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. 2026 Apr 10;17:1791978. doi: 10.3389/fpls.2026.1791978

A Phytophthora infestans CRN1-derived small RNA is predicted to target the potato immune regulator EDS1

Shailja Singh 1,†,‡, Xinyi Hu 1,†,‡, Christina Dixelius 1,*,‡
PMCID: PMC13106546  PMID: 42040283

Abstract

The late blight pathogen, Phytophthora infestans (Pi), causes severe damage to plants in the Solanaceae family. Although knowledge regarding the P. infestans-mediated manipulation of critical components in the plant defense system is growing, many questions remain unanswered. Herein, we aimed to examine the role of Argonaute 1 (AGO1) associated small RNAs in this interaction. Of particular interest was the early communication between the host and the pathogen. To visualize the cellular dynamics underlying potential cross-kingdom RNA trafficking, we first examined the localization and accumulation patterns of plant extracellular vesicles (EVs) and multivesicular bodies (MVBs) using a handful of markers. MVBs were present not only at the plant plasma membrane but also in the germ tube of the invading pathogen. The enrichment of MVBs decreased as the infection process proceeded. At 3.0 days post-inoculation, co-localization between AGO1 from P. infestans and StARA6 was not seen even at the swollen tip of the germ tube. Three Crinkler effector genes encoding small RNAs were found after coimmunoprecipitation, sequencing and extensive bioinformatic analysis. PiCRN1 caused more severe disease compared with PiCRN3, which carries a typical Crinkler (CRN) LFLAK domain. This difference may result from activation of a CRN1-derived siRNA predicted to target the enhanced disease susceptibility 1 (EDS1) gene in potato. To examine whether the observed phenotypic effects can be attributed to any EV cargo from the potato host, we set up a procedure to isolate EVs from P. infestans-infected potato leaves. However, the tiny EV yield obtained during the early infection phase prevented us from such analysis. The understanding of effector cell trafficking and small RNA reprogramming of host plant genes remain elusive in this pathosystem.

Keywords: Argonaute, CRN effectors, EDS1, extracellular vesicles, Phytophthora infestans, potato, small RNAs

Introduction

The membrane-trafficking system, first discovered in mammalian cells, has subsequently been found in most eukaryotes and bacterial species (Couch et al., 2021; Wang et al., 2023). Cellular communication is based on extracellular vesicles (EVs), a heterogeneous group of organelles with different shapes and sizes, enclosed by lipid bilayer membranes that envelope a wide range of cargo considered as waste products to be transported out of the cell (exocytosis) or molecules targeted to other organelles (Stotz et al., 2022). These vesicles also mediate transport in the opposite direction (endocytosis), which includes the cellular import of a range of external molecules, including those from intruders that attempt to invade the plant. All cells can secrete various types of membrane-enclosed vesicles, which are involved in multiple physiological and immune-related processes essential for cell survival (Van Niel and Raposo, 2018). In brief, intracellular trafficking involves steps such as vesicle assembly and processing in the endoplasmic reticulum and Golgi apparatus, followed by their transport to the multivesicular body (MVB) and lysosome/vacuole, with a destination at the plasma membrane (Schlacht et al., 2014; More et al., 2020; Singh et al., 2023). The transport processes in the cell are mediated by the cytoskeleton network, where the so-called motor proteins, together with regulatory proteins and small GTPases, master vesicle movements along the filaments (Chebli et al., 2021; Yuan et al., 2023). The “Ras-like protein from rat brain” or RAB GTPases are the main components in this process (Zerial and McBride., 2001). The corresponding RAB GTPases in Arabidopsis are the ARA proteins, where ARA6 is a commonly used marker for MVB biogenesis (Ueda et al., 2001; Li et al., 2018). MVBs are the central components of endosomal trafficking and EV biogenesis (Cui et al., 2020) and may represent important sites where membrane transport pathways in the host intersect with pathogen invasion (Rutter and Innes, 2017; Cai et al., 2018). Tetraspanins (TETs) are another group of conserved transmembrane proteins in eukaryotic cells, with 17 genes identified in Arabidopsis (Qin et al., 2024). TET8 and TET9 play important roles in the secretion of plant EVs that mediate the trafficking of host small RNAs into fungal pathogens to silence their virulence genes (Cai et al., 2018). The endocytic and endosomal trafficking systems in plants have garnered increasing interest, as it has become evident that both pathogens and plant host molecules can utilize this membrane-based secretion system to invade organisms with whom they are in close contact.

