About

Linda Hanley-Bowdoin is the William Neal Reynolds Distinguished Professor in the Department of Plant and Microbial Biology at North Carolina State University. Her research focuses on plant and microbial biology, particularly on geminiviruses, a large family of plant viruses with circular, single-stranded DNA genomes that replicate through double-stranded intermediates. Her work investigates how geminiviruses depend on host DNA polymerases to replicate and how they replicate in differentiated plant cells that lack detectable levels of these host factors. She is involved in developing resistance strategies against these viruses by working on peptide aptamers and trans-dominant negative mutants to interfere with viral replication, collaborating with researchers in Africa and Asia to implement these technologies in key crops. Additionally, her research extends to characterizing plant origins of replication, understanding their relationship to genome structure, DNA methylation, histone modifications, and transcriptional activity. Her contributions include advancing knowledge of DNA replication and transcription in plants, with a focus on virus-host interactions, genome organization, and resistance mechanisms.

Selected publications

• Development of Modular Geminivirus‐Based Vectors for High Cargo Expression and Gene Targeting in Plants

  Plant Biotechnology Journal · 2025-09-21 · 5 citations

  articleOpen access

  Viral vectors can be useful tools for expressing recombinant proteins as well as delivering gene-editing machinery. Despite their utility, the development and subsequent optimisation of these tools is often a difficult and tedious process. Thus, although considerable work has been done to create useful viral vectors for gene editing and protein expression, there is a lack of understanding of how best to design these vectors for specific applications. For instance, it is often unclear whether the inclusion of heterologous promoter sequences or different viral components will improve cargo expression or replicon accumulation. To address some of these hurdles, we designed a GoldenBraid (GB)-compatible viral vector system based on the geminivirus-beet curly top virus (BCTV). This system allows for simple, modular cloning of a variety of reporter constructs. Making use of this modular cloning strategy, we compared a variety of alternative viral vector architectures. Interestingly, native BCTV promoters outperformed the constitutive 35S promoter, while the removal of the BCTV virion-sense genes promoted reporter expression. Intriguingly, these modifications had no effect on total replicon accumulation. These results show the utility of the new modular BCTV-based vectors for protein expression and gene targeting applications, as well as uncover design principles that may inform future geminivirus-based viral vector architectures. We anticipate that the availability of this new modular system will spark the broad adoption of replicon-based strategies in protein expression and gene editing experiments in plants.

  PublisherOA PDFDOI

• Replication Timing Uncovers a Two-Compartment Nuclear Architecture of Interphase Euchromatin in Maize

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-07-17 · 2 citations

  preprint

  ABSTRACT Genome replication is temporally regulated during S phase, with specific genomic regions replicating at defined times in a process that is known as replication timing (RT). Based on 3D cytology in replicating nuclei, we previously proposed a “mini-domain chromatin fiber RT model” for maize euchromatin that suggested it is subdivided into early-S and middle-S compartments distinguished by chromatin condensation and RT. However, whether this compartmentalization reflects a general nuclear architecture that persists throughout the cell cycle was unclear. To test this model, we conducted two orthogonal assays – Hi-C for genome-wide interaction data and 3D FISH for direct visualization of chromatin organization. Hi-C eigenvalues and insulation scores revealed distinct patterns of early-S regions having negative insulation scores with long-range contacts while middle-S regions showed the opposite. Early-S regions correlated more strongly with epigenomic signatures of open, transcriptionally active chromatin than middle-S regions. 3D oligo FISH painting confirmed that early-S and middle-S regions occupy adjacent but largely non-overlapping nucleoplasmic spaces during all interphase stages, including G1. Our findings redefine the maize euchromatin “A” compartment as having two distinct, closely interspersed subcompartments, early-S and middle-S, underscoring the importance of replication timing (RT) as a defining feature of chromatin architecture and genome organization.

