Biology subjects

Faulkner, C.

Publications and source records attributed to Faulkner, C..

15 recordsLinked to original sources

bioRxiv2026

Colletotrichum higginsianum effector ChEC108 binds a plasmodesmal HMA protein and elicits plant defence

To establish infection, phytopathogens deploy effectors to compromise host defences and facilitate invasive growth. As part of this, the battle for control of symplastic connectivity via plasmodesmata is a key determinant of infection outcomes, yet little is known about how fungal effectors directly exploit these channels, and in turn, how hosts defend them. Here, we have identified ChEC108 as a plasmodesmal-targeting, cell-to-cell mobile effector from the anthracnose fungus, Colletotrichum higginsianum. ChEC108 binds the plasmodesmal protein HEAVY METAL-ASSOCIATED (HMA) ISOPRENYLATED PLANT PROTEIN 6 (HIPP6) from Arabidopsis via a tetrahedral metal ion coordination site with either of its HMA domains. Constitutive in planta expression of ChEC108 induces plasmodesmal closure and the upregulation of defence-associated genes in a manner dependent on its capacity to bind HIPP6. Further, HIPP6 binding impairs cell-to-cell mobility of ChEC108. Alongside the finding that loss of ChEC108 favoured C. higginsianum infection, this suggests ChEC108-HIPP6 interaction at plasmodesmata positively regulates defence.

plant biology↗

bioRxiv2025

Primary metabolism underpins the execution of immune responses in different tissues of the same plant

Cell-surface perception of microbes triggers a range of rapid immune responses in plants that include the induction of cell isolation by plasmodesmal closure, the production of reactive oxygen species, and changes to gene expression. Here, we identify that some of these immune response are differentially executed in leaves of different ages in the same plant. We observed that when compared to mature leaves, young, expanding leaves do not close their plasmodesmata, have a reduced transcriptional response to immune elicitors, and are more susceptible to a bacterial pathogen. Disconnecting leaf age from physiology, we determined that both plasmodesmal closure and the magnitude of transcriptional responses are dependent on whether the leaf is a carbon sink or a carbon source. To probe the relevance of differential regulation of plasmodesmata in sink and source tissues, we forced plasmodesmal closure in young sink leaves and found this perturbed the normal outputs of growth and defence. Thus, we propose that sink leaves do not close their plasmodesmata during immune reponses to prioritise carbon use for growth over defence, and consequently that primary metabolism underpins different immune response profiles in different leaves of the same plant.

plant biology↗

bioRxiv2024

Putative neofunctionalization of a Poales-specific EXO70 clade

EXO70s are uniquely expanded in land plants compared to all other eukaryotic lineages. The functional implications of this expansion and diversification on the conserved role of EXO70 as a subunit of the octameric exocyst complex have remained unresolved. We previously demonstrated barley (Hordeum vulgare) EXO70FX12, a member of the monocot-specific EXO70FX clade, is required for resistance to wheat stripe rust in conjunction with the leucine-rich repeat receptor kinase (LRR-RK) HvPUR1. Through phylogenetic analysis, we identified unique features of the EXO70FX clade, leading us to hypothesize that this clade experienced neofunctionalization. Using structural predictions and protein-protein interaction assays, we demonstrate that HvEXO70FX12 lost the ability to serve as a subunit within the exocyst complex. We predict that the EXO70FX clade has largely lost exocyst association and represents a novel acquisition that emerged during Poales diversification for immunity.

plant biology↗

bioRxiv2024

Re-analysis of mobile mRNA datasets highlights challenges in the detection of mobile transcripts from short-read RNA-Seq data

Short-read RNA-Seq analyses of grafted plants have led to the proposal that large numbers of mRNAs move over long distances between plant tissues, acting as potential signals. The detection of transported transcripts by RNA-Seq is both experimentally and computationally challenging, requiring successful grafting, delicate harvesting, rigorous contamination controls and data processing approaches that can identify rare events in inherently noisy data. Here, we perform a meta-analysis of existing datasets and examine the associated bioinformatic pipelines. Our analysis reveals that technological noise, biological variation and incomplete genome assemblies give rise to features in the data that can distort the interpretation. Taking these considerations into account, we find that a substantial number of transcripts that are currently annotated as mobile are left without support from the available RNA-Seq data. Whilst several annotated mobile mRNAs have been validated, we cannot exclude that others may be false positives. The identified issues may also impact other RNA-Seq studies, in particular those using single nucleotide polymorphisms (SNPs) to detect variants.

