Biology subjects

Misas Villamil, J. C.

Publications and source records attributed to Misas Villamil, J. C..

8 recordsLinked to original sources

bioRxiv2026

Salicylic acid-triggered apoplastic proteolysis releases cryptic phytocytokines with distinct immunogenic functions

Plants rely on an innate immune system to defend against pathogens through various molecular responses. In addition to classical damage- and pathogen-associated molecular patterns (DAMPs and PAMPs), plants produce endogenous signaling peptides termed phytocytokines that amplify and regulate immune responses following stress. Although most characterized phytocytokines originate from dedicated precursor proteins, the contribution of multifunctional proteins to phytocytokine generation remains poorly understood. Here, we show that salicylic acid (SA) rapidly remodels the maize apoplastic peptidome through an early, transient proteolytic program driven by apoplastic serine hydrolases. Time course peptidomics identified fourteen candidate phytocytokines, including two cryptic peptides, PC13 and PC14, released from the stress-associated zinc-finger protein ZmSAP7 and the migration inhibitory factor-like protein ZmMDL1, respectively. Both peptides activated immune-associated gene expression but triggered distinct transcriptional responses and exerted opposing effects on Ustilago maydis infection, with PC13 enhancing resistance and PC14 promoting susceptibility. Biochemical analysis demonstrated that PMSF-sensitive apoplastic serine proteases directly process ZmMDL1 to release PC14. Together, our findings uncover a SA-responsive proteolytic pathway that generates functionally distinct phytocytokines from multifunctional proteins, expanding the repertoire of immune signaling peptides and revealing an additional layer of regulation in plant defense.

plant biology↗

bioRxiv2026

A membrane-anchored inhibitor of papain-like cysteine proteases promotes Pseudomonas root colonization

Pseudomonas species, spanning both beneficial and pathogenic lifestyles, possess conserved mechanisms to modulate plant immunity. Nevertheless, the mechanisms by which commensal bacteria establish and maintain host colonization remain poorly understood. Here, we report the characterization of a Pseudomonas chagasin-like protease inhibitor (Cpi1), conserved across pseudomonads representing a novel class of membrane-anchored PLCP inhibitor. Unlike previously described secreted protease inhibitors, P. putida Cpi1 is a lipoprotein localized to the bacterial surface and outer membrane vesicles (OMVs), positioning it to selectively inhibit immune-related papain-like cysteine proteases (PLCPs) during host interactions. Functional assays demonstrated inhibition of maize PLCP activity in the nanomolar range, while cpi1 deletion and chagasin motif mutants exhibited significantly impaired early root colonization, particularly in the meristematic and elongation zones. Besides, lack of cpi1 resulted in an altered structure of a maize root-associated synthetic community. We hypothesize that, Cpi1 may protect critical bacterial surface proteins from cleavage by inhibiting plant proteases and thereby modulate the release of MAMPs, dampening host immune responses. Moreover, the release of Cpi1 via OMVs could further extend its function within the root periphery and the apoplast. Together, our results uncover a conserved, membrane-anchored mechanism among pseudomonads for subverting plant immunity and establishing host-microbe interactions.

microbiology↗

bioRxiv2024

Quantitative detection of plant signaling peptides utilizing ELISA

Plant signaling peptides, also known as phytocytokines, are involved in a number of signaling mechanisms, including cell-to-cell communication during plant development and immunity. The detection of small peptides in plant tissues is challenging and often relies on time-consuming and cost-intensive approaches. Here, we present an ELISA-based assay as a rapid and cost-effective method for the detection of naturally released peptides in plant tissues. Our ELISA-based method was developed to detect Zip1, a 17-amino-acid phytocytokine derived from Zea mays that elicits salicylic acid signaling in maize leaves. Using a custom peptide-antibody, we designed an experimental pipeline to achieve peptide specificity, selectivity and sensitivity allowing the detection of the Zip1 peptide in complex biological samples. As a proof of concept, we transfected maize protoplasts to overexpress the precursor molecule PROZIP1 and treated maize leaves with salicylic acid to induce native PROZIP1 expression and Zip1 release. Using ELISA, we were able to quantify native Zip1 signals with a detection limit in the nanogram range, which allowed us to detect different Zip1-containing peptides in plant material. This method can be adapted for the detection and quantification of a variety of plant signaling peptides.

plant biology↗

bioRxiv2022

Combination of transcriptomic, proteomic and degradomic profiling reveals common and distinct patterns of pathogen-induced cell death in maize

Regulated cell death (RCD) is crucial for plant development, as well as in decision-making in plant-microbe interactions. Previous studies revealed components of the molecular network controlling RCD, including different proteases. However, the identity, the proteolytic network as well as molecular components involved in the initiation and execution of distinct plant RCD processes, still remain largely elusive. In this study, we analyzed the transcriptome, proteome and N-terminome of Z. mays leaves treated with the Xanthomonas effector avrRxo1, the mycotoxin Fumonisin B1 (FB1), or the phytohormone salicylic acid (SA) to dissect plant cellular processes related to cell death and plant immunity. We found highly distinct and time-dependent biological processes being activated on transcriptional and proteome levels in response to avrRxo1, FB1 and SA. A correlation analysis of the transcriptome and proteome identified general, as well as trigger-specific markers for cell death in Z. mays. We found that proteases, particularly papain-like cysteine proteases, are specifically regulated during RCD. Collectivley, this study characterizes distinct RCD responses in Z. mays and provides a framework for the mechanistic exploration of components involved in the initiation and execution of cell death.

