Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Plant Biology
Non Technical Summary
Grafting, the technique of joining two plant parts together, is an ancient technique that is used to propagate plants. Many industries rely on grafting to successfully grow economically significant species, such as apples, where desirable varieties are grafted onto hardy rootstocks. The ability of two different varieties or species to graft is called graft compatibility. While the process of grafting has existed for thousands of years, the mechanisms that underlie graft compatibility remain poorly understood. Due to this, graft compatibility is determined through trial and error. Trial-combinations are not always easily diagnosed as incompatible. A type of graft incompatibility, called delayed incompatibility, occurs when grafts appear to be successful only to fail months or years later. Because woody crops are slow-growing and challenging to use in research, little is known about the molecular basis that leads to graft incompatibility. This project utilizes a non-tree model system: tomato and eggplant grafted as compatible partners and tomato and pepper grafted as incompatible partners. All of these plants are fast growers and easily studied in a research environment.Previous work has shown that compatible grafts require a complex network of molecular and hormonal signaling. This project will investigate the role that these signals play in grafting. We will do this by analyzing known mobile signals and signal transport in both compatible and incompatible grafted plants. We will also assess if necrosis (tissue death) in the graft junction correlates with blocked intercellular transport. Through this project, we aim to advance the understanding of graft biology. A better understanding of graft compatibility will benefit breeders and growers who are negatively affected by the loss of crops during graft incompatibility.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
Many tree and vegetable growers rely on grafting to continually propagate essential varieties. While the process of grafting has existed for thousands of years, the mechanisms that underlie graft compatibility remain poorly understood. Due to this, graft compatibility is often tested through a process of trial and error. Trial-combinations are not always easily diagnosed as incompatible. A type of graft incompatibility, called delayed incompatibility, occurs when grafts appear to be successful only to fail months or years later due to failed vascular connections.This project aims to elucidate the mechanisms that determine compatibility, by utilizing the herbaceous model system:Solanum lycopersicum(tomato),Solanum melongena(eggplant), andCapsicum annum(pepper). The broad goal of this project is to investigate the role of intercellular communication in grafted herbaceous plants.The first objective of this project will focus on the timing and importance of symplastic transport at the graft junction by investigating the formation of plasmodesmata.The second objective of this project will determine if mobile signals are transported across the graft junction. This, in combination with our findings regarding plasmodesmata, will provide insight into molecular trafficking across the graft junction.The third objective of this project will investigate how necrosis impacts the intercellular signaling within the graft interface, especially within incompatible grafted plants.These experiments align with the Plant Health and Production and Plant Products program area priority.
Project Methods
Aim 1: Is plasmodesmata formation at the graft junction interface altered in incompatible grafts?Generation of transgenic tomato, eggplant, and pepper with plasmodesmata (PD) fluorescent markers:PDLP5 is a protein that associates with plasmodesmata. Two constructs will be generated for this experiment: pPDLP5::PDLP5-GFP and pPDLP5::PDLP5- RFP. Each of these will be transformed individually into tomato, eggplant, and pepper. Tomato and pepper are known, incompatible graft partners. Tomato and eggplant are known, compatible graft partners. 5-10 lines for each transformation will be grown to the T2 generation for grafting experiments.Grafting as a tool to track the formation of PD at the graft junction:The graft combinations below will be performed: tomato:tomato, pepper:pepper, eggplant:eggplant, tomato:pepper, pepper:tomato, tomato:eggplant, eggplant:tomato. Red and green puncta will indicate half-PD formation, whereas yellow puncta will form when PD-associated proteins colocalize, meaning that whole PD has developed across the junction. No puncta will show a lack of PD at the graft interface. To determine that the marker line has not affected PD formation or the ability of the crops to graft, all transgenic crops will be self-grafted. To quantify the baseline number of PD formed along the graft junction in self-grafted plants, scions with PDLP5-GPF will be grafted to the same genotype expressing PDLP5-RFP. To quantify the formation of interspecies PD along the graft junction in compatible heterografts, tomato expressing PDLP5-GPF will be grafted to eggplant expressing PDLP5-RFP. To quantify the number of interspecies PD formed in incompatible grafts, tomato