Progress 04/15/18 to 04/14/20
Outputs
Target Audience:Throughout my professional and scientific development during this project, I have reached the following audiences: Undergraduates: In March 2020 I delivered a guest lecture to an undergraduateintroductory virology class on the regulations and use of biotechnology in plant production. Federal agencies and elected officials: I advocated on behalf of plant health andmy professional society (American Phytopathological Society) in March 2020. Plant pathologists and other scientists: Myresearch has been made available to the public and fellow scientists through presentation at an international congress and submission to bioRxiv. I additionally was invited to presenton public engagement as a graduate student to a regional conference (APS Potomac Division Meeting). I continuously engage with my scientific community on Twitter and have been writing articles for my professional society newsletter about science policy subjects that relate to our community. General Public:I have recorded an episode to contribute to the new APS Plantopia podcast and have participated in many Skype A Scientist sessions with various K-12 classrooms across the country. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training Objective 1:Attend at least one workshop relating to symbiosis or science policy. Thiswas previously satisfiedby my attendance at the AAAS CASE workshop, as well as a science communication workshop in June 2018. However, as the American Phytopathological Society (APS) Public Policy Board (PPB) Intern, I attended our fly-in visit to Washington, D.C. in March 2020, where I was able to further develop policy skills and learn more about interacting with different parts of the federal government as a scientist. As part of this experience, I met with various offices within the USDA and other federal agencies. Throughout the trip and beyond Ihaveshared theknowledge I gained with my community of early career researchers through Twitter and other virtual outreach. My internship with the PPB has opened many more opportunities for me including networking with established scientists in my field, as well a participating in other boards including APS' International Year of Plant Health task force and recording a podcast on gene editing in agriculture that will be available to the general public. Training Objective 2:Publish 1-2 manuscripts on the proposed research.A manuscript of the research supported by this grant has been submitted to the preprint server bioRxiv and is currently publicly available. I submitted the manuscript for review at PNAS and have just recently finished minor revisions. Additionally, genomes generated through this project are currently being analyzed in anticipation of preparing another manuscript. Training Objective 3:Present the proposed research at one or more conferences.I previously satisfied this objective by presenting a poster at the International Symbiosis Society Congress in July 2018, and attended the AAAS annual meeting in February 2019 as well. Yet, I have continued to attend meetings and disseminate this work. I presented a poster at the International Congress for Molecular Plant-Microbe Interactions in Glasgow Scotland in July2019. I have received a travel award to present this work at the Plant Health 2020 meeting in Denver, CO in August 2020. Training Objective 4:Graduation of the Ph.D. candidate within the project duration or 6 months of its completion.I defended my Ph.D. in Plant Pathology on April 8th, 2020 and satisfied all degree requirements by the 17th. Additional Training Opportunities: I did not formally present this work atPlant Health 2019 (August 2019, Cleveland, OH)and the APS Potomac Division Meeting(March 2020, Virtual), though I did attend these meetings and make new connections relevant to my professional development and gained insight into my project over the course of interacting with other scientists. I applied for a NIFA postdoctoral fellowship under advisement of Dr. David Baltrus, thus identifying a postdoctoral research position and a new mentor. I was able to visit the University of Arizona to give a seminar on this topic and network with scientists there. I mentored a high school student during the spring of 2018 and co-mentored a visiting college student during the summer of 2018 while she conducted research in our lab. I have continued to have 1-on-1 meetings with my mentor (Dr. Bogdanove) throughout the year and to present at weekly lab meetings, as well as our yearly lab retreat. Ihelped facilitate a Cornell Alliance for Science workshop on gene editing in Hyderabad, India in March 2019. As a faculty member for this workshop for Indian scientists, I helped in the course design and did presentations on science communication and the science of gene editing. I have delivered a guest lecture on biotechnology for an introductory virology class in March 2020. I have served on the Office of Public Relations and Outreach board for APS and on the International Year of Plant Health task force. How have the results been disseminated to communities of interest?I presented this work at the International Symbiosis Society Congress in July 2018 andthe International Congress of Molecular Plant-Microbe Interactions in July 2019. I was recently awarded a travel grant to present this research at the Plant Health 2020 meeting in August. I gave a seminar at the University of Arizona (November 2019) and at Cornell University (April 2020) that covered this project. Finally, a manuscript detailing the results of this project is available through bioRxiv and currently under review at PNAS. