Abstract
Programmed cell death occurring during plant development (dPCD) is a fundamental process integral for plant growth and reproduction. Here, we investigate the connection between developmentally controlled PCD and fungal accommodation in Arabidopsis thaliana roots, focusing on the root cap-specific transcription factor ANAC033/SOMBRERO (SMB) and the senescence-associated nuclease BFN1. Mutations of both dPCD regulators increase colonization by the beneficial fungus Serendipita indica, primarily in the differentiation zone. smb-3 mutants additionally exhibit hypercolonization around the meristematic zone and a delay of S. indica-induced root-growth promotion. This demonstrates that root cap dPCD and rapid post-mortem clearance of cellular corpses represent a physical defense mechanism restricting microbial invasion of the root. Additionally, reporter lines and transcriptional analysis revealed that BFN1 expression is downregulated during S. indica colonization in mature root epidermal cells, suggesting transcriptional control as a means for microbial regulation of host dPCD.
Key findings in bullet points
The process of programmed cell death in root development (dPCD) influences the extent and outcomes of fungal symbiosis
Fungal colonization of the root tip and differentiation zone is restricted by SMB-mediated clearance of dead cells, which preserves the meristem and regulates symbiosis
The plant nuclease BFN1, which is associated with senescence, is exploited by beneficial microbes to facilitate their root accommodation
eLife assessment
This study explores the interplay between fungal colonization and controlled programmed cell death in Arabidopsis thaliana root cells. The authors reveal how this process is affected by corpse clearance in the root cap, highlighting some of the key elements in this process including a root cap-specific transcription factor. With this, the authors have discovered an important relationship between transcriptional regulation of developmentally controlled cell death and the beneficial colonization of plants by fungi. The work thus establishes a solid basis for further studies of plant-microbiome associations along the root axis.
Introduction
Plant roots elongate by producing new cells in the root apical meristem at the root tip. As roots extend further into the soil environment, the newly formed root tissue is naturally exposed to microbial attack, challenging the successful establishment of root systems. However, microbial colonization at the meristematic zone is rarely detected 1, 2. The sensitive tissue of meristematic stem cells is surrounded by the root cap, a specialized root organ that orchestrates root architecture, directs root growth based on gravitropism and hydrotropism, and senses environmental stimuli. In addition, the root cap is presumed to have a protective function in soil exploration 3, 4.
In Arabidopsis thaliana (hereafter Arabidopsis), the root cap consists of two distinct tissues: the centrally located columella root cap at the very root tip and the peripherally located lateral root cap (LRC), which flanks both the columella and the entire root meristem 5. A ring of specific stem cells continuously generates both new LRC cells and root epidermal cells 5. However, despite the constant production of LRC cells, the root cap itself does not grow in size but matches the size of the meristem 3, 6, 7. To maintain size homeostasis, root cap development is a highly regulated process that varies in different plant species. In Arabidopsis, a combination of dPCD and shedding of old cells into the rhizosphere has been described 3, 8. The centrally located columella root cap along with adjacent proximal LRC cells is shed as a cell package, followed by a PCD process 9, 10, 11. In contrast, LRC cells at the distal end of the root tip elongate and reach the edge of the meristematic zone where they undergo dPCD, orchestrated as part of a terminal differentiation program by the root cap-specific transcription factor ANAC33/SOMBRERO (SMB) 6, 8, 12. SMB belongs to a plant-specific family of transcription factors carrying a NAC domain (NAM - no apical meristem; ATAF1 and -2, and CUC2 - cup-shaped cotyledon). SMB promotes the expression of genes involved in the initiation and execution of LRC cell death, including the senescence-associated bifunctional nuclease BFN1 and the putative aspartic protease PASPA3 6, 11. BFN1 localizes in the ER, but upon cell death the protein is released and its nuclease activity ensures rapid and irreversible degradation of RNA and DNA in the nucleus and cytoplasm as part of a rapid cell-autonomous corpse clearance at the root surface 6, 13, 14. Accordingly, DNA and RNA fragmentation in bfn1-1 loss-of-function mutants is delayed 6. Precise timing of cell death and elimination of LRC cells before they fully enter the elongation zone is essential for maintaining root cap size and optimal root growth 6. Loss of SMB activity results in a delayed cell death, causing LRC cells to enter the elongation zone where they eventually die without expression of dPCD executor genes in the root cap 6. Interestingly, the aberrant cell death of LRC cells in the elongation zone of smb-3 mutants is not followed by corpse clearance, resulting in an accumulation of uncleared cell corpses along the entire root surface 6.
Despite its importance in root morphology and plant development, little is known about the importance of dPCD and rapid cell corpse clearance on plant-microbe interactions. To address this question, we tested two well-characterized loss-of-function T-DNA insertion lines, smb-3 and bfn1-1, during colonization with Serendipita indica, a beneficial fungus of the order Sebacinales. As a root endophyte, S. indica colonizes the epidermal and cortex layers of a broad range of different plant hosts, conferring various beneficial effects, including plant growth promotion, protection against pathogenic microbes and increased tolerance to abiotic stresses 15, 16, 17. The colonization strategy of S. indica comprises an initial biotrophic interaction, followed by a growth phase associated with a restricted host cell death that does not, however, diminish the beneficial effects on the plant host. The induction of restricted cell death in the epidermal and cortex layers is a crucial component of the colonization strategy of S. indica and is accompanied by an increased production of fungal hydrolytic enzymes 1, 18. The switch between biotrophic and cell death-associated phase can vary depending on the host system and environmental conditions, but has been postulated to occur approximately 6 to 8 days post inoculation (dpi) in Arabidopsis 18. Although several effector proteins involved in fungal accommodation have been described 19, 20, 21, 22, 23, 24, the exact mechanism by which S. indica manipulates host cell death and the role of dPCD in fungal accommodation in the roots are largely unclear.
