Biology subjects

Whitham, S.

Publications and source records attributed to Whitham, S..

5 recordsLinked to original sources

bioRxiv2026

Turnip mosaic virus-based gRNA delivery system for plant genome editing

Plant virus-based gRNA delivery systems offer a rapid alternative to stable transformation for CRISPR-mediated genome editing, but potyvirus-based platforms in Cas9-expressing plants are still underexplored. Here, we developed a turnip mosaic virus (TuMV)-based system for gRNA delivery in Cas9-expressing Nicotiana benthamiana and tested whether Csy4-mediated gRNA processing could improve editing efficiency. A TuMV construct carrying a gRNA targeting PHYTOENE DESATURASE (NbPDS) induced detectable editing in both infiltrated and systemic tissues, although editing frequencies were low. Incorporation of the bacterial endoribonuclease Csy4 increased editing efficiencies in the two NbPDS genes, raising editing in infiltrated leaves to 7.1-13.8% for NbPDSa and 7.6-23.0% for NbPDSb, while lower but reproducible editing was detectable in systemic leaves. The TuMV-Csy4 platform also supported editing of a second endogenous target, MAGNESIUM CHELATASE SUBUNIT H (NbChlH), and enabled multiplex editing of NbPDS and NbChlH regardless of guide order. Editing efficiencies were consistently higher in infiltrated leaves than in systemic leaves, and no visible photobleaching or chlorosis was observed in systemic tissues despite confirmed molecular editing. To assess the potential for heritable editing, a tRNAIle mobility element was fused to the NbPDS gRNA. Although this construct increased somatic editing, no albino progeny were recovered after screening approximately 20,000 seedlings, indicating that heritable editing was not achieved under these conditions. Together, these results establish TuMV as a platform for Cas9-based gRNA delivery and show that Csy4-mediated processing improves editing efficiency, supports multiplex targeting, and demonstrates the feasibility of potyvirus-based genome editing systems in plants.

plant biology↗

bioRxiv2026

Effects of atmospheric CO2 levels on the susceptibility of maize to diverse pathogens

Rising atmospheric CO2 has profound implications for crop productivity and food security. Based on studies in C3 plants, elevated CO2 (eCO2) can shape plant-pathogen interactions, although the outcomes are often variable. The question of how eCO2 influences immunity and disease development in C4 plants, such as the globally important cereal crop maize (Zea mays L.), has not been systematically examined. We challenged maize plants grown under ambient CO2 (aCO2, 420 ppm) and eCO2 (550 ppm) with bacterial, viral, fungal, and oomycete pathogens. Plants grown in eCO2 were more susceptible to sugarcane mosaic virus, suggesting compromised antiviral defenses, less susceptible to Clavibacter nebraskensis, Exserohilum turcicum, and Colletotrichum graminicola, and susceptibility to Puccinia sorghi and Pythium sylvaticum was unchanged. Reduced susceptibility to C. nebraskensis was associated with enhanced basal immune responses. These results establish a foundation for dissecting eCO2-responsive defense mechanisms, and they highlight a critical need to understand how eCO2 will impact plant responses to microbes, pests, and abiotic stresses under future conditions.

plant biology↗

bioRxiv2024

Elevated CO2 alters soybean physiology and defense responses, and has disparate effects on susceptibility to diverse microbial pathogens

O_LIIncreasing atmospheric CO2 levels have a variety of effects that can influence plant responses to microbial pathogens. However, these responses are varied, and it is challenging to predict how elevated CO2 (eCO2) will affect a particular plant-pathogen interaction. We investigated how eCO2 may influence disease development and responses to diverse pathogens in the major oilseed crop, soybean (Glycine max [L.] Merr.). C_LIO_LISoybeans grown in ambient CO2 (aCO2, 419 parts per million (ppm)) or in eCO2 (550 ppm) were challenged with bacterial, viral, fungal, and oomycete pathogens, and disease, pathogen growth, gene expression and molecular plant defense responses were quantified. C_LIO_LIIn eCO2, plants were less susceptible to Pseudomonas syringae pv. glycinea (Psg) but more susceptible to bean pod mottle virus, soybean mosaic virus, and Fusarium virguliforme. Susceptibility to Pythium sylvaticum was unchanged, although a greater loss in biomass occurred in eCO2. Reduced susceptibility to Psg was associated with enhanced defense responses. Increased susceptibility to the viruses was associated with reduced expression of antiviral defenses. C_LIO_LIThis work provides a foundation for understanding of how future eCO2 levels may impact molecular responses to pathogen challenge in soybean and demonstrates that agents infecting both shoots and roots are of potential concern in future climatic conditions. C_LI

