Transcriptomic and metabolomic analyses of cucumber fruit peels reveal a developmental increase in terpenoid glycosides associated with age-related resistance to Phytophthora capsici

Abstract

The oomycete, Phytophthora capsici, infects cucumber (Cucumis sativus L.) fruit. An age-related resistance (ARR) to this pathogen was previously observed in fruit of cultivar ‘Vlaspik’ and shown to be associated with the peel. Young fruits are highly susceptible, but develop resistance at ~ 10–12 days post pollination (dpp). Peels from resistant (16 dpp) versus susceptible (8 dpp) age fruit are enriched with genes associated with defense, and methanolic extracts from resistant age peels inhibit pathogen growth. Here we compared developing fruits from ‘Vlaspik’ with those of ‘Gy14’, a line that does not exhibit ARR. Transcriptomic analysis of peels of the two lines at 8 and 16 dpp identified 80 genes that were developmentally upregulated in resistant ‘Vlaspik’ 16 dpp versus 8 dpp, but not in susceptible ‘Gy14’ at 16 dpp. A large number of these genes are annotated to be associated with defense and/or specialized metabolism, including four putative resistance (R) genes, and numerous genes involved in flavonoid and terpenoid synthesis and decoration. Untargeted metabolomic analysis was performed on extracts from 8 and 16 dpp ‘Vlaspik’ and ‘Gy14’ fruit peels using Ultra-Performance Liquid Chromatography and Quadrupole Time-of-Flight Mass Spectrometry. Multivariate analysis of the metabolomes identified 113 ions uniquely abundant in resistant ‘Vlaspik’ 16 dpp peel extracts. The most abundant compounds in this group had relative mass defects consistent with terpenoid glycosides. Two of the three most abundant ions were annotated as glycosylated nor-terpenoid esters. Together, these analyses reveal potential mechanisms by which ARR to P. capsici may be conferred.

References

[1] Granke LL, Quesada-Ocampo L, Lamour K, Hausbeck MK . Advances in research on Phytophthora capsici on vegetable crops in the United States. Plant Dis 2012; 96: 1588-1600.

[2] Tian D, Babadoost M . Host range of Phytophthora capsici from pumpkin and pathogenicity of isolates. Plant Dis 2004; 88: 485-489.

[3] Gevens AJ, Ando K, Lamour KH, Grumet R, Hausbeck MK . A detached cucumber fruit method to screen for resistance to Phytophthora capsici and effect of fruit age on susceptibility to infection. Plant Dis 2006; 90: 1276-1282.

[4] Ando K, Hammar S, Grumet R . Age-related resistance of diverse cucurbit fruit to infection by Phytophthora capsici. J Amer Soc Hort Sci 2009; 134: 176-182.

[5] Ando K, Carr KM, Grumet R . Transcriptome analyses of early cucumber fruit growth identifies distinct gene modules associated with phases of development. BMC Genomics 2012; 13: 518-534.

[6] Develey-Rivière M-P, Galiana E . Resistance to pathogens and host developmental stage: a multifaceted relationship within the plant kingdom. New Phytol 2007; 175: 405-416.

[7] Kim YJ, Hwang BK, Park KW . Expression of age-related resistance in pepper plants infected with Phytophthora capsici. Plant Dis 1989; 73: 745-747.

[8] Hoffman LE, Wilcox WF, Gadoury DM, Seem RC . Influence of grape berry age on susceptibility to Guignardia bidwellii and its incubation period length. Phytopathology 2002; 92: 1068-1076.

[9] Kennelly MM, Gadoury DM, Wilcox WF, Magarey PA, Seem RC . Seasonal development of ontogenic resistance to downy mildew in grape berries and rachises. Phytopathology 2005; 95: 1445-1452.

[10] Gadoury DM, Seem RC, Ficke A, Wilcox WF . Ontogenic resistance to powdery mildew in grape berries. Phytopathology 2003; 93: 547-555.

[11] Gee CT, Gadoury DM, Cadle-Davidson L . Ontogenic resistance to Uncinula necator varies by genotype and tissue type in a diverse collection of Vitis spp. Plant Dis 2008; 92: 1067-1073.

[12] Twomey MC, Wolfenbarger SN, Woods JL, Gent DH . Development of partial ontogenic resistance to powdery mildew in hop cones and its management implications. PLoS ONE 2015; 10: 1-24.

[13] Asalf B, Gadoury DM, Tronsmo AM et al. Ontogenic resistance of leaves and fruit, and how leaf folding influences the distribution of powdery mildew on strawberry plants colonized by Podosphaera aphanis. Phytopathology 2014; 104: 954-963.

