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An Improved Nicotiana Benthamiana Strain for Aphid and Whitefly Research

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An Improved Nicotiana Benthamiana Strain for Aphid and Whitefly Research bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.237180; this version posted August 5, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Article Title 2 An improved Nicotiana benthamiana strain for aphid and whitefly research 3 4 Running title 5 Acylsugars protect Nicotiana benthamiana 6 7 8 Author names 9 Honglin Feng1, Lucia Acosta-Gamboa2, Lars H. Kruse3, Alba Ruth Nava Fereira4, Sara Shakir1†, 10 Hongxing Xu1‡, Garry Sunter4, Michael A. Gore2, Gaurav D. Moghe3, Georg Jander1* 11 12 13 Author Affiliations 14 1Boyce Thompson Institute, Ithaca NY, USA 15 2Plant Breeding and Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca NY, 16 14853, USA 17 3Plant Biology Section, School of Integrative Plant Science, Cornell University, Ithaca NY, 14853, USA 18 4Department of Biology, University of Texas San Antonio, San Antonio TX, 78249, USA 19 †Present address: Gembloux Agro-Bio Tech Institute, the University of Liege, Gembloux, Belgium 20 ‡Present address: College of Life Science, the Shaanxi Normal University, Xi’an, China 21 22 *Correspondence: 23 Georg Jander 24 Boyce Thompson Institute 25 Ithaca, NY 14853 26 USA 27 Phone: 607-254-1365 28 Email: [email protected] 29 30 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.237180; this version posted August 5, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 31 Abstract 32 Nicotiana benthamiana is used extensively as a platform for transient gene expression and as a model 33 system for studying plant-virus interactions. However, many tobacco-feeding insects, e.g. Myzus persicae 34 (green peach aphids) and Bemisia tabaci (whiteflies), grow poorly on N. benthamiana, limiting its utility 35 for research on plant-insect interactions. Using CRISPR/Cas9, we generated knockout mutations in two 36 N. benthamiana acylsugar acyltransferase genes, ASAT1 and ASAT2, which contribute to the biosynthesis 37 of insect-deterrent acylsucroses. Whereas ASAT1 mutations reduced the abundance of two predominant 38 acylsucroses, ASAT2 mutations caused almost complete depletion of foliar acylsucroses. Both M. persicae 39 and B. tabaci survived and reproduced significantly better on asat2 mutant plants than on wildtype N. 40 benthamiana. Furthermore, ASAT1 and ASAT2 mutations reduced the water content and increased the 41 temperature of leaves, indicating that foliar acylsucroses can protect against desiccation. Improved aphid 42 and whitefly performance on ASAT2 mutants will make it possible to use the efficient transient 43 overexpression and gene expression silencing systems that are available for N. benthamiana to study 44 plant-insect interactions. Additionally, the absence of acylsugars in ASAT2 mutant lines will simplify 45 transient expression assays for the functional analysis of acylsugar biosynthesis genes from other 46 Solanaceae. 47 48 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.237180; this version posted August 5, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 49 Introduction 50 Nicotiana benthamiana, a species of wild tobacco that is native to Australia, is commonly used by plant 51 molecular biologists as model system for laboratory research. Susceptibility to a wide variety of plant 52 viruses has made N. benthamiana a popular model for fundamental studies of plant-virus interactions 53 (Goodin et al., 2008; Bally et al., 2018). Scientists have developed N. benthamiana as a transgene 54 expression powerhouse by engineering viral vectors to express heterologous genes, including fluorescent 55 reporter genes to visualize cell structures (Bally et al., 2018). Antibodies, biofuel compounds, and other 56 protein and metabolite products have been produced in N. benthamiana (Arntzen, 2015; Powell, 2015). 57 Virus-induced gene silencing (VIGS), which is employed to study gene function in a variety of plant 58 species, was originally developed in N. benthamiana (Hayward et al., 2011). Recently, high-efficiency 59 CRISPR/Cas9 germline gene editing using virus-encoded guide RNA (gRNA) was demonstrated for the 60 first time in N. benthamiana (Ellison et al., 2020). Although N. benthamiana is hyper-susceptible to many 61 plant viruses, it is not a good host for two virus-transmitting Hemiptera, Myzus persicae (green peach 62 aphid) (Thurston, 1961; Hagimori et al., 1993) and Bemisia tabaci (silverleaf whitefly) (Simon et al., 63 2003), that grow well on cultivated tobacco (Nicotiana tabacum). 