Biology subjects

Castillo, A. G.

Publications and source records attributed to Castillo, A. G..

8 recordsLinked to original sources

bioRxiv2026

Extracellular vesicles associate with infectious geminiviral particles in the apoplast of infected plants.

Plant viruses have evolved diverse strategies to facilitate their movement and survival within the host. Among them, geminiviruses co-opt host cellular machinery to replicate and disseminate. Traditionally, viral propagation has been associated with intercellular symplastic trafficking mediated by plasmodesmata and viral movement proteins. However, recent evidence demonstrated that the plant RNA virus turnip mosaic virus (TuMV) components are associated with extracellular vesicles (EVs). EVs are membrane-bound structures secreted to the extracellular space to potentially mediate several plant-pathogen interactions such as cross-kingdom RNA interference or the delivery of stress response proteins. In animals, EVs facilitate viral transmission both within the host and across species, but knowledge about their potential roles in plant viral infection is scarce. In this study, we demonstrate that EVs isolated from geminivirus-infected plants contain complete viral genomes and both capsid and viral movement proteins. Furthermore, these EV fractions were demonstrated to be infectious when mechanically inoculated onto naive plants. This discovery suggests that EVs may serve as alternative carriers for geminivirus components, enabling long-range transport or potentially modulating host immune responses, and highlights geminiviral capacity to transverse membrane boundaries, essential for circulative arbovirus propagation in their insect vectors. Significance StatementViruses are obligate intracellular parasites that shape ecological communities and crucially challenge animal and plant health worldwide. Conversely to animal viruses, plant-infecting viruses rely on plasmodesmata to disseminate through their host and establish systemic infection. Nonetheless, most plant viruses are insect-transmitted whose ecological cycle relies on their insect vector spread. Thus, strategies to cross continuous membrane barriers are essential for their dissemination and may be potentially conserved in plant hosts. Our discovery that infectious viral entities are associated with EVs reveals an alternative pathway for geminiviral movement within plant hosts that could facilitate vector transmission, challenging our long-standing understanding of plant virus biology and expanding the current conception of plant viral pathology.

plant biology↗

bioRxiv2025

Complex Interactions Between Genome Components of Two ToLCNDV Isolates from India and Spain Determine Effective Infection in Tomato

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus whose infectivity and host range depend on the coordinated functions of its DNA-A and DNA-B components. Here, we investigated the replication, movement, and systemic infection capacity of ToLCNDV isolates from India (IN) and Spain (ES) and their pseudo-recombinants in tomato and Nicotiana benthamiana. In tomato, the IN isolate (A-IN/B-IN) systemically infected all plants, whereas the ES isolate (A-ES/B-ES) failed to do so. Pseudo-recombinant analyses revealed that DNA-B from the IN isolate complemented A-ES in trans, enabling systemic spread, while B-ES accumulated poorly and supported systemic infection only inefficiently. Although both A components replicated locally in tomato, DNA-A from the IN isolate was unable or only marginally able to infect systemically in the absence of DNA-B, demonstrating an essential contribution of DNA-B to long-distance movement. In N. benthamiana, both isolates established systemic infections even as monopartite viruses, though with substantially reduced viral titers and attenuated symptoms, indicating that host factors can partially compensate for the absence of DNA-B. Using a DsRed-tagged {Delta}CP mutant of ToLCNDV-ES, we further show that coat protein is required for systemic movement in N. benthamiana, despite the presence of DNA-B-encoded movement functions. Collectively, these results uncover striking host- and isolate-dependent differences in component compatibility and demonstrate that systemic infection by ToLCNDV-IN DNA-A in tomato requires DNA-B, while ToLCNDV-ES additionally depends on coat protein for efficient movement in N. benthamiana.

plant biology↗

bioRxiv2025

Defender or accomplice? Dual roles of plant vesicle trafficking in restricting and enabling geminiviral systemic infection.

