Roadmap to Success: How Oomycete Plant Pathogens Invade Tissues and Deliver Effectors.

Filamentous plant pathogens threaten global food security and ecosystem resilience. In recent decades, significant strides have been made in deciphering the molecular basis of plant-pathogen interactions, especially the interplay between pathogens' molecular weaponry and hosts' defense machinery. Stemming from interdisciplinary investigations into the infection cell biology of filamentous plant pathogens, recent breakthrough discoveries have provided a new impetus to the field. These advances include the biophysical characterization of a novel invasion mechanism (i.e., naifu invasion) and the unraveling of novel effector secretion routes. On the plant side, progress includes the identification of components of cellular networks involved in the uptake of intracellular effectors. This exciting body of research underscores the pivotal role of logistics management by the pathogen throughout the infection cycle, encompassing the precolonization stages up to tissue invasion. More insight into these logistics opens new avenues for developing environmentally friendly crop protection strategies in an era marked by an imperative to reduce the use of agrochemicals.

Phytophthora zoospores display klinokinetic behaviour in response to a chemoattractant.

Microswimmers are single-celled bodies powered by flagella. Typical examples are zoospores, dispersal agents of oomycete plant pathogens that are used to track down hosts and infect. Being motile, zoospores presumably identify infection sites using chemical cues such as sugars, alcohols and amino acids. With high-speed cameras we traced swimming trajectories of Phytophthora zoospores over time and quantified key trajectory parameters to investigate chemotactic responses. Zoospores adapt their native run-and-tumble swimming patterns in response to the amino acid glutamic acid by increasing the rate at which they turn. Simulations predict that tuneable tumble frequencies are sufficient to explain zoospore aggregation, implying positive klinokinesis. Zoospores thus exploit a retention strategy to remain at the plant surface once arriving there. Interference of G-protein mediated signalling affects swimming behaviour. Zoospores of a Phytophthora infestans G⍺-deficient mutant show higher tumbling frequencies but still respond and adapt to glutamic acid, suggesting chemoreception to be intact.

Oomycete Metabolism Is Highly Dynamic and Reflects Lifestyle Adaptations.

The selective pressure of pathogen-host symbiosis drives adaptations. How these interactions shape the metabolism of pathogens is largely unknown. Here, we use comparative genomics to systematically analyze the metabolic networks of oomycetes, a diverse group of eukaryotes that includes saprotrophs as well as animal and plant pathogens, with the latter causing devastating diseases with significant economic and/or ecological impacts. In our analyses of 44 oomycete species, we uncover considerable variation in metabolism that can be linked to lifestyle differences. Comparisons of metabolic gene content reveal that plant pathogenic oomycetes have a bipartite metabolism consisting of a conserved core and an accessory set. The accessory set can be associated with the degradation of defense compounds produced by plants when challenged by pathogens. Obligate biotrophic oomycetes have smaller metabolic networks, and taxonomically distantly related biotrophic lineages display convergent evolution by repeated gene losses in both the conserved as well as the accessory set of metabolisms. When investigating to what extent the metabolic networks in obligate biotrophs differ from those in hemibiotrophic plant pathogens, we observe that the losses of metabolic enzymes in obligate biotrophs are not random and that gene losses predominantly influence the terminal branches of the metabolic networks. Our analyses represent the first metabolism-focused comparison of oomycetes at this scale and will contribute to a better understanding of the evolution of oomycete metabolism in relation to lifestyle adaptation. Numerous oomycete species are devastating plant pathogens that cause major damage in crops and natural ecosystems. Their interactions with hosts are shaped by strong selection, but how selection affects adaptation of the primary metabolism to a pathogenic lifestyle is not yet well established. By pan-genome and metabolic network analyses of distantly related oomycete pathogens and their nonpathogenic relatives, we reveal considerable lifestyle- and lineage-specific adaptations. This study contributes to a better understanding of metabolic adaptations in pathogenic oomycetes in relation to lifestyle, host, and environment, and the findings will help in pinpointing potential targets for disease control. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Fatal attraction: How Phytophthora zoospores find their host.