Here, we show that the accumulation of uncleared LRC cell corpses on the roots of smb-3 mutants triggers hypercolonization by S. indica, especially around the meristematic zone, and delays S. indica-induced root growth promotion. We propose that a tight regulation of host dPCD and rapid and complete root cap clearance play important roles in restricting fungal colonization at the root apical meristem. Furthermore, we show that S. indica downregulates BFN1 in older and differentiated epidermal cells to promote fungal accommodation. Our results emphasize that beneficial microbes have the ability to modify plant dPCD processes to enhance host colonization.
Results
The SMB-mediated clearance of dead cells protects the root meristem and regulates symbiosis
dPCD and corpse clearance are the final steps of LRC differentiation that maintain root cap organ size in root tips of Arabidopsis. This process is orchestrated by the LRC-specific transcription factor SMB and executed by its direct and indirect downstream targets (Fig. 1A). To characterize the role of disrupted dPCD in Arabidopsis LRCs, we analyzed the phenotypic implications of the SMB loss-of-function allele smb-3. We first visualized the extent of cell death in the smb-3 T-DNA insertion line with Evans blue staining, a viability dye that penetrates non-viable and damaged/dying cells 25. Evans blue staining highlighted the presence of uncleared LRC cell corpses along the surface of primary roots of smb-3 mutants, starting at the distal boarder of the meristematic zone (Fig. 1B, C and Fig. S1A). We further characterized smb-3 mutants with Proteostat staining, a fluorescent dye that binds to quaternary protein structures typically found in misfolded and/or aggregated and condensed proteins (hereafter referred to as protein aggregates) 26. Proteostat staining revealed an accumulation of protein aggregates in uncleared dead LRC cells adhering to the roots of smb-3 mutants (Fig. 1D – F). Additional filter trap analysis confirmed increased levels of protein aggregates in smb-3 mutant compared to WT roots (Fig. 1G). We previously observed that aggregated proteins accumulate in WT roots in dying and sloughed columella cell packages but not in young LRC-, meristematic- or healthy differentiated-cells 26. Notably, in smb-3 mutants, neither Proteostat nor Evans blue was detected in young LRC cells covering the meristem or in epidermal cells beneath the uncleared LRC cell corpses along the elongation and differentiation zone. This highlights that loss of SMB activity specifically affects the induction of dPCD in LRC cells at the transition between meristematic and elongation zone (Fig. 1D, F and Fig. S1A). These data show that the delayed cell death of LRC cells of smb-3 mutants in the elongation zone is accompanied by an impaired protein homeostasis (proteostasis), resulting in the accumulation of misfolded and aggregated proteins in uncleared LRC cell corpses.
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smb-3 mutant roots exhibit uncleared cell corpses loaded with misfolded / aggregated proteins.
(A) Schematic representation of lateral root cap (LRC) development in WT and smb-3 mutant plants, impaired in dPCD. (B) Evans blue staining of the differentiation zone in 14-day-old WT and smb-3 roots. Scale indicates 100 μm. (C) Relative quantification of Evans blue staining of the differentiation zone in 14-day-old WT and smb-3 mutant roots (in reference to B). 5 plants per genotype were used, taking 4 images per plant along the main root axis. Statistical relevance was determined by unpaired, two-tailed Student’s t test before normalization (****P < 0.0001). (D) CLSM images of 10-day-old WT and smb-3 mutant roots stained with Proteostat (red) showing the meristematic- and the beginnings of the elongation-zone. Scale indicates 100 μm. (E) Magnification of the differentiation zone of WT and smb-3 mutant roots. Proteostat (red) and Hoechst (blue) channels are shown. Scale indicates 50 μm. (F) Quantification of relative Proteostat fluorescence levels, comparing the differentiation and meristematic zones of WT and smb-3 mutants. 5 x 10-day-old plants were used for each genotype. Statistical significance was determined by one-way ANOVA and Tukey’s post hoc test before normalization (significance threshold: P 0.05). (G) Filter trap and SDS-PAGE analysis with anti-poly-glutamine (polyQ) antibodies of 15-day-old WT and smb-3 mutant roots. The images are representative of two independent experiments.