The vesicle trafficking system enables multidirectional cargo fluxes between endomembrane compartments, ensuring the viability of eukaryotic cells. However, vesicle trafficking plays dual roles during pathogen infections. In plants, the endomembrane system mediates autophagic immune responses but can also be hijacked by pathogens to facilitate successful infections. In this study, we demonstrate that vesicle trafficking machinery acts as a double-edged sword during infection by the geminivirus tomato yellow leaf curl Sardinia virus (TYLCSaV) in Nicotiana benthamiana. Virus-induced gene silencing (VIGS) of eight genes encoding key vesicle trafficking regulators revealed contrasting outcomes. Silencing of NbSAR1 and NbAP-1{gamma} significantly increased systemic geminiviral DNA accumulation, whereas silencing of Nb{delta}-COP, NbARF1, and clathrin genes almost completely abolished infection. Notably, this inhibition is hypothesized to result from direct or indirect impairment in viral movement, as replication remained unaffected by gene silencing. Furthermore, the observed effects affect other geminiviruses, including tomato yellow leaf curl virus (TYLCV) and beet curly top virus (BCTV), but not unrelated pathogens such as the RNA potato virus X (PVX) or the plant pathogenic bacterium Pseudomonas syringae. These findings suggest that while the vacuolar and autophagy branches of the vesicle trafficking system might mediate antiviral autophagic defense responses, the integrity of endocytosis and retrograde transport is essential for systemic geminiviral infection.

plant biology↗

bioRxiv2022

The transcriptional regulator JAZ8 interacts with the C2 protein from geminiviruses and limits the viral infection in Arabidopsis

Jasmonates (JAs) are phytohormones that finely regulate critical biological processes, including plant development and defense. JASMONATE ZIM-DOMAIN (JAZ) proteins are crucial keeping JA-responsive genes in a repressed state. In the presence of JA-Ile, JAZ repressors are ubiquitinated and targeted for degradation by the ubiquitin/proteasome system, allowing the activation of the downstream transcription factors and, consequently, the activation of JA-responsive genes. A growing body of evidence has shown that JA signalling is crucial in defending against plant viruses and their insect vectors. Here, we describe the interaction of C2 proteins from two geminiviruses from the genus Begomovirus, tomato yellow curl Sardinia virus (TYLCSaV) and tomato yellow leaf curl virus (TYLCV), with the transcriptional repressor JAZ8 from Arabidopsis thaliana and its closest orthologue in tomato, SlJAZ9. Both JAZ and C2 proteins colocalize in the nucleus, forming discrete nuclear speckles. Overexpression of JAZ8 did not lead to altered responses to TYLCV infection; however, knock-down of JAZ8 favours the geminiviral infection in plants. Low levels of JAZ8 likely affect the viral infection specifically since JAZ8-silenced plants do not display developmental phenotypes nor present differences in their interaction with the viral insect vector. Our results show that JAZ8 interacts with geminiviral C2 proteins and exerts an anti-geminiviral effect.

plant biology↗

bioRxiv2022

Characterization of the Rhizophagus irregularis multicopper oxidase family indicates that the iron transporter RiFTR1 does not require a ferroxidase partner

The contribution of arbuscular mycorrhizal fungi (AM fungi) to plant iron (Fe) acquisition has been demonstrated in several studies. Recently, it has been shown that AM fungi use a high-affinity reductive pathway for Fe uptake. In the AM fungus Rhizophagus irregularis the ferric reductase RiFRE1 and the Fe permeases RiFTR1 and RiFTR2 have already been characterized. In an attempt to identify the third component of the reductive iron uptake pathway, a genome-wide approach has been used in R. irregularis to find genes encoding ferroxidases of the multicopper oxidase (MCO) gene family. Nine genes putatively encoding MCOs (RiMCO1-9) were identified. A phylogenetic analysis of MCO sequences of fungi from different taxonomic groups revealed that all RiMCOs clustered together in the ferroxidase/laccase group, and none with the Fet3-type ferroxidases. RiMCO1 and RiMCO3 were the only MCO genes displaying a detectable gene expression pattern typical of a high-affinity Fe transport system, indicating that RiMCO1 and RiMCO3 might have a role in the reductive high-affinity Fe uptake system. Moreover, yeast mutant complementation assays showed that the iron permease RiFTR1 can operate without the presence of a ferroxidase, indicating that it is able to transport also ferrous (II) iron.