Oomycete plant pathogens, such as Phytophthora and Pythium species produce motile dispersal agents called zoospores that actively target host plants. Zoospores are exceptional in their ability to display taxis to chemical, electrical and physical cues to navigate the phyllosphere and reach stomata, wound sites and roots. Many components of root exudates have been shown attractive or repulsive to zoospores. Although some components possess very strong attractiveness, it seems that especially the mix of components exuded by the primary host is most attractive to zoospores. Zoospores actively approach attractants with swimming behaviour reminiscent of other microswimmers. To achieve a unified description of zoospore behaviour when sensing an attractant, we propose the following terms for the successive stages of the homing response: reorientation, approaching, retention and settling. How zoospores sense and process attractants is poorly understood but likely involves signal perception via cell surface receptors. Since zoospores are important for infection, undermining their activity by luring attractants or blocking receptors seem promising strategies for disease control.

A Phytophthora infestans RXLR effector targets a potato ubiquitin-like domain-containing protein to inhibit the proteasome activity and hamper plant immunity.

Ubiquitin-like domain-containing proteins (UDPs) are involved in the ubiquitin-proteasome system because of their ability to interact with the 26S proteasome. Here, we identified potato StUDP as a target of the Phytophthora infestans RXLR effector Pi06432 (PITG_06432), which supresses the salicylic acid (SA)-related immune pathway. By overexpressing and silencing of StUDP in potato, we show that StUDP negatively regulates plant immunity against P. infestans. StUDP interacts with, and destabilizes, the 26S proteasome subunit that is referred to as REGULATORY PARTICLE TRIPLE-A ATP-ASE (RPT) subunit StRPT3b. This destabilization represses the proteasome activity. Proteomic analysis and Western blotting show that StUDP decreases the stability of the master transcription factor SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) in SA biosynthesis. StUDP negatively regulates the SA signalling pathway by repressing the proteasome activity and destabilizing StSARD1, leading to a decreased expression of the SARD1-targeted gene ISOCHORISMATE SYNTHASE 1 and thereby a decrease in SA content. Pi06432 stabilizes StUDP, and it depends on StUDP to destabilize StRPT3b and thereby supress the proteasome activity. Our study reveals that the P. infestans effector Pi06432 targets StUDP to hamper the homeostasis of the proteasome by the degradation of the proteasome subunit StRPT3b and thereby suppresses SA-related immunity.

Sterol-Sensing Domain (SSD)-Containing Proteins in Sterol Auxotrophic Phytophthora capsici Mediate Sterol Signaling and Play a Role in Asexual Reproduction and Pathogenicity.

Phytophthora species are devastating filamentous plant pathogens that belong to oomycetes, a group of microorganisms similar to fungi in morphology but phylogenetically distinct. They are sterol auxotrophic, but nevertheless exploit exogenous sterols for growth and development. However, as for now the mechanisms underlying sterol utilization in Phytophthora are unknown. In this study, we identified four genes in Phytophthora capsici that encode proteins containing a sterol-sensing domain (SSD), a protein domain of around 180 amino acids comprising five transmembrane segments and known to feature in sterol signaling in animals. Using a modified CRISPR/Cas9 system, we successfully knocked out the four genes named PcSCP1 to PcSCP4 (for P. capsici SSD-containing protein 1 to 4), either individually or sequentially, thereby creating single, double, triple, and quadruple knockout transformants. Results showed that knocking out just one of the four PcSCPs was not sufficient to block sterol signaling. However, the quadruple "all-four" PcSCPs knockout transformants no longer responded to sterol treatment in asexual reproduction, in contrast to wild-type P. capsici that produced zoospores under sterol treatment. Apparently, the four PcSCPs play a key role in sterol signaling in P. capsici with functional redundancy. Transcriptome analysis indicated that the expression of a subset of genes is regulated by exogenous sterols via PcSCPs. Further investigations showed that sterols could stimulate zoospore differentiation via PcSCPs by controlling actin-mediated membrane trafficking. Moreover, the pathogenicity of the "all-four" PcSCPs knockout transformants was significantly decreased and many pathogenicity related genes were downregulated, implying that PcSCPs also contribute to plant-pathogen interaction. IMPORTANCE Phytophthora is an important genus of oomycetes that comprises many destructive plant pathogens. Due to the incompleteness of the sterol synthesis pathway, Phytophthora spp. do not possess the ability to produce sterols. Therefore, these sterol auxotrophic oomycetes need to recruit sterols from the environment such as host plants to support growth and development, which seems crucial during pathogen-plant interactions. However, the mechanisms underlying sterol utilization by Phytophthora spp. remain largely unknown. Here, we show that a family of sterol-sensing domain-containing proteins (SCPs) consisting of four members in P. capsici plays a key role in sterol signaling with functional redundancy. Moreover, these SCPs play a role in different biological processes, including asexual reproduction and pathogenicity. Our study overall revealed the multiple functions of PcSCPs and addressed the question of how exogenous sterols regulate the development of heterothallic Phytophthora spp. via SSD-containing proteins.

