2025 SIVB Student Award Winners

2025 JOHN S. SONG AWARD

Functional Characterization of Plant Specific Nuclear Factor Y Subunit Alpha (NFYA) Transcription Factor in Creeping Bentgrass (Agrostis stolonifera).

The Nuclear Transcription Factor Y subunit A (NFYA) is a key regulatory component of the NF-Y heterotrimeric complex, crucial for transcriptional regulation in eukaryotes. In plants, the diversification of the NFYA gene family enables specialized functions in growth, development, and stress adaptation. This study focuses on characterizing AsNFYA1 in creeping bentgrass (Agrostis stolonifera), a non-model turfgrass species. Preliminary findings confirm miR169 as a post-transcriptional regulator of AsNFYA1, with 5′ RACE verifying miRNA-mediated mRNA cleavage. Analysis of the AsNFYA1 overexpression (OE) and knockdown (KO) lines indicates that AsNFYA1 positively regulates plant development, as evidenced by enhanced plant growth in OE lines but reduced biomass production in KO lines. To identify direct targets, ChIP-seq will map AsNFYA1 binding sites, linking its regulatory activity to stress-responsive and developmental genes. Additionally, yeast two-hybrid assays and AlphaFold 3 predictions will explore protein-protein interactions, including its role in forming functional NF-Y complexes. This study enhances our understanding of NFYA’s multifaceted roles in plants and offers insights for improving stress resilience in crops through targeted genetic strategies.

Zhaohui Chen, Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634. Abstract Presentation: P-2033

2025 GORDON SATO AND WALLY MCKEEHAN AWARD

Discovery and Initial Characterization of a Diverse Family of Microproteins Derived from Alternative Open Reading Frames in SCN(NaV) Genes

Microproteins (MPs) are a growing class of short peptides that can arise from upstream open reading frames. Recent advances in ribosome profiling have uncovered a myriad of MPs that serve vital functions in ion channel modulation, cell signaling and energetics, and more. However, there remains a need to continue characterizing novel MPs to determine their expression, function, and disease relevance. The SCN(NaV) gene family encodes voltage gated sodium channels that control sodium ion influx and trigger action potentials in excitable cells (e.g. cardiomyocytes and neurons). Our lab recently discovered a MP encoded by SCN5A (SCN5A-MP) that is generated by translation of an alternative open reading frame (altORF). We find that overexpressed SCN5A-MP can be detected in mouse hearts by western blot. In addition, a peptide matching SCN5A-MP sequence has been reported in human heart proteomics data, and western blot on human cardiac tissue lysates reveals bands co-migrating with transgene-derived SCN5A-MP. We subsequently identified several other SCN(NaV) family genes that may also encode MPs via putative altORFs. In follow-up studies, we used C-terminal V5-tagged reporter constructs to demonstrate that these altORFs are indeed translated and to assess where these altORF-derived MPs reside within cells, revealing diverse localizations. Notably, immunoblotting using custom antibodies generated against several of these altORF-derived MPs demonstrates that these are generally unstable. This reiterates the need for in vivo characterizations, particularly since peptide fragments matching these MPs have been identified in prior proteomics investigations done in human tissues. Overall, this work has uncovered the potential for several SCN(NaV) gene family members to encode a diverse panel of MPs, and future studies are needed to further characterize these and assess their potential relevance to a breadth of diseases that have been associated with this important gene family, including arrhythmias, epilepsy, neuropathy, neuromuscular disorders, and sudden death syndromes.

Jasmyn Hoeger, Department of Internal Medicine, Abboud Cardiovascular Research Center, Carver College of Medicine, University of Iowa, Iowa City, IA. Abstract Presentation: A-1000

2025 PHILIP R. WHITE AWARD

A Novel Type I-F CRISPR System Confers Kilobase-scale Genomic Deletions at GC-rich PAMs in Plants

CRISPR-Cas systems have provided remarkably capable tools for editing plant genomes. Type II and Type V CRISPR-Cas nucleases, Cas9 and Cas12a, respectively, are commonly used to generate small, site-specific indels. However, achieving large genomic deletions with high efficiency in plants remains challenging using these canonical CRISPR-Cas systems. Type I CRISPR systems, which encode the multi-effector Cascade machinery, are known to generate large-scale deletions but have received comparatively little attention for plant genome editing. Moreover, several Type I subtypes have been unexplored for use in plants, leaving untapped potential for novel variants possessing nuclease activity at alternative PAMs and enhanced editing efficiency. Aided by genome-database discovery tools, we uncover a Type I-F CRISPR system derived from Methylomonas methanica (MmeCascade). When expressed in transgenic rice plants, the MmeCascade, harboring a unique Cas3-Cas2 fusion, results in large genomic deletions on the order of several kilobases at two genomic target sites containing GC-rich PAMs. Strikingly, deletions elicited by MmeCascade are bidirectional and occur with high editing efficiency. To complement this potent genome editor, we adapt a genome editing analysis tool for compatibility with Oxford Nanopore DNA sequencing of long-range PCR amplicons, complete with target site interrogation and data visualization capabilities. Equipped with this novel Type I-F CRISPR system, engineering large-scale genomic perturbations in plants is achievable with high efficiency, enabling investigations into the functional roles of long non-coding RNAs, cis-regulatory elements, and gene clusters in plants.

