Progress 01/01/24 to 12/31/24
Outputs
Target Audience:During this reporting period we reached two general audiences: First we reported our results to the plant molecular biology and plant pathology scientific communities through presentations in national meetings and at invited seminars (Maize Genetics Meeting in Raleigh NC, talks at Pudue University, University of Nebraska, NC State University) and through publication (described elsewhere). Here the audiences consisted largely of plant scientists - academic, government and industrial researchers and their graduate students. Second, we participated in and organized several outreach events (described below) in which the target audiences were middle and high school science teachers and students in North Carolina. Changes/Problems:In March 2023 Dr. Saet-Byul Kim, whose work inspired this project, movedfrom her position at NCSU where she was being funded by this project as a research associate, to the University ofNebraska where she took up a position as an Assistant professor. We decided to move the research portion of the project toNebraska with Dr. Kim and have now subcontracted $ 399,283 from NCSU to Nebraska which we intended to use to fund apostdoctoral scientist to work on this project. We appreciate the help that NIFA provided to facilitate this subcontract. Dr. Kim's move and setting up her new lab was time-consuming and necessitated a delay in project activities. In addition, both this year andlast year, we had problems finding suitably skilled personnel for the postdoc position. During 2024, weemployed a postdoc for four months on this project, but it became evident that their skills were not suited to this position, and they found employment in another project at UNL that was more suitable to their skill set. Since it was hard to identify a suitable postdoc, we decided to hire a technician to work on this project goals under Dr. Kim's close supervision. This technician,Minji Kang,was hired in November 2024 and has been paid under this proposal. These delays have resulted in us being substantially underspent on this project. We plan to request at least one, and possibly two, year's no-cost extension to enable us to spend the project funds and achieve the project goals. What opportunities for training and professional development has the project provided?This year, the project provided the opportunity to train Libia Gomez-Trejo, a Ph.D. student, and Minji Kang, a technician. This grant supported Libia's acquisition of molecular skills in protein-protein interactions in the maize/rust pathosystem. We recently hired a technician, Minji, to work on this project. She has focused on identifying interacting proteins with Rp1-D and AvrRp1-D in maize and N. benthamiana. At UNL, we participated in Discovery Days on East Campus at UNL, where the public met scientists working on corn diseases. We introduced plant pathology by using different types of bacteria and fungi. At NCSU We held two one day plant science/breeding workshops attended by ~16 middle and high school science teachers each. One workshop was at the Central Crops Research Station in Clayton NC, where teachers met scientists working on corn, soybean and sweet potato breeding and genetics. The other was at the Mountain Horticultural Crops Research Station and Extension Center at Mills River NC In this workshop teachers met with scientists working on, blackberry breeding, breeding of ornamental ornamental plants and with insect and microbial plant pests or apples and tomatoes. In both classes teachers were instructed in cutting edge classroom activities in genetics and were each provided with kits worth ~$250 to demonstrate plant genetics principles in the classroom. As part of a week-long USDA-funded Ag-discovery camp held on the NCSU campus, we also organized and participated in a half day visit by 20 high school students to the Central Crops Research Station in Clayton NC in which, over the course of a morning, the students met with scientists working on maize genetics, soybean breeding and cotton production. This outreach work was part of the outreach efforts funded by this grant and an NSF-funded project; "RESEARCH-PGR: Exploring the Genetic and Mechanistic Bases of Quantitative Disease Resistance in Maize. To organize each of these events, we collaborated with educational professionals at the NCSU Science House. We organized most of the presentations involving university faculty and we funded both workshops, including the provision of classroom supplies. How have the results been disseminated to communities of interest?We published our results in PLOS Pathogens (https://doi.org/10.1371/journal.ppat.1012662). We reported our results to the plant molecular biology and plant pathology scientific communities through presentations in national meetings and at invited seminars (Maize Genetics Meeting in Raleigh NC, talks at Pudue University, University of Nebraska, NC State University). What do you plan to do during the next reporting period to accomplish the goals?We hope to identify the interacting proteins with Rp1-D by Turbo-ID in transgenic maize plants and by co-immunoprecipitation in maize. Using the Alphfold protein structure prediction tool we have shown that the Rp1-D forms likely a pentamer after activation by AvrRp1-D. To confirm this resistosome formation, we plan to perform a blue native PAGE assay. This would be a novel finding in maize
Impacts
What was accomplished under these goals?