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Plants have bacteria, fungi, and other microbes living within and around them that can affect their health. The fungus I study can live within the soil and can infect rice seedlings, sunflowers, and other plants. Interestingly, the fungus, Rhizopus microsporus, relies on a toxin made by a bacterial partner that lives inside of it to cause plant disease. Bacterial-fungal relationships are not very well studied, but can inform our approach to disease control as we discover more fungi have bacterial endosymbionts. My work on thisbacterial-fungal mutualism is a step towards understandinghow bacteria interact with their fungal hosts. We know that this fungus can't reproduce without having its bacterial partner and that the bacterium relies on certain molecular machinery to interact with its host. However, we don't know any specifics about what is exchanged using that machinery. Given that the plant microbiome is filled with fungi and bacteria that interact to impact plant health, understanding what molecular exchange is happening can open new avenues for biocontrol and plant growth promotion by targeting bacteria that associate with and live inside fungi.Bacteria commonly rely on effector proteins that they secrete to influence their hosts and environment. In this project, I am investigating proteins from Rhizopus-associated Burkholderia spp. that resemble transcription activator-like (TAL) effector proteins from plant pathogenic Xanthomonas and Ralstonia spp. This is the first investigation of endofungal bacterial effectors and of TAL effectors targeting a non-plant host. Here I present the detailed accomplishments of my Research Objectives; accomplishment of the Training Objectives is detailed in the following section. Research Objective 1: Assess Type III Secretion-and Nuclear Localization Signal-dependentfungal nuclear localization of Btl19-13 during symbiosis. I created strains of the endohyphal bacterium Burkholderia rhizoxinica (now Mycetohabitans rhizoxinica) that make a yellow fluorophore (YFP) and a Burkholderia Tal-like (BTL) protein, or derivative, that is fused to a red fluorophore (mCherry). I infected these strains back into the wild type host fungus Rhizopus microsporus and used deconvolution microscopy determine that a BTL protein, BTL19-13, is expressed when the bacterium is within the fungus, as indicated by red fluorescent bacteria. I was unable to visualize secretion of the BTL9-13 protein, likely due to interference by the fluorophore. However, taken together with my preliminary datain plantaand in yeast, as well as our phenotyping data from Objective 2, my hypothesis that Btl19-13 is a secreted, nuclear-localizing effector protein is supported enough to not require backup experiments. Research Objective 2:Determine the effect of Btl19-13 on host transcription.I confirmed our mutant strain of B. rhizoxinica has the btl gene knocked out and created a complement strain that is transformed with a plasmid containing the btl gene under its native promoter. I conducted RNAseq on fungi infected with wildtype, mutant, and complement bacteria and determined the impact on the fungal transcriptome of Btl19-13 presence. Unfortunately, most of the differentially expressed genes were hypothetical proteins, so I was unable to infer a molecular mechanism for Btl19-13. However, I did find that fungi infected with the Btl19-13 mutant bacterial strain grew slower on nutrient-rich media amended with sodium dodecyl sulfate (SDS). No other distinguishingphenotypes were observed: bacterial infection ability, fungal growth on other media, fungal sexual and asexual sporulation, etc. SDS is a detergent that causes cell membrane stress, so the biological relevance of this phenotype is unknown and would be the subject of future study.I also created a "rescue" strain that has a plasmid-borne btl gene from another Burkholderia sp., btl18-14, but fungi infected with the btl8-14 strain were not rescued for the SDS growth phenotype implying that BTL proteins have some host specificity or play different roles across isolates. I attempted a Dual Luciferase Assay to investigate the molecular mechanism by which Btl19-13 is able to affect