Progress 05/01/23 to 04/30/24
Outputs
Target Audience:The target audience(s) reached by our efforts during this reporting periodincludecrop specialists at NC State, Virginian Tech, and the University of Florida. In addition, horticulture and agronomic crop producers and crop consultants, specifically those that produce tomato, eggplant, and tomato. We have also interacted withcounty and state officials, NC Cooperative Extension Agents, NC Department of Agriculture Agronomists. and onepostdoctoral scholars. Preliminary results have been shared with the broader scientific community through research and extension talks at conferences. Changes/Problems:The major limiting factor in advancing the proposed research was finding a postdoctoral research associate with the training and expertise to conduct the proposed research objectives. This was particularly challenging because funding for this project and the project itself becameactive during COVID. Furthermore, there was a challenge with award notification that further delayed the project start date.Together, these factors delayed project start time, sample processing, sequencing, and objective completion. During the first year of this project tenure, the PI (Huerta) was awardeda USDA Equipment Grant titled "Think and Seq: Rapid,high-quality long-read sequencing of genomes, transcriptomes, and epigenomes using a PacBio Sequel IIe System for basic and applied agricultural research" (GRANT13659323, Proposal# 2022-06352). This award allowed her to negotiate with the Genomic Sciences Laboratory at NC (Statehttps://research.ncsu.edu/gsl/) to use the equipment as long as the appropriate channel for reserving the Sequel IIe wasfollowed and she prepared samples and acquired reagents and supplies to do her own sequencing on the instrument. Through this grant, Dr. Huerta also negotiated with Pacific Biosciences of California, Inc (PacBio) to get eight freeSMRT Cells (or 2, 4 SMRT Cells 8M each for sequencing on the Sequel® IIe systems). These SMRT Cell 8M trays cost $3,922 a piece, and we could sequence anywhere between 45-98 stains per cell, depending on genome size. To take advantage of this sequencing power, we need to prepare sequencing libraries in-house, which we now have the capacity to do, but we need to purchase the reagents to perform this work. We can easily accomplish this with funds allocated to current services on our initial budget when we proposed sending high molecular weight DNAto SeqCenter for hybrid genome assembly (Pacbio and Illumina), which would be done in the SeqCenter facility in Pittsburgh. Now, wecan perform PacBio sequencing in the Huerta Lab by reallocating funds from current services to supplies and materials. This would enable the Huerta lab to purchase PacBio Binding Kit 3.2 and clean up beads ($1,648 for the kit to prepare24 samples), need a total of eight kits ($13,184), a Sequel IIe sequencing kit per cell (each kit is $874), need a total of four ($3,496). This would allow us to sequence the number we had proposed to sequence plus 50 more isolates and we would still stay within our budget. What opportunities for training and professional development has the project provided?The research team that has performedsample collection, Ralstonia solanacearum species complex stainisolation, characterization, and genomic analysis includes the lead PI, a postdoctoral associate who initiated the work and was offered full-time permanent employment before project completion, and one summer research undergraduate who was sponsored through a Summer REEU program. Postdoctoral scholarsandundergraduatesworking on this project gained professional and technical skills that prepare them for the real world and a career that supports agricultural productivity. The postdoctoral associate hired had strong bioinformatic skills but little background in plant pathology. She gained experience in isolating bacteria from infested tissue, renewing an APHIS PPQ permit for domestic interstate tissue sample movement. She also gained experience and skills inDNA isolation, DNA shearing, library preparation, pooling samples, loading the sample into the instrument, collecting the sequence data, and assembling and annotating the genomes. In addition, 133 WGS of stain in theRalstoniasolanaceraumspecies complex were downloaded from publically available databases to develop the Hidden Markov Model named HRM-Rhs, which we used to extract Rhs toxins from publically available whole genomes