[en] KEY MESSAGE: Nicotiana benthamiana acylsugar acyltransferase (ASAT) is required for protection against desiccation and insect herbivory. Knockout mutations provide a new resource for investigation of plant-aphid and plant-whitefly interactions. Nicotiana benthamiana is used extensively as a transient expression platform for functional analysis of genes from other species. Acylsugars, which are produced in the trichomes, are a hypothesized cause of the relatively high insect resistance that is observed in N. benthamiana. We characterized the N. benthamiana acylsugar profile, bioinformatically identified two acylsugar acyltransferase genes, ASAT1 and ASAT2, and used CRISPR/Cas9 mutagenesis to produce acylsugar-deficient plants for investigation of insect resistance and foliar water loss. Whereas asat1 mutations reduced accumulation, asat2 mutations caused almost complete depletion of foliar acylsucroses. Three hemipteran and three lepidopteran herbivores survived, gained weight, and/or reproduced significantly better on asat2 mutants than on wildtype N. benthamiana. Both asat1 and asat2 mutations reduced the water content and increased leaf temperature. Our results demonstrate the specific function of two ASAT proteins in N. benthamiana acylsugar biosynthesis, insect resistance, and desiccation tolerance. The improved growth of aphids and whiteflies on asat2 mutants will facilitate the use of N. benthamiana as a transient expression platform for the functional analysis of insect effectors and resistance genes from other plant species. Similarly, the absence of acylsugars in asat2 mutants will enable analysis of acylsugar biosynthesis genes from other Solanaceae by transient expression.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Feng, Honglin; Boyce Thompson Institute, Ithaca, NY, 14853, USA
Acosta-Gamboa, Lucia; Plant Breeding and Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, 14853, USA
Kruse, Lars H; Plant Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, 14853, USA ; Michael Smith Laboratories, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada
Tracy, Jake D; Plant-Microbe Biology and Plant Pathology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, 14853, USA ; Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT, 59717, USA
Chung, Seung Ho; Boyce Thompson Institute, Ithaca, NY, 14853, USA
Nava Fereira, Alba Ruth; Department of Biology, University of Texas San Antonio, San Antonio, TX, 78249, USA ; Department of Biological Sciences, Northern Illinois University, Dekalb, IL, 60115, USA
Shakir, Sara ; Université de Liège - ULiège > Département GxABT > Plant Sciences ; Boyce Thompson Institute, Ithaca, NY, 14853, USA
Xu, Hongxing; Boyce Thompson Institute, Ithaca, NY, 14853, USA ; College of Life Science, The Shaanxi Normal University, Xi'an, China
Sunter, Garry; Department of Biology, University of Texas San Antonio, San Antonio, TX, 78249, USA ; Department of Biological Sciences, Northern Illinois University, Dekalb, IL, 60115, USA
Gore, Michael A; Plant Breeding and Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, 14853, USA
Casteel, Clare L; Plant-Microbe Biology and Plant Pathology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, 14853, USA
Moghe, Gaurav D; Plant Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, 14853, USA
Jander, Georg ; Boyce Thompson Institute, Ithaca, NY, 14853, USA. gj32@cornell.edu
Cornell Startup Funds DFG - Deutsche Forschungsgemeinschaft United States Department of Agriculture Biotechnology Risk Assessment Grant NSF - National Science Foundation DARPA - Defense Advanced Research Projects Agency
Funding text :
This research was supported by Cornell startup funds to G.D.M., Deutsche Forschungsgemeinschaft award #411255989 to L.H.K., and United States Department of Agriculture Biotechnology Risk Assessment Grant 2017-33522-27006, US National Science Foundation award IOS-1645256, and Defense Advanced Research Projects Agency (DARPA) agreement HR0011-17-2-0053 to G.J, and US National Science Foundation award #1723926 to C.L.C. G.S. is part of a team supporting DARPA’s Insect Allies program under agreement HR0011-17-2-0055. M.A.G. is part of a team supporting DARPA’s Advanced Plant Technologies program under agreement HR0011-18-C-0146. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies of the U.S. Government.
