Ustilago maydis PR-1-like protein has evolved two distinct domains for dual virulence activities

The diversification of effector function, driven by a co-evolutionary arms race, enables pathogens to establish compatible interactions with hosts. Structurally conserved plant pathogenesis-related PR-1 and PR-1-like (PR-1L) proteins are involved in plant defense and fungal virulence, respectively. It is unclear how fungal PR-1L counters plant defense. Here, we show that Ustilago maydis UmPR-1La and yeast ScPRY1, with conserved phenolic resistance functions, are Ser/Thr-rich region mediated cell-surface localization proteins. However, UmPR-1La has gained specialized activity in sensing phenolics and eliciting hyphal-like formation to guide fungal growth in plants. Additionally, U. maydis hijacks maize cathepsin B-like 3 (CatB3) to release functional CAPE-like peptides by cleaving UmPR-1La’s conserved CNYD motif, subverting plant CAPE-primed immunity and promoting fungal virulence. Surprisingly, CatB3 avoids cleavage of plant PR-1s, despite the presence of the same conserved CNYD motif. Our work highlights that UmPR-1La has acquired additional dual roles to suppress plant defense and sustain the infection process of fungal pathogens.

The corn smut Ustilago maydis is a dimorphic fungus switching from a unicellular haploid cell to pathogenic dikaryotic hyphae when infecting maize 21 .This irreversible morphological shift is essential for U. maydis virulence.During the biotrophic stages of its sexual life cycle, U. maydis requires fine sensing of environmental signals to adapt to changes in plant development and direct hyphal growth toward nutrient sources 22 .It sequentially secretes a pool of effector proteins to shield hyphae from plant attacks, suppress immune responses, and manipulate plant metabolism, ultimately facilitating successful colonization of host plants 23 .Effectors that overcome plant resistance are usually considered the most rapidly evolved genes in coevolutionary arms races.However, it is unknown whether these effector proteins contribute to the integration of plant signals to promote U. maydis virulence.
In this study, we functionally characterize the secreted effector protein UmPR-1La from U. maydis to understand how the structurally conserved CAP domain of a PR-1-like protein evolved an opposite role to plant PR-1 proteins.We demonstrate that UmPR-1La promotes virulence through dual functional activities derived from two distinct motifs.UmPR-1La binds plant-derived phenolics to elicit hyphal-like structures, while a CAPE-like peptide, released from UmPR-1La via the action of a plant cysteine protease, can suppress plant immunity.Our work provides a mechanistic understanding of the fungal parasitism enabled by the dual roles of PR-1-like protein.

U. maydis PR-1-like protein is a virulence factor
Ustilago maydis encodes two CAP-domain containing PR-1-like proteins, designated UmPR-1La (UMAG_01204) and UmPR-1Lb (UMAG_04343).They consist of an N-terminal signal peptide, a serine/threonine (Ser/Thr)-rich extension region, and a C-terminal CAP domain (Fig. 1a).This extension region is also present in three out of the 17 maize PR-1 proteins 24 .The CAP domain sequence alignment showed that UmPR-1La, but not UmPR-1Lb, contains a conserved CNYx motif also found in plant PR-1s (Supplementary Fig. 1a, Fig. 1a).This suggests that a 16-amino acid peptide (PPGNYIGKFKENVSPN) following the motif may be released from UmPR-1La by a protease.Comparing the peptide sequences following the CNYx motif in plant PR-1 and smut fungal PR-1L proteins, a notable conservation was observed in the first seven amino acids (P[P/V]GN[Φ/V][I/V]G) (Fig. 1b).We designate the fungal peptides as CAPE-like peptides (CAPE-L).To understand the evolutionary relationship between smut fungal PR-1Ls versus Saccharomyces cerevisiae PRYs and plant PR-1s, we performed a phylogenetic analysis using CAP domains from several plants and fungal species (Fig. 1c).UmPR-1La and UmPR-1Lb are placed in two separate clades (clades I and II) of smut fungal PR-1L proteins.Both of these clades are distantly related to the plant PR-1 clade III.Clade I, which includes UmPR-1La, is more closely related to the ScPRY clade IV than to the smut fungal clade II.The analysis of the CAP domains infers protein evolution, which shows that the UmPR-1La and UmPR-1Lb proteins belong to two subfamilies of PR-1L proteins expanded in smut fungal pathogens.The expansion and sequence divergence of smut fungal PR-1L proteins suggest that UmPR-1La and UmPR-1Lb may have distinct functional roles.
A qRT-PCR analysis showed that PR-1La and PR-1Lb were not expressed in axenic culture, but were induced during biotrophic development of U. maydis SG200, a solopathogenic haploid strain that completes its life cycle without a mating partner 25 (Fig. 1d and Supplementary Fig. 1b).The deletion of PR-1La, but not PR-1Lb, in SG200 resulted in a decrease in disease symptoms in infected plants (Fig. 1e and Supplementary Fig. 1c).This defect could be rescued by introducing a single copy of PR-1La into the Δpr-1la mutant under the control of its native promoter.Moreover, a similar decrease in virulence was also observed in the double mutant ΔΔpr-1lab.While grown in a liquid medium or induced to form filaments on charcoal agar, the deletion mutants and complemented strains were indistinguishable from SG200 (Supplementary Fig. 1d).These findings suggest UmPR-1La is associated with the in-planta development of U. maydis.
The Ser/Thr-rich region mediates cell surface exposure of CAP domain Glycosylphosphatidylinositol (GPI)-anchored ScPRY3 and C. albicans non-GPI-anchored Rbe1p are cell surface-localized PR-1L proteins 15,26 .It has also been reported that Ser/Thr-rich regions of GPI-anchored glycoproteins could override the role of GPI anchors in surface exposure in a length-dependent manner 27 .We, therefore, hypothesized that the Ser/Thr-rich region of non-GPI anchored UmPR-1L and ScPRY1 proteins localizes CAP domains to the cell surface.As the expression of U. maydis PR-1L genes was not detected in axenic culture, we constitutively expressed both UmPR-1L proteins with C-terminal-HA-tags in SG200 under the control of the otef promoter.For a comparative assessment of localization by fluorescence microscopy, an ScPRY1-mCherry fusion protein was also constitutively expressed in yeast AH109 cells using the ADH1 promoter.Both UmPR-1La and ScPRY1-mCherry localized to the surface of SG200 filaments and AH109 cells, respectively (Fig. 2a, b).To determine the localization of UmPR-1La on filamentous cell walls after plasmolysis, we utilized the haploid AB33 strain.This strain harbors the compatible bW1/bE2 genes controlled by the nitrate-inducible nar1 promoter, enabling filamentous growth in a liquid nitrate minimal medium 28 .After subjecting the AB33_PR-1La filaments (overexpressing HA-tagged PR-1La) to plasmolysis, the AF594-immunostained PR-1La proteins were not located inside the plasmolysis-expanded region (Fig. 2c).Instead, they showed co-localization with wheat germ agglutinin (WGA)-AF488 staining chitins on the surface of the region, confirming the presence of UmPR-1La on the cell walls.
In contrast, UmPR-1Lb proteins were not found on the cell surface due to their instability.Only truncated fragments were detected after secretion (Fig. 2a, d).Interestingly, when ScPRY1 was fused with the signal-peptide (SP) of UmPR-1La and constitutively expressed in U. maydis, it did not localize to the cell surface, despite being expressed and secreted (Fig. 2a, d).Possibly the PRY1 ligand required for cell surface binding is absent in U. maydis or inaccessible to ScPRY1.The cell-surface localization of ScPRY1 in U. maydis was restored by fusing the N-terminus of UmPR-1La containing the SP and Ser/Thr-rich region to the ScPRY1 CAP-domain (N PR-1La -CAP PRY1 ) (Fig. 2a).The analysis indicated that the Ser/Thr-region is sufficient to mediate the cell surface attachment of UmPR-1La in U. maydis.
To further investigate how PR-1La binds to the surface of U. maydis cells, we purified each recombinant protein PR-1La (PR-1La24-279; FL), the Ser/Thr region (PR-1La24-153; S/T), and the CAPdomain (PR-1La  ; CAP) and incubated with SG200 filaments. Only FLand S/T proteins were bound to the cell surface (Fig. 2e).This finding further supports the notion that the N-terminal Ser/Thrextension region is necessary and sufficient to mediate the cell wall localization of PR-1La.Interestingly, the localization of UmPR-1La in the bud necks and growing tips of U. maydis sporidial cells coincided with sites of chitin and chitosan deposition 29 (Fig. 2a).This suggests that UmPR-1La proteins may associate with fungal cell walls via binding to chitin/chitosan.Alternatively, it may interact with the cell wall proteins.