plant biology↗

bioRxiv2024

Plasmodesmal closure elicits stress responses

Plant cells are connected to their neighbors via plasmodesmata facilitating the exchange of nutrients and signaling molecules. During immune responses, plasmodesmata close, but how this contributes towards a full immune response is unknown. To investigate this, we developed two transgenic lines with which we could induce plasmodesmal closure independently of immune elicitors, using the over-active CALLOSE SYNTHASE3 allele icals3m and the C-terminus of PDLP1 to drive callose deposition at plasmodesmata. Induction of plasmodesmal closure increased the expression of stress responsive genes, salicylic acid accumulation and resistance to Pseudomonas syringae DC3000. More homogeneous plasmodesmal closure using icals3m also led to the accumulation of starch and sugars, decreased leaf growth, as well as hypersusceptibility to Botrytis cinerea. Based on the profile of responses, we conclude that plasmodesmal closure itself activates stress signaling, raising questions of what signals mediate this response and whether these responses occur in all circumstances when plasmodesmata close.

plant biology↗

bioRxiv2023

Cell-specialized chloroplast signaling orchestrates photosynthetic and extracellular reactive oxygen species for stress responses

Cellular responses to abiotic stress involve multiple signals including secondary messengers such as reactive oxygen species (ROS) and Ca2+, phytohormones such as abscisic acid (ABA) and chloroplast-to-nucleus retrograde signals such as 3-phosphoadenosine 5-phosphate (PAP). Mechanism(s) by which these messengers, produced in different subcellular compartments, intersect for cell regulation remain enigmatic. Previously we showed that the chloroplast retrograde signal PAP, similar to ABA, induces an increase in ROS levels in guard cells (Pornsiriwong et al, 2017). Here we demonstrate a mechanistic link enabling ABA and PAP to coordinate both chloroplast and plasma membrane ROS production. In whole leaves, PAP alters various ROS-related processes including plasmodesmal permeability as well as responses to ozone and the bacterial elicitor flg22, but mainly initiates processes that quench ROS during oxidative stress. Conversely, we show in guard cells, both PAP and ABA induce an increase in ROS levels in both chloroplasts via photosynthetic electron transport, and the apoplast via the RESPIRATORY BURST OXIDASE HOMOLOG (RBOH). Both subcellular ROS sources were necessary for ABA- and PAP-mediated stomatal closure. However, PAP signaling diverges from ABA by activating RBOHD, instead of RBOHF, for apoplastic ROS mediated stomatal closure. We identified three calcium-dependent protein kinases (CPKs) as the post-translational activators of RBOHD-mediated ROS production. CPK13, CPK32, and CPK34 were transcriptionally induced by PAP and concurrently activate RBOHD and the slow anion channel SLAC1 by phosphorylating two Serine (S) residues, including S120 which is also targeted by the core ABA signaling kinase OPEN STOMATA 1 (OST1). Consequently, overexpression of the PAP-induced CPKs rescues stomatal closure in ost1. Our data identify stomatal chloroplasts, to be nodes in the multifaceted cellular stress response networks as they are both sources and mediators of ROS and retrograde signals such PAP. Thus, chloroplasts are not just mediators of photosynthesis in response to, for example, excess light, but can serve as critical nodes in the multifaceted cellular stress response networks in specialized cells via retrograde signals, providing support to the concept of sensory plastids. Significance StatementThe chloroplast is an environmental sensor for stresses such as excess light and drought via the activation of photosynthetic-mediated retrograde signals. However, how does it function in specialized cells for which carbon fixation is secondary? Here we show the chloroplast is an important node to coordinate multiple plant signaling pathways in response to stresses such as drought. The chloroplast retrograde signal 3-phosphoadenosine 5-phosphate (PAP) plays multiple roles in reactive oxygen species (ROS) signaling and homeostasis. While PAP suppresses ROS in photosynthetic tissue, PAP instead induces guard cell ROS in chloroplasts and extracellular space to induce stomatal closure. We decipher how PAP-induced proteins activate both extracellular ROS production and anion channels for stomatal closure, thus providing a mechanism by which chloroplasts provide a strategic complement to canonical hormonal pathways in regulating plant physiological responses in specialized cells.