transcription and see if it was an activator like Xanthomonas and Ralstonia TAL effectors. Unfortunately, the Dual Luciferase Assay appeared to be flawed in design based on the behavior of negative controls. So I changed to a GUS assay, which is commonly used to investigate the impact of TAL effectors on promoter gene regulationin planta. I employeda minimal promoter with or without a BTL19-13 binding element driving gene expression of GUS and co-infiltrated Agrobacterium tumefaciens with a GUS construct and a TAL effector/BTL19-13 expression construct into Nicotiana benthamiana. The activity of GUS is then measured as an indirect way to look at expression changes. BTL19-13 was unable to upregulate GUS activity and similarly unable to downregulateGUS activity by binding competition with an activator. Taken together,my data supports the hypothesis that BTL9-13 is an effector protein that impacts the host by altering host transcription, though by an unknown mechanism that differs fromXanthomonasandRalstoniaTAL effectors which are activators in the same assayin planta. Research Objective 3:Survey the distribution of BTLs across a collection of Rmm-Brh symbioses.I acquired a global collection of 14 isolates and we didSouthern blots to determine btl gene presence. All isolates had at leastbtlgene fragments in the Southern blot, indicating a level of conservation of btl genes, though they varied in size and genomic context. There are 3 previously sequenced genomes that are of high enough quality to identifybtlgenes so I identified and analyzed thebtlgenes from these genomes for common promoter and sequence elements. Additionally, I sequenced 7 more genomes which I am continuing to work on and anticipate publishing these to make them an available resource and to share the insights intobtlgene diversity.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Carter, M.E., Carpenter, S.C.D., Dubrow, Z.D., Sabol, M.S., Rinaldi, F.C., Lastovetsky, O.A., Mondo, S.J., Pawlowska T.E., and A.J. Bogdanove. (2020) "A TAL effector-like protein of an endofungal bacterium increases the stress tolerance and alters the transcriptome of the host." Under Review at PNAS. bioRxiv. https://doi.org/10.1101/2020.03.04.968529.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Carter, M.E. et al. (2019) "Thinking outside the plant: TAL effector-like proteins in a bacterial-fungal symbiosis." IS-MPMI Congress 2019.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Carter, M.E. et al. (2018) "TAL effector-like proteins in Burkholderia-Rhizopus microsporus symbiosis." International Symbiosis Society Congress 2018.
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2020
Citation:
Carter, M.E. et al. (2020) "Endofungal Mycetohabitans spp. have TAL-like proteins that can alter the transcriptome and stress tolerance of their host". Plant Health 2020.
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Progress 04/15/18 to 04/14/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?I have attended 3 professional society meetings so far: theInternational Symbiosis Society Congress in Oregon in July 2018, the International Congress of Plant Pathology in Massachusetts in August 2018, and the American Association for the Advancement of Science meeting in Washington, D.C., in February 2019. These meetings have allowed me to broaden my network and learn about related research areas and science policy. Just prior to the fellowshipfunding being awarded, I attended the AAAS CASEscience policy workshop in March 2018 as I detailed in my Training Plan,satisfying Training Objective 1. Additionally, I attended a science communication workshop facilitated by the Cornell Alliance for Science in June 2018 and helped facilitate aCornell Alliance for Scienceworkshop on gene editing in Hyderabad, India in March 2019. As a faculty member for this workshop for Indian scientists, I helped in the course design and did presentations on science communication and the science of gene editing. Over the summer of 2018 I was selected to be the Early Career Intern for the American Phytopathological Society and have served one of the two years of my term in that position. I had time for this internship because my fellowship allowed me to devote so much time to my thesis research. This position has increased my understanding of how professional societies interact with the federal government, including policy makers and grant agencies. I am more prepared to become a tenure-track professor who is active in science and agricultural policy. I mentored a high school student during the spring of 2018 and co-mentored a visiting college student during the summer of 2018 while she conducted research in our lab. I have continued to have 1-on-1 meetings with my mentor (Dr. Bogdanove) throughout the year and to present at weekly lab meetings, as well as our yearly lab retreat. How have the results been disseminated to communities of interest?I presented this