The late blight disease, caused by the oomycete pathogen Phytophthora infestans, remains a major problem, particularly for potato and tomato crops, worldwide (Nowicki et al., 2012). This pathogen is heterothallic, with the capacity to produce a large number of spores (Fry, 2008), and its genome is dense, containing numerous potential effector candidates (Haas et al., 2009). Since 1876, when de Bary (1876) first presented his study on this pathogen, different stages of host plant infection by the pathogen have been studied. However, several fundamental questions, including when and what types of effectors are delivered during the initiation and progress of the late blight disease in the potato tissue, have not been fully answered. In brief, the infection starts with germinating spores that land on a plant host and form an appressorium-like swelling at the end of the germ tube (Whisson et al., 2016; Boevink et al., 2020). Small appressoria are formed, and enzymes are secreted to degrade the plant cuticle and cell walls. After they enter the plant cell, intercellular hyphae start growing from the infection vesicle, ramify the tissue, and form special structures called haustoria. The specific tight physical connection observed between haustoria, and the plant endomembrane system suggests that haustoria play a central role in the exchange of molecules between the two organisms with different effectors (Wang et al., 2018). The P. infestans genome harbors more than 500 predicted RXLR effectors, named after the conserved amino acid motif, which follows the signal peptide in proteins (Haas et al., 2009; Pais et al., 2018). However, the RXLR effectors represent only a small share of the wide range of molecules secreted by this pathogen (Dagdas et al., 2018). Crinkler (CRN) effectors constitute an additional large group of 196 predicted genes and 255 pseudogenes (Haas et al., 2009). The spores of P. infestans encyst on the plant surface, followed by their germination, germ tube growth, and host tissue penetration. Subsequently, P. infestans forms haustoria in the invaded host cells during the early biotrophic phase of the infection. Studies on secreted effectors from P. infestans have often been centered on this specialized structure (Wang et al., 2017; Boevink et al., 2020).

In this study, we aimed to investigate potential cross-kingdom events that occur early in the plant–P. infestans interaction, before haustoria formation. We found plant EVs in the germ tubes of P. infestans before their entry into the host. Data from PiAGO1 co-precipitation and small RNA sequencing provided information on siRNAs from CRN effectors. Of particular interest was CRN1, which lacks a typical LFLAK motif but promoted infection in Nicotiana benthamiana. This function could most likely be explained based on a CRN1-derived siRNA that is predicted to target the enhanced disease susceptibility 1 (EDS1) gene, which codes for the EDS1 protein, a central component in the immune system.

Materials and methods

Plasmids

The potato (Solanum tuberosum, St) orthologs of the genes coding for Arabidopsis Rab/Ypt GTPase ARA6 (Ueda et al., 2001) and TET8 (Boavida et al., 2013) were amplified from the leaves of the cultivar Desirée and inserted in the Gateway plasmids pGWB561 downstream of the 35S promoter or in pGWB555 with the same promoter and carrying a red fluorescent protein (RFP) tag. The primers used in the cloning experiments are listed in Supplementary Table 1. A plasmid harboring StAGO1a with a DNA fragment encoding the green fluorescent protein (GFP) tag was also included in the study (Persson Hodén et al., 2021). The predicted CRN genes, i.e., CRN1, CRN2, and CRN3, were cloned from the P. infestans strain 11388 and inserted into the Gateway plasmid pGWB506 with a GFP tag.

Plant materials and pathogen strains

Nicotiana benthamiana and potato cultivar ‘Desirée’ were cultivated as described previously (Li, 2011; Jahan et al., 2015). Two P. infestans strains were used, i.e., 11388 and 88069. The culture conditions and methodology used for inoculating P. infestans were as outlined earlier (Vetukuri et al., 2011; Jahan et al., 2015).

Agroinfection and confocal microscopy

For all analyses, we used 6.0-week-old N. benthamiana wild-type plants (Hu et al., 2022). Leaves were agroinfiltrated with pGWB506 containing 35S::PiAGO1-GFP, 35S::StAGO1a-GFP or pGWB561 harboring 35S::StTET8-RFP, 35S::StARA6-RFP, 35S::PiCRN1-GFP, 35S::PiCRN2-GFP, and 35S::PiCRN3-GFP. The same leaves were inoculated with the P. infestans strain 11388, 1.0-day post-agroinfiltration. Responses were monitored daily and recorded using an LSM800/LSM780 NLO confocal microscope (Zeiss, Oberkochen, Germany). The excitation/detection wavelengths for GFP and RFP were 488/499–547 nm and 488/532–588 nm, respectively. The confocal analysis on the different materials was repeated at least four times.

Quantitative reverse transcription–polymerase chain reaction

Total RNA was isolated from potato leaves, and EVs were collected using the RNeasy Plant Mini Kit (Qiagen, Sollentuna, Sweden) and TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). cDNA synthesis was performed using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). The analysis was performed on three independent biological replicates, with 30 leaves per sample, using the Maxima SYBR/Fluorescein qPCR Master Mix (Thermo Fisher Scientific, USA). For pairwise comparisons, a two-tailed Student’s t-test was used. Statistical significance thresholds were set at P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***). Replicate numbers and statistical details are provided in the figure legends.

