Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1160 - Pathology | 100% |
Huanglongbing (HLB) is a widespread and devastating citrus disease that has severely impacted citrus production in the United States. It is characterized by hard, misshapen, and bitter fruit with yellowing of the shoots and leaves. HLB is associated with the phloem-limited, insect transmitted bacterium Canditatus Liberibacter asiaticus (CLas). The inability to culture CLas has made it difficult to study the plant-microbe interactions. All commercial citrus are susceptible to CLas and there is no known resistant variety. Infected citrus trees are asymptomatic for 1-2 years post-infection and die within 3-5 years; highlighting the need for improved detection and management methods. A better understanding of how CLas manipulates citrus innate immunity would allow for the development of enhanced pathogen management. To gain insight to how CLas is manipulating citrus immunity, we sought to identify and validate dynamic changes in protein abundance where CLas resides, the phloem. Navel oranges were mock or graft inoculated with the California CLas strain (HHCA). At ten months post inoculation, crude phloem was extracted from five uninfected and five infected navel trees. Preliminary results demonstrated that proteins that had reduced expression were involved in general housekeeping and photosynthesis, while those with induced expression were part of the defense response. A subset of significantly induced proteins included various proteases such as glycosidases, serine, cysteine, and aspartic proteases. Homologous proteins act as immune-related proteases in other crops such as tomato, and maize and are pathogen effectors targets. We seek to identify the secreted cysteine proteases that are involved in immunity in citrus and aspects of the citrus immunity that are manipulated by CLas. This knowledge will enable insight into HLB disease progression and lead to better HLB management strategies that can inhibit the plant-microbe interactions.
This research proposal will address the program area of plant health and production and the challenge area of food security. The goal of the project is to identify the role of secreted cysteine proteases in citrus during Candidatus Liberibacter asiaticus (CLas) infection. In other crops like tomato and maize, homologous proteases are known to be immune related and are targeted by pathogen effectors. Further interrogation of these proteases during huanglongbing (HLB) development would provide insight into CLas pathogenesis. There are many sutdies to alleviate disease symptoms to increase fruit yield, but there are few that focus on molecular mechanisms of disease.With the information gained from this research, better HLB management strategies can be development to interrupt the key plant-pathogen interactions. The specific objectives are to:
Objective 1: Identify immune-related proteases induced and activated in response to pathogen perception: To identifyimmune-related cysteine proteases in citrus, uninfected navels that are grown in a research greenhouse will be utilized. Both the general immune response and pathogen perception will be induced.Upon defense activation and pathogen perception, we will monitor the expression and activation of the cysteine proteases that were previously identified as significantly changing in thenavel phloem (preliminary data). The expression of the proteases will be assayed by qPCR and theactivation will be monitored by activity-based protein profiling (ABPP) coupled with mass spectrometry.ABPP is a powerful tool that utilizes probes specific to a protein class.The probe covalently binds to active proteases enabling detection and identification. The general immune response will be activated using an analog of the plant defense hormone salicylic acid (SA), and pathogen perception will be mimicked by treating with the bacterial flagellin epitope (flg22) of either CLas or Pseudomonas syringae. This will determine that proteases are involved during the general immune response and pathogen recognition. Common hallmarksof induced defense responses include an oxidative burst and an increase in marker gene expression.Oxidative burst will be assayed using a fluorescence microplate assay that measures reactive oxygen species. Marker gene expression will be validated by qPCR.Objective 2: Investigate the ability of CLas to suppress citrus defense response: Here, we will compare uninfected and infected navel oranges from both the greenhouse and the field to determine aspects of the plant immune response that are inhibited by CLas. The expression and activity of the secreted cysteine proteases will be monitored by qPCR and ABPP coupled with mass spectrometry, respectively. The results of these experiments will determine the most responsive proteases that are both induced and activated by CLas. Potentially, there will be a few members that will be inhibited by CLas. Dampened defense responses will be examined by infiltrating flg22 into both uninfected and infected navel trees as well as spray inoculation with SA. Reduced oxidative burst and reduced marker gene expression will be used to evaluate whether general defense response and PTI are compromised due to CLas infection.
Target Audience
Target audiences: The HLB community are the target audience. The results that we attained can provide targets for improved HLB management strategies for citrus growers. Efforts: During this reporting period, my efforts were directed towards outreach and mentorship. I held a leadership role in the Kids into Discovering Science (KiDS) program. I organized volunteers to teach weekly modules in a rural and marginalized community. I have started mentoring an undergraduate student towards the end of this reporting period.
Changes / Problems
Nothing Reported
Training & Professional Development
During the project, the project funded my attendance to the International Congress of Plant Pathology (ICPP) in Boston MA. I gave a poster presentation that allowed me to connect with other scientists and gain valuable feedback. I had a one-on-one meeting with a potential post-doctoral advisor Dr. Barbara Valent. Additionally, I was able to meet and connect with other scientists who work on similar pathogens. Immediately after, I attended the NIFAFellows meeting in Washington D.C. At this meeting, I connected with other fellows and discovered alternative career pathways that I had not previously considered.