  PublisherDOI

• SEGS-1 episomes generated during cassava mosaic disease enhance disease severity

  Frontiers in Plant Science · 2025-01-10 · 2 citations

  articleOpen accessSenior authorCorresponding

  Cassava is an important root crop that is produced by smallholder farmers across Sub-Saharan Africa. Cassava mosaic disease (CMD), which is caused by a group of cassava mosaic begomoviruses (CMBs), is one of the most devastating diseases of cassava. A previous study showed that SEGS-1 (sequences enhancing geminivirus symptoms), which occur both in the cassava genome and as episomes during CMD, can increase CMD disease severity and overcome host resistance. In this report, we examined the effects of exogenously applied SEGS-1 on the incidence of CMB infection, symptom severity, and viral DNA copy number in five cassava cultivars that ranged from highly susceptible to highly resistant to CMD. These studies revealed that the effect of SEGS-1 is cultivar dependent. Susceptible cultivars developed severe CMD with or without exogenous SEGS-1, while exogenous SEGS-1 increased disease severity in cultivars carrying CMD2 resistance, which is conferred by a single locus, but not CMD1 resistance, which is polygenic. Analysis of infected plants in the absence of exogenous SEGS-1 revealed that some, but not all, cultivars form SEGS-1 episomes during CMD. The presence of endogenous SEGS-1 episomes in TME14, a CMD2 resistant cultivar, correlated with CMD severity. In contrast, TME3, a closely related CMD2 cultivar, did not produce endogenous SEGS-1 episomes and was more resistance than TME14. The different capacities of TME3 and TME14 to form SEGS-1 episomes is unlikely due to sequence differences in and around their genomic SEGS-1 loci. The functional regions of SEGS-1 were mapped using TME3 to sequences flanking the episome junction, but the junction itself was not required for activity. All cassava cultivars have SEGS-1 sequences in their genomes that have the potential to negatively impact the development of stable CMD resistance by cassava breeding programs.

  PublisherOA PDFDOI

• Replication Timing Uncovers a Two-Compartment Nuclear Architecture of Interphase Euchromatin in Maize

  Research Square · 2025-03-12

  preprintOpen access
  PublisherOA PDFDOI

• Comparison of transcriptional activity profiling by metabolic labeling or nuclear <scp>RNA</scp> sequencing

  The Plant Journal · 2025-08-01

  articleOpen access

  SUMMARY The application of high‐throughput sequencing to cellular transcriptome profiling (RNA‐seq) has enabled significant advances in our understanding of gene expression in plants. However, conventional RNA‐seq data reports mainly cytoplasmic transcript abundance rather than actual transcription rates. As a result, it is less sensitive to detect unstable and low‐abundance nuclear RNA species, such as long non‐coding RNAs, and is less directly connected to chromatin features and processes such as DNA replication. To bridge this gap, several protocols have been established to profile newly synthesized RNA in plants and other eukaryotes. These protocols can be technically challenging and present their own difficulties and limitations. Here we analyze newly synthesized nuclear RNA metabolically labeled in vivo with 5‐ethynyl uridine (EU‐nuclear RNA) in maize ( Zea mays L.) root tips and compare it with the entire nuclear RNA population. We also compare both nuclear RNA preparations to conventional RNA‐seq analysis of cellular RNA. The transcript abundance profiles of protein‐coding genes in nuclear RNA and EU‐nuclear RNA were tightly correlated with each other ( R 2 = 0.767), but quite distinct from that of cellular RNA ( R 2 = 0.170 or 0.293). Nuclear and EU‐nuclear RNA reads are frequently mapped across entire genes, including introns, while cellular reads are predominantly mapped to mature transcripts. Both nuclear and EU‐nuclear RNA exhibited a greater ability to detect both protein‐coding and non‐coding expressed genes.