To assess the effects of impaired dPCD processes in the smb-3 mutant root cap on plant- microbe interactions, we measured colonization rates of S. indica. We quantified extraradical colonization using the chitin-binding fluorescent marker Alexa Fluor 488 conjugated to Wheat Germ Agglutinin (WGA-AF 488) as a proxy for fungal biomass. We compared staining intensities of WGA-AF 488 between S. indica-colonized WT and smb-3 roots. S. indica showed a clear hypercolonization phenotype along the main root axis of smb-3 mutants (Fig. 2A, B). On WT roots, S. indica preferentially colonized the differentiation zone, leaving the meristematic and elongation zone largely uncolonized, whereas mycelial growth was clearly detectable at the root tips of smb-3 plants (Fig. 2A and Fig. S1B) 1, 2. Intraradical colonization by S. indica was quantified by comparing fungal and plant single-copy housekeeping marker genes using quantitative PCR (qPCR), after washing of roots to remove outer fungal mycelium. The results showed a significant increase in intraradical fungal accommodation in smb-3 mutants compared to WT roots (Fig. 2C). To assess the biological implications of hypercolonization, we measured S. indica-induced root growth in WT and smb-3 mutants. While S. indica consistently and significantly increased root length in WT plants at 8, 10 and 14 dpi, increased length of smb-3 mutant roots was only observable at later stages of colonization, indicating a delayed growth promotion phenotype (Fig. 2D). Detailed cytological analysis confirmed that S. indica grew extensively around the meristematic zone of smb-3 mutants but not of WT roots (Fig. 3A, B) and showed increased colonization of smb-3 mutants in the differentiation zone (Fig. 3C). We further observed that S. indica was accommodated in cells that were subject to cell death and protein aggregation in the smb-3 background (Fig. 3B, D, E). Together, these findings indicate that loss of SMB activity in the root cap results in an accumulation of uncleared LRC cell corpses that promotes fungal colonization from the meristematic to the differentiation zone. Therefore, we postulate that the continuous clearance of root cap cells in WT roots is important to limit microbial colonization along the entire root axis and prevent microbial colonization in the meristematic zone.
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smb-3 mutants display extraradical hypercolonization and increased intraradical colonization by S. indica.
(A) Representative images show extraradical colonization of 10-day-old WT and smb-3 mutant seedlings (seed inoculated). S. indica was stained with WGA-AF 488. Roots were scanned and captured with a LI-COR Odyssey M imager using the bright field (BF) and Alexa Fluor 488 channel (green). White arrowheads indicate colonization of the root tip in the smb-3 mutant background. Scale indicates 5 mm. (B) Relative quantification of WGA-AF 488 signal as proxy for extraradical colonization on smb-3 mutants and WT roots (in reference to A). The statistical comparison was made by two-tailed Student’s t test for unpaired samples (****P < 0.0001) using 10 plants. (C) Measurement of intraradical colonization in WT and smb-3 mutant roots were performed by qPCR. Roots from 7 biological replicates were collected and washed to remove extra-radical hyphae, using approximately 30 seedlings for each genotype per replicate. The graph is normalized to WT for relative quantification of colonization. Statistical analysis was done via two-tailed Student’s t test for unpaired samples (*P = 0.0466). (D) Root length measurements of WT and smb-3 mutants, with mock or S. indica treatments (seed inoculated) over a two-week time-period show S. indica-induced growth promotion in WT roots at all time points. Growth promotion of smb-3 mutants during S. indica colonization was delayed and only observed at later stages of colonization but not during earlier time points. To assess growth promotion, 3 biological replicates for both genotypes were used, each with around 40 plants per replicate. Root length was measured at 8, 10 and 14 dpi and statistical analysis was performed via one-way ANOVA and Tukey’s post hoc test (significance threshold: P = 0.05).
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Cytological analyses of S. indica-colonized smb-3 mutants and WT roots.
For CLSM analyses, 7-day-old seedlings were inoculated with S. indica spores and roots were analyzed at 10 dpi. (A) Representative images of the meristematic zone of Arabidopsis WT and smb-3 mutants during S. indica colonization. WGA-AF 488 stain (green) was used to visualize fungal structures. Transmitted light (TL) images are also shown. Scale indicates 100 μm (B) Magnification of a smb-3 mutant root tip colonized with S. indica. Asterisks indicate penetration of hyphae into dead cells stained with propidium iodide (PI – magenta). Scale indicates 100 μm. (C) Representative images of the differentiation zone of WT and smb-3 mutants colonized with S. indica and stained with WGA-AF 488 and PI. Scale indicates 100 μm. (D) Representative images of the meristematic zone of WT and smb-3 mutant root tips inoculated with S. indica, stained with WGA-AF 488 and Proteostat (red). Scale indicates 100 μm (E) Magnification of the root differentiation zone of smb-3 mutants showing S. indica colonization, stained with WGA-AF 488, Hoechst and Proteostat. Penetration of fungal hyphae into uncleared cell corpses is marked with asterisks. Dotted yellow line indicates LRC cell corpse. Scale indicates 50 µm. (F) Representative images of the differentiation zone of S. indica-colonized WT and smb-3 roots at 10 dpi, stained with Evans blue. Scale indicates 100 μm.
Interestingly, Evans blue cell death staining of S. indica-inoculated smb-3 mutants displayed a clearing of LRC cell corpses from the surface of smb-3 mutant roots over time, while mock-treated smb-3 mutant roots remained littered with LRC cell corpses (Fig. 3F and Fig. S1C). This observation indicates that S. indica is able to degrade uncleared cell corpses, which likely provide additional nutrients that fuel fungal hypercolonization in the smb-3 mutant background.