Convergent evolution of immune receptors underpins distinct elicitin recognition in closely related Solanaceous plants.

Elicitins are a large family of secreted proteins in Phytophthora. Clade 1 elicitins were identified decades ago as potent elicitors of immune responses in Nicotiana species, but the mechanisms underlying elicitin recognition are largely unknown. Here we identified an elicitin receptor in Nicotiana benthamiana that we named REL for Responsive to ELicitins. REL is a receptor-like protein (RLP) with an extracellular leucine-rich repeat (LRR) domain that mediates Phytophthora resistance by binding elicitins. Silencing or knocking out REL in N. benthamiana abolished elicitin-triggered cell death and immune responses. Domain deletion and site-directed mutagenesis revealed that the island domain (ID) located within the LRR domain of REL is crucial for elicitin recognition. In addition, sequence polymorphism in the ID underpins the genetic diversity of REL homologs in various Nicotiana species in elicitin recognition and binding. Remarkably, REL is phylogenetically distant from the elicitin response (ELR) protein, an LRR-RLP that was previously identified in the wild potato species Solanum microdontum and REL and ELR differ in the way they bind and recognize elicitins. Our findings provide insights into the molecular basis of plant innate immunity and highlight a convergent evolution of immune receptors towards perceiving the same elicitor.

An actin mechanostat ensures hyphal tip sharpness in Phytophthora infestans to achieve host penetration.

Filamentous plant pathogens apply mechanical forces to pierce their hosts surface and penetrate its tissues. Devastating Phytophthora pathogens harness a specialized form of invasive tip growth to slice through the plant surface, wielding their hypha as a microscopic knife. Slicing requires a sharp hyphal tip that is not blunted at the site of the mechanical interaction. How tip shape is controlled, however, is unknown. We uncover an actin-based mechanostat in Phytophthora infestans that controls tip sharpness during penetration. Mechanical stimulation of the hypha leads to the emergence of an aster-like actin configuration, which shows fast, local, and quantitative feedback to the local stress. We evidence that this functions as an adaptive mechanical scaffold that sharpens the invasive weapon and prevents it from blunting. The hyphal tip mechanostat enables the efficient conversion of turgor into localized invasive pressures that are required to achieve host penetration.

Phytophthora capsici sterol reductase PcDHCR7 has a role in mycelium development and pathogenicity.

The de novo biosynthesis of sterols is critical for the majority of eukaryotes; however, some organisms lack this pathway, including most oomycetes. Phytophthora spp. are sterol auxotrophic but, remarkably, have retained a few genes encoding enzymes in the sterol biosynthesis pathway. Here, we show that PcDHCR7, a gene in Phytophthora capsici predicted to encode Δ7-sterol reductase, displays multiple functions. When expressed in Saccharomyces cerevisiae, PcDHCR7 showed the Δ7-sterol reductase activity. Knocking out PcDHCR7 in P. capsici resulted in loss of the capacity to transform ergosterol into brassicasterol, which means PcDHCR7 has the Δ7-sterol reductase activity in P. capsici itself. This enables P. capsici to transform sterols recruited from the environment for better use. The biological characteristics of ΔPcDHCR7 transformants were compared with those of the wild-type strain and a PcDHCR7 complemented transformant, and the results showed that PcDHCR7 plays a key role in mycelium development and pathogenicity of zoospores. Further analysis of the transcriptome indicated that the expression of many genes changed in the ΔPcDHCR7 transformant, which involve in different biological processes. It is possible that P. capsici compensates for the defects caused by the loss of PcDHCR7 by remodelling its transcriptome.

Molecular sensors reveal the mechano-chemical response of Phytophthora infestans walls and membranes to mechanical and chemical stress.