  PublisherOA PDFDOI

• A <scp>TIR</scp> ‐ <scp>NLR</scp> gene from Arabidopsis Pla‐1 confers resistance to geminivirus infection

  The Plant Journal · 2025-12-01 · 1 citations

  articleOpen access

  Geminiviruses are single-stranded DNA viruses that infect many plant species and cause serious losses in agronomically important crops. An earlier study showed that the Arabidopsis thaliana ecotype Pla-1 is resistant to infection by diverse geminivirus species and mapped the major resistance locus Geminivirus Resistance of Pla-1 1 (GRP1) to chromosome 1. In this study, we fine-mapped the GRP1 locus to a 0.6-Mb region and showed that its strength is gene-dosage-dependent. We also uncovered two minor resistance loci, GRP2 and GRP3, that mapped to chromosomes 3 and 5, respectively, and showed that GRP3 resistance is dependent on GRP1. RNA-Seq analysis of plants inoculated with the geminivirus, cabbage leaf curl virus (CaLCuV, Begomovirus brassicae), showed that AT1G31540, which is located in the GRP1 region and encodes a Toll/interleukin-1 receptor (TIR) type nucleotide-binding leucine-rich repeat receptor (NLR), is upregulated in Pla-1 compared to the susceptible Col-0. AT1G31540 specifies two TIR-NLR isoforms that contain non-synonymous codon differences between the two Arabidopsis ecotypes. Expression of the longer Pla-1 isoform, which includes a dual-segment leucine-rich repeat domain and an integrated domain at the C terminus, conferred CaLCuV resistance to Col-0, resulting in reduced viral DNA accumulation and no leaf chlorosis. In contrast, expression of the shorter isoform, which lacks the second leucine-rich repeat segment and the integrated domain, did not confer resistance. This study established that effector-triggered, TIR-NLR-mediated plant innate immunity contributes to geminivirus defense responses and identified a new host genetic resource to combat these important plant viral pathogens.

  PublisherOA PDFDOI

• A comparison of genomic methods to assess DNA replication timing

  Scientific Reports · 2025-05-22 · 4 citations

  articleOpen accessSenior author

  Replication timing (RT), the temporal order in which genomic regions replicate, is considered a functional feature of multiple cellular processes and chromatin organization. Two approaches to measure RT are the Repli-seq and DNA copy number (also called S/G1) methods. We previously adapted Repli-seq using 5-ethynyl-2'- deoxyuridine (EdU) pulse-labeling and bivariate flow sorting, and while the approach offers high resolution and exposes heterogeneity in timing, the S/G1 method is a simpler, faster and less resource-intensive assessment. Here we modified the S/G1 technique by using EdU labeling (EdU-S/G1) to facilitate better separation of replicating from non-replicating nuclei during flow sorting, which enables the collection of a more pure sample of G1-phase nuclei. When comparing the three methods we found that profiles from the S/G1 and EdU-S/G1 methods are highly correlated with each other and with Repli-seq profiles for early replication. We also found that the EdU-S/G1 approach offers a better representation of replication in early and late S phase than the conventional S/G1 method. However, the high reproducibility of RT profiles among all three methods indicates that considerations of cost and sample availability can drive the decision of which method to choose.

  PublisherOA PDFDOI

• SEGS-1 episomes generated during cassava mosaic disease influence disease severity