work at the International Symbiosis Society Congress in July 2018, which was my first public presentation on this project and satisfiedTraining Objective 2. Academics workingin other symbiotic systems were present and I was able to make connections with other scientists working on bacterial symbionts of other organisms. My abstract was recently accepted for presentation at the International Congress of Molecular Plant-Microbe Interactions in Glasgow, Scotland in July 2019. I will be making a poster to attend this meeting as well. What do you plan to do during the next reporting period to accomplish the goals?Research Objectives: Research Objective 1:Assess Type III Secretion- and Nuclear Localization Signal-dependent fungal nuclear localization of BTL19-13 during symbiosis I will continue to work with the strains I have developed in the last year and will focus on imaging these strains to get a clearer picture of BTL localization in vivo. I am trained on two microscopes and will be using both to try to get the best chance of detecting mCherry signal. I will consult with the Biotechnology Resource Center's Imaging Facility here on Cornell Campus and the members of the Pawlowski lab, both experts in imaging, for tips and troubleshooting if I am unable to work out a protocol on my own. Research Objective 2:Determine the effect of BTL19-13 on host transcription I will carry out the Dual luciferase assay using the strains developed in the past year. I believe this will be quickly accomplished now that the cloning is complete, and the assay is fairly standard. The RNA-seq experiment is finished but I am working on what outputs would make sense for poster and paper figures, as well as gene ontology analysis with R tools to look for enrichment. However, given that the BTL could be targeting any gene with an appropriate EBE, it may be difficult or impossible to narrow down to a reasonable list of candidate genes at this time. Research Objective 3: Survey the distribution of BTLs across a collection of Rmm-Brh symbioses I will get the last isolates that I am interested in testing so that I have a global collection of 10-12 bacteria. The Southern blot will be repeated using all strains. If time and money allow, I may genome sequence strains that are not already sequenced, to determine the sequence of the TAL effectors, but that is the lowest priority item at this time. Training Objectives: Publications (Training Objective 2) will be satisfied in year two of the fellowship based on the data I am currently collecting. These publications will be prepared as a final chapter(s) to my thesis and will be submitted prior to the defense of my thesis.I had a thesis committee meeting in March 2019 and am on track to defend my thesis inApril of next year, thus satisfying Training Objective 4 at the end of the grant period. The other training objectives have already been satisfied, though I will be presenting this work at IC-MPMI in July, further satisfying Training Objective 3.
Impacts
What was accomplished under these goals?
The microbiome is a current hot topic and not just the microbiomes of humans. Plants have tons of bacteria, fungi, and other microbes living within and around them that can affect how healthy the plant stays. The fungus I study can live within the soil, but can also infect rice seedlings, sunflowers, and other plants. Interestingly, the fungus needs a bacterial partner that lives inside of it, to make a rice plantsick. The bacterium makes a toxin that it gives to the fungus to kill the rice plant, releasing food and nutrients for both of the microbial partners. Relationships like this bacterial-fungal one are not very well studied, but can clearly inform our approach to disease control if many fungi have bacterial partners like this. Even partners that don't produce toxins but help their fungal hosts in other ways can be detrimental to plant health. Over the past year, I have continued my work on a bacterial-fungal mutualism to learn more about how bacteria interact with their fungal hosts. We know that this fungus can't reproduce without having its bacterial partner and that the bacterium relies on certain molecular machinery to interact with its host. However, we don't know any specifics about what is exchanged using that machinery. Given that the plant microbiome is filled with fungi and bacteria that interact to impact plant health, understanding what molecular exchange ishappening can open new avenues for biocontrol and plant growth promotion by targeting bacteria that associate with and live inside fungi. This fundamental research has a lot of potential, but we have to start learning about systems like the one studied in this grant. Accomplishment of the Training Objectives is detailed in the following text boxes. Research Objective 1:Assess Type III Secretion- and Nuclear Localization Signal-dependent fungal nuclear localization of BTL19-13 during symbiosis I have