Phytophthora infestans, causal agent of late blight in potato and tomato, remains challenging to control. Unravelling its biomechanics of host invasion, and its response to mechanical and chemical stress, could provide new handles to combat this devastating pathogen. Here we introduce two fluorescent molecular sensors, CWP-BDP and NR12S, that reveal the micromechanical response of the cell wall-plasma membrane continuum in P. infestans during invasive growth and upon chemical treatment. When visualized by live-cell imaging, CWP-BDP reports changes in cell wall (CW) porosity while NR12S reports variations in chemical polarity and lipid order in the plasma membrane (PM). During invasive growth, mechanical interactions between the pathogen and a surface reveal clear and localized changes in the structure of the CW. Moreover, the molecular sensors can reveal the effect of chemical treatment to CW and/or PM, thereby revealing the site-of-action of crop protection agents. This mechano-chemical imaging strategy resolves, non-invasively and with high spatio-temporal resolution, how the CW-PM continuum adapts and responds to abiotic stress, and provides information on the dynamics and location of cellular stress responses for which, to date, no other methods are available.

Silencing susceptibility genes in potato hinders primary infection of Phytophthora infestans at different stages.

Most potato cultivars are susceptible to late blight disease caused by the oomycete pathogen Phytophthora infestans. A new source of resistance to prevent or diminish pathogen infection is found in the genetic loss of host susceptibility. Previously, we showed that RNAi-mediated silencing of the potato susceptibility (S) genes StDND1, StDMR1 and StDMR6 leads to increased late blight resistance. The mechanisms underlying this S-gene mediated resistance have thus far not been identified. In this study, we examined the infection process of P. infestans on StDND1-, StDMR1- and StDMR6-silenced potato lines. Microscopic analysis showed that penetration of P. infestans spores was hampered on StDND1-silenced plants. On StDMR1- and StDMR6-silenced plants, P. infestans infection was arrested at a primary infection stage by enhanced cell death responses. Histochemical staining revealed that StDMR1- and StDMR6-silenced plants display elevated ROS levels in cells at the infection sites. Resistance in StDND1-silenced plants, however, seems not to rely on a cell death response as ROS accumulation was found to be absent at most inoculated sites. Quantitative analysis of marker gene expression suggests that the increased resistance observed in StDND1- and StDMR6-silenced plants relies on an early onset of SA- and ET-mediated signalling pathways. Resistance mediated by silencing StDMR1 was found to be correlated with the early induction of SA-mediated signalling. These data provide evidence that different defense mechanisms are involved in late blight resistance mediated by functional impairment of different potato S-genes.

Uncovering the Role of Metabolism in Oomycete-Host Interactions Using Genome-Scale Metabolic Models.

Metabolism is the set of biochemical reactions of an organism that enables it to assimilate nutrients from its environment and to generate building blocks for growth and proliferation. It forms a complex network that is intertwined with the many molecular and cellular processes that take place within cells. Systems biology aims to capture the complexity of cells, organisms, or communities by reconstructing models based on information gathered by high-throughput analyses (omics data) and prior knowledge. One type of model is a genome-scale metabolic model (GEM) that allows studying the distributions of metabolic fluxes, i.e., the "mass-flow" through the network of biochemical reactions. GEMs are nowadays widely applied and have been reconstructed for various microbial pathogens, either in a free-living state or in interaction with their hosts, with the aim to gain insight into mechanisms of pathogenicity. In this review, we first introduce the principles of systems biology and GEMs. We then describe how metabolic modeling can contribute to unraveling microbial pathogenesis and host-pathogen interactions, with a specific focus on oomycete plant pathogens and in particular Phytophthora infestans. Subsequently, we review achievements obtained so far and identify and discuss potential pitfalls of current models. Finally, we propose a workflow for reconstructing high-quality GEMs and elaborate on the resources needed to advance a system biology approach aimed at untangling the intimate interactions between plants and pathogens.

A slicing mechanism facilitates host entry by plant-pathogenic Phytophthora.

Phytophthora species, classified as oomycetes, are among the most destructive plant pathogens worldwide and pose a substantial threat to food security. Plant pathogens have developed various methods to breach the cuticle and walls of plant cells. For example, plant-pathogenic fungi use a 'brute-force' approach by producing a specialized and fortified invasion organ to generate invasive pressures. Unlike in fungi, the biomechanics of host invasion in oomycetes remains poorly understood. Here, using a combination of surface-deformation imaging, molecular-fracture sensors and modelling, we find that Phytophthora infestans, Phytophthora palmivora and Phytophthora capsici slice through the plant surface to gain entry into host tissues. To distinguish this mode of entry from the brute-force approach of fungi that use appressoria, we name this oomycete entry without appressorium formation 'naifu' invasion. Naifu invasion relies on polarized, non-concentric, force generation onto the surface at an oblique angle, which concentrates stresses at the site of invasion to enable surface breaching. Measurements of surface deformations during invasion of artificial substrates reveal a polarized mechanical geometry that we describe using a mathematical model. We confirm that the same mode of entry is used on real hosts. Naifu invasion uses actin-mediated polarity, surface adherence and turgor generation to enable Phytophthora to invade hosts without requiring specialized organs or vast turgor generation.