The senescence associated plant nuclease BFN1 is exploited by beneficial microbes to facilitate root accommodation
To further explore the role of root dPCD during S. indica accommodation in Arabidopsis, we performed transcriptome analysis, tracking developmental cell death-marker gene expression during different colonization stages 27. The major regulator in LRCs, SMB, showed no significant changes in expression during fungal colonization (Fig. 4A, C). However, a marked decrease in the expression of BFN1 was observed at 6 dpi in Arabidopsis (Fig. 4B, C). To validate the RNA-Seq analysis, we performed whole-root qPCR of WT mock- and colonized-roots, confirming BFN1 downregulation at the onset of cell death in S. indica-colonized plants (Fig. 4D).
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BFN1 is downregulated during interaction with S. indica.
RNA-Seq expression profiles of (A) SMB and (B) BFN1 in Arabidopsis roots mock-treated or inoculated with S. indica at 3, 6 and 10 dpi. The log2-transformed TPM values are shown and the lines indicate average expression values among the 3 biological replicates. Asterisk indicates significantly different expression (adjusted p-value < 0.05) (C) The heat map shows the expression values (TPM) of Arabidopsis dPCD marker genes with at least an average of 1 TPM across Arabidopsis roots mock-treated or inoculated with S. indica at 3, 6 and 10 dpi. The TPM expression values are log2 transformed and row-scaled. Genes are clustered using spearman correlation as distance measure. Each treatment displays the average of three biological replicates. The dPCD gene markers were previously defined (Olvera-Carrillo et al., 2015). (D) BFN1 expression in WT Arabidopsis during S. indica colonization at 8 and 11 dpi. RNA was isolated from 3 biological replicates for qPCR analysis, comparing BFN1 expression with an Arabidopsis ubiquitin marker gene. (E) Representative CLSM images of the differentiation zone of mock- and S. indica- colonized pBFN1::NLS-tdTOMATO reporter roots at 7dpi. The tdTOMATO signal (magenta) represents BFN1 expression and S. indica was stained with WGA-AF 488 (green). Scale indicates 100µm. (F) Whole seedling scans of mock- and S. indica-treated pBFN1::NLS-tdTOMATO plants taken with a LI-COR Odyssey M imager at 7dpi. Images show BFN1 expression via tdTOMATO signal alone, BFN1 expression together with S. indica stained by WGA-AF 488 and S. indica colonization alone. Scale indicates 5mm.
While SMB expression is restricted to the LRC, BFN1 exhibits a broader expression pattern across various cell types and tissues, such as root cap cells, cells adjacent to emerging lateral root primordia, differentiating xylem tracheary elements, as well as senescent leaves, and abscission zones of flowers and seeds 28, 29. This widespread expression establishes BFN1 as a key player in the general regulation of dPCD and senescence processes in various tissues in Arabidopsis. To visualize the extent of BFN1 downregulation upon S. indica colonization in different zones of the root, we used a transgenic BFN1 promoter-reporter line (pBFN1::NLS-tdTOMATO) 11. In agreement with the previously described GUS reporter lines 28, we detected activation of the BFN1 promoter via accumulation of the fluorescent tdTOMATO signal in the nucleus of root cap and xylem cells in mock-treated roots (Fig. S2A). Additionally, we observed promoter activation in epidermal root cells of the differentiation zone (Fig. 4E). In the distal region of the differentiation zone in young epidermal cells, the tdTOMATO signal was observed in nuclei, while in the basal region of the differentiation zone in older epidermal cells, the tdTOMATO signal appeared to be dispersed (Fig S2). These findings indicate ongoing nuclear membrane degradation and cell death in the older part of the root, independent of fungal colonization and suggest activation of BFN1 during root epidermal cell aging/senescence. Next, we inoculated the pBFN1 reporter lines with S. indica and observed a reduction in promoter activity in epidermal cells that were in contact with the fungus compared with mock-treated roots (Fig. 4E, F). BFN1 expression and nuclear localization in the root cap or xylem was not affected by S. indica colonization (Fig. S2B). This indicates that the downregulation of BFN1 by S. indica occurs mainly in epidermal cells of the differentiation zone and is regulated independently of SMB and its activity in the root cap.
To assess the phenotypic effects of BFN1 downregulation, we analyzed Arabidopsis bfn1-1 KO mutants using the cell death and protein aggregates markers, Evans blue and Proteostat. Staining of bfn1-1 mutants with Evans blue showed an increase of cell remnants in the epidermal cell layer of the differentiation zone (Fig. 5A, B), consistent with a proposed delay of dPCD and cell corpse clearance by the BFN1 activity. Furthermore, while WT roots were devoid of protein aggregates, bfn1-1 mutants exhibited aggregates along the primary root axis, starting at the transition between elongation and differentiation zone. The meristematic zone remained free of protein aggregates (Fig. 5C, D). These data suggest that the lack of BFN1 activity in the root cap, xylem, and senescent epidermis creates a general cellular stress in the roots that affects proteostasis in the differentiation zone (Fig. S3). Similar to bfn1-1, WT roots colonized by S. indica showed increased Evans blue and Proteostat signal in the differentiation zone. Aggregated proteins were detected in colonized and adjacent non-colonized cells along the differentiation zone, suggesting a non-cell autonomous host response to the fungus (Fig. S4).