created strains of the endohyphal bacteriumBurkholderia rhizoxinicathat make a yellow fluorophore (YFP) and aBurkholderiaTal-like (BTL) protein, or derivative, that is fused to a red fluorophore (mCherry). I have infected these strains back into the wild type host fungusRhizopus microsporusand are currently using deconvolution and confocal microscopy to track the yellow bacteria and their red proteins. The YFP is constitutively expressed and the BTL:mCherry hybrid is driven by the native BTL promoter, so it serves as an expression reporter as well. Preliminary imaging has shown that the BTL protein is expressed when the bacterium is within the fungus, but can only be seen within the bacterium. This may be due to an incompatibility of the mCherry protein and the secretion system, or not enough mCherry signal outside of the bacterium. I am further refining out microscopy protocol and slide preparation to better understand. Research Objective 2:Determine the effect of BTL19-13 on host transcription RNAseq:I confirmed our mutant strain ofB. rhizoxinica has the btl gene knocked out and created a complement strain that is transformed with a plasmid containing the btl gene under its native promoter. I also created a "rescue" strain that has a plasmid-borne btl gene from another strain ofB. rhizoxinicathat we cloned. These four strains, mutant, wild type, complement, andrescue, were reinfected into the host fungus. Fungal growth was collected and used to extract RNA for sequencing. I did 3 biological replicates per strain and did eukaryotic polyA RNA enrichment. Sequencing data was bioinformaticallyprocessed and we found that the presence of the btl gene resulted in transcriptionally up- and down-regulated genes. I compared the wild type/mutant expression changes with the complement/mutant expression changes and have found 265 differentially expressed genes in common. Many genes appear to be involved in fungal cell morphology and development, as well as transcription factors and transporters. I am currently analyzing for enriched gene ontology terms and looking at potential BTL binding sites within the promoters. Dual luciferase assay: I have constructed a series of plasmids for a dual luciferase assay in Nicotiana benthamiana using Agrobacterium-mediated transformation. Each of these plasmids has the Renilla luciferase gene driven by the constitutive 35S promoter and the Firefly luciferase gene driven by the rice promoter for the OsSULTR3;6 transporter. This promoter was chosen because our lab has used it previously in luciferase activities and it is naturally targeted by a Xanthomonas TAL effector. The effector binding element (EBE) for the Xanthomonas TAL effector was replaced in the promoter with a restriction enzyme cut site. Plasmids were digested at this cut site or one of two others, then ligated with oligonucleotides sequences of the BTL EBE or a scrambled sequence, creating a series of 6 vectors to use for testing the effect of the BTL protein on gene expression. These plasmids are Gateway Destination vectors, so I shuttled the btl gene or a designer TAL effector (dTALE) gene in to the plasmids and transformed the products into Agrobacterium tumefacians. Preliminary experiments are underway to tune the assay using the dTALE positive control and empty vector. Research Objective 3: Survey the distribution of BTLs across a collection of Rmm-Brh symbioses Southern blots: After extensive troubleshooting, I was unable to use western blots with a custom BTL antibody to detect BTL proteins within the symbiosis. I have instead moved to Southern blots of the genomic DNA isolated from Burkholderia, as previously detailed as a backup plan. Six Burkholderia isolates were provided by Dr. Teresa Pawlowska, 3 of which have been previously sequenced. I received 10 NRRL isolates of Rhizopus microsporus from the USDA culture collection and screened them for bacteria. Four isolates did contain Burkholderia spp. as confirmed by PCR and sequencing with Burkholderia-specific primers. All ten strains were used for a Southern blot and all contained at least one band indicating the presence of a btl gene. We will be repeating this Southern blot with more strains from the CBS culture collection and with a different digest of the genomic DNA to try to clarify how many btl genes were in each strain. Unfortunately, we are unable to amplify and sequence the btl genes as they do not contain enough sequence similarity for primer design, unlike Xanthomonas tal genes.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Carter, M. et al. (2018) "TAL effector-like proteins in Burkholderia-Rhizopus microsporus symbiosis." International Symbiosis Society Congress 2018.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
Carter, M. et al. (2019) "Thinking outside the plant: TAL effector-like proteins in a bacterial-fungal symbiosis." IS-MPMI Congress 2019.
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