UmPR-1La protects hyphae against plant toxic phenolics
We next investigated the biological relevance of the CAP domain at the fungal cell wall.ScPRY1 and ScPRY2 protect cells against eugenols in a caveolin-binding motif (CBM)-dependent manner 12,14 , and UmPR-1La was relatively close to ScPRYs in our phylogenetic analysis (Fig. 1c).
Therefore, we hypothesized that UmPR-1La could shield cells from toxic plant compounds.Based on sequence alignment of the CBMs from known lipid-binding PR-1L proteins 19 , we identified three aromatic residues at positions 1, 3, and 8 of the CBM, which are crucial for sterol/phenolic binding in ScPRY1 12 and are conserved in UmPR-1La (Fig. 3a).In contrast, UmPR-1Lb does not exhibit conservations at positions 3 and 8.The analysis suggests that UmPR-1La may bind to sterols or phenolics, potentially protecting U. maydis cells from their toxicity.To test this idea, we treated SG200 cells expressing PR-1L proteins with eugenols, which maize also produces 30 .Only cells expressing UmPR-1La proteins induced pseudohyphal structures and survived (Fig. 3b).In contrast, cells producing UmPR-1Lb, ScPRY1, or chimeric proteins (N PR-1La -CAP PRY1 ) did not form pseudohyphae and lost the ability to grow after eugenol treatment.
These observations led us to investigate whether S. cerevisiae cells could also induce pseudohyphae in response to eugenols.Despite surviving the eugenol treatment, the S. cerevisiae cells were unable to develop pseudohyphal structures, even when overexpressing ScPRY1-mCherry (Supplementary Fig. 3).The result suggests that UmPR-1La possesses an additional specialized function to induce pseudohyphal structures in U. maydis, which is absent in ScPRY1, and this structure is not needed to protect S. cerevisiae cells.This finding also explains why the PRY1 and chimeric proteins, which contain the PRY1 CAP domains capable of binding eugenols 14 , fail to shield U. maydis from eugenols, despite the conservation of the protective role of PR-1-like proteins between U. maydis and S. cerevisiae.
We further mutated the aromatic residues at positions 3 and 8 of the UmPR-1La CBM to alanine (PR-1La F212A Y217A ; FY*).UmPR-1La (FY*) was The protein accession numbers are provided.Plant CAPE peptides that have been identified and reported are indicated 8,9 .Ph Pseudozyma hubeiensis, Ss Sporisorium scitamineum, Sr Sporisorium reilianum, Uh Ustilago hordei, Ub Ustilago bromivora.c Phylogenetic analysis of plant PR-1 and PR-1-like proteins from smut fungi and yeast.Protein sequences are retrieved from the NCBI, aligned using Clustal Omega, and curated with trimAl.Analysis was carried out in MEGA 7.0 using the Maximum Likelihood method with the WAG+G+I model and 1000 bootstrap replicates.Clade I and II: smut fungal PR-1Ls; Clade III: Plant PR-1s; IV: yeast.Kb Kalmanozyma brasiliensis, Mp Melanopsichium pennsylvanicum, Man Moesziomyces antarcticus, Map Moesziomyces aphidis, Ut Ustilago trichophora, Sg Sporisorium graminicola.d qRT-PCR analysis of PR-1La expression.Total RNA was extracted from SG200-infected maize leaves harvested at the indicated days post-infection (dpi), and from cells cultured in liquid culture (A.C).Peptidylprolyl Isomerase (PPI) gene was used for normalization.The PR-1La expression level in A.C. was set to 1.0.Values are mean ± standard deviation (SD) of three biological replicates.e Virulence assay of Δpr-1l and the complementation strain.Δpr1la_PR-1La: Δpr1la was complemented by introducing a single allele of PR-1La under the control of its native promoter.Disease symptoms were scored at 12 dpi according to the severity depicted in the color code.The number of infected plants and the average disease index (DI) from four independent infections are shown.Significant differences between samples were determined by a two-tailed unpaired t-test (*P < 0.05; **P < 0.01; ***P < 0.001).ns no significance.Source data are provided as a Source Data file.
Fig. 2 | UmPR-1La is a cell-surface-associated protein.a UmPR-1La localizes to the surface of filaments and the bud-neck of sporidial cells.Hydroxyl fatty acid-induced SG200 expressing C-terminal HA-tagged proteins under the otef promoter formed filaments on a hydrophobic parafilm surface.Immunolocalization was performed using anti-HA and anti-IgG-AF488 antibodies to locate HA-tagged proteins.SG200_PR-1La: produced the signal-peptide (SP)-containing full-length protein (279 aa; 31.5 kDa); SG200_PR-1La(FY*): produced PR-1La(FY*) proteins with the two aromatic residues at positions 3 and 8 of the caveolin binding motif replaced with alanine (see Fig. 3a); SG200_PR-1Lb: produced the SP-containing full-length protein (480 aa; 54.2 kDa); SG200_PRY1: produced the proteins (32.2 kDa) containing UmPR-1La's SP (green strip box) fused to PRY1 (20-299 aa); SG200_N PR-1La -CAP PRY1 : produced the chimera proteins (31 kDa) containing the N-terminus of UmPR1-La (1-133 aa, including SP and S/T region (green box)) fused to the PRY1's CAP-domain (159-299 aa; orange box).Bars, 10 μm.aa: amino acid.b PRY1-mCherry localizes to the periphery of AH109 cells.Cells expressing PRY1-mCherry proteins driven by the ADH1 promoter were cultured on YPD agar and subjected to fluorescence detection.Bars: 10 μm.c UmPR-1La colocalizes with chitins.AB33 (negative control) and AB33_PR-1La strains expressing C-terminal HA-tagged PR-1La under the otef promoter, were grown in nitrate minimal medium to induce filaments 71 .Immunolocalization of PR-1La was performed using anti-HA and anti-IgG-AF594, followed by chitin staining with WGA-AF488.Plasmolysis was performed before fluorescence detection.Plasmolysis-enlarged regions (*).d Secretion of PR-1L, PRY1, and chimeric proteins.SG200 expressing HA-tagged proteins under the otef promoter were grown in YEPSL liquid medium until reaching an OD 600 of 0.6.Proteins from cell pellets and TCA-precipitated supernatants were prepared for immunoblotting analysis.Tubulin served as a cytosolic protein control, and the Coomassie blue staining (CBS)-membranes were used to verify loading.e PR-1La binds to the filamentous surface via its N-terminus.Immunolocalization of PR-1LaHis on SG200filaments using anti-His and anti-IgG-AF488 antibodies.Bars, 10 μm.A diagram illustrates the positions of full-length and truncated PR-1La proteins.The protein purity is shown in Supplementary Fig. 2a.All Fig. 2 experiments were performed independently at least three times, yielding consistent outcomes.Source data are provided as a Source Data file.
expressed and secreted in SG200, showing the similar localization pattern as unmutated UmPR-1La (Figs. 2a and 3c).However, it lost the ability to induce pseudohyphal structures to protect SG200 cells against eugenols and rescue the virulence of Δpr-1la (Fig. 3d-e).These results indicate that UmPR-1La promotes fungal virulence by initiating protective hyphal-like structures to safeguard cells against toxic compounds in a CBM-dependent manner.Collectively, these findings also imply that the activation of protective UmPR-1La-shielded hyphallike structures necessitates cell-wall localization, phenolic binding, and signal transduction.Failure to complete any of these steps will hinder the formation of protective structures crucial for U. maydis virulence.