The major goal of this project is to extend our understanding of the control of the defense response mediated by Rp1-D focusing on pathogen derived components. We will also identify and characterize pathogen components involved in pathogenesis.. It should be noted that this project was co-funded by NIFA and NSF. Here we are reporting the NIFA funded portion of the project. NSF funded work is taking place in Wyoming under the direction of PI Dr.Eun-Sook Park. We collaborate closely with Dr. Park . We had several achievements this year, despite some recruiting problems (see "Changes/Problems" section below). We were able to show that the transcript expression level during maize infection of AvrRp1-D allele AvrRp1-DIA16 identified from the Puccinia sorghi isolate IA16, to which Rp1-D does not confer resistance, was approximately three-fold lower than the level of expression of AvrRp1-D from the avirulent isolate IN2, to which Rp1-D does confer resistance. The proteins encoded by these two alleles differ by only a single amino acid and we had previously shown that both proteins were able to trigger Rp1-D dependent HR at approximately similar levels. The lower expression of the virulent allele AvrRp1-DIA16 suggests that its expression level may not be enough to trigger a resistance response and associated HR cell death through interacting with Rp1-D. In collaboration with Dr. Matt Helm (USDA-ARS), we were able to demonstrate nuclear localization of AvrRp1-D. However, observations of differing localizations and different HR-inducing activities of a series of AvrRp1-D deletion constructs , suggested that nuclear localization was not necessary to induce Rp1-D-mediated cell death in Nicotiana benthamiana. The nuclear localization of AvrRp1-D indicates that AvrRp1-D may regulate gene expression to manipulate the defense response. Our collaborator, Dr. Park's group has generated evidence of the DNA-binding potential of Avr-Rp1D and they are investigating the interactions of this protein with transcription factors. We designed a set of vectors for the expression of Turbo-ID tagged Rp1-D and Avr-Rp1D (plus control constructs) in maize and have sent them to the Wisconsin crop innovation center (WCIC) for construction and transformation into maize. As planned last year, we published a paper describing the identification and characterization of AvrRp1-D in the journal Plos Pathogens, one of the top journals in our field. The paper also described how to isolate rust haustoria, generate haustorial cDNA libraries and use them for the identification of effectors and avirulence genes.
Publications
- Type:
Other Journal Articles
Status:
Published
Year Published:
2024
Citation:
Kim S-B, Kim K-T, In S, Jaiswal N, Lee G-W, Jung S, Rogers A, G�mez-Trejo LF, Gautam S, Helm M, Ahn H-K, Lee H-Y, Read QD, Woo J, Holan KL, Whitham SA, Jones JDG, Choi D, Dean R, Park E, Balint-Kurti P(2024) Use of the Puccinia sorghi haustorial transcriptome to identify and characterize AvrRp1-D recognized by the maize Rp1-D resistance protein. PLOS Pathogens 20(11): e1012662. https://doi.org/10.1371/journal.ppat.1012662
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Progress 01/01/23 to 12/31/23
Outputs
Target Audience:During this reporting period we reached two general audiences: First we reported our results to the plant molecular biology and plant pathology scientific communities through presentations in national meetings (Molecular Plant Microbe interactions meeting in Providence RI, Maize Genetics Meeting in St. Louis MO). Here the audiences consisted largely of plant scientists - academic, government and industrial researchers and their graduate students. Second, we participated in and organized several outreach events (described below) in which the target audiences were middle and high school science teachers and students in North Carolina and Maryland Changes/Problems:This year we had a major change to the project. In March 2023 Dr. Saet-Byul Kim, whose work inspired this project, moved from her position at NCSU where she was being funded by this project as a research associate, to the University of Nebraska where she took up a position as an Assistant professor. We decided to move the research portion of the project to Nebraska with Dr. Kim and have now subcontracted $ 399,283 from NCSU to Nebraska which we intend to use to fund a postdoctoral scientist to work on this project. We appreciate the help that NIFA provided to facilitate this subcontract. Approximately $ 127K unspent funds also remain at NCSU where it will be used to fund field-based aspects of the research as well as our outreach activities and our commitments to Dr. Dean's summer salary. Dr. Kim's move and setting up her new lab was time-consuming and necessitated a delay in project activities. In addition, we have not been successful in recruiting a qualified postdoc to work in Nebraska on this project. Dr. Kim has not yet spent any