and allowed us to test our model. REEU undergraduate gained valuable research experience and exposure to the science behind agriculture. This undergraduate worked closely with Dr. Huerta to isolate bacteria from infested and infected tissue, purify strains, and learn molecular techniques to characterize bacterial plant pathogens. Through this project,Dr. Huerta has had the opportunity to build collaborative relationships with two research teams in Mexico. The first collaboration is with the Universidad Politécnica de Morelos (UPEMOR), Morellos, in the state of Mexico, in Mexico where she was invited to become an affiliate faculty member, allowing her to be on student committees at this university. The second collaboration in Mexico is with Cinvestav Irapuato in Guanajuato, where she also served as a student committee member and is supporting research endeavors at this research center. At both universities, Dr. Huerta is serving as a technical advisor and knowledge hub to empowerscholars to characterize theRalstoniasolanaceraumspecies complex population in different states across Mexico. How have the results been disseminated to communities of interest?Preliminary results from ongoing research for this grant have been disseminated via research talks and extension notes. Knowledge gained from our research is shared with the public, USDA officials, and the scientific community. This is important to safeguard agricultural productivity in NC and the nation. This includes: Invited seminars at research institutions Huerta, A.I. Macro and Micro-Community Dynamics that Impact Adaptation, Evolution, and Resilience. Department of Plant Pathology, UW-Madison, WI., May 2023. Huerta, A.I. Rhs-toxin Mediated Competitive Fitness Impacting Plant Pathogenic Bacterial Survival. Department of Plant and Soil Sciences, University of Delaware, Newark, DE, March 2023. Contributed Oral presentations at Confrences Gómez, Andrea M., Ameen, S., Huguet-Tapia, J., Huerta, A.I.Bacterial warfare: Proteinaceous toxins repertoires in plant pathogenic bacteria. Plant Health 2023, Denver, Co., August 2023. Schmidt, K., Liao, Y-Y, Huerta, A.I. TagM and its effects on T6SS and the plants we hold dear. 2022 Summer Undergraduate Research & Creativity Symposium, Raleigh, NC, July 2023. What do you plan to do during the next reporting period to accomplish the goals?Hire a new postdoc or technician who can lead strain characterization of the remaining50Ralstonia solanacearumspecies complex stains that remain uncharacterized in our collection and weresampled in the summer/fall of 2024. Isolate another 25strains from South Carolina, Georgia, and Virginia for an additional 75 strains. This will complete our goal of sampling different fields affected by bacterial wilt and different crops to understand better the current genetic diversity ofRalstoinia solanacearumspecies complex stains. Characterizing these strains will enable us to perform WGS onthem and we will be able to conductphenotypic assays to complete the proposed objectives. This includes Objecves (1B).Generate an inhibition spectrum for each strain in the collection that measures the inhibitory and susceptibility phenotype against a panel of genetically diverse strains in the RSSC at different temperatures.Goal 3:Investigate the correlation between Rhs-toxin abundance, geographic location, and competitive fitness.Investigate the correlation between Rhs-toxin abundance, geographic location, and competitive fitness. Objective (3A) Test the role of Rhs-toxins in K60's ability to inhibit the growth of RSSC strain, other soilborne pathogens, and biological control agents. (3B) Correlate Rhs-toxin diversity with host range using both in-vitro and in-vivo experiments. We are perfectly positioned to move forward with proposed research objectives because a lot of the bioinformatic pipeline development to sequence strain, assemble, and annotate genomes has been achieved. Furthermore, we have developed the code to mine bacterial genomes for Rhs toxins and to generate networks and corrlation. We just need the final set of stains that represent the geographic diversity ofRalstoniaacross the Southeast. To the end, Dr. Huera is collaborating with Inga Meadows and Dr. Daisy Ahumada, extension specialists at NC State. With Hehe Want at Clemson University:Drs. Mathew Parrat and Jeff Jones at the University of Florida and Yuan Zeng at Virginia Tech to achieve this goal.
Impacts
What was accomplished under these goals?