Isolation of plant extracellular vesicles

Potato EVs were isolated from apoplastic washing fluid. The isolation procedure used was based on a combination of previously described methods (Rutter and Innes 2017; Huang et al., 2021). In brief, 6.0-week-old plants were inoculated with the P. infestans strains 88069 or 11388 (Åsman et al., 2016; Vetukuri et al., 2011). Sixty uninfected (control) and infected leaves were collected 5.0 days post-inoculation (dpi). Their surfaces were carefully cleaned using sterile water. Apoplastic washing fluid was collected after three centrifugation steps. First, the collected wash samples were centrifuged at a speed of 900 × g to remove cell debris, followed by ultracentrifugation at 40, 000 × g and 100, 000 × g to obtain the P40 and P100 fractions, respectively. The EV pellet was resuspended in 1 x PBS for further analysis. Samples with potential EVs were first monitored using transmission electron microscopy (TEM). To further assist in the evaluation, the samples were stained with DiOC6 (3, 3′-dihexyloxacarbocyanine iodide; Otsuga et al., 1998), a lipophilic dye that selectively labels the endoplasmic reticulum, vesicle membranes, and other membrane-bound organelles (Rutter and Innes, 2017) and checked for fluorescent signals. The isolated EVs were prepared for the qRT–PCR analysis. Infection using P. infestans strains was performed as described earlier (Persson Hodén et al., 2021).

TEM analysis

The TEM analysis of potato EVs was performed following Corona et al. (2023). For negative staining, a 5.0 µl drop of the EV fraction (P100) was carefully placed onto a carbon-coated copper mesh grid (TED PELLA INC., (Caspilor AB Lidingö, Sweden), followed by gentle washes with sterile water. A 2.0% uranyl acetate solution was used for staining. Air-dried grids were examined under a Tecnai G2 Spirit BioTwin 80 kV transmission microscope from FEI (Hilsboro, USA) at the Biological Visualization (BioVis) platform, Uppsala University, Sweden.

Bioinformatics and statistical analyses

The datasets for StAGO1a sequences were derived from a small RNA pull-down assay of infected and uninfected plants. Libraries were prepared using the Illumina TruSeq Small RNA Kit and sequenced on an Illumina NextSeq 2000 platform at SciLifeLab (Solna, Sweden). In parallel, data from degradome analysis, and small RNA datasets from P. infestans combined with information on effectors, tasiRNA loci and potato resistance genes were all integrated and run in a convolutional neural network using a smartPARE R package. Details on materials and analysis can be found in Persson Hodén et al., 2021. The sequence data can be found at the NCBI Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE163382.

Results

To visualize the cellular dynamics underlying potential cross-kingdom RNA trafficking, we first examined the localization and accumulation patterns of plant EVs and MVBs at different cellular levels during the infection of N. benthamiana leaves by P. infestans. Based on the enrichment of the StARA6 and StTET8 markers, we inferred that both MVBs and EVs increased upon P. infestans infection compared with those in the uninfected control conditions (Supplementary Figures 1A–D). This activation of the endosomal trafficking and secretion machinery in response to pathogen attack was expected, as similar observations have been reported from the Arabidopsis–Pseudomonas Pst DC3000 system, followed by other examples (Wang et al., 2014; Yuen et al., 2023).

Next, we examined the cellular localization of StAGO1a after agroinfiltration. We found that StAGO1a-GFP co-localized with the StARA6-RFP marker (Figures 1A–D). MVBs were seen not only at the plant plasma membrane but also in the germ tube of the invading pathogen (Figure 1E). The latter prompted us to look closely at events that occur before 5.0 dpi. At 3.0 dpi, the co-localization between PiAGO1 and StARA6 was not seen even at the swollen tip of the germ tube (Figure 2A). The enrichment of MVBs decreased as the infection process proceeded (Figures 2B–D). Based on the StAGO1a pull-down and sequencing data (Persson Hodén et al., 2021), we only identified siRNAs originating from P. infestans that are predicted to target host genes in potato. No potato siRNAs predicted to target P. infestans genes were detected (Supplementary Tables S2, S3). Notably, three CRN effectors were found among the listed candidates for the P. infestans siRNA source. The three CRN genes were individually cloned and tagged with GFP. The P. infestans strains were combined with the StTET8-RFP marker in assays using N. benthamiana. The enrichment of potato EVs after the expression of CRN1 and CRN2, compared with that after the expression of CRN3, was noticed (Figure 3).

Figure 1.

Confocal fluorescence imaging panels of Nicotiana benthamiana leaf cells showing extracellular vesicle marker StARA6-RFP (red) and Argonaute proteins StAGO1a-GFP or PiAGO1-GFP (green) under non-infected and Phytophthora infestans-infected conditions. Panels A-D compare marker combinations with and without infection. Panel E shows fungal mycelial structures on the leaf surface. Red, green, and merged channels are displayed. White arrows indicate punctate structures consistent with extracellular vesicles. Scale bars represent 10 μm.