plant biology↗

bioRxiv2022

Maize phytocytokines and microbial-patterns trigger antagonistic features in co-incidence with wounding and fungal pathogens

Phytocytokines are signalling peptides that activate immune responses and alarm bystander cells of danger. The pathways of phytocytokine perception and activation are thought to be shared between exogenous danger signals such as microbe-associated molecular patterns (MAMPs) and endogenous, passively released, damage-associated molecular patterns (DAMPs). However, downstream responses triggered by danger molecules and their effect in plant survival is still largely unknown. Here, we have identified three biologically active maize orthologues of phytocytokines previously described in other plants. The maize phytocytokines show common features with MAMPs, including the induction of immune related genes and activation of papain-like cysteine proteases. In contrast to MAMPs, the phytocytokines do not promote cell death in the presence of wounding. In infection assays with fungal pathogens of two different life styles we found that phytocytokines affect the development of disease symptoms, likely due to the activation of phytohormonal pathways. Collectively, our results show that phytocytokines and MAMPs trigger unique and antagonistic features of immunity. We propose a model in which phytocytokines activate immune responses partially similar to MAMPs but in contrast to microbial signals, they act as danger and survival molecules to the surrounding cells. Future studies will focus on the components determining the divergence of signalling outputs upon phytocytokine activation.

plant biology↗

bioRxiv2022

Exploiting effector - host interactions using TurboID-based proximity labeling

Plant pathogens secrete effectors, which target host proteins to facilitate infection. The Ustilago maydis effector UmSee1 is required for tumor formation in the leaf during infection of maize. UmSee1 interacts with maize SGT1 and blocks its phosphorylation in-vivo. In the absence of UmSee1, U. maydis cannot trigger tumor formation in the bundle sheath. However, it remains unclear which host processes are manipulated by UmSee1 and the UmSee1-SGT1 interaction to cause the observed phenotype. Proximity-dependent protein labeling involving the turbo biotin ligase tag (TurboID) for proximal labeling of proteins is a powerful tool for identifying the protein interactome. We have generated transgenic U. maydis that secretes biotin ligase-fused See1 effector (UmSee1-TurboID-3HA) directly into maize cells. This approach, in combination with conventional co-immunoprecipitation allowed to identify additional UmSee1 interactors in maize cells. Collectively, our data identified three ubiquitin-proteasome pathway-related proteins (ZmSIP1, ZmSIP2, ZmSIP3) that either interact with or are close to UmSee1 during host infection of maize with U. maydis. ZmSIP3 represents a cell cycle regulator which degradation appears to be promoted in the presence of UmSee1. Our data provide a possible explanation for the requirement of UmSee1 in tumor formation during U. maydis - Zea mays interaction.

plant biology↗

bioRxiv2022

Maize immune signalling peptide ZIP1 evolved de novo from a retrotransposon

Plants are subjected to different types of threats that require appropriate physiological responses to counteract them. Signalling peptides are produced under specific conditions and elicit physiological changes. PROZIP1 encodes such a signalling peptide, Zip1, that induces salicylic acid defence responses in maize (Zea mays) leading to a better protection against biotrophic pathogens. Despite salicylic acid pathway being conserved amongst plants, we observed that Zip1 only occurs in the Zea genus. PROZIP1s evolution is associated with transposons, as it resides in the terminal repeat of a retrotransposon from the Gyma family. We traced back the mutations that were encountered by this transposon and found that PROZIP1 emerged de novo in Zea. This emergence likely occurred less than 728,000 years ago. In conclusion, we describe the evolution of a recently emerged plant immune signalling peptide from a transposon sequence.

plant biology↗

bioRxiv2020

Epichloe festucae in mutualistic association with Lolium perenne suppresses host apoplastic cysteine protease activity

Plants secrete various defence-related proteins into the apoplast, including proteases. Papain-like cysteine proteases (PLCPs) are central components of the plant immune system. To overcome plant immunity and successfully colonise their hosts, several plant pathogens secrete effector proteins inhibiting plant PLCPs. We hypothesized that not only pathogens but also mutualistic microorganisms interfere with PLCP-meditated plant defences to maintain endophytic colonisation with their hosts. Epichloe festucae forms mutualist associations with cool season grasses and produces a range of secondary metabolites that protect the host against herbivores. In this study, we performed a genome wide identification of Lolium perenne PLCPs, analysed their evolutionary relationship and classified them into nine PLCP subfamilies. Using activity-based protein profiling, we identified four active PLCPs in the apoplast of L. perenne leaves that are inhibited during endophyte interactions. We characterized the L. perenne cystatin LpCys1 for its inhibitory capacity against ryegrass PLCPs. LpCys1 inhibits LpCP2, indicating that LpCys1 might play a role in the suppression of PLCP activity during the interaction with E. festucae. However, since the activity of other L. perenne PLCPs is not sensitive to LpCys1 we propose that additional inhibitors are involved in the suppression of apoplastic PLCPs during E. festucae infection.

plant biology↗