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BFN1 downregulation promotes fungal accommodation.
(A) Microscopy images of the differentiation zone of 14-day-old WT and bfn1-1 mutant roots, stained with Evans blue. Scale indicates 100 μm. (B) Quantification of Evans blue staining (in reference to A), comparing 14-day-old WT and bfn1-1 mutants. 10 plants were used for each genotype, taking 4 pictures along the main root axis per plant. ImageJ was used to calculate the mean grey value to compare relative staining intensity. Statistical significance was determined using an unpaired, two-tailed Student’s t test before normalization (*** P < 0.0001). (C) Proteostat staining of 10-day-old WT and bfn1-1 mutant root tips. Scale indicates 100 μm. (D) Quantification of Proteostat staining (in reference to C) using 4 to 5 10-day-old WT and bfn1-1 mutants. Statistical analysis was performed via one-way ANOVA and Tukey’s post hoc test before normalization (significance threshold: P ≤ 0.05). (E) Extraradical colonization of 10-day-old WT and bfn1-1 mutant plants, seed-inoculated with S. indica and stained with WGA-AF 488 (green). Roots were scanned and captured with a LI-COR Odyssey M imager. Arrowheads indicate the position of the uncolonized root tips. Scale indicates 5 mm. (F) Relative quantification of extraradical colonization of bfn1-1 mutant and WT roots, using WGA-AF 488 signal as a proxy for fungal biomass (in reference to E). Statistical comparisons were made by unpaired, two-tailed Student’s t test for unpaired samples (**P < 0.01) (G) Intraradical colonization of WT and bfn1-1 mutants was measured via qPCR. Roots from 7 biological replicates were collected and washed to remove outer extraradical mycelium, using approximately 30 plants per time point and replicate for each genotype. Each time point was normalized to WT for relative quantification of colonization. Statistical analysis was performed via one-way ANOVA and Tukey’s post hoc test (significance threshold: P 0.05).
To investigate the biological relevance of BFN1 downregulation during S. indica root colonization, we quantified extraradical fungal growth using the WGA-AF 488 stain. When comparing staining intensities of S. indica-inoculated bfn1-1 mutants and WT seedlings, we observed a significantly stronger fluorescence signal at the roots of bfn1-1 mutants, indicating a higher extraradical fungal colonization along the differentiation zone (Fig. 5E, F and Fig. S3D). However, similar to WT roots, bfn1-1 mutants did not exhibit fungal colonization around the meristematic zone as observed in smb-3-colonized roots (Fig. 5E). Quantification of intraradical colonization by qPCR, after removal of outer fungal mycelium, showed a significant increase of S. indica biomass in bfn1-1 mutants at later stages of interaction (Fig. 5G). Together, these results emphasize that downregulation of BFN1 during colonization is beneficial for intra- and extraradical fungal accommodation in the differentiation zone.
Next, we investigated the impact of other beneficial microbes on dPCD by examining transcriptional responses in Arabidopsis roots colonized by different organisms. These included S. vermifera, an orchid mycorrhizal fungus closely related to S. indica, and two bacterial synthetic communities derived from either Arabidopsis roots or the rhizosphere of Hordeum vulgare 16. In all three interactions, BFN1 expression was consistently decreased in Arabidopsis roots (Fig. S5A). Additionally, RNA-Seq analysis of Arabidopsis dPCD marker genes during S. vermifera colonization confirmed the downregulation of BFN1 (Fig. S5 B, C). Our findings indicate that microbes may benefit from delayed post-mortem corpse clearance after dPCD in host plants and suggest that beneficial microbes may have evolved mechanisms to manipulate dPCD pathways to increase colonization (Fig. 6).
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dPCD and its proposed effects on plant-microbe interactions.
The root cap protects and covers the stem cells of the root apical meristem. Its size in Arabidopsis is maintained by a high cellular turnover of root cap cells. While the columella root cap is shed from the root tip, a dPCD machinery marks the final step of LRC differentiation and prevents LRC cells from entering into the elongation zone. Induction of cell death by the transcription factor SMB is followed by irreversible DNA fragmentation and cell corpse clearance, mediated by the nuclease BFN1, a downstream executor of dPCD (Fendrych et al., 2014). The absence of dPCD induction in the smb-3 knockout mutant leads to a delay in LRC differentiation and allows LRC cells to enter the elongation zone, where they die uncontrolled, resulting in an accumulation of LRC cell corpses along the differentiation zone. In a WT background, the fungal endophyte S. indica colonizes the differentiation zone of Arabidopsis roots and can also be found in shed columella cell packages. The impaired dPCD of the smb-3 mutant phenotype results in a hypercolonization of Arabidopsis, along the differentiation zone as well as the meristematic zone, highlighting that the continuous clearance of root cap cells is necessary for restricting microbial accommodation at the meristematic zone. Loss of the downstream dPCD executor BFN1 does not affect fungal colonization in the meristematic zone but increases accommodation by S. indica in the differentiation zone, where BFN1 appears to be involved in dPCD of senescent epidermal cells and undergoes downregulation during S. indica colonization.