[14] Asalf B, Gadoury DM, Tronsmo AM, Seem RC, Stensvand A . Effects of development of ontogenic resistance in strawberry leaves upon pre- and postgermination growth and sporulation of Podosphaera aphanis. Plant Dis 2016; 100: 72-78.

[15] Whalen MC . Host defence in a developmental context. Mol Plant Pathol 2005; 6: 347-360.

[16] Shibata Y, Kawakita K, Takemoto D . Age-related resistance of Nicotiana benthamiana against hemibiotrophic pathogen Phytophthora infestans requires both ethylene- and salicylic acid-mediated signaling pathways. Mol Plant-Microbe Interact 2010; 23: 1130-1142.

[17] Cao Y, Ding X, Cai M et al. The expression pattern of a rice disease resistance gene Xa3/Xa26 is differentially regulated by the genetic backgrounds and developmental stages that influence its function. Genetics 2007; 177: 523-533.

[18] Zhao J, Fu J, Li X, Xu C, Wang S . Dissection of the factors affecting development-controlled and race-specific disease resistance conferred by leucine-rich repeat receptor kinase-type R genes in rice. Theor Appl Genet 2009; 119: 231-239.

[19] Ando K, Carr KM, Colle M, Mansfeld BN, Grumet R . Exocarp properties and transcriptomic analysis of cucumber (Cucumis sativus) fruit expressing age-related resistance to Phytophthora capsici. PLoS ONE 2015; 10: e0142133.

[20] Ando K, Grumet R . Transcriptional profiling of rapidly growing cucumber fruit by 454-pyrosequencing analysis. J Am Soc Hortic Sci 2010; 135: 291-302.

[21] Colle M, Straley E, Makela SB, Hammar SA, Grumet R . Screening the cucumber plant introduction collection for young fruit resistance to Phytophthora capsici. HortScience 2014; 49: 244-249.

[22] Bolger AM, Lohse M, Usadel B . Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 2014; 30: 2114-2120.

[23] Li Z, Zhang Z, Yan P, Huang S, Fei Z, Lin K . RNA-Seq improves annotation of protein-coding genes in the cucumber genome. BMC Genomics 2011; 12: 540-551.

[24] Huang S, Li R, Zhang Z et al. The genome of the cucumber, Cucumis sativus L. Nat Genet 2009; 41: 1275-1281.

[25] Trapnell C, Roberts A, Goff L et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 2012; 7: 562-578.

[26] Anders S, Pyl PTP, Huber W . HTSeq-A Python framework to work with high-throughput sequencing data. Bioinformatics 2015; 31: 166-169.

[27] Love MI, Huber W, Anders S . Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014; 15: 550-571.

[28] Jones P, Binns D, Chang H-Y et al. InterProScan 5: Genome-scale protein function classification. Bioinformatics 2014; 30: 1236-1240.

[29] Alexa A, Rahnenfuhrer J, Lengauer T . Improved scoring of functional groups from gene expression data by decorrelating GO graph structure. Bioinformatics 2006; 22: 1600-1607.

[30] Supek F, Bošnjak M, Škunca N, Šmuc T . REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS ONE 2011; 6: e21800.

[31] Ekanayaka EAP, Celiz MD, Jones AD . Relative mass defect filtering of mass spectra: a path to discovery of plant specialized metabolites. Plant Physiol 2015; 167: 1221-1232.

[32] Colle M. Cucumber (Cucumis sativus L.) fruit development: factors influencing fruit size, shape and resistance to Phytophthora capsici. PhD dissertation, Michigan State University, East Lansing, MI, USA, 2015.

[33] Neilson EH, Goodger JQD, Woodrow IE, Møller BL . Plant chemical defense: at what cost? Trends Plant Sci 2013; 18: 250-258.

[34] Huot B, Yao J, Montgomery BL, He SY . Growth-defense tradeoffs in plants: a balancing act to optimize fitness. Mol Plant 2014; 7: 1267-1287.

[35] Panter SN, Hammond-Kosack KE, Harrison K, Jones JDG, Jones DA . Developmental control of promoter activity is not responsible for mature onset of Cf-9B-mediated resistance to leaf mold in tomato. Mol Plant Microbe Interact 2002; 15: 1099-1107.

[36] Meng X, Zhang S . MAPK cascades in plant disease resistance signaling. Annu Rev Phytopathol 2013; 51: 245-266.