UmPR-1La senses plant phenolics and elicits pseudohyphae formation
The formation of pseudohyphae in UmPR-1La-expressing cells suggests that UmPR-1La plays a role in sensing plant signals to activate the transition of nonpathogenic to pathogenic growth, which is crucial for U. maydis virulence.In maize, G-type lignin biosynthesis initiates with ferulic acids derived from the shikimate phenylpropanoid pathway.These ferulic acids undergo several intermediate conversions before ultimately producing lipophilic eugenols 30,31 (Fig. 4a).The discovery of ScPRY1's binding to various structurally related steroids 32 has led us to speculate that UmPR-1La might respond to eugenol precursors exhibiting different levels of antifungal activity 33 .SG200_PR-1La cells responded to ferulic acid (FA), coumaric acid (CouA), and coniferyl alcohol (CA) by forming long and branching pseudohyphal structures, similar to the morphology observed in eugenol-treated cells (Fig. 4b and Supplementary Fig. 4a).In contrast, UmPR-1La(FY*) cells showed no induction of pseudohyphae upon exposure to the tested compounds.These cells were insensitive to FA and CouA, continuing to produce healthy sporidial cells and increasing in cell density (Fig. 4b and Supplementary Fig. 4a).However, UmPR-1La(FY*) cells treated with CA displayed an unhealthy appearance with swollen and abnormal morphology (Supplementary Fig. 4a).Despite these changes, CAtreated UmPR-1La(FY*) cells exhibited a higher survival rate compared to cells treated with eugenol.The phenolic-treatment analysis indicates that carboxylic phenolics, such as CouA and FA, are less toxic to U. maydis.Despite the varying levels of toxicity, U. maydis responds to structurally related phenolics and triggers pseudohyphae via UmPR-1La's CBM.
We next investigated whether Sr10279, an ortholog of UmPR-1La from smut fungus Sporisorium reilianum containing both highly conserved CBM and CNYx motifs (Supplementary Fig. 1a), could stimulate hyphal-like formation in U. maydis and restore the Δpr-1la virulence.Proteins from cell pellets and TCA-precipitated supernatants of SG200 cells expressing HA-tagged proteins were prepared for immunoblotting analysis.Tubulin was used as an internal control for a non-secreted cytosolic protein (1:8,000 dilution, Sigma #T6199).Coomassie-blue staining of membrane served as a means of verifying loading.Similar results were observed in two independent biological experiments.e Virulence assay of SG200, Δpr-1la, and the complementation strain Δpr-1la_PR-1La(FY*). Δpr-1la_PR-1La(FY*): Δpr-1la was complemented by introducing a single allele of PR-1La(FY*) under the control of its native promoter.Disease symptoms of infected plants were evaluated at 12 dpi.The number of infected plants from three independent infections is shown above each column.The average DI is reported, and asterisks indicate statistical significance compared to SG200 as determined by a two-tailed unpaired Student's t test (p < 0.05).Source data are provided as a Source Data file.
The C-terminal HA-tagged Sr10279 proteins expressed under the otef promoter localized to the filamentous surface of SG200_SP PR-1La -Sr10279; however, when exposed to FA, they did not form hyphal-like structures (Fig. 4c, d).Furthermore, introducing a single copy of the Sr10279 allele, regulated by UmPR-1La's promoter, failed to rescue the virulence phenotype of Δpr-1la (Fig. 4e).This discrepancy can be attributed to the potential divergence in downstream signal reception partners between Sr10279 and UmPR-1La, despite their CAP domains likely having highly conserved function.The partner of UmPR-1La is likely unable to interact with Sr10279 and PRY1, leading to their failure in inducing hyphal-like structures in U. maydis.While Sr10279 is incapable of substituting UmPR-1La's role in eliciting hyphal-like structures in U. maydis, it may be capable of inducing hyphal-like structures in S. reilianum cells.
To investigate the direct binding of PR-1La to phenolics, we performed fluorescence spectroscopy to measure the change in intrinsic tryptophan fluorescence of recombinant PR-1La and PR-1La(FY*) proteins upon binding to FA and CA.Following incubation with fixed or various concentrations of FA, the intrinsic fluorescence intensity of PR-1La decreased in an FA-concentration-dependent manner, while it remained unchanged for PR-1La(FY*) proteins (Fig. 4f and Supplementary Fig. 4b).In the case of CA, both versions of the PR-1La proteins  c, d). e S. reilianum Sr10279 fails to restore Δpr-1la virulence.Δpr-1la_Sr10279: a single allele of Sr10279 containing the UmPR-1La's SP and under the control of UmPR-1La promoter was introduced into Δpr-1la.The number of infected plants from three independent infections is indicated above each column.The DI is reported, and asterisks indicate statistical significance compared to SG200 as determined by a two-tailed unpaired t-test (p < 0.05).f Detection of PR-1La binding to ferulic acids.The fluorescence intensities of recombinant PR-1LaHis proteins were measured after incubation with or without 5 µM FA in the sodium acetate buffer (pH 5.5) for 15 min.Buffer control: sodium acetate buffer.FA control: the FA containing buffer.Tryptophan residues in PR-1La proteins were selectively excited at 290 nm, and the emission spectra were recorded in the 310-450 nm range.Values represent mean ± sd from three independent measurements in arbitrary units (arb.units).These measurements were conducted using two separate preparations of purified proteins.Source data are provided as a Source Data file.
responded to CA by causing a shift in the fluorescence peak.However, the peak intensity of PR-1La(FY*) was slightly reduced relative to PR-1La (Supplementary Fig. 4c).Notably, the reduction in fluorescence was not due to protein instability during the incubation (Supplementary Fig. 4d).Our finding is in line with the previous report showing that mutations in the CBM of PRY1 affect binding to cholesteryl acetate but not cholesterol 12 .Similarly, our result suggests that U. maydis PR-1La directly binds to ferulic acids but may have different binding affinity among the related compounds.
Cathepsin-B-like protease releases CAPE-La peptides from UmPR-1La The projection of CAP domains at the cell surface, which fulfills a dual function in detecting phenolics and providing protection, has the potential to expose the CNYx motif to an unidentified protease.This exposure could facilitate the release of UmCAPE-La (PPGNYIGKF-KENVSPN) peptides into the plant apoplast and contributes to U. maydis virulence.To investigate the cleavage of UmPR-1La, we collected the apoplastic fluid from SG200-infected leaves, which was subjected to LC-MS/MS analysis to identify the peptides.Despite multiple attempts, we failed to detect endogenous UmCAPE-La peptide, possibly due to the low expression of PR-1La.As an alternative approach, we explored UmPR-1La cleavage by incubating the culture supernatant of SG200_PR-1La strain with the apoplastic fluid from salicylic-acid (SA)-inoculated maize leaves, along with different protease inhibitors (Supplementary Fig. 5a).The stability of UmPR-1La proteins was enhanced in the presence of the cysteine protease inhibitor E64.Increasing the amount of E64 effectively blocked the cleavage of UmPR-1La (Fig. 5a), indicating that UmPR-1La is potentially cleaved by a plant cysteine protease.
We next investigated the cleavage by another cysteine protease, Cathepsin B-like 3 (CatB3; ONM57676.1),which is expressed in the early stage of infection and strongly induced from 4dpi and onwards (Supplementary Fig. 5b).When treated with CatB3, the intensity of fulllength UmPR-1La proteins was reduced but no truncated fragments were detected, indicating a different cleavage pattern compared to Xcp2 (Fig. 5b, c and Supplementary Fig. 5d).This suggests that cleavage occurred specifically at the C-terminus, potentially generating UmCAPE-La peptides.The activity of CatB3 was blocked by E64 (Fig. 5b), and alanine substitutions at either Cys121 or both Cys121 and His276 of CatB3's catalytic active site 35 prevented the cleavage of UmPR-1La (Fig. 5c).In the case of plant PR-1s, we surprisingly discovered that CatB3 cleaved recombinant PR-1La but not plant PR-1 proteins (Fig. 5d).In line with Fig. 5d, the ZmCAPE-derived short tryptic peptide PPGNFR was not detected in the CatB3-cleavage PRB1-3 sample by targeted LC-MS/MS analysis (Supplementary Fig. 6b).However, the short tryptic peptide PPGNYIGK of UmCAPE-La was consistently identified in two independent experiments using CatB3-cleaved recombinant PR-1La and UmPR-1La samples (Fig. 5e; Supplementary Fig. 6b).These findings suggest that CatB3 specifically cleaves after the CNYD motif of UmPR-1La, resulting in the release of UmCAPE-La peptides.

