and Plant Pathology, Institute of and is beginning to be widely is an emerging challenge. For example, Agricultural Sciences, Banaras Hindu University, Varanasi, adopted. One of the major chal- many sources of technical variation exist India 2 Department of Plant Science, University of Manitoba, lenges faced by this rapidly growing between transcriptomes, arising due to Winnipeg, MB R3T 2N2, Canada field lies in data analysis. Li et al. now differences in capture efficiency, amplifica3 Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão present Sinova, a single-cell analyt- tion biases, and sequencing depth, makPreto, SP, Brazil ical platform offering temporal, spa- ing it difficult to detect discrete biological 4 Molecular Glycobiotechnology Group, Discipline of Biochemistry, School of Natural Sciences, National tial, and regulatory reconstruction of differences between single cells. Moreover, in any approach, whether bulk or University of Ireland Galway, Galway, Ireland developmental processes. single cell, continuous monitoring of gene *Correspondence: [email protected] (V.K. Gupta). expression within the same cells over time http://dx.doi.org/10.1016/j.tibtech.2016.04.002 Recent technological advances have is not possible. Finally, to capture expresenabled the analysis of genome-wide sion profiles, single cells must be dissociReferences 1. Lombardo, L. et al. (2016) New technologies for insect- mRNA expression within single cells. ated from tissue, resulting in a loss of resistant and herbicide-tolerant plants. Trends Biotechnol. These developments are proving to be spatial and temporal information. These 34, 49–57 2. Wang, Y. et al. (2014) Simultaneous editing of three pivotal and broadly applicable over many issues present barriers to using single-cell homoeoalleles in hexaploid bread wheat confers heritable biological disciplines. Previous analyses of RNA-seq to systematically describe bioresistance to powdery mildew. Nat. Biotechnol. 32, 947– gene expression were restricted to the logical processes and identify the regula951 3. Pumplin, N. and Voinnet, O. (2013) RNA silencing suppres- population level, lacking resolution by tors driving them. sion by plant pathogens: defence, counter-defence and counter-counter-defence. Nat. Rev. Microbiol. 11, 745– averaging results over many cells, and 760 obscuring signals from rare cell types. In response to these challenges, Li et al. 4. Sarma, B.K. and Singh, H.B. (2014) Harnessing transgeHowever, single-cell RNA sequencing [4] developed a new single-cell analytical nerational plant immunity. Curr. Sci. 107, 1941–1942 5. Dangl, J.L. et al. (2013) Pivoting the plant immune system can identify novel, rare cell types. For methodology, Sinova. With this platform, from dissection to deployment. Science 341, 746–751 example, a previously undiscovered cell they interrogate the development of the 6. García, J.A. and Pallás, V. (2015) Viral factors involved in species within the intestine was recently mouse growth plate, a structure required plant pathogenesis. Curr. Opin. Virol. 11, 21–30 7. Safarnejad, M.R. et al. (2011) Antibody-mediated resis- uncovered via single-cell RNA-seq, a feat for bone elongation and regeneration. The tance against plant pathogens. Biotechnol. Adv. 29, that would otherwise have proven impos- postnatal growth plate comprises cells at 961–971 sequential stages of differentiation, the sible with previous approaches [1]. 8. Ramírez, V. et al. (2011) MYB46 modulates disease susexact regulation of which remains largely ceptibility to Botrytis cinerea in Arabidopsis. Plant Physiol. 155, 1920–1935 The past year has heralded a new phase in unknown at a systematic level. In this 9. Goddard, R. et al. (2014) Enhanced disease resistance caused by BRI1 mutation is conserved between Brachy- single-cell technology. Analysis of hun- study, the expression profiles of 217 single podium distachyon and barley (Hordeum vulgare). Mol. dreds of cells within a study has become cells were captured from this developPlant Microbe Interact. 27, 1095–1106 standard, although remaining limitations mental structure, using the Fluidigm C1 10. Alam, M. et al. (2015) Overexpression of a rice heme activator protein gene (OsHAP2E) confers resistance to on ease, cost, and scale have been a platform. This simultaneous capture overpathogens, salinity and drought, and increases photobarrier to the broad application of sin- comes much interexperimental technical synthesis and tiller number. Plant Biotechnol. J. 13, 85– gle-cell approaches. These impediments variation, and such an unsynchronized 96 11. Salas, C.E. et al. (2015) Biologically active and antimicrobial have recently been addressed via the real- population contains cells representing peptides from plants. Biomed Res. Int. 102129 2015 ization of highly parallel single-cell expres- many stages of development, enabling 12. Boller, T. and Felix, G. (2009) A renaissance of elicitors: perception of microbe-associated molecular patterns and sion profiling [2,3]. Of particular note, both temporal and spatial reconstruction danger signals by pattern-recognition receptors. Annu. Macosko et al. [2] used a simple, cost- of differentiation trajectories from a popuRev. Plant Biol. 60, 379–406 effective, and robust microfluidic method lation ‘snapshot’.
