Performing Department
PLANT SOIL MICROBIAL
Non Technical Summary
Destructive vineyard pathogens wreak havoc on wine, juice, and table grape industries world-wide as the pathogens and the cultivated grape host (V. vinifera, V. labrusca, and hybrids) continue in an evolutionary-arms race. Researchers across the globe have investigated the critical pathogens in their agricultural regions to best protect this specialty crop but are challenged with changes in fungicide resistance, improved pathogen virulence, and speciation. This proposal focuses on better understanding the dynamics of the Plasmopara viticola (grapevine downy mildew) cryptic species complex to aid and improvedowny mildew management.The goals of this study are to identify genetic and environmental differences in the three economically relevant cryptic species to set the foundation for management research involving these cryptic species.The two research objectives of this NIFA Predoctoral research proposal are to 1) determine differences in gene expression among P. viticola cryptic species by performing an infection time-course comparison using RNAseq analysis and 2) compare the effector repertoire of P. viticola clades vinifera and riparia to clade aestivalis to increase understanding of host-pathogen interactions and how these interactions may play a role in informing disease management among the recently identified cryptic species. Objective 1 aims to find the differences between the cryptic species while Objective 2 aims to compare to discuss how these differences impact pathogen infection and success.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The major goals of this project include two (2) research objectives and three (3) training/career goals to improve genetic and infectivity knowledge involving the recently identified cryptic species of the grapevine pathogenPlasmopara viticola as well as refine the scientific, interpersonal and communication, and written skills of the Project Director. The project targets theAFRI Farm Bill Program Priority Area of "Plant Health and Production and Plant Products".Further characterization of the grapevine downy mildew crypticspecies (cl. riparia, aestivalis, and vinifera)will provide insight into infection and how it can be mitigated. Identification of effectors secreted from these organisms opensthe door to future analysis for use in breeding of resistance genes within commercial grape species that can reduce levels of infection in wine, juice, table, and raisin grapes worldwide--thus improving crop production.Research ObjectivesObjective 1:Perform an infection time-course comparison between P. viticola cryptic species using RNAseq analysis to determine differences in gene expression.Objective 2:Compare the effector repertoire of P. viticola clades vinifera and riparia to clade aestivalisTraining/Career goalsT&C Goal 1:Further develop molecular biology and computational skills.T&C Goal 2:Develop as a scientific leader and mentor.T&C Goal 3:Build upon collaborative efforts and science communication.
Project Methods
Efforts:To understand the interactions between P. viticola and the recently identified commercially relevant cryptic species (cl. aestivalis, riparia, and vinifera), I will perform RNAseq during pathogen challenge to explore 1) gene expression differences between cryptic species and infection stage and 2) effector presence and expression levels between the P. viticola crypticObj. 1: Perform an infection time-course comparison between P. viticola cryptic species using RNAseq analysis to determine differences in gene expression: I hypothesize that there will be gene expression differences between the three cryptic species evaluated (cl. aestivalis, riparia, and vinifera) across the various infection stages on the same host species. Plants of Vitis interspecific hybrid cv. Vignoles will be grown using hydroponics within a growth chamber (protocol actively used and managed in the Miles laboratory) to generate fully expanded leaves to be used for the infection time course experiment. Prior to the experiment, live cultures of the obligate pathogens will be maintained. Three isolates, one per cryptic species (cls. riparia, aestivalis, and vinifera) will be used for the time-course comparison. One leaf per time point will be inoculated using 20 uL drops of inoculum concentrated to1x104sporangia/mL. Five time points will be performed: 0-, 24-, 48-, 72-, and 144-hours post-inoculation (hpi). These time points represent different stages of pathogen challenge and infection: prior to infection, early infection (host penetration and colonization), and late infection (and sporulation). Three biological replicates will be performed per cryptic species. Leaf punches will be taken at the appropriate time point, flash-frozen using liquid nitrogen, and stored in a -80°C freezer until RNA extraction. The RNAseq library will be prepared in collaboration with the MSU Genomics Core and sequenced using the NovaSeq shortread sequencing platform at MSU's RTSF Genomics Core. The resulting sequencing reads will be processed using MSU's High Performance Computing Center (HPCC) and aligned to the reference genome (cl. aestivalis)17. Then data will be analyzed using R statistical software packages (e.g., edgeR, for differential analysis) to calculate relative gene expression and identify differentially expressed genes. Expected outcomes: The data generated from this experiment will be used to identify differences in gene expression based on cryptic species tested, classes of gene expression (analysis using GO (gene ontology) terms) by cryptic species, and grouping of gene expression by infection stage.Obj. 2: Compare the effector repertoire of P. viticola clades vinifera and riparia to clade aestivalis: I hypothesize that the effector expression profile during infection will differ among cryptic species; isolates of the less aggressive cl. riparia (10) will have fewer effectors as compared to cls. aestivalis and vinifera. To identify the effectoromes of P. viticola cls. riparia, aestivalis, and vinifera, samples will be taken at selected time points post-inoculation and RNAseq will be performed. This objective will utilize the dataset generated in Obj. 1, with the data processing and analyses performed separately. Three of the five time points will be analyzed when making comparisons between the three cryptic species effectoromes (24-, 48-, and 72-hpi). Samples will be collected, and RNAseq data will be generated and processed as described in Obj. 1. The predicted proteins of P. viticola cl. aestivaliswill be used to generate a comparison against the three tested cryptic species. While it is assumed that the previously annotated genome using P. viticola cl. aestivalis will be representative of the cl. aestivalis isolated used in thisstudy, they will be still be compared against one another. Both apoplastic and cytoplasmic effectors will be targeted in this analysis. To identify these effectors, the novel pipeline EffectorO18 will be used. The EffectorO pipeline utilizes machine learning to identify and predict effectors found specifically in oomycetes. This pipeline will identify previously known effectors as well as potentially identifying novel effectors used to evade host immunity--a critical aspect considering this proposal's study of cryptic species. Expected outcomes: The effectorome of each cryptic species (cl. aestivalis, vinifera, and riparia) when infecting the same host (Vitis interspecific hybrid cv. Vignoles) will be characterized from this experiment. A description of effectors expressed at early-, mid-, and late- infection will be identified and expression levels compared across time points. It is hypothesized that there will be similarities, or core effectors, found across the respective infection time points. The data and conclusions generated from Obj. 2 will provide the framework for further study into infection mechanisms used by each cryptic species of P. viticola throughout infection. These effectors can be functionally characterized to identify R-genes within grape hosts to be utilized in breeding pipelines.Evaluations:The progress made regarding the proposed research and training objectives will be evaluated weekly with my primary mentor; bi-monthly meetings with my collaborating mentor; and reports to my advisory group via bi-annual meetings. During this proposed timeline, research deliverables to disseminate completed parts of the proposed project include: a research presentation at a minimum of one conference, an anticipated minimum of one first author publication in a peer reviewed plant pathology journal, and a research presentation at one MSU departmental seminar. In addition, career development deliverables include participation in workshops offered at APS Plant Health 2025 and the Grapevine Downy and Powdery Mildew (GDPM) Conference 2025, mentorship of an undergraduate researcher to develop professional and laboratory skills, and increased collaboration relationships and skills from travel to the Collaborating Mentor's lab. Annual evaluations from my primary mentor as well as my dissertation committee will be used to assess my progress on the proposed project and professional development as pertains to my three T&C goals.