Biology subjects

Iyer-Pascuzzi, A. S.

Publications and source records attributed to Iyer-Pascuzzi, A. S..

15 recordsLinked to original sources

bioRxiv2025

Lf2 is a knotted homeobox regulator that modulates leaflet number in soybean

Variation in leaf complexity modulates light capture and is a target for crop enhancement. Soybean typically has compound leaves with three leaflets each, but a spontaneous mutation, designated lf2, possesses seven leaflets, offering a means to dissect the molecular mechanisms specifying leaflet number and assess its potential for soybean improvement. However, the developmental and genetic bases of the lf2 mutation remain unknown. Here, we characterize the seven-leaflet phenotype and identify the mutation responsible for the phenotypic changes. Microscopic examination of leaf emergence sites revealed that the seven-leaflet phenotype arises in a two-step process: five leaflets form initially followed by secondary leaflet initiation at the margins of the central leaflet. Genetic mapping delineated lf2 to a [~]2.5 Mb region at the start of chromosome 11. Fortuitously, integration of pedigree analysis with comparative analysis of genomic sequences from the region pinpointed a 2-bp deletion in the coding sequence of a gene, which is homologous to the Arabidopsis KNAT7 encoding a KNOTTED1-LIKE HOMEOBOX 2 transcription factor, as the sole candidate for Lf2. The deletion is predicted to result in disruption of the putative DNA-binding homeodomain. Expression of the wild-type allele of the candidate gene in the seven-leaflet lf2 mutant restored the three-leaflet phenotype, while disruption of the wild-type allele through CRISPR-Cas9 editing induced extra leaflet formation. This study advances our understanding of leaflet formation in legumes and provides a template for utilizing compound leaf architecture to optimize photosynthetic efficiency and yield in soybean.

plant biology↗

bioRxiv2025

Agent-based computational modeling of the stochastic dynamic behavior of actin filaments recapitulates the homeostatic cortical array in plant epidermal cells

The homeostatic cortical actin array in plant cells plays important roles in fundamental processes, including intracellular transport, secretion, cell expansion, and cytoplasmic streaming. In response to diverse chemical and mechanical signals, the cortical array can remodel within minutes to assume new configurations or altered filament abundance. The homeostatic cortical actin array of some plant epidermal cells comprise sparsely distributed individual actin filaments and actin bundles, which enables tracking and quantitative analysis of dynamic properties over many minutes at high spatiotemporal resolution. Previous studies using quantitative live-cell imaging, small molecule inhibitors, and genetic mutations reveal the robust dynamic steady state of the cortical actin array, with individual filaments showing a behavior termed stochastic dynamics. Compared to experimental findings, computational efforts focused on the plant actin cytoskeleton are lacking, although computational models have the potential to define underlying mechanisms of actin array homeostasis and remodeling. Here, we used an agent-based computational model to reproduce the stochastic dynamic behavior of individual actin filaments in epidermal cells with consideration of key governing factors, including the nucleation, polymerization, depolymerization, severing, capping, and branching of filaments. Our model was able to reproduce experimental observations with respect to the abundance and length of filaments as well as the rates or frequencies of dynamic events. This model can be used to study the role of myosin motors and other actin-binding proteins, as well as the effects of signaling events and fluxes in cellular second messengers, on actin dynamics in plant cells.

plant biology↗

bioRxiv2025

Vascular anatomy changes in tomato stems grown under simulated microgravity

Plants grown in spaceflight exhibit differences in physiology and morphology compared to those grown on Earth. While changes in gravity are a major environmental change, other space-related stressors make it difficult to identify the microgravity-specific responses. These knowledge gaps can be filled by ground-based microgravity simulators that randomize the perceived gravity vector, but these approaches have primarily been limited to smaller plants and seedlings. This study reports a set of meter-scale 2D clinostats that support the growth of plants beyond the seedling stage. Tomato plants were grown in five sequential trials under upright rotating control and clinorotated "simulated microgravity" conditions. We found that simulated microgravity impacted plant growth in each trial, but the response varied by trial. Analysis of environmental co-variates across trials revealed that temperature significantly contributed to variation in plant growth. Further, our results show that moderate heat stress can promote plant growth under simulated microgravity. Thus, this work demonstrates the potential of meter-scale clinostats to uncover interactions between the environmental and simulated microgravity, which alter plant growth.