U. maydis deploys UmCAPE-La to suppress plant immunity
To determine the functional importance of UmCAPE-La in modulating plant immunity to promote fungal virulence, we analyzed the expression of maize immune-related genes during the early stages of U. maydis infection.qRT-PCR analysis revealed that the expressions of plant PR-1, PR-2, and PR-5 genes were significantly induced upon infection by the ΔΔpr-1lab strain compared to the wild type SG200, suggesting an additional role of UmPR-1L in subverting plant immunity (Fig. 6a).
We next investigated the role of UmCAPE-La peptides in regulating plant immunity by assessing disease development and defense gene expression in maize seedlings co-inoculated with ΔΔpr-1lab and synthetic peptides.The application of UmCAPE-La peptides enhanced susceptibility to the ΔΔpr-1lab infection, and conversely suppressed defense-related gene expression (Fig. 6b, c), suggesting the specific role of UmPR-1La in promoting fungal virulence by suppressing plant immunity via UmCAPE-La peptides.In contrast, the application of ZmCAPE peptides did not effectively reduce disease symptoms of ΔΔpr-1lab compared to the mock control (Fig. 6b).This contrasts with previous studies showing a boost in plant SA-dependent immunity by CAPE peptides 8,9 , which could potentially be attributed to the timing or method of ZmCAPE delivery.
To investigate this further, we generated the complementation strains replacing UmPR-1La's CAPE-La sequence with either the UmPR-1Lb's CAPE-Lb or ZmCAPE sequence (Fig. 6d).The Δpr-1la_PR-1La(CAPE-Lb) strain, carrying UmCAPE-Lb peptides, partially restored the virulence of Δpr-1la, resulting in an intermediate phenotype that showed no significant difference from SG200 or Δpr-1la.In comparison to Δpr-1la_PR-1La(FY*) cells which could not initiate hyphal-like structures and restore the virulence of Δpr-1la, this finding suggests that shifting to hyphal growth is the primary function of UmPR-1La.Conversely, Δpr-1la_PR-1La(ZmCAPE), with ZmCAPE peptides, significantly reduced the U. maydis virulence, leading to the development of tiny tumors with a size of less than 1 mm.This suggests a role of ZmCAPE in plant defense, and the spatial and temporal delivery of ZmCAPE peptides by U. maydis could effectively boost plant immunity to suppress fungal virulence.The substantial decrease in virulence caused by Δpr-1la_PR-1La(ZmCAPE) could be partly rescued by applying UmCAPE-La, but not by a scrambled peptide of UmCAPE-La (Fig. 6e).These findings highlight the contrasting roles of U. maydis CAPE-La and plant CAPE in regulating plant immunity.