Single-Cell RNA-Seq Steps Up to the Growth Plate
Trends in Biotechnology, July 2016, Vol. 34, No. 7
525
cross-species manipulations, there is a movement afoot in the EU, especially in many of the MS that have exercised the opportunity to renationalize decision about GM crops, to outright reject these technologies. This knee-jerk reaction to a real opportunity to accelerate crop productivity, lower the ecological footprint of food production, and improve the lot of many food-insecure farmers and families around the world is a shame. The burden of increasing global food security cannot be in the hands of the few GM crop-adopting countries, led by Argentina, Australia, Brazil, Canada and the USA, but must be shared equally between all industrialized countries. The commercialization of new, higher yielding crops needs all leading food producers to engage. The EU cannot abrogate its duty to use its wealth and resources on behalf of humanity.
This latest move away from science- Resources based regulation in the EU will have i www.fao.org/fileadmin/templates/wsfs/docs/ wide-ranging effects beyond the EU itself, expert_paper/How_to_Feed_the_World_in_2050.pdf effects that will complicate international ii www.ictsd.org/bridges-news/bridges/news/ efforts to achieve global food security. eu-confirms-provisional-agreement-on-nationalWhile numerous GM-adopting nations gmo-bans iii http://ec.europa.eu/research/biosociety/pdf/ have positively responded to the chala_decade_of_eu-funded_gmo_research.pdf lenge of the FAO, the EU continues to iv http://giannini.ucop.edu/media/are-update/files/ ignore the impact of their domestic articles/v12n2_5.pdf choices on this crucial global issue. The EU has secure food supplies and is ignor- 1Department of Agriculture and Resource Economics, ing the cost its politically motivated tech- University of Saskatchewan, 51 Campus Drive, Saskatoon, S7N 5A8, Canada nology choice has on food insecure 2Johnson-Shoyama Graduate School of Public Policy, consumers and countries. The EU has University of Saskatchewan, 101 Diefenbaker Place, failed in its responsibility to its own citizens Saskatoon, S7N 5B8, Canada and to those in developing countries by *Correspondence: [email protected] (S.J. Smyth). ignoring science and evidence. http://dx.doi.org/10.1016/j.tibtech.2016.04.003
Concluding Remarks While the opportunity to use GM technologies in the EU seems to be lost, there is a bigger opportunity that the world cannot afford to waste. A host of new plant breeding techniques and technologies are poised to enter the crop variety development sector. They offer an exciting opportunity to reverse the trend to weaker yield growth. While many of these new technologies and techniques do not involve
References 1. Klümper, W. and Qaim, M. (2014) A meta analysis of the impact of genetically modified crops. PLoS ONE 9, 1–7 2. Kathage, J. and Qaim, M. (2012) Economic impacts and impact dynamics of Bt (Bacillus thuringiensis) cotton in India. PNAS 109, 11652–11656 3. Vitale, J. et al. (2014) Cotton. In Handbook on Agriculture, Biotechnology and Development (Smyth, S.J. et al., eds), pp. 604–620, Edward Elgar 4. Karplus, V.J. (2014) China. In Handbook on Agriculture, Biotechnology and Development (Smyth, S.J. et al., eds), pp. 112–125, Edward Elgar 5. Bennet, R. et al. (2006) The economic impact of genetically modified cotton on South African smallholders: yields, profits and health effects. J. Dev. Studies 42, 662–677
6. Yorobe, J.M. and Smale, M. (2012) Impacts of Bt maize on smallholder income in the Philippines. Agbioforum 15, 152–162 7. Alston, J.M. et al. (2014) The size and distribution of the benefits from the adoption of biotech soybean varieties. In Handbook on Agriculture, Biotechnology and Development (Smyth, S.J. et al., eds), pp. 728–751, Edward Elgar 8. Huang, J. et al. (2010) A decade of Bt cotton in Chinese fields: assessing the direct effects and indirect externalities of Bt cotton adoption in China. Sci. China Life Sci. 53, 981–991 9. Smyth, S.J. et al. (2011) Changes in herbicide use after the adoption of HR canola in Western Canada. Weed Tech. 25, 492–500 10. Kouser, S. and Qaim, M. (2011) Impact of Bt cotton on pesticide poisoning in smallholder agriculture: a panel data analysis. Ecol. Econ. 70, 2105–2113 11. Hobbs, J.E. et al. (2014) The perils of zero tolerance: technology management, supply chains and thwarted globalization. Int. J. Tech. Globalization 7, 203–216
