Wheat defenses display both sustained and time-of-day-specific responses to Rhopalosiphum padi (bird cherry-oat aphid) infestation over a 24-hour period

Wheat defenses display both sustained and time-of-day-specific responses to Rhopalosiphum padi (bird cherry-oat aphid) infestation over a 24-hour period | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Wheat defenses display both sustained and time-of-day-specific responses to Rhopalosiphum padi (bird cherry-oat aphid) infestation over a 24-hour period Yoshiahu Goldstein, Jinlong Han, Daniel Kunk, Let Kho Hao, Vamsi J. Nalam, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7114751/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 6 You are reading this latest preprint version Abstract Wheat is constantly challenged by destructive pests, such as herbivores, even as it adapts to shifting environmental factors. While plant responses to herbivore infestation (hours post-infestation, hpi) are studied, how these responses are modulated by daily light-dark cycles remains poorly understood. Here, we investigate the effects of Rhopalosiphum padi aphid infestation on Svevo wheat using an RNA-seq approach, analyzing responses at multiple time points over a 24-hour infestation period. Both persistently regulated and hpi-specific plant responses were identified. Gene Ontology (GO) terms related to terpene synthesis and oxidoreductase were consistently enriched throughout the infestation period, whereas ethylene and jasmonic acid synthesis were enriched at specific hpi. Notably, daytime infestation specifically enriched GO terms for protein phosphorylation and motifs of WRKY transcription factors, indicating a crucial role for the circadian clock in shaping these defense responses. Additionally, highly correlated clustered genes revealed unique pathways, including those involved in terpene synthesis and amino acid catabolic processes. Understanding these initial single-day responses to aphid infestation may provide valuable insights into the complex plant-aphid interaction and contribute to the development of targeted defensive strategies. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Full Text Supplementary Files S1DEtranscriptionfactorsofalltimepoints.xlsx S2Deseq2DEgenesofalltimepoints.xlsx S3MotifenrichementatallHPIs.xlsx S4WGCNAclusters.xlsx S5HighlycorrelatedclustersWGCNAandspecificDE.xlsx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Major revisions 03 Sep, 2025 Reviewers agreed at journal 31 Jul, 2025 Reviewers invited by journal 19 Jul, 2025 Editor invited by journal 14 Jul, 2025 Editor assigned by journal 14 Jul, 2025 First submitted to journal 13 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7114751","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":487737524,"identity":"07b11070-9d41-4ec0-b6c3-4ce3333dc9a4","order_by":0,"name":"Yoshiahu Goldstein","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIie2QMQ6CMBSGnwssDawlIeoROqEDhykLLjVxcmaSQfQsHIHERJY6mtB0YcLFAxDDYKs7dDSx3/D3H96X9hXAYvlNHKAAIdSZ6mRtriDglVawoQJaaag+DRRS513bvmLkice97HcY/PxIxxXOVyQ5pyiQbC8K9TDMb+W40jAHJ8UFEcnSBimF4O2Usum+iuCpGMwUGmHaK6Vxr9LoloDzCCeZ2oUzR4YEo8ldPPVjQT/Ec6+uO/FUxc9P48qyUjE76IrIJ0fHNYtM56DDbSenLRaL5T95A8YITY8jlwoqAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0001-2757-2290","institution":"Ben-Gurion University of the Negev - Sede Boqer Campus","correspondingAuthor":true,"prefix":"","firstName":"Yoshiahu","middleName":"","lastName":"Goldstein","suffix":""},{"id":487737525,"identity":"fb25267d-4070-43a7-9b45-6a82e6ef0205","order_by":1,"name":"Jinlong Han","email":"","orcid":"","institution":"Colorado State University","correspondingAuthor":false,"prefix":"","firstName":"Jinlong","middleName":"","lastName":"Han","suffix":""},{"id":487737526,"identity":"82117097-712d-4680-a7fc-105aed7ef495","order_by":2,"name":"Daniel Kunk","email":"","orcid":"","institution":"Colorado State University","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Kunk","suffix":""},{"id":487737527,"identity":"6843bc4c-ae6e-49f9-aead-6bb5573c771c","order_by":3,"name":"Let Kho Hao","email":"","orcid":"","institution":"Ben-Gurion University of the Negev - Sede Boqer Campus","correspondingAuthor":false,"prefix":"","firstName":"Let","middleName":"Kho","lastName":"Hao","suffix":""},{"id":487737528,"identity":"ecbc2870-d050-4cd1-8647-3e1d3052db33","order_by":4,"name":"Vamsi J. Nalam","email":"","orcid":"","institution":"Colorado State University","correspondingAuthor":false,"prefix":"","firstName":"Vamsi","middleName":"J.","lastName":"Nalam","suffix":""},{"id":487737529,"identity":"0485bca4-5ca8-4a7b-b4ef-4c39df8a6dca","order_by":5,"name":"Vered Tzin","email":"","orcid":"","institution":"Ben-Gurion University of the Negev - Sede Boqer Campus","correspondingAuthor":false,"prefix":"","firstName":"Vered","middleName":"","lastName":"Tzin","suffix":""}],"badges":[],"createdAt":"2025-07-13 17:30:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7114751/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7114751/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88945240,"identity":"a18171cf-fd6f-4b6d-9c9b-b04727a5d395","added_by":"auto","created_at":"2025-08-13 04:50:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":84216,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental setup and RNA-seq analysis. (a) Wheat (Svevo) plants were infested with approximately 40 R.padi aphids at 4 ZT (12:00). Samples were collected every 4 hours from both infested and control plants, with aphids carefully removed from the leaves prior to collection. The samples were promptly frozen in liquid nitrogen and stored at -80°C for future analysis. (b) The number of differentially expressed (DE) genes at each hours post infestation (hpi). Collections at 4, 8, 12, and 24 hpi were taken during the day, while 16 and 20 hpi were collected at night. Red bars represent up-regulated genes while blue bars represent down-regulated genes.