Arntzen C (2015) Plant-made pharmaceuticals: from ‘Edible Vaccines’ to Ebola therapeutics. Plant Biotechnol J 13:1013–1016
Arrendale RF, Severson RF, Sisson VA, Costello CE, Leary JA, Himmelsbach DS, Vanhalbeek H (1990) Characterization of the sucrose ester fraction from Nicotiana glutinosa. J Agric Food Chem 38:75–85
Bally J, Jung H, Mortimer C, Naim F, Philips JG, Hellens R, Bombarely A, Goodin MM, Waterhouse PM (2018) The rise and rise of Nicotiana benthamiana: a plant for all reasons. Ann Rev Phytopathol 56 56:405–426
Bertani G (1951) Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300
Bombarely A, Rosli HG, Vrebalov J, Moffett P, Mueller LA, Martin GB (2012) A draft genome sequence of Nicotiana benthamiana to enhance molecular plant-microbe biology research. Mol Plant-Micr Int 25:1523–1530
Bos JI, Prince D, Pitino M, Maffei ME, Win J, Hogenhout SA (2010) A functional genomics approach identifies candidate effectors from the aphid species Myzus persicae (green peach aphid). PLoS Genet 6:e1001216
Capella-Gutierrez S, Silla-Martinez JM, Gabaldon T (2009) trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25:1972–1973
Cardoso MZ (2008) Herbivore handling of a plant’s trichome: The case of Heliconius charithonia (L.) (Lepidoptera: Nymphalidae) and Passiflora Lobata (Killip) Hutch. (Passifloraceae). Neotropical Entomol 37:247–252
Casteel CL, Yang C, Nanduri AC, De Jong HN, Whitham SA, Jander G (2014) The NIa-Pro protein of turnip mosaic virus improves growth and reproduction of the aphid vector, Myzus persicae (green peach aphid). Plant J 77: 653–663
Chortyk OT, Pomonis JG, Johnson AW (1996) Syntheses and characterizations of insecticidal sucrose esters. J Ag Food Chem 44:1551–1557
Chortyk OT, Severson RF, Cutler HC, Sisson VA (1993) Antibiotic activities of sugar esters isolated from selected Nicotiana species. Biosci Biotechnol Biochem 57:1355–1356
Clay N, Adio A, Denoux C, Jander G, Ausubel F (2009) Glucosinolate metabolites required for an Arabidopsis innate immune response. Science 323:95–101
D’Auria JC (2006) Acyltransferases in plants: a good time to be BAHD. Curr Opin Plant Biol 9: 331–340
Egan AN, Moore S, Stellari GM, Kang BC, Jahn MM (2019) Tandem gene duplication and recombination at the AT3 locus in the Solanaceae, a gene essential for capsaicinoid biosynthesis in Capsicum. Plos One 14:e0210510
Egea I, Albaladejo I, Meco V, Morales B, Sevilla A, Bolarin MC, Flores FB (2018) The drought-tolerant Solanum pennellii regulates leaf water loss and induces genes involved in amino acid and ethylene/jasmonate metabolism under dehydration. Sci Rep 8:2791
Ellison EE, Nagalakshmi U, Gamo ME, Huang PJ, Dinesh-Kumar S, Voytas DF (2020) Multiplexed heritable gene editing using RNA viruses and mobile single guide RNAs. Nat Plants 6:620–624
Elzinga DA, De Vos M, Jander G (2014) Suppression of plant defenses by a Myzus persicae (green peach aphid) salivary effector protein. Mol Plant Microbe Interact 27: 747–756
Fan P, Leong BJ, Last RL (2019) Tip of the trichome: evolution of acylsugar metabolic diversity in Solanaceae. Curr Opin Plant Biol 49:8–16
Fan P, Miller AM, Schilmiller AL, Liu X, Ofner I, Jones AD, Zamir D, Last RL (2016) In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network. Proc Natl Acad Sci USA 113:E239–E248
Feng H, Jander G (2021) Rapid screening of emopenMyzus persicaeemclose (green peach aphid) RNAi targets using emopenTobacco rattle virusemclose. Methods Mol Biol 2360:105–117