Letter
Enhancing Plant Disease Resistance without R Genes Birinchi Kumar Sarma,1 Harikesh Bahadur Singh,1 Dilantha Fernando,2 Roberto Nascimento Silva,3 and Vijai Kumar Gupta4,* Crop plants encounter constant biotic challenges, and these challenges have historically been best managed with resistance (R) genes. However, the rapid evolution of new pathogenic strains along with the nonavailability or nonidentification of R genes in cultivated crop species against a large number of plant pathogens have led researchers to think beyond R genes. Biotechnological tools have shown promise in dealing with such challenges. Technologies such as transgenerational plant immunity, interspecies transfer of pattern recognition receptors (PRRs), pathogen-derived resistance (PDR), gene regulation, and expression of antimicrobial peptides (AMPs) in host plants from other
Trends in Biotechnology, July 2016, Vol. 34, No. 7
523
plant species have led to enhanced receptor-like proteins (RLPs); signals Similarly, disruption of the main brassinosdisease resistance and increased from potential pests/pathogens received teroid receptor (BRI1, brassinosteroid by the PRR result in a conformational insensitive 1) in both Brachypodium disfood security. Lombardo et al. recently reviewed new approaches to implementing R genes for insect and herbicide resistance in plants [1]. Similar approaches are also relevant for pathogen resistance in plants. Here, we summarize alternative approaches to engineer disease resistance in plants. In addition to genome editing [2] and antisense technologies [3], several other promising technologies have also emerged as potential instruments for countering the limitations of R gene-mediated resistance in plants against pathogens and insect herbivores. Technologies such as transgenerational epigenetics (TE) in plants could be employed to improve some traits in a shorter period of time. TE can be defined as an epigenetic change that persists across one or more subsequent generations. TE can also be utilized to manage plant pathogens, insect herbivores, and other abiotic stresses such as mechanical injuries. The TE blockage in the expression of some particular genes in response to environmental stimuli, due to either DNA methylation or histone modifications, may accelerate the plant's ability to restrict pest/ pathogen development [4]. When outside stimuli induce such blockages in plant gene expression, loss of expression may be permanent in that generation and passed to the next generation in the same state through seeds. Plants thereby pass on the perceived environmental threats of both biotic and abiotic stresses to offspring. Additionally, interspecies transfer of receptors has also shown promise in managing biotic stresses. Broad-spectrum resistance in plants can be developed by transferring pattern recognition receptors (PRRs) between plant species [5]. Engineering broad-spectrum disease resistance through transferring PRRs has shown promise, and the impact is expected to be long-lasting. These PRRs are either receptor kinases (RKs) or
524
Trends in Biotechnology, July 2016, Vol. 34, No. 7
change in the receptor, leading to the activation of downstream defense signaling genes. Transferring PRRs from related species, or wild relatives, that can perceive a pathogen threat from one plant cultivar or species to another and thereby increasing the level of resistance in a susceptible plant cultivar or species against a specific pathogen.
