Genomic Approaches to Unravel Virulence and Resistance Determinants of Vector-transmitted Viruses in Tomato

PI: S.P. Dinesh-Kumar（加州大学戴维斯分校）CoPIs: Robert Gilbertson， Diane Ullman和Ilias Tagkopoulos（加州大学戴维斯分校）西红柿是世界上第二大蔬菜作物。美国和全世界的番茄生产受到两种破坏性昆虫传播病毒的威胁——番茄斑点枯萎病毒（TSWV）和番茄黄卷叶病毒（TYLCV）。这些病毒的管理是困难的，往往严重依赖于使用杀虫剂来控制病媒。杀虫控制往往是无效的，对人类健康和环境有负面影响，并可能导致选择抗杀虫剂形式的病媒。在番茄中，Ty1基因座和Sw5基因座分别是TYLCV和TSWV的主要遗传抗性来源，两者都已渗入到商品番茄品种中。然而，对于Ty1-和sw5介导的耐药机制知之甚少。该项目将为植物对昆虫传播病毒感染的抗性和易感性的本质及其相互作用机制提供新的见解。这些研究将为在感染和/或宿主防御过程中，RNA （TSWV）和DNA （TYLCV）病毒是否靶向相似或不同的途径提供见解。然后，该项目将利用这些研究的结果来优化和验证一种名为mirna诱导基因沉默（MIGS）的新方法，该方法可用于评估番茄的基因功能。如果成功，该项目的结果可能会导致针对这些病毒的新一代抗病毒策略。该项目还将每年为萨克拉门托城市学院的本科生和戴维斯高中的高中生提供为期10周的培训项目。野生番茄亲缘基因Sw5和Ty1基因座向栽培番茄的遗传抗性分别提供了对TSWV和TYLCV的最佳保护来源。本项目的研究人员假设Sw5-和ty1介导的TSWV和TYLCV的识别触发了涉及常见和特异性免疫信号网络的早期和可识别的转录变化。此外，这些病毒在易感番茄基因型上的感染诱导转录变化，从而促进病毒的毒力。为了深入了解转录变化，研究人员将使用RNAseq方法鉴定番茄TSWV和TYLCV抗性和易感反应期间差异表达的转录物。计算方法将用于分析RNASeq数据，以确定在TSWV和TYLCV抗性和易感相互作用中差异调节的关键共同和特异性基因。该项目将通过针对已知候选基因，优化和验证转基因番茄基因功能研究的MIGS方法。优化后的MIGS方法将用于评价选定的差异表达基因在番茄TYLCV和TSWV抗性和敏感互作中的功能。该项目的结果可能会为设计新一代控制TYLCV和TSWV以及其他重要植物病原体的策略提供见解。所有数据和生物资源将向公众开放。所有RNAseq数据都将通过GEO存储和获取。所有MIGS载体和候选目标基因构建可根据要求通过Addgene （https://www.addgene.org/）和ABRC （http://www.biosci.ohio-state.edu/~plantbio/Facilities/abrc/abrchome.htm）获得。来自转基因番茄系的种子将通过加州大学戴维斯分校的番茄遗传资源中心（http://tgrc.ucdavis.edu/）获得。

PI: S.P. Dinesh-Kumar (University of California-Davis)CoPIs: Robert Gilbertson, Diane Ullman, and Ilias Tagkopoulos (University of California-Davis)Tomato is the second most important vegetable crop in the world. Tomato production in the US and worldwide is threatened by two devastating insect-transmitted viruses - tomato spotted wilt virus (TSWV) and tomato yellow leaf curl virus (TYLCV). Management of these viruses is difficult and often relies heavily on the use of insecticides to control the vectors. Insecticidal control is often ineffective, has negative human health and environmental effects, and can lead to selection of insecticide-resistant forms of the vectors. In tomato, the Ty1 locus for TYLCV and the Sw5 locus for TSWV are major genetic resistance sources, and both have been introgressed into commercial tomato varieties. However, very little is known about the mechanisms involved in Ty1- and Sw5-mediated resistance. This project will provide new insights into the nature of and interplay between mechanisms of plant resistance and susceptibility to infection by insect-transmitted viruses. These studies will provide insights into whether similar or different pathways are targeted by RNA (TSWV) versus DNA (TYLCV) viruses during infection and/or host defense. The project will then use the results from these studies to optimize and validate a new approach called miRNA-induced gene silencing (MIGS) that can be used to assess gene function in tomato. If successful, the outcomes of this project will likely lead to a new generation of antiviral strategies against these viruses. The project will also provide a 10-week training program every year for undergraduate students from Sacramento City College and high school students from Davis Senior High School.The genetic resistance mediated by Sw5 and Ty1 loci introgressed from wild tomato relatives into cultivated tomato provides the best source of protection against TSWV and TYLCV, respectively. The investigators of this project hypothesize that Sw5- and Ty1-mediated recognition of TSWV and TYLCV trigger early and discernable transcriptional changes that involve common and specific immune signaling networks. In addition, infection of these viruses on susceptible tomato genotypes induces transcriptional changes that promote virus virulence. To gain insights into the transcriptional changes, the investigators will use RNAseq approach to identify differentially expressed transcripts during TSWV and TYLCV resistance and susceptible responses in tomato. Computational methods will be used to analyze RNASeq data to identify key common and specific genes that are differentially regulated during TSWV and TYLCV resistance and susceptible interaction. The project will optimize and validate the MIGS approach for gene function studies in transgenic tomato by targeting known candidate genes. The optimized MIGS approach will be used to evaluate function of selected differentially expressed genes during TYLCV and TSWV resistance and susceptible interactions in tomato. Results from the project will likely provide insights into designing new generation of strategies to control not only TYLCV and TSWV but also other important plant pathogens. All data and biological resources will be accessible to the public. All RNAseq data will be deposited and available through GEO. All MIGS vector and candidate target gene constructs will be available upon request and through Addgene (https://www.addgene.org/) and ABRC (http://www.biosci.ohio-state.edu/~plantbio/Facilities/abrc/abrchome.htm). Seeds from transgenic tomato lines will be available through the Tomato Genetics Resource Center at UC Davis (http://tgrc.ucdavis.edu/).