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7114751/v1/b433faf88e803981fd8b4fc2.png"},{"id":88945246,"identity":"5bcc0137-57ac-432e-92eb-46c4f0e386c2","added_by":"auto","created_at":"2025-08-13 04:50:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":139865,"visible":true,"origin":"","legend":"\u003cp\u003eGene ontology (GO) enrichment bubble plot of molecular functions at each hpi timepoint. GO enrichment analysis was performed on the DE genes of each hpi timepoint. The top 10 GO terms with the most significant p-values for each timepoint were selected. Bubble size represents the number of DE genes contributing to the enrichment, while color intensity reflects the level of p-value significance. Enrichment analysis was performed using the gProfiler web tool, with the Triticum turgidum genome from EnsemblPlants as the reference and a BH-FDR cutoff of 0.05.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7114751/v1/5a6143aab88c10e0cd477774.png"},{"id":88945242,"identity":"89667eec-dbce-4420-b825-29d4249f1423","added_by":"auto","created_at":"2025-08-13 04:50:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":178503,"visible":true,"origin":"","legend":"\u003cp\u003eGene ontology (GO) enrichment bubble plot of biological processes at each hpi timepoint. GO enrichment analysis was performed on the DE genes of each hpi timepoint. The top 10 GO terms with the most significant p-values for each timepoint were selected. Bubble size represents the number of DE genes contributing to the enrichment, while color intensity reflects the level of p-value significance. Enrichment analysis was performed using the gProfiler web tool, with the Triticum turgidum genome from EnsemblPlants as the reference and a BH-FDR cutoff of 0.05.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7114751/v1/655521ce4bbdd9a62ed28621.png"},{"id":88945884,"identity":"c9795570-1bd9-48bd-89ee-171f0e17858f","added_by":"auto","created_at":"2025-08-13 04:58:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":120084,"visible":true,"origin":"","legend":"\u003cp\u003eTranscription factor family enrichment at each hpi timepoint. (a) Bubble plot of transcription factor (TF) family enrichment at each hpi timepoint. TFs were identified using the PlantTFDB tool, and enrichment analysis of TF families was performed for each timepoint, using all expressed genes as the background. Bubble size represents p-value significance, while color intensity reflects the log2 odds ratio. An FDR cutoff of 0.05 was applied. (b) Heatmap of the log2 fold change of DE WRKY genes. Fold changes were calculated using DESeq2, comparing aphid-infested conditions to control.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7114751/v1/f4b6f7b425987e7e99831fca.png"},{"id":88945885,"identity":"78e719ca-3304-41f8-be51-118838b12ff1","added_by":"auto","created_at":"2025-08-13 04:58:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":285813,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of highly correlated aphid-responsive gene groups. (a)-(c) Gene expression levels of highly correlated gene clusters were analyzed. First, genes were clustered using WGCNA, and aphid-associated modules were identified. These modules were then tested for enrichment of specific DE timepoints using Fisher’s exact test, with a 0.05 FDR cutoff. A total of 22 unique groups were identified across the clusters. The clustered groups were further analyzed through GO term enrichment using gProfiler and motif enrichment using the AME tool of the MEME suite, applying a 0.05 adjusted p-value. Three highlighted clustered groups are presented here.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7114751/v1/a7bbffbd6bb459e4290d0596.png"},{"id":89063837,"identity":"721f8fc2-27a6-4613-b486-65a5656c1d46","added_by":"auto","created_at":"2025-08-14 10:08:48","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":759914,"visible":true,"origin":"","legend":"","description":"","filename":"SubmissionManuscriptHPIPMB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7114751/v1_covered_011cf589-e551-448e-bf64-2ae062ccb800.pdf"},{"id":88945887,"identity":"d6bd93bd-4c52-4845-b886-fdd4f99db107","added_by":"auto","created_at":"2025-08-13 04:58:34","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":36490,"visible":true,"origin":"","legend":"","description":"","filename":"S1DEtranscriptionfactorsofalltimepoints.