plant biology↗

bioRxiv2023

A soybean rust effector protease suppresses host immunity and cleaves a 3-deoxy-7-phosphoheptulonate synthase

The devastating soybean rust (SBR) pathogen, Phakopsora pachyrhizi, encodes many secreted proteins, but only two have been functionally characterized for their roles in rust virulence. Here, we demonstrate that transient expression of P. pachyrhizi effector candidate 15 (PpEC15), an aspartic protease, leads to enhanced bacterial growth in planta, suppression of callose deposition, reduced expression of plant defense-related marker genes and suppression of pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species (ROS). Stable expression of PpEC15 in soybean suppresses PAMP-induced ROS production and enhances bacterial growth, indicating that, collectively, PpEC15 suppresses host and non-host innate immune responses. Yeast-two-hybrid and proximity labeling identified putative PpEC15 interacting partners including a peptide-chain release factor (PCRF), a NAC83 (NAM, ATAF, and CUC) transcription factor, and a DAHP (3-deoxy-7-phosphoheptulonate) synthase. We further show that PpEC15 can cleave DAHP but does not cleave PCRF or NAC83. Virus-induced gene silencing of NAC83, PCRF and DAHP altered PAMP-induced ROS production and salicylic acid production, indicating that these proteins may be involved in immune signaling. Collectively, our data show that PpEC15 is conserved across P. pachyrhizi isolates and other economically important rust species and is involved in the suppression of plant basal defense responses. Understanding the role of PpEC15 in P. pachyrhizi virulence will provide a foundation for designing targeted intervention strategies to generate rust-resistant crops.

plant biology↗

bioRxiv2023

Clathrin Light Chains are essential in negative regulation of cell death and immunity in Arabidopsis through interacting with autophagy pathway

Clathrin plays a critical role in clathrin-mediated endocytosis (CME) in plants, and it is required for autophagy in mammals. However, the functional interconnection of clathrin with autophagy has not been firmly established in plants. Here, we demonstrate that loss of function of clathrin light chain (CLC) subunit 2 and 3 results in salicylic acid (SA)- and H2O2-dependent accelerated senescence and activated defense responses in Arabidopsis, which are hallmarks of the autophagy-related gene (ATG) mutants. Similar to atg mutants, the clc2-1clc3-1 double mutant has enhanced sensitivity to both carbon and nitrogen starvation and enhanced resistance to biotrophic bacterial and fungal pathogens. In addition, the autophagy flux was significantly reduced in the roots of clc2-1clc3-1 mutant plants relative to Col-0 plants under carbon starvation conditions. Furthermore, our Yeast-2-hybrid (Y2H) and Luciferase complementation assays showed that CLC2 directly interacted with ATG8h and ATG8i. Mutations within the unique ATG8-interacting motif (AIM) of CLC2 as well as at the LIR/AIM-docking site (LDS) of ATG8h abolished the interaction between CLC2 and ATG8h. As anticipated, both GFP-ATG8h/GFP-ATG8i and CLC2 were subjected to autophagic degradation in the vacuoles. Together, our data revealed that the accelerated senescence and activated immune responses observed in Arabidopsis clc2-1clc3-1 mutant plants result from impaired autophagy, and CLC2 participates in autophagy through direct interactions with ATG8h and ATG8i in an AIM1- and LDS-dependent manner. Our results unveil a previously unidentified link between the function of CLCs and autophagy.

plant biology↗