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-06-12

  preprintOpen accessSenior authorCorresponding

  Cassava is an important root crop that is produced by smallholder farmers across Sub-Saharan Africa. Cassava mosaic disease (CMD), which is caused by a complex of cassava mosaic begomoviruses (CMBs), is one of the most devastating diseases of cassava. A previous study showed that SEGS-1 (sequences enhancing geminivirus symptoms), which occurs in both the cassava genome and as an episome during CMD, can increase CMD disease severity and overcome host resistance. In this report, we examined the effects of exogenously applied SEGS-1 on the incidence of CMB infection, symptom severity, and viral DNA copy number in five cassava cultivars that ranged from highly susceptible to highly resistant to CMD. These studies revealed that the effect of SEGS-1 is cultivar dependent. Susceptible cultivars developed severe CMD in the absence or presence of exogenous SEGS-1, while exogenous SEGS-1 increased disease severity in cultivars carrying CMD2 but not CMD1 resistance. Analysis of infected plants in the absence of exogenous SEGS-1 revealed that some, but not all cultivars, form SEGS-1 episomes during CMD. The presence of endogenous SEGS-1 episomes in TME14, a CMD2 resistant cultivar, correlated with CMD severity. In contrast, TME3, a closely related CMD2 cultivar, did not produce endogenous SEGS-1 episomes and showed more resistance than TME14. DNA sequence analysis indicated that the different capacities of TME3 and TME14 to form SEGS-1 episomes is unlikely due to sequence differences in and around their genomic SEGS-1 loci. Because of its inability to form episomes, TME3 was used to map the functional regions of SEGS- 1 to sequences flanking the epsiome junction, but junction itself was not required for activity. Together, these experiments provided insight into the functional form of SEGS-1 in cassava and the effect of cassava genotype on SEGS-1 activity.

  PublisherOA PDFDOI

• Early Detection of Plant Virus Infection Using Multispectral Imaging and Machine Learning

  Plant Health Cases · 2024-07-31 · 2 citations

  article

  Abstract Climate change-resilient crops like cassava are projected to play a key role in 21st-century food security. However, cassava production in East Africa is limited by RNA viruses that cause cassava brown streak disease (CBSD). CBSD typically causes subtle or no symptoms on stems and leaves, while destroying the root tissue, which means farmers are often unaware their fields are infected until they have a failed harvest. The subtle symptoms of CBSD have made it difficult to study the spread of the disease in fields. We will use an engineering advancement, our active multispectral imager (MSI), to rapidly determine the infection status of plants in the field in Tanzania. The MSI observes leaves using many different wavelengths, and the resulting light spectra are interpreted by machine learning models trained on cassava leaf scans. Under laboratory conditions, the MSI detects CBSD infection with 95% accuracy at 28 days post-infection, when plants have no visible symptoms. Our multinational team is studying and modeling the spread of CBSD to assess the efficacy of using the MSI to detect and remove infected cassava plants from fields before CBSD can spread. In addition to improving the food security of people who eat cassava in sub-Saharan Africa, our technology and modeling framework may be useful in diseases of other vegetatively propagated crops such as banana/plantain, potato, sweet potato, and yam. Information © The Authors 2024

  PublisherDOI

• Development of modular geminivirus-based vectors for high cargo expression and gene targeting in plants

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-06-29 · 4 citations

  preprintOpen access

  Abstract Viral vectors can be useful tools for expressing recombinant proteins as well as delivering gene-editing machinery. Despite their utility, the development and subsequent optimization of these tools is often a difficult and tedious process. Thus, although considerable work has been done to create useful viral vectors for gene editing and protein expression, there is a lack of understanding of how best to design these vectors for specific applications. For instance, it is often unclear whether the inclusion of heterologous promoter sequences or different viral components will improve cargo expression or replicon accumulation. To address some of these hurdles, we designed a GoldenBraid (GB)-compatible viral vector system based on the geminivirus – Beet curly top virus (BCTV). This system allows for simple, modular cloning of a variety of reporter constructs. Making use of this modular cloning strategy, we compared a variety of alternative viral vector architectures. Interestingly, native BCTV promoters outperformed the constitutive 35S promoter, while the removal of the BCTV virion-sense genes promoted reporter expression. Intriguingly, these modifications had no effect on total replicon accumulation. These results show the utility of the new modular BCTV-based viral vectors for protein expression and gene targeting applications, as well as uncover design principles that may inform future geminivirus-based viral vector architectures. We anticipate that the availability of this new modular system will spark the broad adoption of replicon-based strategies in protein expression and gene editing experiments in plants.

  PublisherOA PDFDOI

Awards & honors

• William Neal Reynolds Distinguished Professor