Progress 06/15/20 to 06/14/23
Outputs
Target Audience:The target audience for this research project included the members of the Agricultural College at Colorado State University, the greater scientific community of plant pathologists, and all members of the agricultural industry including farmers and disease management companies. Part of my PhD Dissertation contained results from this project and thus was described to members of Colorado State University and a small group of the public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? During this project, I was able to translate my technical skillset from experiments conducted in the plant model species, Arabidopsis thaliana, to theagriculturally relevant species canola. After graduation, I aimed to obtain a job in the agricultural industry sector therefore developing this skillset was vital to making me a competitive candidate. In Arabidopsis, most genetic tools are easily accessible. For instance, you are able to order transgenic seeds in any gene of interest and a large number of existing protocols for many different laboratory techniques. Working in B. napus was very humbling to me because there were very few tools or protocols developed so Ihad to retrain my thought processes on how to answer experimental questions and troubleshoot through seemingly simple, but difficult protocols. I truly believe that without this grant, I would have never gained these experiences and it has truly put me in a better position to obtain a job outside of academia. During this project,I attended two virtual conferences hosted by the American Phytopathological Society (APS) and the American Society of Plant Biologists (ASPB). Even though I attended both of these conferences virtually due to COVID-19 I was able to attend seminars that increased my knowledge of current research within plant pathology and within the greater plan sciences community. Addressed in past Yearly Reports, my original project proposal has collaborators at Cargill where I had planned on working with them on other Brassica cultivars and different pathogens having an impact on Cargill's farming consumers. However, due to COVID-19 and sustained limited accessibility to their facility, I was unable to complete the proposed experiments. However, I was able to have multiple conversations where we discussed experimental design, greenhouse facilities, and equipment available. I found these conversations very informative on how research is conducted outside of academia, especially in terms of how information is shared between industry and academia. Although I was unable to physically do the work proposed for this collaboration, these conversations prepared me for future conversations with other agricultural industries in terms of how experiments are more team-focused than individual contributors and cautiousness about whom information is shared.? How have the results been disseminated to communities of interest? ?During the project term, I was able to share both the preliminary and final results of this project with other members of my graduate program and my department. Further, during my PhD Dissertation public seminar, I presented the final results of this project to members of Colorado State University within and outside of plant pathology as well as other members outside of science and research in general. I did publish a review in Frontiers of Plant Science titled "Cytokinin regulation of source-sink relationships in plant growth and plant-pathogen interactions." This review covers the role of CK in the synthesis and movement of carbohydrates andamino acids in plants. It also discusses how this role CK has on altering the movement of photoassimilates related to plant immunity in different pathosystems. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This research project has accomplished the characterization of Cytokinin-Induced Priming(CIP) against the hemibiotrophic bacterial pathogen, Pseudomonas syringae pv. maculicola (Psm) by discovering the most effective concentration in which cytokinin (CK) should be applied to decrease bacterial growth, the timeframe in which CK reduces susceptibility, and the growth effects due to CK treatments. The application of CK concentrations from 1nM to 100mM showed as the concentration of CK increased, susceptibility against Psm decreased. However, when CK was applied at a concentration greater than 100mM, visible areas of cell death were seen prior to the pathogen infiltration. Thisindicatesthat the millimolar range of CK induces spontaneous cell death which would not be a favorable strategy to agricultural applications. Therefore, I determined 100uM CK application, resulting in decreased susceptibility without spontaneous cell death was the most beneficial concentration in which CK should be applied. Following one application of 100uM CK, B. napus plants showed a decrease in bacterial growth when treated plants were challenged with Psm 48 to 72 hours after the hormone treatment. Prior to 48 hours and after 72 hours, the CK application increased susceptibility. For any new proposed disease management strategy to be adopted by the agricultural community, the growth effects of CIP needed to be assessed. Following one application of CK, no growth effects were seen in treated B. napus plants. Because I determined that CIP was effective at reducing susceptibility 72 hours after application, I wanted to determine if there were growth effects due to maintaining plants in a CK-primed state constantly by treating plants with CK every 3 days. After 8 weeks of growth, CK-treated plants did