Biology subjects

Bart, R. S.

Publications and source records attributed to Bart, R. S..

7 recordsLinked to original sources

bioRxiv2026

Multiple approaches for CRISPR-based targeting of DNA methylation to promoters of bacterial and viral susceptibility genes in cassava

Targeted epigenetic modifications of specific gene regulatory regions have the potential to confer beneficial traits for crop improvement. Two recently developed CRISPR/Cas9-based epigenome editing tools were tested in transgenic cassava to target cytosine methylation to the promoter region of MeSWEET10a, a necessary gene for infection by the Cassava bacterial blight pathogen, Xanthomonas phaseoli pv. manihotis. The two systems leverage unique methyltransferases, and each induced distinct DNA methylation profiles at the targeted site, decreased effector-triggered MeSWEET10a expression, and attenuated water-soaking symptoms in inoculated leaves. Further, DNA methylation was simultaneously targeted, in addition to MeSWEET10a, to two susceptibility genes for Cassava brown streak virus. Relative levels of de novo DNA methylation at the three loci were inversely correlated with DNA methylation-antagonizing H3K4me3 marks. Finally, an initial assessment of DNA methylation after one generation indicated specific inheritance of CpG methylation that was unstable in the absence of the methyltransferase systems.

plant biology↗

bioRxiv2025

Targeting dCas9-SunTag to a susceptibility gene promoter is sufficient for CRISPR interference

Cassava production in sub-Saharan Africa is severely impacted by diseases. Most pathogens require interaction with host susceptibility factors to complete their life cycles and cause disease. Targeted DNA methylation is an epigenetic strategy to alter gene expression in plants and we previously reported that a zinc-finger fused to DMS3 could establish methylation at the promoter of MeSWEET10a, a bacterial susceptibility gene, and this resulted in decreased disease. Here, we attempt a similar strategy for cassava brown streak disease. This disease is caused by the ipomoviruses CBSV and UCBSV. These viruses belong to the family Potyviridae, which has been shown extensively to require host eIF4E-family proteins to infect plants and cause disease. We previously found that cassava plants with simultaneous knockout mutations in two eIF4E genes, nCBP-1 and nCBP-2, resulted in decreased susceptibility to CBSD. Here, we report successful simultaneous targeting of both promoters with methylation using a dCas9-DMRcd-SunTag system. However, in contrast to our previous work with MeSWEET10a, controls indicate that CRISPR interference is occurring in these lines and is sufficient for reduction of gene expression. Future research will use genetic crosses to segregate away the DNA methylation reagents and, if DNA methylation proves heritable, assess whether methylation alone is sufficient increase resistance to CBSD.

plant biology↗

bioRxiv2022

Haplotype resolved DNA methylome of African cassava genome

Cytosine DNA methylation is involved in biological processes such as transposable element (TE) silencing, imprinting, and X chromosome inactivation. Plant methylation is mediated by MET1 (mammalian DNMT1), DRM2 (mammalian DNMT3), and two plant-specific DNA methyltransferases, CMT2 and CMT3 (Law and Jacobsen, 2010). De novo DNA methylation in plants is established by DRM2 via the plant specific RNA-directed DNA methylation (RdDM) pathway that depends on two DNA-dependent RNA polymerases, Pol IV and Pol V (Gallego-Bartolome et al., 2019; Law and Jacobsen, 2010; Stroud et al., 2013). The DNA methylome of cassava has been previously documented based on its haploid collapsed genome (Wang et al., 2015). Since the cassava genome is highly heterozygous, DNA methylome analysis of the haplotype-collapsed genome misses many features of the methylome. With the development of long read sequencing and chromosomal conformation capture techniques, haplotype resolved genomes are available for highly heterozygous genomes (Mansfeld et al., 2021; Qi et al., 2022; Zhou et al., 2020), which provides high-quality reference genomes facilitating studies of haplotype resolved DNA methylomes.

plant biology↗

bioRxiv2022

A comparison of ImageJ and machine learning based image analysis methods to measure cassava bacterial blight disease severity