plant biology↗

bioRxiv2022

Plasmodesmal connectivity in C4 Gynandropsis gynandra is induced by light and dependent on photosynthesis

O_LIIn leaves of C4 plants the reactions of photosynthesis become restricted between two compartments. Typically, this allows accumulation of C4 acids in mesophyll cells and subsequent decarboxylation in the bundle sheath. In C4 grasses proliferation of plasmodesmata between these cell types is thought to increase cell-to-cell connectivity to allow efficient metabolite movement. However, it is not known if C4 dicotyledons also show this enhanced plasmodesmal connectivity and so whether this is a general requirement for C4 photosynthesis is not clear. How mesophyll and bundle sheath cells in C4 leaves become highly connected is also not known. C_LIO_LIWe investigated these questions using 3D- and 2D- electron microscopy on the C4 dicotyledon Gynandropsis gynandra as well as phylogenetically close C3 relatives. C_LIO_LIThe mesophyll-bundle sheath interface of C4 G. gynandra showed higher plasmodesmal frequency compared with closely related C3 species. Formation of these plasmodesmata was induced by light. Pharmacological agents that perturbed chloroplast development or photosynthesis reduced the number of plasmodesmata, but this inhibitory effect could be reversed by the provision of exogenous sucrose. C_LIO_LIWe conclude that enhanced formation of plasmodesmata between mesophyll and bundle sheath cells is wired to the induction of photosynthesis in C4 G. gynandra. C_LI

plant biology↗

bioRxiv2022

A PDLP-NHL3 complex integrates plasmodesmal immune signaling cascades

The plant immune system relies on the perception of molecules that signal the presence of a microbe threat. This triggers signal transduction that mediates a range of cellular responses via a collection of molecular machinery including receptors, small molecules, and enzymes. One response to pathogen perception is the restriction of cell-to-cell communication by plasmodesmal closure. We previously found that while chitin and flg22 trigger specialized immune signaling cascades in the plasmodesmal plasma membrane, both execute plasmodesmal closure via callose synthesis at the plasmodesmata. Therefore, the signaling pathways ultimately converge at or upstream of callose synthesis. To establish the hierarchy of signaling at plasmodesmata and characterize points of convergence in microbe elicitor-triggered signaling, we profiled the dependence of plasmodesmal responses triggered by different elicitors on a range of plasmodesmal signaling machinery. We identified that, like chitin, flg22 signals via RBOHD to induce plasmodesmal closure. Further, we found that PDLP1, PDLP5 and CALS1 are common to microbe- and SA-triggered responses, identifying PDLPs as a candidate signaling nexus. To understand how PDLPs relay a signal to CALS1, we screened for PDLP5 interactors and found NHL3, which is also required for chitin-, flg22- and SA-triggered plasmodesmal responses and PDLP-mediated activation of callose synthesis. We conclude that a PDLP-NHL3 complex acts as an integrating node of plasmodesmal signaling cascades, transmitting multiple immune signals to activate CALS1 and plasmodesmata closure. Significance StatementPlants close plasmodesmata to restrict cell-to-cell communication after pathogen perception and in response to a range of stresses. All these stimuli trigger callose deposition at the plasmodesmal neck, suggesting a convergence of signaling. We have defined the hierarchy of molecular components and signals required to mediate plasmodesmal closure in immune responses, identifying a PDLP-NHL3 complex as a critical node that integrates multiple signaling cascades to regulate plasmodesmata.