Discussion
In this study, we investigated the functional link between dPCD and microbial accommodation in roots. Impaired dPCD in the Arabidopsis smb-3 and bfn1-1 mutants increased colonization by the beneficial endophyte S. indica. The smb-3 mutants display hypercolonization along the entire primary root, suggesting that the extra sheet of cell corpses surrounding smb-3 roots provide additional and easily accessible nutrients that fuel fungal colonization. In fact, we observed a clearing effect with progressive colonization stages. Furthermore, hypercolonization of Arabidopsis, caused by the loss of dPCD in the root cap, mitigates the beneficial effects of S. indica by delaying the induction of growth promotion. Most notably, we observed hypercolonization of the meristematic zone of smb-3 mutants (Fig. 6). The root apical meristem embedded in this zone of the root tip is essential for root growth, as all primary root tissue originates from these continuously dividing and differentiating stem cells 30. This sensitive tissue is surrounded by the root cap, which protects it from external stresses 31. The phenotype of smb-3 mutants shows resemblance to the human skin disease hyperkeratosis. In healthy human skin, a pool of stem cells produces layers of cells that divide, differentiate, die, and are shed. Such developmental programs form a physical and dynamic barrier against environmental factors. Microbes attempting to establish themselves are consistently removed by skin exfoliation 32. Patients with hyperkeratosis show an accumulation of dead cells on the outer skin layer, making them more susceptible to microbial infection 33. Likewise, malfunctions within regulated cell death in mammal gut epithelial cells produce death-induced nutrient release (DINNR) that can fuel bacterial growth and infection and could cause a variety of disorders such as inflammatory diseases 34. The importance of an intact root cap in plant-microbe interactions is further highlighter by the fact that the physical removal of root caps in maize plants leads to increased colonization of the root tip by the plant growth promoting rhizobacterium (PGPR) Pseudomonas fluorescens 35 and to changes in the rhizosphere microbiome composition along the root axis 36. Moreover, it has been suggested that border cells released from the root cap may distract root-feeding nematodes from attacking plant roots 37. Our results provide strong evidence that root cap size maintenance in the form of constant root cap cell turnover in Arabidopsis acts as a dynamic barrier, analogous to epidermal cell turnover in animals. It thus represents a sophisticated physical mechanism to prevent or reduce intracellular microbial colonization near the root meristematic tissue and contributes to the maintenance of a beneficial interaction with root endophytes.
Mutation of BFN1 displayed significantly increased colonization in the differentiation zone but not in the meristematic zone (Fig. 6). The differences in colonization patterns to the smb-3 mutants likely reflect spatial expression and localization patterns of SMB and BFN1 activity throughout Arabidopsis roots. While the expression of the transcription factor SMB is restricted to the LRC, the senescence-associated nuclease BFN1 is expressed in different tissues undergoing dPCD and senescence below and above ground 28, 29. Here, we show that BFN1 is additionally expressed in differentiated root epidermal cells that undergo nuclear degradation during root maturation. This suggests an age-dependent dPCD in the outer epidermal layer of Arabidopsis roots where expression of BFN1 possibly pre-dates cortical and epidermal abscission during secondary root growth and contributes to clearance of cell corpses during periderm emergence 38. This process resembles root cortex senescence (RCS) and cell death (RCD) in grass species such as wheat, barley, and corn, which typically start in the epidermis and spread toward the endodermis 39.
Additionally, we observe differences in the presence and distribution of protein aggregates in smb-3 and bfn1-1 mutants. In smb-3 mutants protein aggregates are restricted to LRC cells adhering to the primary root. In contrast, bfn1-1 mutation results in scattered cell death in the epidermal cell layer, which is additionally littered with protein aggregates along the differentiation zone, regardless of the occurrence of cell death.
While we show that dPCD protect the meristem from microbial colonization, we propose that some adapted microbes manipulate host dPCD processes by affecting the transcriptional expression of BFN1 to facilitate accommodation in the root. Whether active interference by fungal effector proteins, fungal-derived signaling molecules or a systemic response of Arabidopsis roots underlies BFN1 downregulation by S. indica remains to be investigated. It was recently demonstrated that small active metabolites produced either by Toll/interleukin-1 receptor (TIR)-containing leucine-rich repeat (NLR) receptors 40 or by fungal-derived enzymes 19 through hydrolysis of RNA/DNA can mediate host cell death. This raise the exiting possibility that RNAse and DNAse activities of BFN1 may be involved in the production of small active nucleotide-derived metabolites that affect cell death, cell corpse clearance, proteostasis and fungal accommodation in the differentiation zone. This hypothesis should be followed up by metabolomic and proteomic approaches. Notably, we have shown that other beneficial microbes such as the closely related fungus S. vermifera and synthetic bacterial communities isolated from Arabidopsis and H. vulgare 16 also downregulate BFN1 in Arabidopsis. These findings emphasize the presence of a conserved pathway influenced by diverse beneficial microbes to downregulate BFN1 expression in epidermal tissue to facilitate symbioses.
In conclusion, our data show that tight regulation of host dPCD in epidermal- and root cap-tissue plays an important role in restricting fungal colonization. From a microbial perspective, dPCD pathways represent a potential intersection for promoting symbiotic interactions and possibly nutrient availability (Fig. 6). These results shed light on the complex relationship between PCD and microbial accommodation in plant roots, offering valuable insights into the development of plants that establish more efficient partnerships with beneficial microbes.