of the project funds sub-contracted to her. These delays mean that almost halfway through the project we have only spent about 25% of the funds. In the coming year we hope to recruit a qualified postdoc and Ph.D. student to work on the project in Nebraska. If this proves to be impossible, we will consider using project funds to recruit a technician in Dr. Kim's lab to work on the project goals under her close supervision. We also plan to request a no-cost extension on funding at the appropriate time. What opportunities for training and professional development has the project provided?The main opportunity for training provided by the project this year has been the training Dr. Kim has received as a new faculty member. This grant provided Dr. Kim with resources that enabled her to get her new position and continue to facilitate her development as an independent scientist leading a project with collaborators in Wyoming, Indiana, Nebraska, North Carolina, Germany and the UK. Since many of our project activities have been delayed by Dr. Kim's move (see "Changes/Problems " section below), training of other postdocs or students on this grant has not been possible this year. However, we anticipate that we will resume these activities in 2024. We held two one day plant science/breeding workshops attended by ~16 middle and high school science teachers each. One workshop was at the Central Crops Research Station in Clayton NC, where teachers met scientists working on corn, soybean and sweet potato breeding and genetics. The other was at theMountain Horticultural Crops Research Station and Extension Center at Mills River NC In this workshop teachers met with scientists working on tomato, and ornamental breeding and with insect and microbial plant pests. In both classes teachers were instructed in cutting edge classroom activities in genetics and were each provided with kits worth ~$250 to demonstrate plant genetics principles in the classroom. As part of a week-long USDA-funded Ag-discovery camp held on the NCSU campus, we also organized and participated in a half day visit by 20 high school students to the Central Crops Research Station in Clayton NC in which, over the course of a morning, the students met with scientists working on maize genetics, soybean breeding and cotton production. We also participated in a teacher training class held at the University of Maryland College Park in which 6 teachers participated in a plant pathology and genetics experiment over three sessions. Dr. Balint-Kurti travelled to MD for one session and participated virtually in the other two. This outreach work was part of the outreach efforts funded by this grant and an NSF-funded project; "RESEARCH-PGR: Exploring the Genetic and Mechanistic Bases of Quantitative Disease Resistance in Maize. In each of these events, we collaborated with educational professionals; the NCSU Science House for all the events at NC and Dr. Dan Levin (Associate Clinical Professor.) for the event in MD. We organized most of the presentations involving University faculty and we funded both workshops, including the provision of classroom supplies. How have the results been disseminated to communities of interest?We reported our results to the plant molecular biology and plant pathology scientific communities through presentations in national meetings (International Society for Molecular Plant Microbe Interactions meeting in Providence, RI, Maize Genetics Meeting in St. Louis, MO). Dr. Kim and Dr. Park has both presented their work to the "genetics of maize microbe interaction group", a group of >100 researchers who meet every two months on-line. What do you plan to do during the next reporting period to accomplish the goals?We hope to characterize further the Rp1-D/AvrRp1-D interaction, including identifying the subcellular location where it takes place. We have shown that AvrRp1-D primarily localizes in the nucleus and that Rp1-D appears to require nuclear localization for function. If we can show that the Rp1-D/AvrRp1-D interaction occurs in the nucleus, this would be a novel finding in the field. We also plan to transiently express AvrRp1-D in maize using Foxtail mosaic virus. If the overexpression of AvrRp1-D triggers cell death in maize, the result can be a piece of critical evidence showing that AvrRp1-D is activated by Rp1-D. To identify the proteins in the Rp1-D/AvrRp1-D complex, immunoprecipitation of AvrRp1-D interacting proteins in maize will be conducted. Dr. Park's group in Wyoming willgenerate Rp1-D and Rp1-dp7 transgenic N. benthamiana plants to facilitate biochemical assays including the identification of Rp1-D interacting proteins before and after activation. They will also work to identify the localization dynamics of AvrRp1-D and will use Chromatin Immunoprecipitation sequencing to identify AvrRp1-D binding DNA sequences. They will continue screening effector candidates for suppressors of plant immunity.