Fobes JF, Mudd JB, Marsden MP (1985) Epicuticular lipid accumulation on the leaves of Lycopersicon pennellii (Corr.) D’Arcy and Lycopersicon esculentum Mill. Plant Physiol 77:567–570
Gaquerel E, Kotkar H, Onkokesung N, Galis I, Baldwin IT (2013) Silencing an N-acyltransferase-like involved in lignin biosynthesis in Nicotiana attenuata dramatically alters herbivory-induced phenolamide metabolism. Plos One 8:e62336
Gitelson A, Merzlyak MN (1994) Spectral reflectance changes associated with autumn senescence of Aesculus hippocastanum L. and Acer platanoides L. leaves - spectral features and relation to chlorophyll estimation. J Plant Physiol 143:286–292
Glas JJ, Schimmel BCJ, Alba JM, Escobar-Bravo R, Schuurink RC, Kant MR (2012) Plant glandular trichomes as targets for breeding or engineering of resistance to herbivores. Int J Mol Sci 13:17077–17103
Gong P, Zhang J, Li H, Yang C, Zhang C, Zhang X, Khurram Z, Zhang Y, Wang T, Fei Z, Ye Z (2010) Transcriptional profiles of drought-responsive genes in modulating transcription signal transduction, and biochemical pathways in tomato. J Exp Bot 61:3563–3575
Goodin MM, Zaitlin D, Naidu RA, Lommel SA (2008) Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions. Mol Plant Microbe Interact 21:1015–1026
Hagimori M, Matsui M, Matsuzaki T, Shinozaki Y, Shinoda T, Harada H (1993) Production of somatic hybrids between Nicotiana benthamiana and Nicotiana tabacum and their resistance to aphids. Plant Sci 91:213–222
Jacobs TB, LaFayette PR, Schmitz RJ, Parrott WA (2015) Targeted genome modifications in soybean with CRISPR/Cas9. Bmc Biotechnol 15:16
Karabourniotis G, Kotsabassidis D, Manetas Y (1995) Trichome density and its protective potential against ultraviolet-B radiation damage during leaf development. Canadian J Botany Revue Canadienne De Botanique 73:376–383
Kim J, Kang K, Gonzales-Vigil E, Shi F, Jones AD, Barry CS, Last RL (2012) Striking natural diversity in glandular trichome acylsugar composition is shaped by variation at the Acyltransferase2 locus in the wild tomato Solanum habrochaites. Plant Physiol 160:1854–1870
Kroumova AB, Wagner GJ (2003) Different elongation pathways in the biosynthesis of acyl groups of trichome exudate sugar esters from various solanaceous plants. Planta 216:1013–1021
Kroumova ABM, Zaitlin D, Wagner GJ (2016) Natural variability in acyl moieties of sugar esters produced by certain tobacco and other Solanaceae species. Phytochemistry 130:218–227
Landis JB, Miller CM, Broz AK, Bennett AA, Carrasquilla-Garcia N, Cook DR, Last RL, Bedinger PA, Moghe GD (2021) Migration through a major Andean ecogeographic disruption as a driver of genetic and phenotypic diversity in a wild tomato species. Mol Biol Evol 38:3202–3219
Laue G, Preston CA, Baldwin IT (2000) Fast track to the trichome: induction of N-acyl nornicotines precedes nicotine induction in Nicotiana repanda. Planta 210:510–514
Leong BJ, Hurney SM, Fiesel PD, Moghe GD, Jones AD, Last RL (2020) Specialized metabolism in a nonmodel nightshade: Trichome acylinositol biosynthesis. Plant Physiol 183:915–924
Leong BJ, Lybrand DB, Lou YR, Fan PX, Schilmiller AL, Last RL (2019) Evolution of metabolic novelty: A trichome-expressed invertase creates specialized metabolic diversity in wild tomato. Sci Adv 5:eaaw3754
Liu H, Ding YD, Zhou YQ, Jin WQ, Xie KB, Chen LL (2017) CRISPR-P 2.0: an improved CRISPR-Cas9 tool for genome editing in plants. Mol Plant 10:530–532