Materials and methods
Fungal strains and growth conditions
Fungal experiments were performed with Serendipita indica strain DSM11827 (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). S. indica was grown on complete medium (CM) containing 2% (w/v) glucose and 1.5% (w/v) agar (Hilbert et al., 2012). Fungal material was grown at 28°C in the dark for 4 weeks before spore preparation. For additional experiments, S. vermifera (MAFF305830) was used and grown on MYP medium containing 1.5% agar at 28°C in darkness for 3 weeks before mycelium preparation for root inoculation.
Plant material and growth conditions
Seeds of Arabidopsis thaliana wild-type (WT) ecotype Columbia 0 (Col-0) and T-DNA insertion mutants (bfn1-1 [GK-197G12] and smb-3 [SALK_143526C]) in Col-0 background were used for experiments. Seeds were surface sterilized in 70% ethanol for 15 min and 100% ethanol for 12 min, stratified at 4°C in the dark for 3 days and germinated and grown on ½ MS medium (Murashige-Skoog Medium, with vitamins, pH 5.7) containing 1% (w/v) sucrose and 0.4% (w/v) Gelrite under short-day conditions (8 h light, 16 h dark) with 130 μmol m-2 s-1 light and 22°C/18 °C.
Fungal inoculation
One-week-old seedlings were transferred to 1/10 PNM (Plant minimal Nutrition Medium, pH 5.7) plates without sucrose, using 15 to 20 seedlings per plate. Under sterile conditions, spores of S. indica were scraped from agar CM plates using 0.002% Tween water (Roth), washed two times with ddH2O and pipetted in a volume of 2 ml on plant roots and surrounding area in a concentration of 5x105 spores per plate. ddH2O was used for inoculation of mock plants. For S. vermifera inoculation, mycelium was scrapped from plates in ddH2O, washed and added to Arabidopsis roots in a volume of 1 ml of a stock solution of 1 g / 50 ml.
In case of experiments using seeds inoculation with S. indica, Arabidopsis seeds were surface sterilized, incubated with fungal spore solution at 5x105 concentration for 1 hour and plated on ½ MS plates (without sucrose).
Evans blue staining
For the visualization of cell death in Arabidopsis roots a modified protocol by 25 was used. Roots were washed three times in ddH2O to remove lose external fungal mycelium and then stained for 15 min in 2 mM Evans blue (Sigma-Aldrich) dissolved in 0.1 M CaCl2 pH 5.6. Following, roots were washed extensively with ddH2O for 1 hour and a Leica M165 FC microscope was used for imaging. To quantify Evans blue staining intensity, ImageJ was used to invert the pictures, draw out individual roots and measure and compare mean grey values.
Extraradical colonization assays
To quantify extraradical colonization of S. indica on Arabidopsis, seed-inoculated plants were grown for 10 days. Inoculated and mock-treated seedlings were stained directly on plate by pipetting 2 ml of 1X PBS solution containing Alexa Fluor 488 (5 ul/mL) conjugated with Wheat Germ Agglutinin (WGA-AF 488, Invitrogen). After 2 min of incubation, the roots were washed twice on the plate with 1X PBS solution. The stained seedlings were transferred to a fresh ½ MS square plate. In order to perform a correct and focused scan of the plate with the roots, it was checked that the solid MS medium was flat and even and had no unevenness. To scan the plate, we used an Odyssey M Imaging System (LI-COR Biosciences) and the LI-COR Acquisition Software 1.1 (LI-COR Biosciences). In the software, we selected custom assay and then membrane. We selected the area in the plate to be scanned and selected the channels 488 (for WGA-Alexa Flour 488) and RGB trans (for bright field). We defined the focus offset between 3.5 mm and 4.0 mm (depending on the thickness of the MS medium). For resolution we selected 10 µm when scanning two plants or 100 µm when scanning several plants. Quantification of WGA-AF 488 fluorescence was performed using EmpiriaStudio Software (LI-COR Biosciences).
RNA extraction (intraradical colonization assay)
To measure intraradical colonization via RNA extraction and PCR, plants were harvested at three time points around 7, 10 and 14 dpi. The roots were extensively washed with ddH2O and tissue paper was used to carefully wipe off mycelium on the surface of the roots. After cleaning the roots were shock frozen in liquid nitrogen and RNA was extracted with TRIzol (Invitrogen, Thermo Fisher Scientific, Schwerte, Germany). After a DNase I (Thermo Fisher Scientific) treatment according to the manufacturer’s instructions to remove DNA, RNA was used to generate cDNA through the utilization of the Fermentas First Strand cDNA Synthesis Kit (Thermo Fisher Scientific).
Quantitative RT-PCR analysis
The quantitative real time-PCR (qRT-PCR) was performed using a CFX connect real time system (BioRad) with the following program: 95 °C 3min, 95 °C 15s, 59 °C 20s, 72 °C 30 s, 40 cycles and melting curve analysis. Relative expression was calculated using the 2- ΔΔCT method (Livak and Schmittgen 2001). qRT-PCR primers can be found in Table S1.