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Co-localization of extracellular vesicles with Argonautes (AGOs) in Nicotiana benthamiana with and without Phytophthora infestans infection. (A)StARA6-RFP marker co-localized with StAGO1a-GFP, without infection, (B)StARA6-RFP marker co-localized with StAGO1a-GFP upon P. infestans infection. (C)StARA6-RFP and PiAGO1-GFP markers without infection, (D)StARA6-RFP and PiAGO1-GFP markers upon P. infestans infection. (E)StARA6-RFP and PiAGO1-GFP markers upon P. infestans infection. The confocal images are here focused on the growth of the mycelia of P. infestans on the leaf surface. Scale bars represent 10 μm. White arrows indicate the localization of extracellular vesicles (EVs). Confocal images are representative of at least four independent experiments.

Figure 2.

Time-course confocal imaging of StARA6-RFP (red) and PiAGO1-GFP (green) localization in Nicotiana benthamiana during Phytophthora infestans infection from 3 to 6 days post-infection. Panels A-D represent sequential time points. Fluorescence and merged channels are shown. White arrows indicate punctate structures consistent with extracellular vesicles. Scale bars represent 20 μm.

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Co-localization of StARA6-RFP marker with PiAgo1-GFP during Phytophthora infestans infection in Nicotiana benthamiana over a 3–6 days post-infection (dpi) time course shown in (A) to (D). Scale bar represents 20 μm. The white arrows indicate the localization of extracellular vesicles (EVs). Confocal images shown are representative of at least four independent experiments.

Figure 3.

Imaging of Nicotiana benthamiana leaf tissue expressing extracellular vesicle marker StTET8-RFP (red) and GFP-tagged PiCRN proteins (green). Panel A shows non-infected tissue. Panels B-D show infected samples with PiCRN1-GFP at 4 days post-infection, PiCRN2-GFP at 3 days post-infection, and PiCRN3-GFP at 3 days post-infection. Low- and high-magnification views with merged channels are included. Fluorescence signals are visible in plant cells and at the host–pathogen interface. Scale bars range from 10 to 50 μm.

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Confocal microscopy of the transiently expressed potato extracellular vesicle (EV) marker StTET8-RFP and green fluorescent protein (GFP)-tagged PiCRN proteins in Nicotiana benthamiana. (A) No infection. Scale bar represents 50 μm. (B–D) Images showing (B)PiCRN1-GFP at 4.0 days post-infection (dpi), (C)PiCRN2-GFP at 3.0 dpi, (D)PiCRN3-GFP at 3.0 dpi with Phytophthora infestans. Scale bars represent 10 μm. Panels A and C show low-magnification overviews, while (B) and (D) show higher-magnification views of the host-pathogen interface. Images are representative of at least four independent experiments. PiCRN1-GFP images were captured at 4.0 dpi to better visualize localization at the host–pathogen interface.

Among the three CRN proteins, CRN3 is the only candidate that carries a typical LFLAK domain. We then checked whether cargo from plant EVs could potentially impact pathogen virulence by infecting N. benthamiana leaves with a wild-type P. infestans strain with plasmids carrying either PiCRN1 or PiCRN3 (Figure 4). Two observations were made: PiCRN1, promoted infection when applied externally, and StTET8 (potato EVs) suppressed the extent of the infected area (Figures 4A, B). Similar phenotypic responses were seen in the case of PiCRN3 (Figures 4C, D). However, PiCRN1 caused more severe disease compared with that caused by PiCRN3 (Figures 4B, D). This difference may be associated with the presence of an siRNA identified within the CRN1 sequence that is predicted to target the EDS1 gene in potato (PGSC0003DMT400083030) (Supplementary Table 3), suggesting a potential cross-kingdom small RNA-mediated interactions. To further explore whether the observed phenotypic effects could involve EV-associated cargo from the potato host, we established a procedure to isolate EVs from P. infestans-infected potato leaves (Supplementary Figure 2A). The final pellet from layer P100 was analyzed for EV size using TEM. The diameters of the EVs varied between 36.6 nm and 46.8 nm (Supplementary Figures 2B–D). Subsequently, the pellet contents were subjected to DiOC6 staining using the fluorescent dye DiOC6 to visualize vesicle membranes (Supplementary Figure 2E). Immunogold labelling of canonical EV markers was attempted, but technical issues during the blocking step prevented clear signal detection. Next, presence of Pi-miR8788 (Hu et al., 2022) in the different EV fractions were analyzed. In addition to being present in the initial leaf samples, this miRNA was enriched in the P100 fraction when both the 11388 and 88069 strains were used, but particularly when strain 11388 was used for infection (Supplementary Figures 2F, G). Subsequently, Pi-siRNA 5′-AACTACTCCATGAATGTCTCC-3′ and Pi-siRNA 5′-TTGGAGAGATGGAAAGACGG-3′, predicted to target the 40S ribosomal protein S8 and EDS1 gene, respectively, were analyzed (Supplementary Figures 3A, B). The latter was only found in the initial leaf samples, whereas Pi-siRNA 5′-AACTACTCCATGAATGTCTCC-3′ was enriched in the EV fractions. This result suggests that the targeting of EDS1 by small RNAs may require sufficient time to consolidate correct membrane proteins or other helpers and avoid degradation, and successfully enter the host tissue, most likely through a separate secretion pathway.