tachyon and barley enhanced disease resistance against some cereal fungal pathogens [9]. Further, when the rice heme activator protein (HAP) gene (OsHAP2E), which is known to regulate plant growth, development, and stress responses, was overexpressed, it not only enhanced disease resistance to a fungal and a bacterial pathogen but also conferred rice plants resistance to abiotic stresses such as Genetic engineering and biotechnology drought and salinity [10]. can open doors for counteracting infection; one strategy is introducing genes In addition, generating antimicrobial pepfrom various sources into plants, which tides (AMPs) in plants to fight pathogens could generate disease resistance with has also become an interesting tool to none of the species boundaries that apply reduce crop losses. AMP chitin-binding to conventional methodologies. The capability plays a crucial role in antifungal expression of structural viral nucleic acid activity. The AMP antiviral effect depends sequences (e.g., coat protein, movement on different factors, such as the direct protein, or replicase protein genes) in interaction with the viral envelope, displants, known as pathogen-derived resis- rupting or destabilizing it; competition tance (PDR), generally offers a broader with viruses for the host membrane, prerange of resistance even to the related venting the viral connection with particuviruses. The technique is effective against lar cell receptors; and prevention of a low level of inoculums but, as with most expression of viral genes in the earlier viral proteins, this strategy can elicit R infection stages, affecting the propagagene-driven effector triggered immunity tion and the viral infection [11]. Interest(ETI), causing hypersensitive response ingly, some endogenous peptides are (HR), a mechanism leading to rapid cell even perceived as danger signals and a death surrounding a pathogen infection stereotypical defense response is that limits the pathogen spread [6]. Bioen- induced in host against the invading gineering against viral pathogens is partic- pathogens [12]. Technological intervenularly important as only around 40 R tions may lead to greater utilization of genes have been identified so far against such peptides in reducing pathogen more than 1000 identified plant viruses. progress in hosts. Further, plantibodies have also been developed to protect plants, which could Recent technological advancements in be constructed to focus on any pathogen, biological sciences have thus created sequestering the antigens that are fre- the future possibility for disease resistance quently required to complete the infection in crop plants to those pathogens against cycle, thus preventing diseases [7]. which neither R genes nor available chemicals have been effective. Moreover, overSimilarly, regulating genes that are not con- reliance on R genes for disease resistance sidered to have direct roles in governing has become counterproductive in several host resistance may also lead to enhanced instances. Therefore, new technologies disease resistance against both pathogens are desirable as they are proving to be and insect herbivores. Downregulating cel- viable, complementary, or supplementary lulose synthase (CesA) increases Arabi- to R genes, environmentally acceptable, dopsis resistance to Botrytis cinerea [8]. and ecologically feasible alternatives to R
to capture thousands of single-cell trangene-mediated resistance. Although, in Spotlight most cases detailed work is necessary scriptomes from an individual experimento make these technologies commercially tal sample. Together with the reduced viable, technologies such as PDR and viral cost of sequencing, these developments antisense are already well established in are paving the way for single-cell RNA-seq certain crops and are in the marketplace. to soon become a laboratory staple. Once the newer technologies are perSamantha A. Morris1,@,* fected, they could be used to increase Enabled by these methodological advanfood production and reduce hunger by ces, it is becoming commonplace to gentackling pests and pathogens with great erate single-cell transcriptome data for Single-cell RNA sequencing (RNA- tens of thousands of cells. How to approspecificity. seq) technology is hitting its stride priately and effectively analyze these data 1 Department of Mycology and Plant Pathology, Institute of and is beginning to be widely is an emerging challenge. For example, Agricultural Sciences, Banaras Hindu University, Varanasi, adopted. One of the major chal- many sources of technical variation exist India 2 Department of Plant Science, University of Manitoba, lenges faced by this rapidly growing between transcriptomes, arising due to Winnipeg, MB R3T 2N2, Canada field lies in data analysis. Li et al. now differences in capture efficiency, amplifica3 Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão present Sinova, a single-cell analyt- tion biases, and sequencing depth, makPreto, SP, Brazil ical platform offering temporal, spa- ing it difficult to detect discrete biological 4 Molecular Glycobiotechnology Group, Discipline of Biochemistry, School of Natural Sciences, National tial, and regulatory reconstruction of differences between single cells. Moreover, in any approach, whether bulk or University of Ireland Galway, Galway, Ireland developmental processes. single cell, continuous monitoring of gene *Correspondence: [email protected] (V.K. Gupta). expression within the same cells over time http://dx.doi.org/10.1016/j.tibtech.2016.04.002 Recent technological advances have is not possible. Finally, to capture expresenabled the analysis of genome-wide sion profiles, single cells must be dissociReferences 1. Lombardo, L. et al. (2016) New technologies for insect- mRNA expression within single cells. ated from tissue, resulting in a loss of resistant and herbicide-tolerant plants. Trends Biotechnol. These developments are proving to be spatial and temporal information. These 34, 49–57 2. Wang, Y. et al. (2014) Simultaneous editing of three pivotal and broadly applicable over many issues present barriers to using single-cell homoeoalleles in hexaploid bread wheat confers heritable biological disciplines. Previous analyses of RNA-seq to systematically describe bioresistance to powdery mildew. Nat. Biotechnol. 32, 947– gene expression were restricted to the logical processes and identify the regula951 3. Pumplin, N. and Voinnet, O. (2013) RNA silencing suppres- population level, lacking resolution by tors driving them. sion by plant pathogens: defence, counter-defence and counter-counter-defence. Nat. Rev. Microbiol. 11, 745– averaging results over many cells, and 760 obscuring signals from rare cell types. In response to these challenges, Li et al. 4. Sarma, B.K. and Singh, H.B. (2014) Harnessing transgeHowever, single-cell RNA sequencing [4] developed a new single-cell analytical nerational plant immunity. Curr. Sci. 107, 1941–1942 5. Dangl, J.L. et al. (2013) Pivoting the plant immune system can identify novel, rare cell types. For methodology, Sinova. With this platform, from dissection to deployment. Science 341, 746–751 example, a previously undiscovered cell they interrogate the development of the 6. García, J.A. and Pallás, V. (2015) Viral factors involved in species within the intestine was recently mouse growth plate, a structure required plant pathogenesis. Curr. Opin. Virol. 11, 21–30 7. Safarnejad, M.R. et al. (2011) Antibody-mediated resis- uncovered via single-cell RNA-seq, a feat for bone elongation and regeneration. The tance against plant pathogens. Biotechnol. Adv. 29, that would otherwise have proven impos- postnatal growth plate comprises cells at 961–971 sequential stages of differentiation, the sible with previous approaches [1]. 8. Ramírez, V. et al. (2011) MYB46 modulates disease susexact regulation of which remains largely ceptibility to Botrytis cinerea in Arabidopsis. Plant Physiol. 155, 1920–1935 The past year has heralded a new phase in unknown at a systematic level. In this 9. Goddard, R. et al. (2014) Enhanced disease resistance caused by BRI1 mutation is conserved between Brachy- single-cell technology. Analysis of hun- study, the expression profiles of 217 single podium distachyon and barley (Hordeum vulgare). Mol. dreds of cells within a study has become cells were captured from this developPlant Microbe Interact. 27, 1095–1106 standard, although remaining limitations mental structure, using the Fluidigm C1 10. Alam, M. et al. (2015) Overexpression of a rice heme activator protein gene (OsHAP2E) confers resistance to on ease, cost, and scale have been a platform. This simultaneous capture overpathogens, salinity and drought, and increases photobarrier to the broad application of sin- comes much interexperimental technical synthesis and tiller number. Plant Biotechnol. J. 13, 85– gle-cell approaches. These impediments variation, and such an unsynchronized 96 11. Salas, C.E. et al. (2015) Biologically active and antimicrobial have recently been addressed via the real- population contains cells representing peptides from plants. Biomed Res. Int. 102129 2015 ization of highly parallel single-cell expres- many stages of development, enabling 12. Boller, T. and Felix, G. (2009) A renaissance of elicitors: perception of microbe-associated molecular patterns and sion profiling [2,3]. Of particular note, both temporal and spatial reconstruction danger signals by pattern-recognition receptors. Annu. Macosko et al. [2] used a simple, cost- of differentiation trajectories from a popuRev. Plant Biol. 60, 379–406 effective, and robust microfluidic method lation ‘snapshot’.
Single-Cell RNA-Seq Steps Up to the Growth Plate
Trends in Biotechnology, July 2016, Vol. 34, No. 7
525