show differential growth effects as compared to unprimed plants. CK-treated plants had increased shoot weight, petiole length, and shoot height with decreased root weight and few lateral roots. These results show a negative impact on growth when B. napus plants are in a sustained primed state, therefore, more optimization is needed tobalance growth with increasing plant defense in a CK-primed plant. Unfortunately, I was awarded this grant prior to the COVID-19 pandemic which slowed or halted all in-person research at Colorado State University for months preventing me from reaching the long-term goals of this research project including creating trangenic B. napus lines that would allow me to assess the necessity and sufficiency of endogenous CK signaling during CIP due to the introduction of genes that would allow for the induction of CK biosynthesis or degradation. Modifying the second objective, I used a chemical inhibitor of endogenous CK signaling, PI-55, to prevent CK signing after CK application during CIP. My results showed that PI-55 treated plants have similar susceptibility to Psm as untreated plants following CK-priming, suggesting the chemical inhibitor was not effective at preventing endogenous CK signaling. To assess this hypothesis, I observed the expression of CK signaling genes in plants treated with PI-55 before treating plants with CK. Unfortunately, the expression of CK signaling genes did not show differential expression in plants pretreated withPI-55 or its control treatment. Therefore, further exploration of the necessity of endogenous CK signaling was not possible because more optimization of PI-55 is needed. For the third objective aimed at uncovering the molecular mechanism behind CIP a transcriptome analysis was proposed to understand the changes in gene expression due to the CK treatment after the application and the following pathogen challenge. I was unable tocomplete these experiments in canola, however, I did complete a similar transcriptomic experiment in Arabidopsis thalianaagainst Pseudomonas syringae pv. tomato. I uncovered that 48 hours after CK treatment, there are no differentially expressed genes due to the CK application. However, following the pathogen challenge, defense genes were up-regulated and associated with systemic defense responses. Although during this research project, I was unable to assess transcriptome changes in B. napus,I wouldexpect the results seen in Arabidopsis thaliana to be similar due to both being in the Brassicaceae family and the similar responses I have seen during the characterization of CIP in both species. The results of this study further the understanding of how the application of cytokinin increases plant immunity. Moreover, these results providemore foundational knowledge for future areas of themanipulation of the plant immune system to decrease susceptibility to pathogens. The results of this study demonstrate that CK can be used to increase plant immunity. Still, more research needs to be done to optimize this processif cytokinin is used as a new agrochemical in order to balance the increased immunity at a detriment to plant growth.
Publications
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Progress 06/15/21 to 06/14/22
Outputs
Target Audience:For this research project, the target audience is the scientific community of plant pathologists and the greater agricultural community. The knowledge gained within this reporting period furthered the understanding of priming plant defenses using the plant hormone, cytokinin. More specifically this research project will allow other plant pathologists to understand how cytokinin application to Brassica napusincreases plant defense to Pseudomonas syringae pv. maculicolaand the associatedgrowth effects. Changes/Problems:The following are the approved modifications to the objectives granted by the No-Cost-Extension: "Due to research being halted and severely restricted during the Covid-19 pandemic, I am requesting that the scope of Objective 2 and Objective 3 of my NIFA Pre-Doctoral Fellowship (2019-07070) be modified. These requested modifications will change the methods within the project objectives with no changes being made to the goal of the objectives nor main project aim. Further, these requested modifications will maintain the NIFA program's objectives. Objective 2: Determine the necessity and sufficiency of endogenous CK (cytokinin) content on (Cytokinin-lnduced Priming) CIP in Brassica napus. In order to accomplish this objective, I was to create two B. napus transgenic lines that either increase or decrease endogenous CK through the introduction of genes that synthesize or degrade CK, respectively. Creating the transgenic lines proved to be technically difficult with the added complexity that access to my university's facilities was limited due to Covid-19. Moreover, creation of stable transgenic B. napus lines requires that seeds be stored in the cold for extended time periods that was not possible with the Covid-19-caused limitations. As stated in the project Objective 2, an alternative approach was proposed where a CK inhibitor (PI-55) would be employed if transgenic lines were unable to be created. This methodology has been deployed and is effective at halting CK signaling in B. napus. However, this alone is not enough to determine if endogenous CK content is sufficient to cause the reduction in susceptibility due to priming by CK. Therefore, I propose altering the methods in Objective 2 to include CK hormone quantification by mass spectrometry during CIP. CK levels wilt be analyzed from B. napus