Biology subjects

McDowell, J.

Publications and source records attributed to McDowell, J..

6 recordsLinked to original sources

bioRxiv2025

Advancing RNAi-Based Strategies Against Downy Mildews: Insights Into dsRNA Uptake and Gene Silencing

Downy mildew (DMs) dieases are caused by destructive obligate pathogens with limited control options, posing a significant threat to global agriculture. RNA interference (RNAi) has emerged as a promising, environmentally sustainable strategy for disease management. In this study, we evaluated the efficacy of dsRNA-mediated RNAi in suppressing key biological functions in DM pathogens of Arabidopsis thaliana, pea and lettuce DM pathogens, Hyaloperonospora arabidopsidis (Hpa), Peronospora viciae f. sp. pisi (Pvp) and Bremia lactucae (Bl), respectively. We specifically targeted the cellulose synthase 3 (CesA3) and the beta tubulin (BTUB) genes. Silencing CesA3 impaired spore germination and infection across multiple species, while BTUB silencing reinforced the potential of dsRNA-mediated inhibition. Reduction in gene expression levels correlated well with the sporulation assays confirming the effectiveness of dsRNA-mediated gene silencing. We used dsRNAs that were chemically synthesized, in vitro transcribed (IVT) or produced in E. coli. We found that the length and concentration of these dsRNAs significantly affected uptake efficiency, spore germination, and sporulation, with higher concentrations enhancing inhibitory effects. Confocal microscopy using Cy-5-labelled short-synthesized dsRNA (SS-dsRNA) provided direct evidence of spore uptake, confirming the potential of SS-dsRNA for pathogen control. However, species-specific sequence variations influenced dsRNA efficacy, underscoring the importance of target sequence design. Multiplexed RNAi impacted silencing synergisticly, further reducing germination and sporulation in Hpa. Additionally, we demonstrated that SS-dsRNA-mediated gene silencing is sustained over time, with a significant reduction in gene expression level at 4, 7, 10 and 11dpi. This indicates the durability and efficacy of this approach. Taken together, these findings demonstrate the potential of dsRNA-mediated gene silencing as a precision tool for managing DM pathogens.

plant biology↗

bioRxiv2024

Synchronization of Circadian Clock Gene Expression in Arabidopsis and Hyaloperonospora arabidopsidis and its Impact on Host-Pathogen Interactions

Organisms across all kingdoms have an internal circadian clock running in 24h cycles. This clock affects a variety of processes, including innate immunity in plants. However, the role of pathogen circadian clocks had not been extensively explored. We previously showed that light can influence infection of the oomycete Hyaloperonospora arabidopsidis (Hpa, downy mildew disease) on its natural host Arabidopsis thaliana. Here, we identified Hpa orthologs of known circadian clock genes (CCGs) Drosophila TIMELESS (TIM) and Arabidopsis Sensitive to Red Light Reduced 1 (AtSRR1) genes. Expression of both HpaTIM and HpaSRR1 showed a circadian rhythm when Hpa was exposed to constant light. Contrastingly, these two genes were negatively regulated by constant dark exposure. Furthermore, the expression patterns of HpaTIM and HpaSRR1 correlate with those of AtCCA1 and AtLHY, indicating a synchronisation of biological clock genes between the host and the pathogen. In addition, screening mutants of Arabidopsis Clock Regulated Genes (AtCRGs) with three virulent Hpa isolates revealed that mutations in AtCRGs influenced HpaTIM and HpaSRR1 expression and Hpa development, indicating a functional link between the plant biological clock and virulence. Moreover, sporulation of Hpa was reduced by targeting HpaTIM and HpaSRR1 with short synthesized small interfering RNAs, indicating that the pathogen clock is also relevant to virulence. We propose that plant and pathogen clocks are synchronized during infection and that proper regulation of both clocks are genetically necessary for pathogen virulence.

plant biology↗

bioRxiv2022

Global connectivity and networks of marine reserves

Cooperation between countries in managing and protecting shared marine resources is beneficial both ecologically and economically, but how best to establish the cooperation needed at a global scale is largely unknown. Here, we used hydrodynamic modelling to identify ecologically connected networks of marine reserves (MRs) and evaluated these networks with socio-economic indicators. Most of the networks are homogenous with similar levels of development, shared languages, and other cultural values. However, we found that 17% (11/66) of the largest networks (>20 MRs) span multiple countries. These heterogenous networks are composed of countries with different economic, political, and cultural views. Countries that control more, larger marine reserves also have a more even ratio of source reserves to sinks. We discuss that, while such economic and cultural homogeneity might lead to more efficient ecological management in the short term, heterogeneous networks may prove to be more resilient in the longer term, when climate change will modify marine connectivity.