BackgroundMethods to accurately quantify disease severity are fundamental to plant pathogen interaction studies. Commonly used methods include visual scoring of disease symptoms, tracking pathogen growth in planta over time, and various assays that detect plant defense responses. Several image-based methods for phenotyping of plant disease symptoms have also been developed. Each of these methods has different advantages and limitations which should be carefully considered when choosing an approach and interpreting the results. ResultsIn this paper, we developed two image analysis methods and tested their ability to quantify different aspects of disease lesions in the cassava-Xanthomonas pathosystem. The first method uses ImageJ, an open-source platform widely used in the biological sciences. The second method is a few-shot support vector machine learning tool that uses a classifier file trained with five representative infected leaf images for lesion recognition. Cassava leaves were syringe infiltrated with wildtype Xanthomonas, a Xanthomonas mutant with decreased virulence, and mock treatments. Digital images of infected leaves were captured overtime using a Raspberry Pi camera. The image analysis methods were analyzed and compared for the ability to segment the lesion from the background and accurately capture and measure differences between the treatment types. ConclusionsBoth image analysis methods presented in this paper allow for accurate segmentation of disease lesions from the non-infected plant. Specifically, at 4-, 6-, and 9-days post inoculation (DPI), both methods provided quantitative differences in disease symptoms between different treatment types. Thus, either method could be applied to extract information about disease severity. Strengths and weaknesses of each approach are discussed.

plant biology↗

bioRxiv2022

Mutations in DNA polymerase δ subunit 1 mediate CMD2-type resistance to Cassava Mosaic Geminiviruses

Cassava mosaic disease suppresses cassava yields across the tropics. The dominant CMD2 locus confers resistance to the cassava mosaic geminiviruses. It has been reported that CMD2-type landraces lose resistance after regeneration through de novo morphogenesis. As full genome bisulfite sequencing failed to uncover an epigenetic mechanism for loss of resistance, we performed whole genome sequencing and genetic variant analysis and fine-mapped the CMD2 locus to a 190 kilobase interval. Data suggest that CMD2-type resistance is caused by a nonsynonymous, single nucleotide polymorphism in DNA polymerase {delta} subunit 1 (MePOLD1) located within this region. Virus-induced gene silencing of MePOLD1 in a Cassava mosaic disease-susceptible cassava variety produced a recovery phenotype typical of CMD2-type resistance. Analysis of other CMD2-type cassava varieties identified additional resistance alleles within MePOLD1. MePOLD1 resistance alleles represent important genetic resources for resistance breeding or genome editing, and elucidating mechanisms of resistance to geminiviruses.

plant biology↗

bioRxiv2021

Increased signal to noise ratios within experimental field trials by regressing spatially distributed soil properties as principal components.

Environmental variability poses a major challenge to any field study. Researchers attempt to mitigate this challenge through replication. Thus, the ability to detect experimental signals is determined by the degree of replication and the amount of environmental variation, noise, within the experimental system. A major source of noise in field studies comes from the natural heterogeneity of soil properties which create micro-treatments throughout the field. To make matters worse, the variation within different soil properties is often non-randomly distributed across a field. We explore this challenge through a sorghum field trial dataset with accompanying plant, microbiome and soil property data. Diverse sorghum genotypes and two watering regimes were applied in a split-plot design. We describe a process of identifying, estimating, and controlling for the effects of spatially distributed soil properties on plant traits and microbial communities using minimal degrees of freedom. Importantly, this process provides a tool with which sources of environmental variation in field data can be identified and removed, improving our ability to resolve effects of interest and to quantify subtle phenotypes. IMPORTANCEData from field experiments are notoriously noisy. Proper field designs with high replication aid in mitigating this challenge, yet true biological correlations are still often masked by environmental variability. This work identifies soil property composition as a spatially distributed source of variance to three types of characteristics: plant phenotype, microbiome composition, and leaf traits. We show that once identified, spatial principal component regression was able to account for these effects so that more precise estimates of experimental factors were obtained. This generalizable method is applicable to diverse field experiments.

systems biology↗

bioRxiv2020

Visualizing cassava bacterial blight at the molecular level using CRISPR-mediated homology-directed repair

Research on a few model, plant-pathogen systems has benefitted from years of tool and resource development. This is not the case for the vast majority of economically and nutritionally important plants, creating a crop improvement bottleneck. Cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv. manihotis (Xam), is an important disease in all regions where cassava (Manihot esculenta Crantz) is grown. Here we describe development of a tool for molecular-level visualization of CBB dynamics in vivo. Using CRISPR-mediated homology-directed repair (HDR), we generated plants containing scarless insertion of a GFP reporter at the CBB susceptibility (S) gene MeSWEET10a. Activation of MeSWEET10a-GFP by Xam was subsequently visualized at transcriptional and translational levels. Development of this tool was facilitated by a time-saving, adaptable strategy for identifying successful products of HDR, currently a limiting factor in plant research. This strategy has the potential to enable such research in other systems, improving the practicality of HDR-based experimentation.

plant biology↗