leaf tissue following a spray treatment with a control or CK solution as well as after infiltration with Pseudomonas syringae pv. maculicola (Psm) ES4326 in the pretreated plants. In order to determine necessity of endogenous CK levels during CIP, the CK inhibitor, PI-55, will also be incorporated into the experiment in order to deplete CK signaling during CIP. Proposed changes to Objective 2 will include the cost of the hormone quantification to be completed by my university's Bioanalysis and Omics facility which has estimated the cost at $4,100. Objective 3: Uncovering the molecular mechanism behind CIP in B. napus. The original methodology in Objective 3 was to determine if priming by CK caused alterations in chromatin accessibility through the use of Assay for Transposase Accessibility Chromatin followed by high-throughput sequencing (ATAC-Seq). However, our laboratory tried a similar experiment (using our laboratory funding) in Arabidopsis thaliana that proved to be ineffective. However, the transcriptome analysis proposed in Objective 3 will provide insights into the molecular mechanisms underlying CIP in B. napus ensuring the fulfillment of the original Objective 3 aim. The unused funds originally allocated to the ATAC-Seq experiment ($5,000) will be used to for both the hormone quantification experiment proposed in the modified Objective 2 and molecular biology supplies needed for the completion of the project as well as additional funding needed for publication costs incurred.?" What opportunities for training and professional development has the project provided?Although I was unable to work in-person, I was able to virtuallymeet with the collaborators at Cargill. During these meetings, I gained insight into the agricultural industry as compared to academia. Understanding the logistics of how the industry operates like documentation, non-disclosure agreements, and safety training was beneficial to me because I have the goal of obtaining a job outside of academia when I graduate. I was also able to learn abouttheir growth facilities which are tailored to Brassica species which allowed me to gain knowledge in how it's different than a general plant greenhouse. How have the results been disseminated to communities of interest?I presented the results of this study internally at Colorado State University to fellow graduate students within my Cell and Molecular Biology graduate program. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, I will begin the modified objectives approved by the No-Cost-Extension. These include a new objective 2 and 3. Objective 2 will still aim to determine the necessity andsufficiency of endogenous cytokinin during priming. To do this, I will use a chemical inhibitor of cytokinin-signal to halt endogenous signaling prior to the application of cytokinin and subsequent pathogen infiltration. I will also quantify endogenous cytokinin hormone levels by mass spectrometry during priming by cytokinin with and without the cytokinin signaling inhibitor. Objective 3 will aim to understand the effect that cytokinin-induced priming has on B. napustranscriptome during priming and pathogen challenge. This will allow me to understand similar types of genes that have modified expression levels due to the cytokinin treatment that will allow me to begin to understand the molecular mechanism behind priming.
Impacts
What was accomplished under these goals?
During this reporting period, I have characterized CIP in Brasssica napus.I determined that Brassica napus plants pretreated with 100uM cytokinin 48 hours before infiltration with Pseudomonas syringae pv. maculicolaresults in decreased bacterial quantities as compared to plants pretreated with a control solution. These results show that cytokinin can prime canola by reducing susceptibility to a bacterial pathogen. In order to determine how long the reduction in susceptibility toPsm following one application of cytokinin, plants were infiltrated 3 hours, 24 hours, 48 hours, 72 hours, and 1 week with Psm. My results show that cytokinin reduces susceptibility to Psm between 48 and 72 hours after applicationI also determined that following one cytokinin treatment, B. napus plants display reduced susceptibility to Psm. To understand the growth effects due to cytokinin treatment, one treatment with cytokinin results in no difference in overall growth as compared to control-treated plants. Other growth characteristics were measured like root and shoot fresh and dry weight, petiole length, shoot height, and root length. To understand the growth effects of maintaining a constant primedstate,plants were treated with cytokinin every 3 days. After 8 weeks, cytokinin-treated plants showed an increase in shoot weight with a decrease in root weight. Petiole length and shoot height were greater in cytokinin-treated plants as compared to control-treated plants. These results show that multiple cytokinin treatments have a negative effect on vegetative growth although more research will be needed to understand if there is an effect on reproductive parts of the plant. I had planned to work with collaborators at Cargill to understand if CIP could alter susceptibility against other plant pathogens in canola cultivars important to Cargill. However, Cargill still had their COVID-19 protocols in place that did not allow people outside of their company to work within their facility. Near the end of this reporting period, I requested and was granteda No-Cost-Extension where I changed the project objectives. These changes in objectives are described in other sections of this report.