Filter trap analysis
Filter trap assays were performed as previously described 26, 41. Protein extracts were obtained using native lysis buffer (300 mM NaCl, 100 mM HEPES pH 7.4, 2 mM EDTA, 2% Triton X-100) supplemented with 1x plant protease inhibitor (Merck). Cell debris was removed by several centrifugation steps at 8,000 x g for 10 min at 4 °C. The supernatant was separated, and protein concentration determined using the Pierce BCA Protein Assay Kit (Thermo Fisher). A cellulose acetate membrane filter (GE Healthcare Life Sciences) and 3 filter papers (BioRad, 1620161) were immersed in 1x PBS and placed in a slot blot apparatus (Bio-Rad) connected to a vacuum system. The membrane was equilibrated by 3 washes with equilibration buffer (native buffer containing 0.5% SDS). 300, 200 and 100 µg of the protein extract were mixed with SDS at a final concentration of 0.5% and filtered through the membrane. The membrane was then washed with 0.2% SDS and blocked with 3% BSA in TBST for 30 minutes, followed by 3 washes with TBST. Incubation was performed with anti-polyQ [1:1000] (Merck, MAB1574). The membrane was washed 3 times for 5 min and incubated with secondary antibodies in TBST 3% BSA for 30 min. The membrane was developed using the Odyssey M Imaging System (LI-COR Biosciences). Extracts were also analyzed by SDS-PAGE and western blotting against anti-Actin [1:5000] (Agrisera, AS132640) to determine loading controls.
Confocal laser scanning microscopy (CLSM) and Proteostat staining quantification
CLSM images were acquired using either the FV1000 confocal laser scanning microscope (Olympus) or a Meta 710 confocal microscope with laser ablation 266 nm (Zeiss). All images were acquired using the same parameters between experiments. Excitation of WGA-AF 488 was done with an argon laser at 488 nm and the emitted light was detected with a hybrid detector at 500-550 nm. Proteostat was excited at 561 nm and the signal was detected between 590-700 nm. Hoechst was excited with a diode laser at 405 nm and the emitted light was detected with a hybrid detector at 415-460 nm.
Proteostat staining
For the detection of aggregated proteins, we used the Proteostat Aggresome detection kit (Enzo Life Sciences). Seedlings were stained according to the manufacturer’s instructions. Seedlings were incubated with permeabilizing solution (0.5% Triton X-100, 3 mM EDTA, pH 8.0) for 30 minutes at 4°C with gentle shaking. The seedlings were washed twice with 1X PBS. Then the plants were incubated in the dark with 1x PBS supplemented with 0.5 µl/ml Proteostat and 0.5 µl/ml Hoechst 33342 (nuclear stain) for 30 min at room temperature. Finally, the seedlings were washed twice with 1x PBS and mounted on a slide for CLSM analysis or in mounted in fresh MS plates for LI-COR analysis. Quantification of Proteostat fluorescence was performed using Fiji software or EmpiriaStudio Software (LI-COR Biosciences).
Transcriptomic analysis
Arabidopsis Col-0 WT roots were inoculated with S. indica. Arabidopsis roots were harvested from mock-treated plants and inoculated plants at four different time points post inoculation: 1, 3, 6 and 10 dpi. Three biological replicates were used for each condition. The RNA-seq libraries were generated and sequenced at US Department of Energy Joint Genome Institute (JGI) under a project proposal (Proposal ID: 505829) 42, 43. For each sample, stranded RNA-Seq libraries were generated and quantified by qRT-PCR. RNA-Seq libraries were sequenced with Illumina sequencer. Raw reads were filtered and trimmed using the JGI QC pipeline. Filtered reads from each library were aligned to the Arabidopsis genome (TAIR10) using HISAT2 44 and the reads mapped to each gene were counted using featureCounts 45. Samples harvested at 1 dpi were omitted from the analysis because we decided to focus on the time points at which the interaction between Arabidopsis and S. indica is well established. Differential gene expression analysis was performed using the R package DESeq2 46. Genes with an FDR adjusted p-value < 0.05 were considered as differentially expressed genes (DEGs).
Acknowledgements
We thank the imaging facilities of CECAD (A. Schauss and C. Jüngst) and CEPLAS (P.S. Tan) for their assistance with CLSM. We thank Lisa Mahdi for conducting the experiments and providing the samples used for the RNA-seq analysis. We further would like to thank Yu Zhang, Sravanthi Tejomurthula, Daniel Peterson, Vivian Ng & Igor Grigoriev and their work performed in the work proposal (10.46936/10.25585/60001292) conducted by the U.S. Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy operated under Contract No. DE-AC02-05CH11231. AZ, NC and EL acknowledge support by the German Research Foundation (DFG) - Excellence Strategy of the Federal Republic of Germany - EXC-2048/1 - project ID 390686111. AZ and NC acknowledge support by the SFB-1403-414786233 and DV by the German Excellence Strategy-CECAD, EXC 2030-390661388.
Contributions
Conceptualization: NC, EL, MN, AZ; Methodology: NC, EL, CDQ; Investigation: NC, EL, CDQ, DV, MN, AZ; Visualization: NC, EL, CDQ; Project supervision: AZ; Writing original draft: NC, EL, AZ with the help of all authors.
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