Figure 4.

Images of Nicotiana benthamiana leaves showing different expression conditions with and without Phytophthora infestans infection. Panels A and C show non-infected leaves, and panels B and D show infected leaves at 5 days post-infection. Each panel contains three labeled regions representing empty vector, effector expression (PiCRN1 or PiCRN3), and co-expression with StTET8. Lesion-like areas are visible in infected samples. Scale bars represent 1 cm.

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PiCRN1 compromises the immune response compared to PiCRN3. (A) 1. Empty vector, 2. PiCRN1 plasmid, and 3) PiCRN1 and StTET8. No infection. (B) 1. Empty vector, 2. PiCRN1 plasmid, and 3) PiCRN1 and StTET8. Phytophthora infestans infection. (C) 1. Empty vector, 2. PiCRN3 plasmid, and 3) PiCRN3 and StTET8. No infection. (D) 1. Empty vector, 2. PiCRN3 plasmid, and 3) PiCRN3 and StTET8. Phytophthora infestans infection. Images were captured at 5.0 days post-infection (dpi). Scale bars represent 1.0 cm.

Discussion

In this study, we monitored host-derived EVs and MVBs during the infection of plant leaves by P. infestans. An enrichment of these small membrane-bound vesicles in the host tissue was seen at an early stage of P. infestans infection. Similar observations have been made for several other plant pathogens, such as the powdery mildew fungus and Colletotrichum species (Rubiato et al., 2022; Koch et al., 2025). The role of membrane-bound vesicles in pre- and post-invasive host defense responses has been suggested in multiple studies on syntaxins, which are members of the SYP12 clade (Rubiato et al., 2022). We expected the syntaxin-related genes in potato to be the targets of small RNAs from P. infestans; however, no such candidates were found in the PiAGO1 or StAGO1a datasets (Hu et al., 2022; Persson Hodén et al., 2021). The miR8788 from P. infestans, which targets the potato α/β hydrolase-type gene (StABH1) located in the plasma membrane, has been suggested to participate in translocation processes during the infection process (Hu et al., 2022). This miRNA was also present in the isolated EVs in the present study. Numerous plant processes are affected by the large repertoires of Phytophthora effectors (Fabro, 2022). It is well-documented that P. infestans secretes cell wall-degrading and -modifying enzymes, as well as RXLR effectors that translocate from haustoria into plant cells, where they localize in several organelles (Wang et al., 2017, 2019). CRN effectors are proteins composed of a variety of domains in different orders (Haas et al., 2009). They are a widespread class of proteins not only in oomycetes, but they also are present in several kingdoms (Zhang et al., 2016), whereof several species do not form haustoria. Thus, the translocation process into plant cells may involve more than one single route (Amaro et al., 2017). Likewise, how CRNs enter host nuclei and interfere with DNA where they often are implicated in cell death processes (Torto et al., 2003). The finding that Phytophthora sojae CRN63 induces, whereas PsCRN115 suppresses cell death (Liu et al., 2011), demonstrates the multifunctional capacity of this effector group. Besides endogenous CRN gene regulation in P. parasitica siRNAs (Jia et al., 2017) cross-kingdom events by CRN effectors encoding a siRNA that potentially targets a host gene implicated in plant defense has so far not been reported.

The finding that CRN1 lacks a signal peptide but can promote infection can most likely be attributed to the siRNA-mediated targeting of the EDS1 gene, which impairs the activation of host resistance genes. Interestingly, while the selective enrichment of Pi-miR8788 and 40S ribosomal protein siRNAs in EVs suggests a specialized trafficking system for certain small RNAs, the absence of the EDS1-targeting siRNA from these fractions indicates that P. infestans utilizes diverse delivery mechanisms. These data discriminate our observations from nonspecific co-pelleting, implicating a highly coordinated, selective EV-mediated delivery mechanism. While small RNA enrichment is clear, determining whether these species are encapsulated within the EV lumen or associated with the vesicle surface will provide critical mechanistic insight through RNase and protease protection assays.

However, it remains to be elucidated in greater detail how the wealth of RXLR and CRN effectors activate or interact with small RNAs in this pathogen and host plant species.

Conclusion

During early infection, plant multivesicular bodies accumulate in P. infestans germ tubes. Pi-miR8788 together with a siRNA predicted to target a potato 40S ribosomal gene are selectively enriched in EV fractions, indicating that P. infestans exploits host vesicle pathways for targeted small RNA delivery. StTET8-enriched plant EVs suppress infection, whereas CRN-derived EDS1-targeting siRNAs, which were not enriched in EV fractions, employ alternative mechanisms, revealing a sophisticated strategy of pathogenic manipulation of host defenses.

Acknowledgments

The authors thank Kristian Persson-Hodén for support with the bioinformatics analysis.