Phytophthora infestans RXLR effector AVR1 disturbs the growth of Physcomitrium patens without affecting Sec5 localization.

Plant pathogens often exploit a whole range of effectors to facilitate infection. The RXLR effector AVR1 produced by the oomycete plant pathogen Phytophthora infestans suppresses host defense by targeting Sec5. Sec5 is a subunit of the exocyst, a protein complex that is important for mediating polarized exocytosis during plant development and defense against pathogens. The mechanism by which AVR1 manipulates Sec5 functioning is unknown. In this study, we analyzed the effect of AVR1 on Sec5 localization and functioning in the moss Physcomitrium patens. P. patens has four Sec5 homologs. Two (PpSec5b and PpSec5d) were found to interact with AVR1 in yeast-two-hybrid assays while none of the four showed a positive interaction with AVR1ΔT, a truncated version of AVR1. In P. patens lines carrying ß-estradiol inducible AVR1 or AVR1ΔT transgenes, expression of AVR1 or AVR1ΔT caused defects in the development of caulonemal protonema cells and abnormal morphology of chloronema cells. Similar phenotypes were observed in Sec5- or Sec6-silenced P. patens lines, suggesting that both AVR1 and AVR1ΔT affect exocyst functioning in P. patens. With respect to Sec5 localization we found no differences between ß-estradiol-treated and untreated transgenic AVR1 lines. Sec5 localizes at the plasma membrane in growing caulonema cells, also during pathogen attack, and its subcellular localization is the same, with or without AVR1 in the vicinity.

Mining oomycete proteomes for metalloproteases leads to identification of candidate virulence factors in Phytophthora infestans.

Pathogens deploy a wide range of pathogenicity factors, including a plethora of proteases, to modify host tissue or manipulate host defences. Metalloproteases (MPs) have been implicated in virulence in several animal and plant pathogens. Here we investigated the repertoire of MPs in 46 stramenopile species including 37 oomycetes, 5 diatoms, and 4 brown algae. Screening their complete proteomes using hidden Markov models (HMMs) trained for MP detection resulted in over 4,000 MPs, with most species having between 65 and 100 putative MPs. Classification in clans and families according to the MEROPS database showed a highly diverse MP repertoire in each species. Analyses of domain composition, orthologous groups, distribution, and abundance within the stramenopile lineage revealed a few oomycete-specific MPs and MPs potentially related to lifestyle. In-depth analyses of MPs in the plant pathogen Phytophthora infestans revealed 91 MPs, divided over 21 protein families, including 25 MPs with a predicted signal peptide or signal anchor. Expression profiling showed different patterns of MP gene expression during pre-infection and infection stages. When expressed in leaves of Nicotiana benthamiana, 12 MPs changed the sizes of lesions caused by inoculation with P. infestans; with 9 MPs the lesions were larger, suggesting a positive effect on the virulence of P. infestans, while 3 MPs had a negative effect, resulting in smaller lesions. To the best of our knowledge, this is the first systematic inventory of MPs in oomycetes and the first study pinpointing MPs as potential pathogenicity factors in Phytophthora.

The Mevalonate Pathway Is Important for Growth, Spore Production, and the Virulence of Phytophthora sojae.

The mevalonate (MVA) pathway in eukaryotic organisms produces isoprenoids, sterols, ubiquinone, and dolichols. These molecules are vital for diverse cellular functions, ranging from signaling to membrane integrity, and from post-translational modification to energy homeostasis. However, information on the MVA pathway in Phytophthora species is limited. In this study, we identified the MVA pathway genes and reconstructed the complete pathway in Phytophthora sojae in silico. We characterized the function of the MVA pathway of P. sojae by treatment with enzyme inhibitor lovastatin, deletion of the geranylgeranyl diphosphate synthase gene (PsBTS1), and transcriptome profiling analysis. The MVA pathway is ubiquitously conserved in Phytophthora species. Under lovastatin treatment, mycelial growth, spore production, and virulence of P. sojae were inhibited but the zoospore encystment rate increased. Heterozygous mutants of PsBTS1 showed slow growth, abnormal colony characteristics, and mycelial morphology. Mutants showed decreased numbers of sporangia and oospores as well as reduced virulence. RNA sequencing analysis identified the essential genes in sporangia formation were influenced by the enzyme inhibitor lovastatin. Our findings elucidate the role of the MVA pathway in P. sojae and provide new insights into the molecular mechanisms underlying the development, reproduction, and virulence of P. sojae and possibly other oomycetes. Our results also provide potential chemical targets for management of plant Phytophthora diseases.