systems biology↗

bioRxiv2021

A split GFP system to enhance spatial and temporal sensitivity of translating ribosome affinity purification (TRAP)

Translating ribosome affinity purification (TRAP) utilizes transgenic plants expressing a ribosomal protein fused to a tag for affinity purification of ribosomes and the mRNAs that they are translating. These actively translated mRNAs (translatome) can be interrogated by qPCR or RNAseq. Condition- or cell-specific promoters can be utilized to isolate the translatome of specific cell types, at different growth stages and/or in response to environmental variables. While advantageous for revealing differential expression, this approach may not provide sufficient sensitivity when activity of the condition/cell-specific promoter is weak, when ribosome turnover is low in the cells of interest, or when the targeted cells are ephemeral. In these situations, expressing tagged ribosomes under the control of these specific promoters may not yield sufficient polysomes for downstream analysis. Here, we describe a new TRAP system that employs two transgenes: one is constitutively expressed and encodes a ribosomal protein fused to one fragment of a split GFP; the second is controlled by a stimulus-specific promoter and encodes the second GFP fragment fused to an affinity purification tag. In cells where both transgenes are active, the purification tag is attached to ribosomes by bi-molecular folding and assembly of the split GFP fragments. This approach provides increased sensitivity and better temporal resolution because it labels pre-existing ribosomes and does not depend on rapid ribosome turnover. We describe the optimization and key parameters of this system, and then apply it to a plant-pathogen interaction in which spatial and temporal resolution are difficult to achieve with current technologies. SignificanceTranslating ribosome affinity purification (TRAP) has been modified to allow with increased sensitivity the isolation of RNA from sets of cells in which the activity of condition/cell-specific promoters is weak, ribosome turnover is low, or cells whose nature is ephemeral. Based on the use of a split linker constituted of the GFP driven by a pathogen-inducible promoter, this new TRAP system enabled efficient isolation of translated RNA from pathogen-infected leaf cells.

molecular biology↗

bioRxiv2020

Effect of light and darkness on the growth and development of downy mildew pathogen Hyaloperonospora arabidopsidis.

Disease development in plants requires a susceptible host, a virulent pathogen, and a favourable environment. Oomycete pathogens cause many important diseases and have evolved sophisticated molecular mechanisms to manipulate their hosts. Day length has been shown to impact plant-oomycete interactions but a need exists for a tractable reference system to understand the mechanistic interplay between light regulation, oomycete pathogen virulence, and plant host immunity. Here we present data demonstrating that light is a critical factor in the interaction between Arabidopsis thaliana and its naturally occurring downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). We investigated the role of light on spore germination, mycelium development, sporulation and oospore formation of Hpa, along with defence responses in the host. We observed abundant Hpa sporulation on compatible Arabidopsis under day lengths ranging from 10 to 14 hours. Contrastingly, exposure to constant light or constant dark suppressed sporulation. Exposure to constant dark suppressed spore germination, mycelial development and oospore formation. Interestingly, exposure to constant light stimulated spore germination, mycelial development and oospore formation. A biomarker of plant immune system activation was induced under both constant light and constant dark. Altogether, these findings demonstrate that Hpa has the molecular mechanisms to perceive and respond to light and that both the host and pathogen responses are influenced by the light regime. Therefore, this pathosystem can be used for investigations to understand the molecular mechanisms through which oomycete pathogens like Hpa perceive and integrate light signals, and how light influences pathogen virulence and host immunity during their interactions.

microbiology↗

bioRxiv2019

An oomycete RXLR effector triggers antagonistic plant hormone crosstalk to suppress host immunity

Understanding the mechanisms through which pathogens alter plant cell networks is essential for understanding plant-pathogen interactions and will inform efforts to reduce crop diseases. Oomycetes secrete diverse effector proteins into plant cells. The mechanisms through which these effectors promote virulence are largely unknown. We show that the HaRxL10 effector protein from the Arabidopsis thaliana pathogen Hyaloperonospora arabidopsidis (Hpa) targets a transcriptional repressor (JAZ3) involved in jasmonic acid (JA) signalling. This manipulation activates a regulatory cascade that inhibits salicylic acid (SA) signalling, which normally restricts Hpa infection. This virulence mechanism is functionally equivalent to but mechanistically distinct from activation of the antagonistic JA-SA hormone crosstalk by the bacterial JA-mimicking toxin coronatine and by bacterial Type III effectors. These results reveal a key role for JAZ3 in plant immunity and emphasize that JA-SA crosstalk is an Achilles heel in the plant immune system, vulnerable to manipulation by diverse microbes.

plant biology↗