molecular biology↗

bioRxiv2022

Peripheral membrane proteins modulate stress tolerance by safeguarding cellulose synthases

Controlled primary cell wall remodeling adapts plant growth under stressful conditions, but how these changes are conveyed to adjust cellulose synthesis is unknown. Here, we identify the Tetratricopeptide Thioredoxin-Like (TTL) proteins as new members of the cellulose synthase complex (CSC) and describe their unique and hitherto unknown dynamic association with the CSC under cellulose-deficient conditions. We find that TTLs are essential for maintaining cellulose synthesis under salinity stress, establishing a stress-resilient cortical microtubule array, and stabilizing CSCs at the plasma membrane. To fulfill these functions, TTLs interact with Cellulose Synthase1 (CESA1) and engage with cortical microtubules to promote their polymerization. We propose that TTLs function as bridges connecting stress perception with dynamic regulation of cellulose biosynthesis at the plasma membrane. One Sentence SummaryTTLs are peripheral membrane proteins that maintain the integrity of the cellulose synthase complex upon adverse conditions.

plant biology↗

bioRxiv2020

Organization and control of the ascorbate biosynthesis pathway in plants

The enzymatic steps involved in O_SCPLOWLC_SCPLOW-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler, SW pathway) has been well established and here we comprehensively analyze the subcellular localization, potential physical interactions of SW pathway enzymes and assess their role in control of ascorbate synthesis. Transient expression of GFP-fusions in Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana) mutants complemented with genomic constructs showed that while GME is cytosolic, VTC1, VTC2, VTC4, and O_SCPLOWLC_SCPLOW-GalDH have cytosolic and nuclear localization. While transgenic lines GME-GFP, VTC4-GFP and O_SCPLOWLC_SCPLOW-GalDH-GFP driven by their endogenous promoters accumulated the fusion proteins, the functional VTC2-GFP protein is detected at low level using immunoblot in a complemented vtc2 null mutant. This low amount of VTC2 protein and the extensive analyses using multiple combinations of SW enzymes in N. benthamiana supported the role of VTC2 as the main control point of the pathway on ascorbate biosynthesis. Interaction analysis of SW enzymes using yeast two hybrid did not detect the formation of heterodimers, although VTC1, GME and VTC4 formed homodimers. Further coimmunoprecipitation (CoIP) analysis indicted that consecutive SW enzymes, as well as the first and last enzymes (VTC1 and O_SCPLOWLC_SCPLOW-GalDH), associate thereby adding a new layer of complexity to ascorbate biosynthesis. Finally, metabolic control analysis incorporating known kinetic characteristics, showed that previously reported feedback repression at the VTC2 step confers a high flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration. One sentence summaryMetabolic engineering, genetic analysis and functional mutant complementation identify GDP-O_SCPLOWLC_SCPLOW-galactose phosphorylase as the main control point in ascorbate biosynthesis in green tissues.

molecular biology↗

bioRxiv2018

TTL proteins scaffold brassinosteroid signaling components at the plasma membrane to optimize signal transduction in plant cells

Brassinosteroids (BRs) form a group of steroidal hormones essential for plant growth, development and stress responses. Here, we report that plant-specific TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins are positive regulators of BR signaling functioning as scaffold for BR signaling components in Arabidopsis. TTL3 forms a complex with all core components involved in BR signaling, including the receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1), the transcription factor BRASSINAZOLE RESISTANT1 (BZR1) and the phosphatase BRI1-SUPPRESSOR1 (BSU1), but excluding the co-receptor BAK1. TTL3 is mainly localized in the cytoplasm, but BR treatment increases its localization at the plasma membrane, where it strengthens the association with BR signaling components. Consistent with a role in BR signaling, mutations in TTL3 and related TTL1 and TTL4 genes cause reduced BR responsiveness. We propose a mechanistic model for BR signaling, in which cytoplasmic/nuclear BR components bound to TTL proteins are recruited to the plasma membrane upon BR perception, which in turn allows the assembly of a BR signaling complex, leading to the de-phosphorylation and nuclear accumulation of the transcription factors BZR1 and BES1.

molecular biology↗