Joshua Clem, Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD. Abstract Presentation: P-1006

2025 JOSEPH F. MORGAN AWARD AND 2025 CELLULAR TOXICOLOGY AWARD

Reinforcing Plant Innate Immunity Through Ectopic Expression of Host Defense Peptides

The enhancement of plant innate immunity is an effective strategy to improve crop resistance to diseases. Small cationic antimicrobial peptides called host defense peptides (HDPs) are naturally expressed by organisms in response to invading microorganisms. The ectopic expression of potent HDPs in susceptible plant varieties can be used for engineering plants with improved resistance to a broad spectrum of pathogens. In this study, HDPs of plant origins were investigated for their antimicrobial and cytotoxic activities and subsequently introduced in cultivated potato (Solanum tuberosum L., cultivar Desiree) to improve its disease resistance. Five HDPs were tested individually and in combinations for antimicrobial activity against a range of economically important fungal and bacterial pathogens in vitro. Cytotoxicity was assessed by challenging potato mesophyll protoplasts and mammalian cells in colorimetric viability assays. Single HDP genes and their combinations were introduced into potato by Agrobacterium-mediated transformation. Transgene integration events were confirmed by PCR analysis, gene copy-number assessed by qPCR, gene transcription measured by RT-qPCR, and peptide expression verified by western analysis. In the in vitro bioassays, HDPs SM-985, Ib-AMP 1Q, shepherin 1, and P4650 inhibited pathogen growth when used as single peptides. The most effective three-peptide combinations completely inhibited fungal growth at concentrations of 20–50 µM and bacterial growth at 2.5 µM. HDPs interacted synergistically in combinations, lowering minimum inhibitory concentrations by up to 83%. Observed cytotoxicity was low. The combination of SM-985, Ib-AMP 1Q and shepherin 1 was selected for plant transformation due to its significant antimicrobial activity and lack of cytotoxicity to plant and mammalian cells. Fifty-eight transgenic plant lines have been generated, with delayed disease progression observed as compared with controls. This study will identify novel HDPs for engineering disease resistance in crops and contribute to improving food security.

Nick Schimpf, Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, T1K3M4, CANADA. Abstract Presentation: P-2010

2025 STUDENT TRAVEL AWARD

Peptide REF1 Enhances Efficient Transformation and Impacts the Regeneration in Agrobacterium-mediated Citrus Epicotyl Transformation

Citrus is one of the most widely cultivated fruit crops globally. However, conventional breeding has faced significant challenges in enhancing disease resistance due to the prolonged juvenile phase and other limitations. CRISPR-mediated genome editing offers a promising approach for genetic improvement in plants. However, generating biallelic or homozygous mutants in sweet orange remains challenging due to low transformation and regeneration efficiencies. Interestingly, a plant elicitor peptide (Pep), REGENERATION FACTOR1 (REF1) is known to promote the regeneration and transformation efficiency in tomato. Here, we aim to test whether REF1 can enhance transformation and regeneration efficiencies in citrus. For this purpose, we have synthesized CsREF, CgREF, and SgREF based on their protein sequences in Citrus sinensis, C. paradisi and tomato. Next we tested whether CsREF, CgREF, and SgREF at concentrations of 1 nM, 10 nM, and 100 nM can improve Agrobacterium-mediated transformation. Our results showed that all three REFs can enhance transformation and regeneration. Notably, 100 nM CsREF yielded the highest transformation efficiency, increasing it by 4.8-fold compared with the control. In sum, REF application can enhance transformation and regeneration efficiencies in citrus, thus facilitating genetic study or genetic improvements of citrus.

Yu Feng, Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, Lake Alfred, FL  Abstract Presentation: P-2000

2025 STUDENT TRAVEL AWARD

Genome-wide Analysis of the SPL Gene Family in Lettuce and Its Role in miR156/SPL Module-mediated Plant Development and Regeneration.

Lettuce (Lactuca sativa) is a globally significant leafy vegetable. This study identified 22 SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes in the lettuce genome, with 14 containing recognition sites for microRNA156, suggesting post-transcriptional regulation. Each LsSPL protein possesses the conserved SBP domain and is predicted to localize in the nucleus. Analysis of public RNA-seq datasets revealed tissue-specific expression patterns of the 22 LsSPL genes, with five predominantly expressed in leaves, four in roots, and three in stems, suggesting their distinct roles in plant development. Overexpression of lettuce miRNA156c (miR156-OX) resulted in reduced leaf size and delayed flowering, while suppression (miR156-STTM) resulted in increased leaf size. Surprisingly, cotyledon explants from miR156-OX lines exhibited a 1.9-fold increase in shoot regeneration compared to wild-type, whereas miR156-STTM lines exhibited a 54.3% decrease. A similar enhancement in in-vitro shoot regeneration was observed in ectopic miR156-overexpression tomato lines, indicating a conserved mechanism. Quantitative RT-PCR analysis confirmed the downregulation of LsSPL13A.1, LsSPL13A.2, and LsSPL12.2 in miR156-OX lines and their upregulation in miR156-STTM lines after 5 days of callus induction, implicating their specific roles in in vitro organogenesis and plant regeneration. This comprehensive analysis provides valuable insights into the SPL gene family and the miR156-SPLs regulatory network, specifically highlighting its role in regeneration. These findings have potential applications in enhancing plant growth, development, and biotechnology.

Keila Rodriguez, Crop Transformation Center, Horticulture Science Department, University of Florida, Gainesville, FL and Mid-Florida Research and Education Center, University of Florida, Apopka, FL. Abstract Presentation: P-2048