Major goals accomplished during this reporting period: Goal 1: Ralstona solanacearum species complex isolate collection:(1A) Collected, characterized, and profiled the phenotypic diversity of 118strains in the Ralstonai solanacearum species complexendemic to the Southeastern United Statesisolated from tomato, blueberry, pepper, and eggplant.The Huerta Lab procured 39 strains from national collections, specifically from the international collection of Caitilyn Allen at UW-Madison and David Norman at the University of Florida. These historical strains will beused as control and to monitorpopulation shifts across states in the Southeast.We also isolated 78 strains from blueberry, tomato, and eggplantsduring this reporting period. All isolates procured or isolated by the Huerta Lab were obtained and isolated following the protocol described andsubmitted to APHIS PPQ, allowing Interstate Movement and work in the lab and growth chamber of Regulated organisms by 7 CFR 330. Specifically, all isolates were characterized by running a conventional PCR that can differentiate the R3b2 select agent strains ofR. solanacearumfrom the non-select agents. This PCR amplifiesboth a 280 bp band that corresponds the "universal"R. solanacearumfragment (primer pais 759/760) and a 357-bp fragment that is found only in R3b2 stains (primer pair 630/631. All stains procured from collections and isolated in-house were not select agents. After confirming that no Ralstonaiastains in our possession were R2b2, we sub-classified all the stains into phylotypes using the multiple PCR reaction of Fegan and Prios (2005). After phylotype characterization, the sequevar designation was also assigned through PCR amplification, sequencing, and phylogenetic analysis of the conserved endonuclease (egl) genes following the methods of Fegan and Prior (2005). Goal 2: GenomeSequencing of strains/ Rhs toxin extraction/ Genome mining: (2A)We conducted whole genome sequencing (WGS) of 34Ralstonia solanacearumspecies complex strains that we isolated from blueberry, tomato, and eggplant in the Huerta lab. Sequencing was performed using Pacbio sequencing technology, leveraging the acquisition of a new PacBio Sequel IIe through theleadership of PI Huerta, who led a team of five scientists in writing and submittinga successfulUSDA Equipment Grant titled "Think and Seq: Rapid,high-quality long-read sequencing of genomes, transcriptomes, and epigenomes using a PacBio Sequel IIe System for basic and applied agricultural research" (GRANT13659323, Proposal# 2022-06352). Huerta Lab members conducted all aspects of sample preparation, including DNA isolation, DNA shearing, library preparation, pooling samples, loading the sample into the instrument, collecting the sequence data, and assembling and annotating the genomes. In addition, 133 WGS of stain in the Ralstonai solanaceraumspecies complex were downloaded from publically available databases to develop the Hidden Markov Model named HRM-Rhs, which we used to extract Rhs toxins from publically available whole genomes and allowed us to test our model. Briefly, the HRM-Rhs model was generated after weconducted a comprehensive literature review on functional studies of rhs genes and obtained theRhs amino acidsequences (aa) of these toxins.All sequences used to build the Rhs HMMmodel were manually extracted from the NCBI protein database. 54 Rhs aa sequences from 27 strains belonging to nine distinct bacterial generawere aligned in Geneious Prime version 2021 (www.geneious.com) using the Clustal Omega algorithm with default parameters. The conserved motif that demarcates the hypervariable C-terminal tip from the N-terminal domain, approximately 10 AA in length (PxxxxDPxGL),was extracted and realigned. The realigned sequences were used to build a Hidden Markov Model (HMM) using the build command in HMMER v3.3 (http://hmmer.org). This model was used to confirm the model's accuracy in extracting all rhs genes from all stains used to build the model. This method was then repeated by adding aa to the 5' end of the 10 aa motifs until an HMM was generated that accurately extracted all putative Rhs toxins from bacterial genomes. After manual curation and genome mining, a finalRhs HMMwas built using a 43 AA peptide motif that included the original 10 aa motif that demarcates the conserved N-terminal sequence from the hypervariable motifand 33 additional aa upstream to it. Mining WGS for rhs Genes with the Rhs HMM: An average nucleotide identity analysis was conducted on all downloaded Ralsotnia solanacearum species complex genomes from NCBI. If any genome clustered into an ANI group with 100% similarity, they were considered clones, and all but one were removed from further study. The remaining WGS were translated using a custom Perl script (https://github.ncsu.edu/Huerta-lab/Mine_RhsToxins/blob/main/gb2cds.pl). The Rhs HMM was then used to mine all genomesusing the hmmsearch command of HMMER v3.3 (E-value, 0.05). Returned sequences were passed through the NCBI Conserved Domains database to confirm the presence of RHS domains to be manually curated (Marchler-Bauer et al. 2017). Rhs toxins that lacked a full-length C-terminal tip (<90AA) or were composed just by the motif (i.e., only the 43 AA sequence was retrieved) were removed from further study. Pearl and phyton scripts were then generated to sub-classify Rhs toxin extracted fromRalstoinagenomes into Rhs toxin families and to determine their predicted killing domains. All the pearl and python codes, in addition to the Rhs HMM model, are ready to be used on Southeastern USRalstoina solanacearumspecies complex strains.
Publications
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