Lou YR, Anthony TM, Fiesel PD, Arking RE, Christensen EM, Jones AD, Last RL (2021) It happened again: convergent evolution of acylglucose specialized metabolism in black nightshade and wild tomato. 10.1101/2021.06.08.447545
Luu VT, Weinhold A, Ullah C, Dressel S, Schoettner M, Gase K, Gaquerel E, Xu S, Baldwin IT (2017) O-Acyl sugars protect a wild tobacco from both native fungal pathogens and a specialist herbivore. Plant Physiol 174:370–386
Marchant WG, Legarrea S, Smeda JR, Mutschler MA, Srinivasan R (2020) Evaluating acylsugars-mediated resistance in tomato against Bemisia tabaci and transmission of tomato yyellow leaf curl virus. Insects 11:842
Matsuzaki T, Shinozaki Y, Hagimori M, Tobita T, Shigematsu H, Koiwai A (1992) Novel glycerolipids and glycolipids from the surface-lipids of Nicotiana benthamiana. Biosci Biotechnol Biochem 56:1565–1569
Matsuzaki T, Shinozaki Y, Suhara S, Ninomiya M, Shigematsu H, Koiwai A (1989) Isolation of glycolipids from the surface-lipids of Nicotiana bigelovii and their distribution in Nicotiana species. Agric Biol Chem 53:3079–3082
McKenzie CL, Puterka GJ (2004) Effect of sucrose octanoate on survival of nymphal and adult Diaphorina citri (Homoptera: Psyllidae). J Econ Entomol 97:970–975
McKenzie CL, Weathersbee AA, 3rd, Puterka GJ (2005) Toxicity of sucrose octanoate to egg, nymphal, and adult Bemisia tabaci (Hemiptera: Aleyrodidae) using a novel plant-based bioassay. J Econ Entomol 98: 1242–1247
Meihls LN, Handrick V, Glauser G, Barbier H, Kaur H, Haribal MM, Lipka AE, Gershenzon J, Buckler ES, Erb M, Köllner TG, Jander G (2013) Natural variation in maize aphid resistance is associated with 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside methyltransferase activity. Plant Cell 25:2341–2355
Moghe GD, Leong BJ, Hurney SM, Daniel Jones A, Last RL (2017) Evolutionary routes to biochemical innovation revealed by integrative analysis of a plant-defense related specialized metabolic pathway. Elife 6:e28468
Nadakuduti SS, Uebler JB, Liu X, Jones AD, Barry CS (2017) Characterization of trichome-expressed BAHD acyltransferases in Petunia axillaris reveals distinct acylsugar assembly mechanisms within the Solanaceae. Plant Physiol 175:36–50
Ning J, Moghe GD, Leong B, Kim J, Ofner I, Wang Z, Adams C, Jones AD, Zamir D, Last RL (2015) A feedback-insensitive isopropylmalate synthase affects acylsugar composition in cultivated and wild tomato. Plant Physiol 169:1821–1835
Nonomura T, Xu L, Wada M, Kawamura S, Miyajima T, Nishitomi A, Kakutani K, Takikawa Y, Matsuda Y, Toyoda H (2009) Trichome exudates of Lycopersicon pennellii form a chemical barrier to suppress leaf-surface germination of Oidium neolycopersici conidia. Plant Sci 176:31–37
O’Connell MA, Medina AL, Sanchez Pena P, Trevino MB (2007) Molecular genetics of drought resistance response in tomato and related species. In: Razdan MK, Mattoo AK (eds). Genetic improvement of solanaceous crops Enfield USA, pp 261–283
Penuelas J, Filella I, Biel C, Serrano L, Save R (1993) The reflectance at the 950–970 nm region as an indicator of plant water status. Int J Remote Sens 14:1887–1905
Powell JD (2015) From pandemic preparedness to biofuel production: tobacco finds its biotechnology niche in North America. Agriculture Basel 5:901–917
Ramsey JS, Elzinga DA, Sarkar P, Xin YR, Ghanim M, Jander G (2014) Adaptation to nicotine feeding in Myzus persicae. J Chem Ecol 40:869–877
Ramsey JS, Wilson AC, De Vos M, Sun Q, Tamborindeguy C, Winfield A, Malloch G, Smith DM, Fenton B, Gray SM, Jander G (2007) Genomic resources for Myzus persicae: EST sequencing, SNP identification, and microarray design. BMC Genom 8: 423