plant biology↗

bioRxiv2022

Comparative phyloproteomics identifies conserved plasmodesmal proteins

Plasmodesmata connect neighbouring plant cells across the cell wall. They are cytosolic bridges, lined by the plasma membrane and traversed by endoplasmic reticulum to connect these cell components between cells and tissues. While plasmodesmata are notoriously difficult to extract, tissue fractionation and proteomic analyses have yielded valuable knowledge of their composition. Most proteomic profiles originate from cell suspension cultures in which simple plasmodesmata dominate and have been exclusively generated from dicotyledonous plant species. Here we have generated two novel proteomes to expand tissue and taxonomic representation of plasmodesmata: one from mature Arabidopsis leaves and one from the moss Physcomitrium patens. We have leveraged these and existing data to perform a comparative analysis that, owing to comparing proteomes from an expanded taxonomic tree, allowed us to identify conserved protein families that are associated with plasmodesmata that likely serve as core structural or functional components. Thus, we identified {beta}-1,3-glucanases, C2 lipid-binding proteins and tetraspanins as core plasmodesmal components, with proteins from P. patens and Arabidopsis maintaining plasmodesmal association across diverse species. Our approach has not only identified elements of a conserved, core plasmodesmal proteome, but also demonstrated the added power offered by comparative analysis. Conserved plasmodesmal proteins establish a basis upon which ancient plasmodesmal function can be further investigated to determine the essential roles these structures play in multicellular organism physiology in the green lineages.

plant biology↗

bioRxiv2022

The Hyaloperanospora arabidopsidis effector HaRxL77 is hypermobile between cells and manipulates host defence

Multicellular organisms require dynamic communication between cells, tissues and organs to integrate responses to external and internal signals. In plants, cell-to-cell communication relies in part on plasmodesmata, which connect adjacent cells and allow the exchange of signals and resources. Upon infection by pathogens, plants act to isolate infected cells from non-infected cells by closing plasmodesmata but pathogens can suppress this defence and maintain plasmodesmata in an open state. To address the question of what a pathogen might gain from keeping plasmodesmata open, we screened effectors from the biotrophic Arabidopsis pathogen Hyaloperonospora arabidopsidis (Hpa) for the ability to move cell-to-cell via plasmodesmata in plant tissues. We quantified the mobility of cytoplasmic effectors and identified six that were hypermobile, i.e., can move further than expected for a protein of that size. Of these, HaRxL77 indirectly modifies plasmodesmatal permeability to facilitate hypermobility and suppresses the flg22-induced ROS burst, suggesting that cell-to-cell mobility of effectors allows defence manipulation ahead of the infection front. Thus, this study provides novel insights into how Hpa exploits plasmodesmata-mediated intercellular connectivity to promote infection, characterising a poorly explored element of plant-pathogen interaction. Author SummaryDuring infection, pathogens secrete effectors into host cells to manipulate them for their benefit. Plant cells are connected via plasmodesmata, and pathogen effectors can use these connections to reach uninfected cells. We asked what a pathogen might gain by having effectors that can travel between cells and thus screened Arabidopsis downy mildew effectors for this capability. We found downy mildew produces many cell-to-cell mobile effectors, and that some of these can move further than we might expect. One hypermobile effector, HaRxL77, can open plasmodesmata to allow this increased mobility, although how it does this is not clear. HaRxL77 interferes with several host defence mechanisms, suggesting that cell- to-cell mobility allows effectors to perturb host defence ahead of the infection front.

plant biology↗

bioRxiv2021

Effectors from a Bacterial Vector-Borne Pathogen Exhibit Diverse Subcellular Localization, Expression Profiles and Manipulation of Plant Defense

Climate change is predicted to increase the prevalence of vector borne disease due to expansion of insect populations. Candidatus Liberibacter solanacearum (CLso) is a phloem-limited pathogen associated with multiple economically important diseases in Solanaceous crops. Little is known about the strategies and pathogenicity factors CLso uses to colonize vector and host. We determined the CLso effector repertoire by predicting SEC secreted proteins across four different CLso haplotypes, investigated effector localization in planta, and profiled effector expression in vector and host. The localization of CLso effectors in Nicotiana revealed diverse eukaryotic subcellular targets. The majority of tested effectors were unable to suppress plant immune responses, indicating they possess unique activities. Expression profiling in tomato and the psyllid Bactericera cockerelli indicated CLso differentially interacts with its host and vector and can switch effector expression in response to the environment. This study reveals CLso effectors possess complex expression patterns, target diverse host organelles and the majority are unable to suppress host immune responses. A mechanistic understanding of Lso effector function will reveal novel targets and provide insight into phloem biology.