Impacts
What was accomplished under these goals?
It should be noted that this project was co-funded by NIFA and NSF. Here we are reporting the NIFA funded portion of the project. NSF funded work is taking place inWyoming under the direction of PI Dr.Eun-Sook Park. We collaborate closely with Dr. Park . Where we mentionwork completed in Dr. Park's group below, we clearly specify that it was her groups work. The NSF-funded work is thesubject of a recently-submitted separate report to NSF. While activity on this project was lower than expected this year due to Dr. Kim's move (see "Changes/Problems " section below) we nevertheless achieved several notable findings. In collaboration with Dr. Hee-Kyung Ahn and Jonathan Jones in the UK, we were able to show that Rp1-D oligomerizes upon activation due to interaction with AvrRp1-D, which, in collaboration with Dr. Matt Helm (USDA-ARS) we were able to show that AvrRp1-D suppresses the defense response in Nicotiana benthamiana. These findings are significant. The oligomerization of Rp1-D upon activation suggests that it functions in a similar way to other CC-NLR resistance proteins, though interestingly, the size of the oligomer appears to be substantially smaller than would be expected from a pentamer formation that would be expected based on other studies. The suppression of the N. benthamiana defense response provides us with a clue to the function of Avr-Rp1D as an effector. In Wyoming Dr. Park's group has worked on 234 previously-identified Puccinia sorghi effectors. They have cloned them into appropriate vectors, sequenced them and have started to screen them for immune-response suppressive activity. They have performed structural analysis of Avr-Rp1D and have observed that it has similarity to a DNA-binding domain. We have a draft of a paper on the identification and characterization of Avr-Rp1D, which we hope to submit before the end of the year.
Publications
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Progress 01/01/22 to 12/31/22
Outputs
Target Audience:During this reporting period we reached two general audiences: First we reported our results to the plant molecular biology and plant pathology scientific communities through presentations in the US (Nebraska, North Carolina, Iowa), France (Angers and Montpelier), South Korea (Seoul) and the U.K (Norwich, York , Rothamsted). Here the audiences consisted largely of plant scientists - academic, government and industrial researchers and their graduate students. We also held a one day plant science/breeding workshop attended by 10 middle and high school science teachers. In this workshop the teachers were exposed to the latest plant breeding techniqes and several plant genetics activities for the classroom were demonstrated. Changes/Problems:There were no major changes this year, but themajor change anticpated next year is the relocation of the Dr. Saet-Byul Kim from NCSU , where she is a postdoc to start her new position as an Assistant Professor in the Plant Pathology Dept at the University of Nebraska, Lincoln (UNL). This project was largely based on preliminary data generated by Dr. Kim and she has been the single most signficant contributor to the project both intellectually and in terms of research effort. We plan therefore to move some project activites to UNL in the coming year. We will of course consult with program managers as we do this. What opportunities for training and professional development has the project provided?The postdoc working on the project , Dr. Saet-Byul Kim, was able to develop her skills in project writing (she co-wrote the grant proposal), development and exectution. This included setting up and maintaining collaborations with scientists in Wyoming, South Korea, Germany and the UK. Dr. Kim has recently accepted a job as an assistant professor at the University of Nebraska. The skils she learnt and was able to demonstrate during the first year of the project were an important factor in her being offered the job. In the coming year we hope to transfer some of the project activities to Dr. Kim's new lab in Nebraska to help her continue her development as a new faculty member. How have the results been disseminated to communities of interest?We reported our results to the plant molecular biology and plant pathology scientific communities through presentations