[37] Burkhardt A, Day B . Transcriptome and small RNAome dynamics during a resistant and susceptible interaction between cucumber and downy mildew. Plant Genome 2016; 9: 1-19.

[38] Ron M. The receptor for the fungal elicitor ethylene-inducing xylanase is a member of a resistance-like gene family in tomato. Plant Cell 2004; 16: 1604-1615.

[39] Cantu D, Blanco-Ulate B, Yang L, Labavitch JM, Bennett AB, Powell ALT . Ripening-regulated susceptibility of tomato fruit to Botrytis cinerea requires NOR but not RIN or ethylene. Plant Physiol 2009; 150: 1434-1449.

[40] Dodds PN, Rathjen JP . Plant immunity: towards an integrated view of plant-pathogen interactions. Nat Rev Genet 2010; 11: 539-548.

[41] Stael S, Kmiecik P, Willems P et al. Plant innate immunity--sunny side up? Trends Plant Sci 2015; 20: 3-11.

[42] Wang X, Gao J, Zhu Z et al. TCP transcription factors are critical for the coordinated regulation of ISOCHORISMATE SYNTHASE 1 expression in Arabidopsis thaliana. Plant J 2015; 82: 151-162.

[43] Gao X, Chen X, Lin W et al. Bifurcation of Arabidopsis NLR immune signaling via Ca²⁺-dependent protein kinases . PLoS Pathog 2013; 9: e1003127.

[44] Chen X, Liu J, Lin G, Wang A, Wang Z, Lu G . Overexpression of AtWRKY28 and AtWRKY75 in Arabidopsis enhances resistance to oxalic acid and Sclerotinia sclerotiorum. Plant Cell Rep 2013; 32: 1589-1599.

[45] Babbar N, Oberoi HS, Sandhu SK . Therapeutic and nutraceutical potential of bioactive compounds extracted from fruit residues. Crit Rev Food Sci Nutr 2015; 55: 319-337.

[46] Eisenmann P, Ehlers M, Weinert C et al. Untargeted NMR spectroscopic analysis of the metabolic variety of new apple cultivars. Metabolites 2016; 6: 29.

[47] D’Abrosca B, Scognamiglio M, Corrado L et al. Evaluation of different training systems on Annurca apple fruits revealed by agronomical, qualitative and NMR-based metabolomic approaches. Food Chem 2017; 222: 18-27.

[48] Feder A, Burger J, Gao S et al. A Kelch domain-containing F-Box coding gene negatively regulates flavonoid accumulation in muskmelon. Plant Physiol 2015; 169: 1714-1726.

[49] Pinelo M, Arnous A, Meyer AS . Upgrading of grape skins: significance of plant cell-wall structural components and extraction techniques for phenol release. Trends Food Sci Technol 2006; 17: 579-590.

[50] Domínguez E, Heredia-Guerrero JA, Heredia A . The biophysical design of plant cuticles: an overview. New Phytol 2011; 189: 938-949.

[51] Chen JC, Chiu MH, Nie RL, Cordell GA, Qiu SX . Cucurbitacins and cucurbitane glycosides: structures and biological activities. Nat Prod Rep 2005; 22: 386-399.

[52] Shang Y, Ma Y, Zhou Y et al. Biosynthesis, regulation, and domestication of bitterness in cucumber. Science 2014; 346: 1084-1088.

[53] Qi J, Liu X, Shen D et al. A genomic variation map provides insights into the genetic basis of cucumber domestication and diversity. Nat Genet 2013; 45: 1510-1515.

[54] Kappers IF, Hoogerbrugge H, Bouwmeester HJ, Dicke M . Variation in herbivory-induced volatiles among cucumber (Cucumis sativus L.) varieties has consequences for the attraction of carnivorous natural enemies. J Chem Ecol 2011; 37: 150-160.

[55] Takabayashi J, Dicke M, Posthumus MA . Volatile herbivore-induced terpenoids in plant-mite interactions: variation caused by biotic and abiotic factors. J Chem Ecol 1994; 20: 1329-1354.

[56] Mercke P, Kappers IF, Verstappen FWA, Vorst O, Dicke M, Bouwmeester HJ . Combined transcript and metabolite analysis reveals genes involved in spider mite induced volatile formation in cucumber plants. Plant Physiol 2004; 135: 2012-2024.

[57] Wei G, Tian P, Zhang F et al. Integrative analyses of nontargeted volatile profiling and transcriptome data provide molecular insight into VOC diversity in cucumber plants (Cucumis sativus). Plant Physiol 2016; 172: 603-618.