Rodriguez AE, Tingey WM, Mutschler MA (1993) Acylsugars of Lycopersicon pennellii deter settling and feeding of the green peach aphid (Homoptera, Aphididae). J Econ Entomol 86:34–39
Rodriguez PA, Stam R, Warbroek T, Bos JI (2014) Mp10 and Mp42 from the aphid species Myzus persicae trigger plant defenses in Nicotiana benthamiana through different activities. Mol Plant Microbe Interact 27:30–39
Schiavinato M, Strasser R, Mach L, Dohm JC, Himmelbauer H (2019) Genome and transcriptome characterization of the glycoengineered Nicotiana benthamiana line DeltaXT/FT. BMC Genom 20:594
Schilmiller A, Shi F, Kim J, Charbonneau AL, Holmes D, Jones AD, Last RL (2010) Mass spectrometry screening reveals widespread diversity in trichome specialized metabolites of tomato chromosomal substitution lines. Plant J 62:391–403
Schilmiller AL, Charbonneau AL, Last RL (2012) Identification of a BAHD acetyltransferase that produces protective acyl sugars in tomato trichomes. Proc Natl Acad Sci USA 109:16377–16382
Schilmiller AL, Moghe GD, Fan P, Ghosh B, Ning J, Jones AD, Last RL (2015) Functionally divergent alleles and duplicated loci encoding an acyltransferase contribute to acylsugar metabolite diversity in Solanum trichomes. Plant Cell 27:1002–1017
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675
Shepherd RW, Wagner GJ (2007) Phylloplane proteins: emerging defenses at the aerial frontline? Trends Plant Sci 12:51–56
Simmons AT, Gurr GM, McGrath D, Martin PM, Nicol HI (2004) Entrapment of Helicoverpa armigera (Hubner) (Lepidoptera:Noctuidae) on glandular trichomes of Lycopersicon species. Aust J Entomol 43:196–200
Simon B, Cenis JL, Demichelis S, Rapisarda C, Caciagli P, Bosco D (2003) Survey of Bemisia tabaci (Hemiptera: Aleyrodidae) biotypes in Italy with the description of a new biotype (T) from Euphorbia characias. Bull Entomol Res 93:259–264
Slocombe SP, Schauvinhold I, McQuinn RP, Besser K, Welsby NA, Harper A, Aziz N, Li Y, Larson TR, Giovannoni J, Dixon RA, Broun P (2008) Transcriptomic and reverse genetic analyses of branched-chain fatty acid and acyl sugar production in Solanum pennellii and Nicotiana benthamiana. Plant Physiol 148:1830–1846
Song Z, Li S, Chen X, Liu L, Song Z (2006) Synthesis of sucrose esters. For Stud China 8:26–29
Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30:1312–1313
Stone WD, Pellicore MJ, Hagstrom S, Lawton TJ (2020) HSIviewer: pushbutton hyperspectral image analysis for rapid plant phenotyping. in review
Thompson JD, Higgins DG, Gibson TJ (1994) Clustal-W - Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
Thurston R (1961) Resistance in Nicotiana to the green peach aphids and some other tobacco insect pest. J Econ Entomol 54:946–949
Van Eck J, Keen P, Tjahjadi M (2019) Agrobacterium tumefaciens-mediated transformation of tomato. Methods Mol Biol 1864: 225–234
Van T, Weinhold A, Ullah C, Dressel S, Schoettner M, Gase K, Gaquerel E, Xu SQ, Baldwin IT (2017) O-acyl sugars protect a wild tobacco from both native fungal pathogens and a specialist herbivore. Plant Physiol 174:370–386
Wagner GJ, Wang E, Shepherd RW (2004) New approaches for studying and exploiting an old protuberance, the plant trichome. Ann Bot 93:3–11
Weinhold A, Baldwin IT (2011) Trichome-derived O-acyl sugars are a first meal for caterpillars that tags them for predation. Proc Natl Acad Sci USA 108:7855–7859