plant biology↗

bioRxiv2021

Colletotrichum higginsianum effectors exhibit cell to cell hypermobility in plant tissues and modulate intercellular connectivity amongst a variety of cellular processes

Multicellular organisms exchange information and resources between cells to co-ordinate growth and responses. In plants, plasmodesmata establish cytoplasmic continuity between cells to allow for communication and resource exchange across the cell wall. Some plant pathogens use plasmodesmata as a pathway for both molecular and physical invasion. However, the benefits of molecular invasion (cell-to-cell movement of pathogen effectors) are poorly understood. To begin to investigate this and identify which effectors are cell-to-cell mobile, we performed a live imaging-based screen and identified 15 cell-to-cell mobile effectors of the fungal pathogen Colletotrichum higginsianum. Of these, 6 are "hypermobile", showing cell-to-cell mobility greater than expected for a protein of its size. We further identified 3 effectors that can indirectly modify plasmodesmal aperture. Transcriptional profiling of plants expressing hypermobile effectors implicate them in a variety of processes including senescence, glucosinolate production, cell wall integrity, growth and iron metabolism. However, not all effectors had an independent effect on virulence. This suggests a wide range of benefits to infection gained by the mobility of C. higginsianum effectors that likely interact in a complex way during infection.

plant biology↗

bioRxiv2020

A bootstrap approach is a superior statistical method for the comparison of cell-to-cell movement data

Plasmodesmata are an increasing focus of plant research, and plant physiologists frequently aim to understand the dynamics of intercellular movement and plasmodesmal function. For this, experiments that measure the spread of GFP between cells are commonly performed to indicate whether plasmodesmata are more open or closed in different conditions or in different genotypes. We propose cell-to-cell movement data sets are better analysed by a bootstrap method that tests the null hypothesis that means (or medians) are the same between two conditions, instead of the commonly used Mann-Whitney-Wilcoxon test. We found that that with hypothetical distributions similar to cell-to-cell movement data, the Mann-Whitney-Wilcoxon produces a false positive rate of 17% while the bootstrap method maintains a false positive at the set rate of 5% under the same circumstances. Here we present this finding, as well as our rationale, an explanation of the bootstrap method and an R script for easy use. We have further demonstrated its use on published datasets from independent laboratories.

plant biology↗

bioRxiv2019

Chitin perception in plasmodesmata identifies subcellular, context-specific immune signalling in plants

The plasma membrane (PM) that lines plasmodesmata has a distinct protein and lipid composition, underpinning specific regulation of these connections between cells. The plasmodesmal PM can integrate extracellular signals differently from the cellular PM, but it is not known how this specificity is established or how a single stimulus can trigger independent signalling cascades in neighbouring membrane domains. Here we have used the fungal elicitor chitin to investigate signal integration and responses at the plasmodesmal PM. We found that the plasmodesmal PM employs a receptor complex composed of the LysM receptors LYM2 and LYK4 which respectively change their location and interactions in response to chitin. Downstream, signalling is transmitted via a specific phosphorylation signature of an NADPH oxidase and localised callose synthesis that causes plasmodesmata closure. This demonstrates the plasmodesmal PM deploys both plasmodesmata-specific components and differential activation of PM-common components to independently integrate an immune signal.

plant biology↗

bioRxiv2017

A plant receptor-like kinase promotes cell-to-cell spread of RNAi and is targeted by a virus

RNA interference (RNAi) in plants can move from cell to cell, allowing for systemic spread of an anti-viral immune response. How this cell-to-cell spread of silencing is regulated is currently unknown. Here, we describe that the C4 protein from Tomato yellow leaf curl virus has the ability to inhibit the intercellular spread of RNAi. Using this viral protein as a probe, we have identified the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1) as a positive regulator of the cell-to-cell movement of RNAi, and determined that BAM1 and its closest homologue, BAM2, play a redundant role in this process. C4 interacts with the intracellular domain of BAM1 and BAM2 at the plasma membrane and plasmodesmata, the cytoplasmic connections between plant cells, interfering with the function of these RLKs in the cell-to-cell spread of RNAi. Our results identify BAM1 as an element required for the cell-to-cell spread of RNAi and highlight that signalling components have been co-opted to play multiple functions in plants.

plant biology↗