Funding Statement

The author(s) declared that financial support was received for this work and/or its publication. This study was supported by the Swedish Research Council VR (2015-04259), the Knut and Alice Wallenberg Foundation (KAW 2019.0062), Carl-Trigger’s Foundation (17:122), the Swedish Research Council Formas, and the Swedish University of Agricultural Sciences. Work performed at NGI/Uppsala Genome Center was funded by RFI/VR and Science for Life Laboratory, Sweden.

Footnotes

Edited by: Humberto Prieto, Agricultural Research Institute, Chile

Reviewed by: Ying Wei Khoo, Chinese Academy of Tropical Agricultural Sciences, China

Dilyara Gritsenko, Institute of Plant Biology and Biotechnology (IPBB), Kazakhstan

Data availability statement

Publicly available datasets were analyzed in this study. This data can be found here: Raw and processed sequencing data generated in this study were submitted to the NCBI Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE163382. The smartPARE R package is available online (https://github.com/KristianHoden/smartPARE/), doi:10.5281/zenodo.4495749.

Author contributions

SS: Writing – review & editing, Data curation, Visualization. XH: Writing – original draft, Visualization, Data curation, Methodology. CD: Writing – review & editing, Data curation, Project administration, Conceptualization, Methodology.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher’s note

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2026.1791978/full#supplementary-material

DataSheet1.pdf (3.8MB, pdf)