Discussion
In this study, we have uncovered the multifaceted functions of UmPR-1La in U. maydis adaptation and virulence.The presence of a Ser/Thrrich N-terminal region enables its localization on the cell surface, facilitating the C-terminal CAP domain mediated detection of environmental signals such as phenolics.This sensing mechanism helps fungal cells withstand environmental stress and initiate a shift from vegetative to filamentous growth, which is crucial for their infection strategy and survival within the plant host.Furthermore, the cellsurface projection of the CAP domain facilitates the release of CAPE-L peptides by CatB3 to suppress plant immunity and promote fungal growth and colonization (Fig. 7).Our work highlights that UmPR-1La is a versatile protein that plays a pivotal role in the complex interplay between fungi and their plant hosts.Its ability to present at the cell surface, sense environmental stimuli, integrate signals for adaptation, and manipulate host immunity makes it a crucial factor in fungal adaptation and virulence.
Plants and their fungal pathogens possess the ability to recognize each other and activate complex signaling reactions in both organisms.These fungi rely on sensing and integrating environmental cues to initiate the yeast-to-hyphae switch and direct hyphal growth in plants [36][37][38][39][40] .This transition likely increases contact surface with plant cells, facilitating nutrient uptake.The deletion of PR-1La does not completely hinder U. maydis hyphal formation or fully attenuate disease progression, indicating that fungal cells rely on multiple distinct signaling cascades to initiate filamentous growth within the host, a critical step in fungal pathogenesis.
However, the yeast-to-hyphae transition could be disrupted by plant phenolics, such as ferulic acid 41 .These phenolics, which are the building blocks of lignin, are induced during infection 42 .Breaking down of lignin causes a release of phenolics and leads to their accumulation at infection sites to serve as defense agents to eliminate pathogenic intruders 43 .While the exact origins of PR-1L genes in fungal pathogens remain unclear, their capacity to act as substrates for plant proteases and conservation at CAP domain residues underscore a related role in plant-pathogen interactions during the process of coevolution.The PR-1-like genes evolved to develop new functionalities to confront toxic phenolic compounds encountered during the interactions and transmit signals to trigger hyphal branching inside the host plants.U. maydis hyphae usually orient their growth direction towards maize vascular bundles where they access carbon sources 44,45 .Given that lignin is enriched in the cell walls of maize vascular bundle sheaths, xylem, and sclerenchyma 46 , U. maydis may sense phenolic compounds in addition to nutrient gradient 22 by a means to adjust hyphal growth and direct it toward vascular bundles.
Although ScPRY1 can bind to eugenols, its inability to induce hyphal formation hampers the rescue of U. maydis sporidial cells from toxicity.Unlike ScPRY1, which localizes to the cell periphery of S. cerevisiae to shield cells from eugenols, UmPR-1La accumulates at the cell division site of U. maydis sporidial cells.This localization pattern provides insufficient protection against eugenols.However, this situation could change upon transitioning to hyphal growth, enabling UmPR-1La binding facilitated by ligands on the hyphal wall and increasing the exposure of CAPE-La for cleavage by CatB3.Currently, the intracellular transduction of the signal perceived by UmPR-1La to induce the transition remains unclear.We speculate that phenolic binding induces a conformational change, which could be detected by an unidentified UmPR-1La partner, activating the downstream signaling pathway.Despite Sr10279 possessing conserved aromatic residues in the CBM and the ability to attach to U. maydis hyphal wall, it falls short of replacing UmPR-1La's protective function.We hypothesize that Sr10279 might not interact with the UmPR-1La partner.The The SG200_PR-1La culture supernatant or recombinant PR-1La protein was treated with CatB3, as described in Fig. 5c, d, before subjected to LC-MS/MS analysis.The quantity of short tryptic peptides (PPGNYIGK) was measured in both the -CatB3 and +CatB3 samples using the peak area of fragment ions at specific retention times shown in Supplementary Fig. 6b.A cartoon illustrates the fragment ions of the peptide.Fragment ion b5 extends from the N-terminus, and y6 and y7 ions extend from the C-terminus, while y7++ is a doubly charged ion.The term "intensity" refers to the amplitude of the free induction decay signal.Consistent findings were observed in two independent CatB3-cleavage experiments using recombinant PR-1LaHis and UmPR-1LaHA.Source data are provided as a Source Data file.
potential interacting motif possibly resides in the highly diverse and disordered N-terminal region, with functions that extend beyond its role in facilitating PR-1La protein cell wall localization.Further investigation is necessary to comprehend how N-terminal regions collaborate with CAP domains to participate in the UmPR-1Ladependent signaling pathway.
The Δgpa3 and Δbpp1 mutants exhibit a similar pseudohyphal structure phenotype with UmPR-1La-expressing cells, possibly maydis-delivered ZmCAPEs suppress fungal virulence.Δpr-1la_PR-1La(CAPE-Lb) and Δpr-1la_PR-1La(ZmCAPE): Δpr-1la expressed the PR-1La mutant proteins carrying UmCAPE-Lb (PAGNVEGLFDAQVPAKVQPTPRLRSSCSANERHGS) or ZmCAPE peptides under the control of PR-1La promoter.The indicated strains were inoculated into maize seedlings.A description of each disease category is provided in the Method section.The average DI values determined from at least three independent infections are shown.Significant differences between SG200 and the indicated strains were determined by a two-tailed unpaired Student's t test.Δpr-1la_PR-1La(CAPE-Lb) showed no statistically significant (ns) differences compared to the indicated strains.e UmCAPE-La mitigates the effect of ZmCAPE on fungal virulence.Maize seedlings were inoculated with Δpr-1la_PR-1La(ZmCAPE) along with 0.5 µM of CAPE-La, the scrambled peptide of CAPE-La (YFSKNIKNPPVEGNGP), or water (mock).The average DI values determined from three independent infections are shown.**P < 0.01 denotes significant difference between the CAPE-La and mock inoculation, determined by a two-tailed unpaired Student's t test.Source data are provided as a Source Data file.suggesting the involvement of Gα and Gβ proteins from the cyclic AMP-dependent protein kinase A (cAMP/PAK) pathway 47,48 .However, given that PR-1La lacks a transmembrane domain, it is not clear how the cell surface signals would be perceived and transduced to activate downstream signaling.Several studies have reported the discovery of G proteins in extracellular vesicles (EVs), and that GPCRs regulate EV release to mediate intercellular communication between neighboring and distant cells [49][50][51] .The finding that EVs trigger the yeast to hyphal transition in Candida albicans 51 also supports the possibility that EVs containing signaling proteins such as GPCRs could be involved in transmitting the UmPR-1La signal to switch on filamentous growth.Further study is needed to explore cellular signaling switching to filaments.
Maize Xcp2 and CatB3 are papain-like cysteine proteases that play an important role in apoplastic immunity 34 .This explains why this group of proteases is targeted by pathogens.For example, the U. maydis effector Pit2 inhibits the activity of maize cysteine proteases CP1, CP2, and Xcp2 to prevent the release of Zip peptides and subsequent inactivation of plant SA-dependent signaling 52,53 .However, Pit2 does not inhibit CatB3.In addition, U. maydis also induces the maize cystatin ZmCC9 to inhibit the activities of cysteine proteases, but it has a comparatively weak interaction with CatB3 54 .This evidence, and our observation that CatB3 releases UmCAPE-La, highlights a sophisticated strategy where U. maydis suppresses plant immunity by hijacking maize CatB3 to release a potential functional mimic of ZmCAPE peptide from PR-1La.UmCAPE-La may compete for binding to a yet unknown receptor, leading to inactivation.
It is unclear why CatB3 avoids cleavage of plant PR-1.One speculation is that CatB3 could not access the motif unless a conformational change is induced in PR-1s, which is possible by a specific ligand binding to the PR-1 CAP domains.Alternatively, an unidentified protease is responsible for plant CAPE release from PR-1s.Although AtXcp1 is reported to cleave the CNYD motif and release AtCAPE9 peptides 10 , maize Xcp2, CatB3, and wheat serine protease fail to release monocot CAPEs 9 .Nonetheless, our work opens a new avenue to understand how CatB3 excludes plant PR-1 as its target and whether another protease is responsible for releasing CAPEs in monocots.
Our work provides a mechanistic bridge between understanding how fungal pathogens employ a conserved CAP-domain PR-1L protein to perform a function contradictory to the defense function of PR-1.In the future, identifying the receptor of plant CAPE and fungal CAPE-L peptides will provide more insights into the activation of plant immunity via CAPE-dependent signaling cascades.This will further solve the unresolved questions concerning the role of PR-1 proteins in plant defense.