Publications
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Progress 06/15/20 to 06/14/21
Outputs
Target Audience:The target audience for this research project include the scientific community of plant pathologists, my collaborators at Cargill, the agricultural community, and my undergraduate researcher. The knowledge produced from this project will further the understanding of priming defenses of plant immunity for plant pathologists. Efforts made through publications and presentations of the research at scientific conferences will provide me an avenue to reach this target audience outside of my university. Due to Cargill being a collaborator of this project, I will receive guidance on experimental design and aid on troubleshooting problems that may arise. Further, this collaboration will allow for discussion of the results and possible creation of an agrochemical that could be used in the agricultural industry. With the help of Cargill and Colorado State University, I plan to extend the results of my research to our local agricultural farmers. It is possible that my research will lead to a new method of crop protection by increasing plant immunity. Changes/Problems:With one year left in the project, experiments within objective two pertaining to the creation of CK transgenicB. napuslines will need to be modified. Instead of creating transgenic lines that alter endogenous CK levels, a CK inhibitor will be used to decrease CK signaling in order to determine the necessity of endogenous CK on CIP. This modification will provide sufficient data needed to complete objective two within the time allotted for this research project. What opportunities for training and professional development has the project provided?Within this reporting period, I was able to receive training and mentoring in next generation experiments from the sequencing company we used, my advisor, and other experts within the bioinformatics field. This training was invaluable for my future experiments inB. napusdue to an increase in my technical ability, learning how to conduct bioinformatic analysis, and how to properly interpret the results of a large data set. I also underwent professional development by attending two virtual conferences hosted by the American Phytopathological Society (APS) and the American Society of Plant Biologists (ASPB). Attending both conferences allowed me to attend seminars that increased my understanding of new research within the plant immunity field, learn new techniques that will be useful for this project, and network with other early career scientists and possible future employers. Moreover, as an active member within ASPB, I organized a workshop titled "Careers Beyond Academia," where I invited panelists with careers outside of academia to have small-group discussions with early career scientists. This opportunity allowed me to network with people from different career paths, broadening my scope of possible different opportunities for employment after graduation, as well as provided me with leadership training. How have the results been disseminated to communities of interest?The results of this project have not been directly disseminated to communities of interest through seminars or publications due to the objectives not being completed. However, working with my advisor, I have written and submitted a review toFrontiers in Plant Sciencetitled "Cytokinin regulation of source-sink relationships in plant growth and plant-pathogen interactions." This review covers what physiological impact CK has on the synthesis and movement of carbohydrates and amino acids within many plant species. The review also discusses this role of CK in the context of plant immunity. This manuscript is currently in the review process. What do you plan to do during the next reporting period to accomplish the goals?As stated previously, due to the COVID19 Pandemic, there were significant delays preventing the accomplishment of experiments needed for the completion of the project objectives. Within the next year, characterization of CIP inB. napuswill be completed by conducting experiments to finalize the time effectiveness of CIP, possible growth effects due to CK application, and the breadth of increased immunity to pathogens of different lifestyles due to CIP. Further, the experiments needed to determine the molecular mechanism of CIP inB. napuswill continue through RNA-Seq and ATAC-Seq experiments. With one year left in the project, experiments within objective two pertaining to the creation of CK transgenicB. napuslines will need to be modified. Instead of creating transgenic lines that alter endogenous CK levels, a CK inhibitor will be used to decrease CK signaling in order to determine the necessity of endogenous CK on CIP. This modification will provide sufficient data needed to complete objective two within the time allotted for this research project.
Impacts
What was accomplished under these goals?
Ihave begun to investigate the use of the plant-hormone, cytokinin, as an application to canola plants with the intent to increase the plant immune response to disease-causing bacteria and fungi. Over the past year, I have been characterizing the use of cytokinin application to plants in order to increase plant immunity. These data will provide information about the time after application to which the plant still has an increased immunity, the effects of plant growth due to the cytokinin application, and the resulting broad range of immunity the plant experiences. Further, I have also completed experiments that aim to understand how cytokinin increases plant immunity by obtaininggenome-wide gene expression that are observed after cytokinin application. Increased understanding of how the application of cytokinin increases plant immunity provides more opportunity for manipulation of the plant immune system. This knowledge allows for the creation of plants that are more robust to pathogen attacks resulting in a decrease of crop loss due to disease. Moreover, if cytokinin is used as a new agrochemical, there is potential for a more sustainable and publicly favorable agricultural practice. Due to the COVID19 Pandemic, research conducted at Colorado State University was severely limited and completely halted for months during the 2020-2021 reporting period, thus limiting the amount of time available to achieve the goals of the project. However, experiments within the first project objective to characterize CIP inB. napus have been on-going. I havedetermined the proper concentration of applied CK needed to reduce susceptibility toPseudomonas syringaepv.maculicola(Psm) and have begun to determine the time effectiveness of CIP.The results suggest that as the applied concentration of CK increases, susceptibility toPsmdecreases. However, at an applied concentration greater than 100mM, application of CK causes foliar damage resulting in small lesions indicating a hypersensitive response to CK is occurring. Although more experiments are needed to confirm the results, the reduced susceptibility toPsmhas been seen up to 72 hours after CK application, however extended periods of time after CK treatment have not been tested thus far. Uncovering the molecular mechanism behind CIP stated as the third objective is on-going. Due to the pandemic, I was prevented from completing these experiments in canola, however, I did complete a transcriptomic experiment inArabidopsis thalianato determine the genetic response underlying CIP. This has allowed me to create a pipeline for analysis of the genome-wide gene expression withinB. napusafter CIP. I also expect the results of the RNA-seq project inArabidopsis thalianato be similar to the future RNA-seq experiment inB. napusdue to both being in the Brassicaceae family.
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