References

  1. Amaro T. M. M. M., Thilliez G. J. A., Motion G. B., Huitema E. (2017). A perspective on CRN proteins in the genomics age: evolution, classification, delivery and function revisited. Front. Plant Sci. 8, 99. doi:  10.3389/fpls.2017.00099, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Åsman A. K. M., Fogelqvist J., Vetukuri R., Dixelius C. (2016). Phytophthora infestans Argonaute 1 binds microRNA and small RNAs from effector genes and transposable elements. New Phytol. 211, 993–1007. doi: 10.1111/nph.13946, PMID: [DOI] [PubMed] [Google Scholar]
  3. Boavida L. C., Qin P., Broz M., Becker J. D., McCormick S. (2013). Arabidopsis tetraspanins are confined to discrete expression domains and cell types in reproductive tissues and form homo- and heterodimers when expressed in yeast. Plant Physiol. 163, 696–712. doi:  10.1104/pp.113.216598, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boevink P. C., Birch P. R. J., Turnbull D., Whisson S. C. (2020). Devastating intimacy: the cell biology of plant-Phytophthora interactions. New Phytol. 228, 445–458. doi:  10.1111/nph.16650, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cai Q., Qiao L., Wang M., He B., Lin F. M., Palmquist J., et al. (2018). Plants send small RNAs in extracellular vesicles to fungal pathogen to silence virulence genes. Science 360, 1126–1129. doi:  10.1126/science.aar4142, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chebli Y., Bidhendi A. J., Kapoor K., Greitmann A. (2021). Cytoskeletal regulation of primary plant cell wall assembly. Curr. Biol. 31, R681–R695. doi:  10.1016/j.cub.2021.03.092, PMID: [DOI] [PubMed] [Google Scholar]
  7. Corona M. L., Hurbain I., Raposo G., van Niel G. (2023). Characterization of extracellular vesicles by transmission electron microscopy and immunolabeling electron microscopy. Methods Protoc. 2668, 33–43. doi:  10.1007/978-1-0716-3203-1_4, PMID: [DOI] [PubMed] [Google Scholar]
  8. Couch Y., Buzàs E. I., Di Vizio D., Gho Y. S., Harrison P., Hill A. F., et al. (2021). A brief history of nearly EV-erything - The rise and rise of extracellular vesicles. J. Extracell. Vesicles 10, e12144. doi:  10.1002/jev2.12144, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cui Y., Gao J., He Y., Jiang L. (2020). Plant extracellular vesicles. Protoplasma 257, 3–12. doi:  10.1007/s00709-019-01435-6, PMID: [DOI] [PubMed] [Google Scholar]
  10. Dagdas Y. F., Pandey P., Tumtas Y., Sanguankiattichai N., Belhaj K., Duggan C., et al. (2018). Host autophagy machinery is diverted to the pathogen interface to mediate focal defense responses against the Irish potato famine pathogen. eLife 7, e37476. doi:  10.7554/eLife.37476.021, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. de Bary A. (1876). Researches into the nature of the potato fungus Phytophthora infestans. J. R. Soc Agric. England. II 12, 239–269. [Google Scholar]
  12. Fabro G. (2022). Oomycete intracellular effectors: specialized weapons targeting strategic plant processes. New Phytol. 233, 1074–1082. doi:  10.1111/nph.17828, PMID: [DOI] [PubMed] [Google Scholar]
  13. Fry W. (2008). Phytophthora infestans: the plant (and R gene) destroyer. Mol. Plant Pathol. 9, 385–402. doi:  10.1111/j.1364-3703.2007.00465.x, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Haas B. J., Kamoun S., Zody M. C., Jiang R. H. Y., Handsaker R. E., Cano L. M., et al. (2009). Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans. Nature 461, 393–398. doi:  10.1038/nature08358, PMID: [DOI] [PubMed] [Google Scholar]
  15. Hu X., Persson Hodén K., Liao Z., Åsman A., Dixelius C. (2022). Phytophthora infestans Ago1-associated miRNA promotes potato late blight disease. New Phytol. 233, 443–457. doi:  10.1111/nph.17758, PMID: [DOI] [PubMed] [Google Scholar]
  16. Huang Y., Wang S., Cai Q., Jin H. (2021). Effective methods for isolation and purification of extracellular vesicles from plants. J. Integr. Plant Biol. 63, 2020–2030. doi:  10.1111/jipb.13181, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jahan S. N., Åsman A. K. M., Corcoran P., Fogelqvist J., Vetukuri R. R., Dixelius C. (2015). Plant-mediated gene silencing restricts growth of the potato late blight pathogen Phytophthora infestans. J. Exp. Bot. 66, 2785–2794. doi:  10.1093/jxb/erv094, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jia J., Lu W., Zhong C., Zhou R., Xu J., Liu W., et al. (2017). The 25–26 nt small RNAs in Phytophthora parasitica are associated with efficient silencing of homologous endogenous genes. Front Microbiol. 8, 773. doi:  10.3389/fmicb.2017.00773, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Koch B. L., Rutter B. D., Borniego M. L., Singla-Rastogi M., Gardner D. M., Innes R. W. (2025). Arabidopsis produces distinct subpopulations of extracellular vesicles that respond differentially to biotic stress, altering growth and infectivity of a fungal pathogen. J. Extracell. Vesicles 14, e70090. doi:  10.1002/jev2.70090, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Li X. Y. (2011). Infiltration of Nicotiana benthamiana protocol for transient expression via Agrobacterium. Bio-Protocol 1, e95. doi:  10.21769/BioProtoc.95, PMID: 37752966 [DOI] [Google Scholar]
  21. Li X., Bao H., Wang Z., Wang M., Fan B., Zhum C., et al. (2018). Biogenesis and function of multivesicular bodies in plant immunity. Front. Plant Sci. 9, 979. doi:  10.3389/fpls.2018.00979, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Liu T., Ye W., Ru Y., Yang X., Gu B., Tao K., et al. (2011). Two host cytoplasmic effectors are required for pathogenesis of Phytophthora sojae by suppression of host defenses. Plant Physiol. 155, 490–501. doi:  10.1104/pp.110.166470, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. More K., Klinger C. M., Barlow L. D., Dacks J. B. (2020). Evolution and natural history of membrane trafficking in eukaryotes. Curr. Biol. 30, R553–R564. doi:  10.1016/j.cub.2020.03.068, PMID: [DOI] [PubMed] [Google Scholar]
  24. Nowicki M., Foolad M. R., Nowakowska M., Kozik E. U. (2012). Potato and tomato late blight caused by Phytophthora infestans: An overview of pathology and resistance breeding. Plant Dis. 96, 4–17. doi:  10.1094/PDIS-05-11-0458, PMID: [DOI] [PubMed] [Google Scholar]