U. maydis strain construction, growth conditions, and virulence assays
Zea mays The U. maydis infection was performed using the Honey 236 Taiwan cultivar.The haploid solopathogenic Ustilago maydis SG200 strain was used as a reference strain in this study 25,55 .All U. maydis strains generated and used in this study are listed in Supplementary Table 1.U. maydis strains were cultured on solid potato dextrose agar (PDA) plates (2.4% potato dextrose broth and 2% agar).AB33 derivatives were grown in YEPSL liquid medium (0.4% yeast extract, 0.4% peptone, and 2% sucrose) until reaching an OD 600 of 0.2 at 28 °C, Subsequently, they were shifted to grow in nitrate minimal medium (NM) 56 supplemented with 2% glucose for 8 h to induce b-dependent filaments.For infection, strains were grown in YEPSL liquid medium until reaching an OD 600 of 0.8 at 28 °C.Cells were adjusted to a final OD 600 of 1.0 in H 2 O and injected into stems of 7-day-old maize seedlings.Disease symptoms were assessed and scored at 12 days postinfection (dpi) using the disease rating criteria described in the previously report 25 .The disease index (DI) was calculated as the sum of values from all disease categories, which was obtained by multiplying the number of plants in each category by their corresponding assigned value (Death plant = 11; Heavy tumors on base of stem or stunted growth = 9; Normal tumors (>3 mm) on leave and/or stem = 7; Small tumors (between 1-3 mm) = 5; Tiny tumors (<1 mm) = 3; Chlorosis = 1), and then dividing by the total number of infected plants 57 .Significant differences in disease symptoms between each strain and SG200 or the indicated strain are determined using a two-tailed unpaired Student's t test.

Plasmid and strain construction
Plasmid construction using either Gibson Assembly or standard cloning methods are described in Supplementary Table 2. Primers used in each generated plasmid are listed in Supplementary Table 3.A PCRbased approach was used to generate mutants 58 .For gene integration into the ip locus, plasmids containing a carboxin resistant ip allele (ipR) 59 were linearized with restriction enzymes and subsequently inserted via homologous recombination.The transformation of U. maydis and genomic DNA isolation procedures were performed as described in a previous study 60 .Positive U. maydis transformants were verified by Southern blot analysis.

Gene expression analysis
Total RNAs from infected maize leaves were extracted using QIAGEN RNeasy Plant Mini Kit (Qiagen #74904), followed by DNase-treatment and reverse-transcription before qRT-PCR analysis 61 .The genomic DNA was removed from the total RNA using a TURBO DNA-free™ Kit (Invitrogen# AM1907).The cDNA was reverse-transcribed using a SuperScript® III First-Strand Synthesis SuperMix (Invitrogen# 18080400).The expression of maize GAPDH was used for normalization.The relative expression values of genes were calculated using the 2 -ΔΔCt method 62 .Quantitative real-time PCR analysis was performed using primer pairs for indicated genes listed in Supplementary Table 3.

Immunolocalization of PR-1La
Filamentous cells were induced by spraying sporidial cells with hydroxyl fatty acids on parafilm, as described in the previous report 44 .Parafilms with attached filamentous cells were then incubated with or without 5 µg of purified proteins in 2 ml PBS buffer (pH 7.4) for 4 h at room temperature.Subsequently, immunostaining was performed using anti-His/anti-HA antibody (Yao-Hong Biotech., Taiwan; #YH80003 and #YH80007; 1: 2000 dilution) and Alexa Fluor 488/594 (AF488/594)-conjugated secondary antibody (Invitrogen #A28175; Abcam # AB150116; 1: 2000 dilution) 44 .For the colocalization study in AB33 filaments, the AF594-labeled filaments were stained with 1 µg/ml of wheat germ agglutinin-conjugated Alexa Fluor 488 (WGA-AF488; Invitrogen# W11261) for 10 min in a PBS buffer.The cells were then subjected to a 1-min vacuum in a PBS buffer containing 1 M NaCl before being visualized using microscopy.The fluorescence was observed using Axio Observer fluorescence microscope equipped with Axiocam 702 Monochrome camera (ZEISS, Germany).Images were processed using ZEN 3.2 imaging software (ZEISS).

Protein purification
Plant PR-1His, Xcp2-His, and CatB3 proteins, which contain a signal peptide, were expressed in Nicotiana benthamiana using Agroinfiltration method 63 .Agrobacterium tumefaciens GV3101 strains carrying expression plasmids were infiltrated with needleless syringe to the leaves of 3-weeks-old N. benthamiana plants.To purify CatB3 proteins from the apoplastic fluid, infiltrated leaves harvested at 3 dpi were immersed in an infiltration buffer (25 mM Tris-HCl, 250 mM NaCl, pH 7.5), vacuum-infiltrated for 5 min, and then centrifuged at 1000 g for 5 minutes to collect apoplastic fluid.The apoplastic fluid was concentrated and exchanged with buffer A (25 mM Tris-HCl, 150 mM NaCl, Article https://doi.org/10.1038/s41467-023-41459-4Nature Communications | (2023) 14:5755 pH 7.5) using 10 kDa cutoff filters before being subjected to sizeexclusion chromatography.Fractions containing CatB3 proteins were concentrated and kept in buffer A containing 10 % glycerol.To purify PR-1His and Xcp2His proteins, the apoplastic fluids were exchanged with buffer B (25 mM Tris-HCl, 250 mM NaCl, 15 mM Imidazole, pH 7.5) before being incubated with Ni-NTA agarose beads.The beads were washed with the infiltration buffer, and His-tagged proteins were eluted, buffer-exchanged, and kept in buffer A containing 10% glycerol.
Recombinant His-tagged full-length or truncated PR-1La protein was expressed in E. coli Shuffle T7 cells (NEB #C3026J).To purify recombinant proteins, E. coli cells were disrupted in a lysis buffer (25 mM Tris-HCl, 250 mM NaCl, 20 mM MgCl 2 , 20 mM KCl, 20 mM Imidazole, pH 7.5) supplemented with 0.5% Triton X-100, 0.5 mg/ml lysozyme, 0.1 mg/ml DNase, and 1 mM PMSF before being subjected to Ni-NTA affinity purification as described above, except the beads were washed with the lysis buffer containing 30 mM Imidazole.The eluted proteins were further subjected to size-exclusion chromatography.Purified proteins were kept in buffer A containing 10% glycerol.