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7114751/v1/0a539ae49db75c4890ba7e07.xlsx"},{"id":88945886,"identity":"e021591e-f62d-477c-8db7-a48fc03c3c7f","added_by":"auto","created_at":"2025-08-13 04:58:34","extension":"xlsx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":684680,"visible":true,"origin":"","legend":"","description":"","filename":"S2Deseq2DEgenesofalltimepoints.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7114751/v1/aff8fe1688a99f30fa7f1225.xlsx"},{"id":88945256,"identity":"935ebd04-acb1-4ba3-bb73-efdf9fbe3587","added_by":"auto","created_at":"2025-08-13 04:50:34","extension":"xlsx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":83207,"visible":true,"origin":"","legend":"","description":"","filename":"S3MotifenrichementatallHPIs.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7114751/v1/584dad26a0b18492dcfd2d8c.xlsx"},{"id":88946041,"identity":"ab8dda60-82d8-4580-8683-fd78610fd889","added_by":"auto","created_at":"2025-08-13 05:06:34","extension":"xlsx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":832086,"visible":true,"origin":"","legend":"","description":"","filename":"S4WGCNAclusters.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7114751/v1/0ff48ee125a15af6fc011f3f.xlsx"},{"id":88945249,"identity":"9f3537df-c9c1-4c24-bfe3-e8e81cfee2c3","added_by":"auto","created_at":"2025-08-13 04:50:34","extension":"xlsx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":53137,"visible":true,"origin":"","legend":"","description":"","filename":"S5HighlycorrelatedclustersWGCNAandspecificDE.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7114751/v1/be8ba1b9e35690b4eb711efe.xlsx"}],"financialInterests":"","formattedTitle":"Wheat defenses display both sustained and time-of-day-specific responses to Rhopalosiphum padi (bird cherry-oat aphid) infestation over a 24-hour period","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-molecular-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plan","sideBox":"Learn more about [Plant Molecular Biology](https://www.springer.com/journal/11103)","snPcode":"11103","submissionUrl":"https://submission.nature.com/new-submission/11103/3","title":"Plant Molecular Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7114751/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7114751/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Wheat is constantly challenged by destructive pests, such as herbivores, even as it adapts to shifting environmental factors. While plant responses to herbivore infestation (hours post-infestation, hpi) are studied, how these responses are modulated by daily light-dark cycles remains poorly understood. Here, we investigate the effects of Rhopalosiphum padi aphid infestation on Svevo wheat using an RNA-seq approach, analyzing responses at multiple time points over a 24-hour infestation period. Both persistently regulated and hpi-specific plant responses were identified. Gene Ontology (GO) terms related to terpene synthesis and oxidoreductase were consistently enriched throughout the infestation period, whereas ethylene and jasmonic acid synthesis were enriched at specific hpi. Notably, daytime infestation specifically enriched GO terms for protein phosphorylation and motifs of WRKY transcription factors, indicating a crucial role for the circadian clock in shaping these defense responses. Additionally, highly correlated clustered genes revealed unique pathways, including those involved in terpene synthesis and amino acid catabolic processes. Understanding these initial single-day responses to aphid infestation may provide valuable insights into the complex plant-aphid interaction and contribute to the development of targeted defensive strategies.","manuscriptTitle":"Wheat defenses display both sustained and time-of-day-specific responses to Rhopalosiphum padi (bird cherry-oat aphid) infestation over a 24-hour period","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-13 04:50:29","doi":"10.21203/rs.3.rs-7114751/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-09-03T08:47:01+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-07-31T11:52:17+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-19T07:18:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant Molecular Biology","date":"2025-07-14T15:06:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-14T07:03:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Molecular Biology","date":"2025-07-13T13:30:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-molecular-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plan","sideBox":"Learn more about [Plant Molecular Biology](https://www.springer.com/journal/11103)","snPcode":"11103","submissionUrl":"https://submission.nature.com/new-submission/11103/3","title":"Plant Molecular Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"439e65e1-12f8-4588-abd2-49d1b3d06d49","owner":[],"postedDate":"August 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2025-09-03T12:48:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-13 04:50:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7114751","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7114751","identity":"rs-7114751","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}