64 The poor growth of aphids and whiteflies on N. benthamiana may be attributed in part to 65 glandular trichomes. These epidermal secretory structures on the leaf surface of ~30% of vascular plants 66 (Weinhold and Baldwin, 2011; Glas et al., 2012), have been found to play a crucial defensive role in 67 several ways: as a physical obstacle for insect movement on the plant surface (Cardoso, 2008), 68 entrapment (Simmons et al., 2004), production of volatiles and other defensive metabolites (Laue et al., 69 2000; Schilmiller et al., 2010; Glas et al., 2012), and production of proteins that repel herbivores 70 (phylloplane proteins, e.g. T-phylloplanin) (Shepherd and Wagner, 2007). In addition to their defensive 71 functions, glandular trichomes also protect plants from abiotic stresses such as transpiration water loss 72 and UV irradiation (Karabourniotis et al., 1995). 73 There are two main types of glandular trichomes on N. benthamiana leaves, large swollen-stalk 74 trichomes and small trichomes that are capped by a secretory head with one, two, or four cells (Slocombe 75 et al., 2008). The large trichomes have been shown to secrete phylloplane proteins in N. tabacum. The 76 small trichomes are the most abundant trichomes on tobacco leaf surfaces and secrete exudates, including 77 acylsugars (Wagner et al., 2004; Slocombe et al., 2008). Detached trichomes, a mixture of the large and 78 small trichomes, from N. benthamiana are able to synthesize acylsugars (Kroumova and Wagner, 2003), 79 and the secretory head cells alone are able to synthesize acylsugars in N. tabacum (Kandra and Wagner, 80 1988). 81 Acylsugars, generally sucrose or glucose esterified with aliphatic acids of different chain lengths, 82 are abundant insect-deterrent metabolites produced by Solanaceae glandular trichomes (Arrendale et al., 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.237180; this version posted August 5, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 83 1990; Slocombe et al., 2008; Moghe et al., 2017). Specific acylsugars are associated with aphid-resistant 84 Nicotiana species, while not being detected in more susceptible species in this genus (Hagimori et al., 85 1993). Furthermore, relative to cultivated tomatoes (Solanum lycopersicum), acylsugars provide wild 86 tomatoes (Solanum pennellii) greater resistance against M. persicae (Rodriguez et al., 1993). 87 Diacylsucrose protects crops against tobacco aphids (Myzus persicae nicotianae), B. tabaci, and two- 88 spotted spider mites (Tetranychus urticae) (Chortyk et al., 1996; Alba et al., 2009). The synthetic sucrose 89 octanoate (an analog of Nicotiana gossei sugar esters) is effective in the field against Asian citrus psyllids 90 (Diaphorina citri), citrus leafminer (Phyllocnistis citrella), and a mite complex (including Texas citrus 91 mite, red spider mite, and rust mite) (McKenzie and Puterka, 2004). Interestingly, acylsucroses in 92 Nicotiana attenuata are metabolized to volatile fatty acids by neonate Manduca sexta (tobacco 93 hornworm) larvae, thereby tagging these larvae and attracting predatory ants, Pogonomyrmex rugosus 94 (Weinhold and Baldwin, 2011). 95 Acylsugars and leaf surface lipids more generally may contribute to plant drought tolerance. 96 Transcriptomic studies of drought tolerant wild tomato (Solanum pennellii) populations showed that lipid 97 metabolism genes are among those that are most responsive to drought stress (Gong et al., 2010; Egea et 98 al., 2018). Additionally, acylsugars on the leaf surface in a native S. pennellii population contributed to 99 drought tolerance (Fobes et al., 1985). Acylsugars also are reported to provide protection against drought 100 stress conditions in Solanum chilense (O’ Connell et al., 2007). Similarly, abundant accumulation of 101 acylsugars with C7-C8 acyl groups the in the desert tobacco (Nicotiana obtusifolia) was suggested to 102 provide this species with high drought tolerance for its desert environment (Kroumova et al., 2016). 103 Although the mechanism is not completely understood, it has been proposed that the polar lipids reduce 104 the surface tension of adsorbed dew water, thereby allowing the leaves absorb more condensed water on 105 the surface (Fobes et al., 1985). 106 More recently, enzymes involved in the biosynthesis of acylsugars have been
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