The Genome of Peronospora belbahrii Reveals High Heterozygosity, a Low Number of Canonical Effectors, and TC-Rich Promoters.

Along with Plasmopara destructor, Peronosopora belbahrii has arguably been the economically most important newly emerging downy mildew pathogen of the past two decades. Originating from Africa, it has started devastating basil production throughout the world, most likely due to the distribution of infested seed material. Here, we present the genome of this pathogen and results from comparisons of its genomic features to other oomycetes. The assembly of the nuclear genome was around 35.4 Mbp in length, with an N50 scaffold length of around 248 kbp and an L50 scaffold count of 46. The circular mitochondrial genome consisted of around 40.1 kbp. From the repeat-masked genome, 9,049 protein-coding genes were predicted, out of which 335 were predicted to have extracellular functions, representing the smallest secretome so far found in peronosporalean oomycetes. About 16% of the genome consists of repetitive sequences, and, based on simple sequence repeat regions, we provide a set of microsatellites that could be used for population genetic studies of P. belbahrii. P. belbahrii has undergone a high degree of convergent evolution with other obligate parasitic pathogen groups, reflecting its obligate biotrophic lifestyle. Features of its secretome, signaling networks, and promoters are presented, and some patterns are hypothesized to reflect the high degree of host specificity in Peronospora species. In addition, we suggest the presence of additional virulence factors apart from classical effector classes that are promising candidates for future functional studies.

G protein α subunit suppresses sporangium formation through a serine/threonine protein kinase in Phytophthora sojae.

Eukaryotic heterotrimeric guanine nucleotide-binding proteins consist of α, ß, and γ subunits, which act as molecular switches to regulate a number of fundamental cellular processes. In the oomycete pathogen Phytophthora sojae, the sole G protein α subunit (Gα; encoded by PsGPA1) has been found to be involved in zoospore mobility and virulence, but how it functions remains unclear. In this study, we show that the Gα subunit PsGPA1 directly interacts with PsYPK1, a serine/threonine protein kinase that consists of an N-terminal region with unknown function and a C-terminal region with a conserved catalytic kinase domain. We generated knockout and knockout-complemented strains of PsYPK1 and found that deletion of PsYPK1 resulted in a pronounced reduction in the production of sporangia and oospores, in mycelial growth on nutrient poor medium, and in virulence. PsYPK1 exhibits a cytoplasmic-nuclear localization pattern that is essential for sporangium formation and virulence of P. sojae. Interestingly, PsGPA1 overexpression was found to prevent nuclear localization of PsYPK1 by exclusively binding to the N-terminal region of PsYPK1, therefore accounting for its negative role in sporangium formation. Our data demonstrate that PsGPA1 negatively regulates sporangium formation by repressing the nuclear localization of its downstream kinase PsYPK1.

Time-gated confocal microscopy reveals accumulation of exocyst subunits at the plant-pathogen interface.

Polarized exocytosis is essential for plant development and defence. The exocyst, an octameric protein complex that tethers exocytotic vesicles to the plasma membrane, targets exocytosis. Upon pathogen attack, secreted materials form papillae to halt pathogen penetration. To determine if the exocyst is directly involved in targeting exocytosis to infection sites, information about its localization is instrumental. Here, we investigated exocyst subunit localization in the moss Physcomitrella patens upon pathogen attack and infection by Phytophthora capsici. Time-gated confocal microscopy was used to eliminate autofluorescence of deposited material around infection sites, allowing the visualization of the subcellular localization of exocyst subunits and of v-SNARE Vamp72A1-labelled exocytotic vesicles during infection. This showed that exocyst subunits Sec3a, Sec5b, Sec5d, and Sec6 accumulated at sites of attempted pathogen penetration. Upon pathogen invasion, the exocyst subunits accumulated on the membrane surrounding papilla-like structures and hyphal encasements. Vamp72A1-labelled vesicles were found to localize in the cytoplasm around infection sites. The re-localization of exocyst subunits to infection sites suggests that the exocyst is directly involved in facilitating polarized exocytosis during pathogenesis.