Recipient Organization
UNIVERSITY OF NEVADA
(N/A)
RENO,NV 89557
Performing Department
(N/A)
Non Technical Summary
Potato (Solanum tuberosum) is the third most-consumed food crop in the world grown in roughly 130 countries, 95 of which are developing countries. Potato is considered a food security crop for developing nations, in which potato production has ramped up substantially over the past 30 years. The US remains the fourth largest producer of potatoes in the world. Potato is a top five commodity crop in the PDs' home state of Nevada. Potato has an annual value of $4.2 billion in the USA. Up to 33% of the US potato tuber crop is lost due to postharvest issues like wounding, bruising, and pathogen attack. These losses can reach as high as 50% in developing nations due to factors such as lack of proper storage conditions. The economic importance of potato extends beyond the value of the harvested crop. The global potato processing market is currently valued at $27.4 billion and is expected to grow to $35.4 billion by 2026.It is well known that wound healing and deposition of the corky material suberin in wound sites is of critical importance to the the post-harvest storage life of potatoes. Wound suberin deposition capacity and storage life are positively correlated. That is to say, potato cultivars with better wound suberin deposition capacity tend to have longer storage life. Efforts to improve wound healing capacity of the USA's number one vegetable crop must be informed by understanding the fundamental processes that goven wound healing. This can be accomplished by understanding the genes and signaling processes that govern wound suberin deposition. Thisresearch project aims to do exactly that, to provide an in-depth understanding of the genetic and signaling factors that regulate wound healing in a crop species important fornational and global food security, potato.To accomplish this, cutting-edge genomics and genome editing techniques, such as CRISPR and high-throughput DNA/RNA sequencingwill be employed. Through this work, potato germplasm with altered wound suberin deposition capacity will be generated. The germplasm and information generated through this research will provide a valuable resource for plant breeding efforts aimed at reducing post-harvest potato tuber lossesthat will translatable to other crops that undergo long-term storage (e.g. sweet potato, rutabaga, carrot, etc.).
Animal Health Component
(N/A)
Research Effort Categories
Basic
95%
Applied
(N/A)
Developmental
5%
Goals / Objectives
Wound suberization is critical for preventing major post-harvest losses experienced by the potato industry. In spite of wound suberin'simportance for the potato industry, the molecular signaling events that regulate wound suberin deposition remain unclear. This hold particularly true for transcriptional regulation by transcription factors. Similarly, it is known that suberin is important for defense against devastating post-harvest pathogens such as soft rots yet a specific role for defense-related signaling in modulating suberin accumulation remains essentially uknown.The overarching goal of the project is to provide a better understanding of the transcriptional regulation ofwound suberizationprocesses by transcription factors and pathogen defense-related signaling.Our research will be accomplished through the following Specific Aims:1. Identification and characterization of transcription factors that regulate wound suberin biosynthesis.This will be accomplished through characterization of CRISPR-Cas9 gene-edited potato lines already established by the PDs.2. Determine the role of defense-related signaling in regulating wound suberin biosynthesis.This will be accomplished through studying jasmonic acid-related signaling and soft rot associated defense responses.
Project Methods
Various wet lab and dry lab methods will be employed to achieve our specific aims.Aim1. Identify transcription factors that regulate potato wound suberin biosynthesis and the transcriptional networks to which they pertain.Gene-edited lines targeting specific transcription factorshave been or will be generated through Agrobacterium-mediated transformation of potato using constructs containing multiple guide RNAs (gRNAs) to affect large deletions (~1 Kb) in transcription factors of interest. Suberin-targeted metabolomics will be used to assess the wound healingcapacity of gene-edited lines using using GC-MS or other chromatographic/spectroscopic methods. RNA-seq will be employed to determine perturbations in the transcriptomes of select gene-edited lines. Selection of gene-edited events for each transcription factor will be guided by the aforementioned targeted-metabolomics experiments. DNA Affinity Purification with high throughput sequencing (DAP-seq) will be used to asses the gene targets of each transcription factor under investigation.Aim 2.Determine the role of defense-related signaling in regulating wound suberin biosynthesis. The role of jasmonates in regulating tuber wound suberization will be assessed by pharmacological approaches employing treatment of wounded tubers with methyl Jasmonate (MeJA) or jasmonate-synthesis inhibitor Sodium diethyldithiocarbamate (DIECA). GC-MS will be used to assess suberin deposition in tubers subjected to these treatments. RT-qPCR will be used to measure the expression of suberin-related genes and JA response marker genes. Potato lines defective in the expression of JA signaling genes (e.g. MYC2) will be developedCRISPR-Cas9 gene editing using Agrobacterium-mediated transformation to deliver CAS9 and gRNA-containing T-DNA casettes. These lines wil be subjected to wound healing treatments and assayed for wound suberin deposition using GC-MS and related gene expression using RT-qPCR. Wounded tubers treated with Pectobacterium atrosepticum will be assessed for suberin deposition and related gene expressing usingGC-MS and RT-qPCR, respectively.EffortsEfforts to deliver the knowledge generated from our research will include:Laboratory instruction of graduate students, postdocs, and undergraduate studentsWorkshops for local high school students (FFA) and educators (ag teachers), pandemic permittingPresentation of research at scientific conferences and symposiaPeer-reviewed publicationsPresentation to local communities through venues such as the Nevada Farms ConferenceEvaluationWe will evaluate the success of our project as follows:Number of peer-reviewed publicationsNumber of general public members that were interacted with under this project including high school and undergraduate students trained, audience attendance at presentations of this research in non-scientific forums, and number of K-12 level educators interacted with through outreach effortsNumber of scientists to which the research has been presented to at local, national, and international venues