Dissemination Streams
The publication is available for scientists and the citrus community. Outreach efforts was conducted through the Kids into Discovering Science (KiDS) program. I was the classroom coordinator where I organized volunteers to teach at an elementary in a marginalized community weekly for 10 weeks. The fifth grade students performed an experiment testing bean plant growth in two different soil types. Every week, they learned components of the scientific method. They observed the soil types to formulate a hypothesis, took measurement data of their bean plant growth, made graphs to summarize the data and finalized their conclusions. At the end of the lesson module, a field day was organized where the students were able to connect concepts learned in the classroom with their surroundings.
Next Reporting Steps
Nothing Reported
Target Audience
The target audience of the project reached during this period are both scientistsand growers atthe International ResearchConference on Huanglongbing in Orlando, FL.
Changes / Problems
Nothing Reported
Training & Professional Development
During the first year, The project funded my attendance tothe International Research Conference on Huanglongbing in Orlando, FL. I gave an oral presentation that allowed me to formconnections with other scientistsinterested in my work and potential post-doctoral advisors. I connected with collaborators to outline experiments for a future publication. I also attended a workshop led by the University of California, Davis Graduate Studies on how to optimize my 24 hrs in a day. It allowed me to adjust my schedule to allow for a more productive day.
Dissemination Streams
Nothing Reported
Next Reporting Steps
During the next reporting period, the optimization of experiments to assay other citrus genotypes for immune-related proteases is on-going. Optimization ofqPCR primers toamplify cysteine proteases from other citrus genotypes such as Lisbon Lemon, Rio Red Grapefruit, and Tango Mandarin is required. Activity-based protein profiling (ABPP) willbe perfomed after salicylic acid treatment to assess protease activity during defense activation in comparison to water control. We currently have confirmed PCR + CLas infected Navel oranges. The next stepis to optimize and perform qPCR analyses on defense genes to determine other aspects of immunity that are hampered. For example, expression of defense genes may be reduced during CLas infection. For my training/career development plant, I want to focus on my mentorship skills.The Kids into Discovering Science (KiDS) will commence in January. I will be responsible for organizing volunteers, organizing lessons, and providing lessons to children in underserved communities. The fifth-grade students will learn how to make observations, form a hypothesis, attain and analyze data on the impact of soil type on plant growth. Amanuscript is currently being draftedwhere I anticipate itspublicationby the next reporting period. <br><br>
<br>What was accomplished under these goals? Huanglongbing (HLB) is a devastating citrus disease that has affected the citrus industry worldwide. Candidatus Liberibacter asiaticus, the HLB-associated pathogen,has yet to be grown in a laboratory setting. Therefore, in order to identify how the pathogen progresses during infection,the citrus responses is evaluated. We previously identified citrus proteases that were induced during CLas infection in a study comparing healthy and infected citrus. These citrus proteasesshare similaritiesto immune-related proteases in tomato and maize and have been shown to be involved during defenses against pathogens. Our goal is to (1) identify the immune-related proteases in citrus by activating defenses and mimicking pathogen perception and (2) to investigate the ability of CLas to suppress defense responses in citrus. By elucidating these objectives, better strategies targeting the plant-pathogen interaction can be developed resulting in improved HLB management. Objective 1: identify immune-related proteases induced and activated in response to pathogen perception Currently, we identified citrus cysteine proteases that respond during defense activation by spraying with 2mM of salicylic acid, a known plantdefense activator.We found that compared to water spray control, the expression fora cysteine protease was significantly induced 30 fold by qPCR analyses. The same cysteine proteases werealso induced during CLas infection in our comparative study in both crude phloem extracts from greenhouse-grownWashington Navel Oranges and leaf extracts from Valencia Oranges grown in afield in Texas. These results provide a better understanding of how cysteine proteases are involved during general defense response. Objective 2: Investigate the ability of CLas to suppress citrus defense response: In other crops like maize and tomato, immune-related proteases show a correlation between abundance and activity. During pathogen perception protease abundance is induced as well asactivity. Diverse pathogens can selectively inhibit these proteases to facilitate disease progression. To identifyif CLas inhibits protease activity, acomparative study investigating the change in cysteine protease abundance and activity between infected and uninfected Valencia Navel oranges was conducted. Our current data demonstrates that the abundance and activity of several proteases is increased in infected samples. A specific group of proteases display enhanced abundance but no altered activity during infection. A sharedvirulence protein among currently sequenced CLas strains was able to interact with various cysteine proteases that we identified in our previous proteomic study.Based on these results, CLas targets protease activity to dampen host defense responses. Immune-related proteases in citrus have not been described previously. Even more so, immune related proteases regarding Huanglongbing disease progression has not been investigated. These results have impacted achange in knowledgeby characterizing immune-related proteases in citrus during HLB progression. <br><br><b>Publications</b><br>