  25. Otsuga D., Keegan B. R., Brisch E., Thatcher J. W., Hermann G. J., Bleazard W., et al. (1998). The dynamin-related GTPase, Dnm1p, controls mitochondrial morphology in yeast. J. Cell Biol. 143, 333–349. doi:  10.1083/jcb.143.2.333, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pais M., Yoshida K., Giannakopoulou A., Pel M. A., Cano L. M., Oliva R. F., et al. (2018). Gene expression polymorphism underpins evasion of host immunity in an asexual lineage of the Irish potato famine pathogen. BMC Ecol. Evol. 18, 93. doi:  10.1186/s12862-018-1201-6, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Persson Hodén K., Hu X., Martinez G., Dixelius C. (2021). smartPARE: an R package for efficient identification of true mRNA cleavage sites. Int. J. Mol. Sci. 28, 4267. doi:  10.3390/ijms22084267, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Qin S., Li W., Zeng J., Huang Y., Cai Q. (2024). Rice tetraspanins express in specific domains of diverse tissues and regulate plant architecture and root growth. Plant J. 117, 892–908. doi:  10.1111/tpj.16536, PMID: [DOI] [PubMed] [Google Scholar]
  29. Rubiato H. M., Liu M., O’Connell R. J., Nielsen M. E. (2022). Plant SYP12 syntaxins mediate an evolutionarily conserved general immunity to filamentous pathogens. eLife 11, e73487. doi:  10.7554/eLife.73487, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rutter B. D., Innes R. W. (2017). Extracellular vesicles isolated from the leaf apoplast carry stress-response proteins. Plant Physiol. 173, 728–741. doi:  10.1104/pp.16.01253, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schlacht A., Herman E. K., Klute M. J., Filed M. C., Dacks J. B. (2014). Missing pieces of an ancient puzzle: evolution of the eukaryotic membrane-trafficking system. Cold Spring Harbor Persp. Biol. 6, a016048. doi:  10.1101/cshperspect.a016048, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Singh S., Hu X., Dixelius C. (2023). Dynamics of nucleic acid mobility. Genetics 225, iyad132. doi:  10.1093/genetics/iyad132, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Stotz H. U., Brotherton D., Inal J. (2022). Communication is key: extracellular vesicles as mediators of infection and defence during host–microbe interactions in animals and plants 46, fuab044. doi: 10.1093/femsre/fuab044, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Torto T. A., Li S., Styer A., Huitema E., Testa A., Gow N. A., et al. (2003). EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora. Genome Res. 13, 1675–1685. doi:  10.1101/gr.910003, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ueda T., Yamaguchi M., Uchimiya H., Nakano A. (2001). Ara6, a plant-unique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis thaliana. EMBO J. 20, 4730–4741. doi:  10.1093/emboj/20.17.4730, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Van Niel G., Raposo G. (2018). Shedding light on the cell biology of extracellular vesicles. Nat. Rev. Mol. Cell Biol. 19, 213–228. doi:  10.1038/nrm.2017.125, PMID: [DOI] [PubMed] [Google Scholar]
  37. Vetukuri R. R., Avrova A. O., Grenville-Briggs L. J., Van West P., Söderbom F., Savenkov E. I., et al. (2011). Evidence for involvement of Dicer-like, Argonaute and histone deacetylase proteins in gene silencing in Phytophthora infestans. Mol. Plant Pathol. 12, 772–785. doi:  10.1111/j.1364-3703.2011.00710.x, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wang S., Boevink P. C., Welsh L., Zhang R., Whisson S. C., Birch P. R. J. (2017). Delivery of cytoplasmic and apoplastic effectors from Phytophthora infestans haustoria by distinct secretion pathways. New Phytol. 216, 205–215. doi:  10.1111/nph.14696, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wang S., McLellan H., Bukharova T., He Q., Murphy F., Shi J., et al. (2019). Phytophthora infestans RXLR effectors act in concert at diverse subcellular localisations to enhance host colonisation. J. Exp. Bot. 70, 343–356. doi:  10.1093/jxb/ery360, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wang F., Shang Y., Fan B., Yu J.-Q., Chen Z. (2014). Arabidopsis LIP5, a positive regulator of multivesicular body biogenesis, is a critical target of pathogen-responsive MAPK cascade in plant basal defense. PloS Pathog. 10, e1004243. doi:  10.1371/journal.ppat.1004243, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wang S., Welsh L., Thorpe P., Whisson S. C., Boevink P. C., Birch P. R. J. (2018). The Phytophthora infestans haustorium is a site for secretion of diverse classes of infection-associated proteins. mBio 9, e01216–e01218. doi:  10.1128/mBio.01216-18, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wang Z., Zeng J., Deng J., Hou X., Zhang J., Yan W., et al. (2023). Pathogen-derived extracellular vesicles: emerging mediators of plant-microbe interactions. Mol. Plant-Microbe Interact. 36, 218–227. doi:  10.1094/MPMI-08-22-0162-FI, PMID: [DOI] [PubMed] [Google Scholar]
  43. Whisson S. C., Boevink P. C., Wang S., Birch P. R. J. (2016). The cell biology of late blight disease. Curr. Opin. Microbiol. 34, 127–135. doi:  10.1016/j.mib.2016.09.002, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yuan G., Gao H., Yang T. (2023). Exploring the role of the plant actin cytoskeleton: From signaling to cellular functions. Int. J. Mol. Sci. 24, 15480. doi:  10.3390/ijms242015480, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yuen E. L. H., Shepherd S., Bozkurt T. O. (2023). Traffic control: Subversion of plant membrane trafficking by pathogens. Ann. Rev. Phytopathol. 61, 325–350. doi:  10.1146/annurev-phyto-021622-123232, PMID: [DOI] [PubMed] [Google Scholar]
  46. Zerial M., McBride. H. (2001). Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol. 2, 107–117. doi:  10.1038/35052055, PMID: [DOI] [PubMed] [Google Scholar]
  47. Zhang D., Burroughs A. M., Vidal N. D., Iyer L. M., Aravind L. (2016). Transposons to toxins: the provenance, architecture and diversification of a widespread class of eukaryotic effectors. Nucleic Acids Res. 44, 3513–3533. doi:  10.1093/nar/gkw221, PMID: [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

DataSheet1.pdf (3.8MB, pdf)

Data Availability Statement

Publicly available datasets were analyzed in this study. This data can be found here: Raw and processed sequencing data generated in this study were submitted to the NCBI Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE163382. The smartPARE R package is available online (https://github.com/KristianHoden/smartPARE/), doi:10.5281/zenodo.4495749.

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