expressing PDLP5-GPF will be grafted to pepper expressing PDLP5-RFP.Image analysis of graft junctions:Graft junctions will be cut out, 1 cm on either side of the interface daily, for eight days following grafting, and cleared in ClearSee. Samples will be imaged daily using a Zeiss u880 confocal microscope.Image analysis of PD formation: All images will focus on the same cell types and use the same laser settings. Colocalization and imaging analysis will be performed with consistent parameters on ImageJ.Attendance at a scientific conference:This research will be presented at a national or international scientific conference.Aim 2: Are mobile signals required for successful grafts?Generation of transgenic tomato, pepper, and eggplant with markers for mobile signals:Known mobile signals, CLE42 and PEAR1, are present in vascular tissue. Multi-gene constructs will be built using the MoClo system. The constructs will be transformed and grown to the T2 generation in tomato, eggplant, and pepper.Grafting as a tool to track the transcription and movement of mobile signals CLE42 and PEAR1:The graft combinations below will be performed: tomato:tomato, pepper:pepper, eggplant:eggplant, tomato:pepper, pepper:tomato, tomato:eggplant, eggplant:tomato. To determine that the marker line has not affected the ability of the crops to graft and to quantify the baseline levels of gene expression, all transgenic plants will be self-grafted. To determine protein mobility, each transgenic species will be reciprocally grafted with its respective wild type species. To investigate the effect of hetero-grafting, each transgenic line will be reciprocally grafted with each wild type species. One fluorescent reporter will mark the location of expression. The contrasting fluorophore will mark the mobility of the protein. Both of these signals are already known to be mobile and act non-cell autonomously. Thus, we expect to see mobile protein outside of the region of transcription within the parent tissue. The presence of the mobile signal in the wild type plant will show that mobile signals are capable of crossing the graft junction into the grafted partner.Image analysis of graft junction:Graft junctions will be cut out, 1 cm on either side of the interface daily, for eight days following grafting, and cleared in ClearSee. Samples will be imaged daily using a Zeiss u880 confocal microscope.Image analysis of mobile signal transcription and mobility formation:All images will focus on the same region of the stem and use the same laser settings. Colocalization and imaging analysis will be performed with consistent parameters on ImageJ.Attendance at a scientific conference:This research will be presented at a national or international scientific conference.Generation of publication:Aim 1 and aim 2 will be combined to produce a publication describing the role of intercellular signaling on grafting. This effort will advance knowledge for scientists and breeders.Aim 3: Is necrosis a barrier for communication in grafted plants?Grafting incompatible and compatible plants:The following graft combination will be performed with wild type plants. tomato:tomato, pepper:pepper, eggplant:eggplant, tomato:pepper, pepper:tomato, tomato:eggplant, eggplant:tomato.Tissue printing used to trace reactive oxygen species:Cell death is a common symptom of graft incompatibility. Destruction of tissue leads to reactive oxygen species (ROS). ROS are often quantified using DAB stain, which reacts with ROS and forms a brown precipitate. Because DAB stain is unable to penetrate the stem and cutting the tissue would induce a strong peroxidase response, traditional DAB staining cannot be used. To assay the accumulation of ROS in the stem over time, grafted plants will be harvested at seven, fourteen, and twenty-eight days after grafting. Grafted stems will be cut longitudinally and immediately pressed onto DAB soaked nitrocellulose membranes. Ungrafted stems will be cut and pressed as negative controls, and 20 mM of H2O2 will be added to the DAB soaked nitrocellulose membrane as a positive control. Prints will be developed for two minutes and imaged on a flatbed scanner.Image analysis of DAB signal:Images will be quantitatively analyzed in ImageJ to test for statistical differences in the amount of oxidized DAB on each print between compatible and incompatible graft combinations relative to the un-grafted controls.Attendance at a scientific conference:This research will be presented at a national or international scientific conference.Generation of publication:Aim 3 results will be used to produce a publication regarding the effect of grafting on reactive oxygen species. This effort will improve knowledge on graft incompatibility, and hopefully, begin to build a protocol to test for early graft incompatibility.Key milestones:Successful transient fluorescent PD marker linesSuccessful transient mobile signal marker linesGeneration of fluorescent PD marker lines in tomato, pepper, and eggplantGeneration of mobile signal marker lines in tomato, pepper, and eggplantDAB staining completedDAB staining analyzedBegin ROS publicationGraft fluorescent PD lineGraft mobile signal marker linesCollect all graft junctions and imageAnalyze imagesBegin mobiles signal publication