Fluorescence spectroscopy
In a final volume of 2 ml of buffer containing 50 mM sodium acetate at pH 5.5, the recombinant PR-1LaHis protein (0.25 µM) was mixed with phenolic compounds and incubated for 15 min at room temperature on a rotating wheel.This was followed by measuring the fluorescence intensity of the mixture using Jasco fluorescence spectrophotometer FP-8300 with Spectra Manager software (version 2).The excitation wavelength was set at 290 nm to selectively excite tryptophan residues of PR-1La, and the emission spectra were recorded in the wavelength range of 310-450 nm with a 1 nm interval, using a scan speed of 100 nm/min.

Phenolic treatment
Cells with an initial OD of 0.1 were grown in YEPSL liquid medium containing 1 mM eugenol (Sigma # E51791), 7 mM ferulic acid (Sigma #128708), 5 mM coniferyl alcohol (Sigma #223735), or 5 mM coumaric acid (Sigma #C9008) at a final pH of 6.0, and then the cells were incubated at 28 °C for up to 48 h.

Sample preparation for LC-MS/MS
Protease-cleavage samples were extracted using the suspension trapping (S-Trap) protocol 64 .Briefly, the samples were lysed in a buffer containing 50 mM Tris-HCl (pH 8.0) and 5% SDS, and the protein concentrations was determined using the BCA protein assay (Thermo Fisher Scientific #23225).Before trypsin digestion, disulfide bonds within proteins were reduced with 10 mM Tris(2-carboxyethyl)phosphine hydrochloride and alkylated with 40 mM 2-chloroacetamide (CAA) at 45 °C for 10 min.Each protein sample was digested into peptides by Lys-C and trypsin in an enzyme-toprotein ratio of 1:50 (w/w) within an S-trap micro microcolumn.Following digestion, the peptides were eluted using 40 μl of a solution composed of 50 mM triethyl ammonium bicarbonate, 0.2% formic acid (FA), and 50% (v/v) acetonitrile (ACN).Subsequently, a desalting step was conducted using a Ziptip Pipette Tip and the desalted peptides were dried using a vacuum centrifugation concentrator.The dried peptides were reconstituted in 0.1% (v/v) FA, centrifuged to collect the supernatant, and finally subjected to LC-MS/MS analysis.

LC-MS/MS analysis
For enhanced confidence in peptide identification during LC-MS/ MS analysis, synthetic peptides with stable heavy isotope-labeled lysine or arginine were spiked into the dried peptides, and then dissolved in 10 μl of 0.1% FA.The peptide analysis was conducted using Thermo Fisher Scientific Q-Exactive mass spectrometer coupled with an Ultimate 3000 (Thermo Fisher Scientific), controlled by Xcalibur software (version 4. 3.73.11).A nanoEase M/Z Peptide CSH C18 column (75 µm x 25 cm, 1.7 µm, 130 Å) with a column heater set at 45 °C was employed for peptide separation.Peptides separation was achieved through a linear gradient of 5-25% (Buffer A consisted of 0.1% FA, and buffer B contained 0.1% FA in 100% CAN) over a 60-minute duration, at flow rate of 300 nl/min.The acquisition cycle of the MS data was executed in parallel reaction monitoring (PRM) mode, with a full survey MS scan followed by the mass-to-charge (m/z) range 300-1600.The MS scan was performed with a resolving power of 70,000.The PRM MS/MS acquisitions were performed using a 2.0 Da isolation window, a maximum 250 ms ion injection time, 27% NCE (normalized collision energy), and a resolving power of 35,000.

PRM data analysis
The PRM raw files were analyzed using Skyline (version 22.2) 65 .The fragment spectra predictions for PPGNYIGK and PPGNFR was conducted using Prosit 66 .Protein digestion was set to Trypsin (semi) [KR| P], with a minimum length of peptide set to 6 amino acids.Variable modifications applied included Label:13 C(6)15 N(2)(C-term K) and Label:13C(6)15N(4)(C-term R).Mass accuracy filtering was set to 10 ppm.Ion match tolerance was set at 0.5 m/z, and a minimum of 3 product ions was required for analysis.Both fragment b and y ions were employed for quantification.The peak area of selected fragment ions was extracted for quantification.All data were checked manually for peak selection and retention time.

Data and bioinformatic analyses
Data relevant for CatB3 and Xcp2 gene expression analysis were extracted from the RNAseq dataset available at NCBI Gene Expression Omnibus under accession number GSE103876 23 .To perform phylogenetic analysis, the amino acid sequences of PR-1 and PR-1-like proteins are retrieved from the NCBI database, aligned using Clustal Omega 67 , and trimmed by using trimAl (version 1.4.1) 68.Evolutionary analysis was performed in MEGA 7.0 69 , and the phylogenetic tree was constructed using Maximum Likelihood method with the WAG + G + I model and 1000 bootstrap replicates.MEME (version 5.5.2) 70 was used to obtain the consensus sequence of CAPE and CAPE-like peptides.The scrambled peptide containing an identical amino acid composition to UmCAPE-La, is randomly generated using Scrambled tool in Mimotopes website.

Fig. 1 |
Fig. 1 | The PR-1-like (PR-1L) family proteins in U. maydis.a Schematic drawing displays plant PR-1 and PR-1L protein domain structure.The signal peptide (SP) is depicted as a black stripe, the Ser/Thr-rich (S/T) region as a white box, and the CNYx motif and CAPE/CAPE-like peptide (a red box) are located at the C-terminus of the CAP-domain (green box).Conserved residues shared between plant PR-1's CAPE and PR-1L's CAPE-like peptides are marked in green.Solanum lycopersium SlPR1b (#P04284); Saccharomyces cerevisiae ScPRY1 (#NP012456).b The sequence comparison of plant CAPEs and smut fungal CAPE-like peptides after CNYx motif.The green line indicates the MEME-derived consensus sequence P[P/V]GN[Φ/V][V/I]G.The protein accession numbers are provided.Plant CAPE peptides that have been identified and reported are indicated8,9 .Ph Pseudozyma hubeiensis, Ss Sporisorium scitamineum, Sr Sporisorium reilianum, Uh Ustilago hordei, Ub Ustilago bromivora.c Phylogenetic analysis of plant PR-1 and PR-1-like proteins from smut fungi and yeast.Protein sequences are retrieved from the NCBI, aligned using Clustal Omega, and curated with trimAl.Analysis was carried out in MEGA 7.0 using the Maximum Likelihood method with the WAG+G+I model and 1000 bootstrap replicates.Clade I