in the US (Nebraska, North Carolina, Iowa), France (Angers and Montpelier), South Korea (Seoul) and the U.K (Norwich, York , Rothamsted). The audiences consisted largely of plant scientists - acamdemic, government and industrial researchers and their graduate students. What do you plan to do during the next reporting period to accomplish the goals?We plan to perform immunoprecipitation and proximity-labelling experiments detailed in E1.3 and E.1.4. To identify the proteins interactingwith Rp1-D activated by AvrRp1-D in N. benthamiana, we have generated transgenic tobacco line carrying Rp1-D or Rp1-dp7. We also plan to make a transgenic maize line carrying Rp1-D fused with the a tag. We will use affinity-purification coupled with mass spectroscopy to identify the host proteins targeted by Rp1-D/AvrRp1-D. We plan to determine the components of the Rp1-D/AvrRp1-D signaling complex in (goal E3). To begin with, toexamine the complex, we are performing Blue Native Gel assays. If activated Rp1-D/ Rp1-D21 oligomerizes as expected, the complex will be detected at a higher molecular weight in the protein gel. 250 fungal effectors were already cloned into transientexpress vectors. We plan to clone the remaining 1290 effectors into the same vector for screening experiments detailed in E2.1 and E2.2.1. We expect that these ~ 1500 effectors may cover the majority of secreted proteins from Puccinia sorghi and be a good material to study on further understanding the mechanism of maize/common rust interaction.
Impacts
What was accomplished under these goals?
IMPACT. We identified a protein produced by the fungus that causes the important maize disease, common rust, that is recognized by resistant varieities of maize, triggering an immune response. We are currently analyzing the molecular basis of this recognition, including examining what parts of the protein are recognized and other proteins that might be involved in the recognition or modulating the subsequent response. Ultimately we hope this will lead to a better understanding of the plant defense response in general and will enable the more effective deployment of plant disease resistance mechansism in cultivated crops. E.1.1 Validation and characterization of the AvrRp1-D gene Our goal first year was to validate and characterize AvrRp1-D recognized maize Rp1-D. We determined AvrRp1-D (547-5) induced Rp1-D-dependent cell death both in N. benthamiana and in maize protoplasts Crucially , we also determined that a number of close homologs of Rp1-D, that do not confer resistance, also do not recognize AvrRp1-D. E.1.2 Characterize the AvrRp1-D/Rp1-D molecular interaction. We generated ten deletion constructs of AvrRp1-D to identify the domain interacting with Rp1-D. Co-expression of these constructs with Rp1-D data suggested that N-terminus region of AvrRp1-D was responsible for the recognition of Rp1-D. Our collaborators at the University of Wyoming performed yeast two hybrid (Y2H) analyses to investigate the direct interaction between Rp1-D and AvrRp1-D (Figure 3). They found that AvrRp1-D interacts with the resistance proteinsRp1-D, Rp1-dp2 and Rp1-dp7 in the yeast system. Since Rp1-dp7 did not induce cell death with AvrRp1-D in tobacco, this result was unexpected. To further characterize this interaction, they generated a further set of deletion constructs expressing partsof Rp1-D and Rp1-dp7 and performed Y2H. The LRR2 domains of both Rp1-D and Rp1-dp7 interacted with AvrRp1-D. However, AvrRp1-D interacted with the LRR3 domain of Rp1-D, but not with the equivalent domain of Rp1-dp7, suggesting that LRR3 domain (the C -terminal most region of the leucine rich repeat domain)is critical for conferring specificity of immune recognition E.2.2Identify, characterize CDI effectors We identified a P. sorghi gene (designated 4A12) that caused cell death in maize. Wefused 4A12 with green fluorescent proteinto observe the localization in transgenic tobacco line carrying nuclear marker H1B fused red fluorescent protein. Red fluorescence was detected in nuclei and GFP:4A12 signal overlapped with red fluorescence suggesting localization of nuclei.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Balint-Kurti, Peter, and Saet-Byul Kim. "Close encounters in the corn field." Molecular Plant 15.5 (2022): 802-804.
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