Fig. 4 |
Fig. 4 | Phenolic binding of UmPR-1La triggers pseudohyphae formation.a Structures of intermediate phenolic compounds produced in the G-type lignin pathway.The phenolic's toxicity level is determined by the sensitivity of U. maydis to the compound.b UmPR-1La triggers pseudohyphae formation in response to other phenolics.SG200 and its derivatives expressing the indicated HA-tagged proteins under the otef promoter were grown in YEPSL medium with or without 7 mM ferulic acid (FA) for 24 h.Cell morphology images of the indicated strains were captured, and the OD 600 were measured after 24 h.Values indicate mean ± sd from three independent biological assays.Bars, 20 μm.c Sr10279 localizes to the filamentous surface.SG200_Sr10279: the UmPR-1La's SP was fused to C-terminal HA-tagged Sr10279 (accession# CBQ70610) and expressed under the otef promoter in SG200.Immunolocalization of Sr10279 on SG200_Sr10279 filaments was performed using an anti-HA and anti-IgG-AF488 antibodies.Bars, 10 μm.d Sr10279 fails to trigger pseudohyphae formation.The morphology of cells expressing the indicated proteins, grown in FA-supplemented YEPSL medium for 24 h at 28 °C, was captured.Bars, 20 μm.Similar results were observed in three independent biological experiments (c, d). e S. reilianum Sr10279 fails to restore Δpr-1la virulence.Δpr-1la_Sr10279: a single allele of Sr10279 containing the UmPR-1La's SP and under the control of UmPR-1La promoter was introduced into Δpr-1la.The number of infected plants from three independent infections is indicated above each column.The DI is reported, and asterisks indicate statistical significance compared to SG200 as determined by a two-tailed unpaired t-test (p < 0.05).f Detection of PR-1La binding to ferulic acids.The fluorescence intensities of recombinant PR-1LaHis proteins were measured after incubation with or without 5 µM FA in the sodium acetate buffer (pH 5.5) for 15 min.Buffer control: sodium acetate buffer.FA control: the FA containing buffer.Tryptophan residues in PR-1La proteins were selectively excited at 290 nm, and the emission spectra were recorded in the 310-450 nm range.Values represent mean ± sd from three independent measurements in arbitrary units (arb.units).These measurements were conducted using two separate preparations of purified proteins.Source data are provided as a Source Data file.

Fig. 5 |
Fig. 5 | UmCAPE-La peptide is released from UmPR-1La by CatB3.a, b E64 blocks the cleavage of PR-1La.The SG200_PR-1La culture supernatant (labeled as UmPR-1LaHA) was incubated: (a) with the apoplastic fluid (AF) proteins from SAinoculated maize, along with varying amounts of DMSO-dissolved E64 or an equivalent volume of DMSO, (b) with CatB3 in the presence of either 5 µl of E64 (final conc. of 0.5 mM) or DMSO.A representative blot from one of two independent experiments is shown.c The catalytic-site mutants of CatB3 fail to cleave UmPR-1La.The immunoblot showed the results before and after 2-hr incubations of SG200_PR-1La culture supernatant with CatB3 or its mutants.CatB3 C121A : the alanine substitution at Cys121.CatB3 C121A H276A : the alanine substitutions at Cys121 and His276.A representative blot from one of two independent experiments is shown.The loadings were verified using separated silver-stained PAGEs.d CatB3 does not cleave plant PR-1s.Maize PRB1-3 and tomato PR1b (C-terminal His-tagged proteins), and maize CatB3 (no tag) were purified from the apoplast of N. benthamiana.Fulllength PR-1LaHis proteins was purified from E. coli.The CatB3-digested proteins were separated on SDS-PAGE and stained with Coomassie-blue or analyzed by immunoblotting.Asterisks: the CatB3 protein bands.Black arrowheads: plant PR-1 proteins.Black open arrows: PR-1La proteins.Red open arrows: truncated PR-1La proteins.A representative blot from one of three independent experiments is shown.e Detection of UmCAPE-La peptides through targeted LC-MS/MS analysis.The SG200_PR-1La culture supernatant or recombinant PR-1La protein was treated with CatB3, as described in Fig.5c, d, before subjected to LC-MS/MS analysis.The quantity of short tryptic peptides (PPGNYIGK) was measured in both the -CatB3 and +CatB3 samples using the peak area of fragment ions at specific retention times shown in Supplementary Fig.6b.A cartoon illustrates the fragment ions of the peptide.Fragment ion b5 extends from the N-terminus, and y6 and y7 ions extend from the C-terminus, while y7++ is a doubly charged ion.The term "intensity" refers to the amplitude of the free induction decay signal.Consistent findings were observed in two independent CatB3-cleavage experiments using recombinant PR-1LaHis and UmPR-1LaHA.Source data are provided as a Source Data file.

Fig. 6 |
Fig. 6 | UmCAPE-La enhances plant susceptibility by suppressing plant response.a, c Relative expression of maize PR genes at early infection.Maize seedlings inoculated with (a) SG200 and ΔΔpr-1lab without any peptides, or (c) ΔΔpr-1lab with 0.2 µM synthetic peptide CAPE-La (PPGNYIGKFKENVSPN).RNAs prepared from infected leaves harvested at indicated time point were subjected to qRT-PCR analysis.Expression levels of PR genes were normalized to the GAPDH expression.The normalized PR gene expression in (a) SG200 or (c) ΔΔpr-1lab was set to 1. Values represent mean ± sd of three independent infections.Significant differences between two samples were determined by a two-tailed unpaired Student's t test (*P < 0.05; **P < 0.01; ***P < 0.001).PR1 (NM001147273); PR2 (HM021761); PR5 (U82201).b UmCAPE-La enhances the ΔΔpr-1lab virulence.Maize seedlings were inoculated with ΔΔpr-1lab along with 0.2 µM CAPE-La or ZmCAPE (from ZmPRB1-3).Disease symptoms were scored at 12 dpi.Total numbers of infected plants and the average DI determined from three independent infections are indicated above the respective columns.Statistical significance relative to the mock control were assessed using a two-tailed unpaired Student's t test.d U.

Fig. 7 |
Fig. 7 | Hypothetical model for the PR-1La function.Upon host entry, U. maydis employs UmPR-1La to sense phenolics, initiating signals for shielded hyphal branching as a defense against toxic phenolics.This sensing mechanism promotes fungal hyphal growth towards phenolic-enriched vascular bundles.Additionally, the exposure of UmCAPE-La on the cell surface facilitates its cleavage by CatB3 and subsequent release.UmCAPE-La presumably competes with ZmCAPE for binding to an unidentified receptor, suppressing plant immunity and enhancing U. maydis virulence.Created with BioRender.com.