plant biology↗

bioRxiv2025

Local traveling waves of cytosolic calcium elicited by defense signals or wounding are propagated by distinct mechanisms

Cytosolic Ca2+ signatures with specific spatiotemporal patterns play crucial roles in plant responses to biotic and abiotic stresses. Perception of microbe- or damage-associated molecular patterns (MAMPs or DAMPs) initiates signaling cascades that represent the first layer of plant defense against pathogens known as pattern-triggered immunity (PTI). During PTI, MAMP/DAMP-induced cytosolic Ca2+ fluxes serve as essential messengers in the initiation and transmission of defense signals at the cellular, whole organ, and systemic levels. However, the specific patterns of these Ca2+ signatures in response to different pathogen cues, and the mechanisms that encode them, remain largely unexplored. In this study, we quantitatively assessed Ca2+ signatures at the single-cell level as well as the local traveling Ca2+ waves induced by global treatment of Arabidopsis cotyledons with MAMPs or DAMPs. We demonstrated that MAMPs induced distinct local spatiotemporal Ca2+ responses in epidermal pavement cells, with Ca2+ traveling waves consistently initiated from a subset of cells and spreading in an approximately radial pattern. These local traveling waves propagated at a slow but constant speed of approximately 1 {micro}m/s and spread to a limited number of neighboring cells. In contrast, wound-induced traveling waves displayed a diffusion-like decay pattern that moved rapidly away from the wounded cell but with diminishing speed over time and distance. Mathematical modeling supported a calcium-induced calcium release mechanism that could recapitulate the constant wave speed induced by MAMPs. These findings contribute to a deeper understanding of plant defense-related Ca2+ signaling mechanisms as well as how defense responses are spatially restricted within tissues. One Sentence SummaryA slow, local traveling wave of calcium is initiated from a subset of epidermal cells during plant immune signaling.

cell biology↗

bioRxiv2024

Tomato roots exhibit distinct, development-specific responses to bacterial-derived peptides

O_LIPlants possess cell-surface recognition receptors that detect molecular patterns from microbial invaders and initiate an immune response. Understanding the conservation of pattern-triggered immunity within different plant organs and across species is crucial to its sustainable and effective use in plant disease management but is currently unclear. C_LIO_LIWe examined the activation and immune response patterns of three pattern recognition receptors (PRRs: SlFLS2, SlFLS3, and SlCORE) in different developmental regions of roots and in leaves of multiple accessions of domesticated and wild tomato (Solanum lycopersicum and S. pimpinellifolium) using biochemical and genetic assays. C_LIO_LIRoots from different tomato accessions differed in the amplitude and dynamics of their immune response, but all exhibited developmental-specific PTI responses in which the root early differentiation zone was the most sensitive to molecular patterns. PRR signaling pathways also showed distinct but occasionally overlapping responses downstream of each immune receptor in tomato roots. C_LIO_LIThese results reveal that each PRR initiates a unique PTI pathway and suggest that the specificity and complexity of tomato root immunity are tightly linked to the developmental stage, emphasizing the importance of spatial and temporal regulation in PTI. C_LI

plant biology↗

bioRxiv2024

Genome-informed trophic classification and functional characterization of virulence proteins from the maize tar spot pathogen Phyllachora maydis

Phyllachora maydis is an ascomycete foliar fungal pathogen and the causal agent of tar spot in maize. Though P. maydis is considered one of the most economically important foliar pathogens of maize, our general knowledge of the trophic lifestyle and functional role of effector proteins from this fungal pathogen remains limited. Here, we utilized a genome-informed approach to predict the trophic lifestyle of P. maydis and functionally characterized a subset of candidate effectors from this fungal pathogen. Leveraging the most recent P. maydis genome annotation and the CATAStrophy pipeline, we show this fungal pathogen encodes a predicted Carbohydrate-active enzymes (CAZymes) repertoire consistent with that of biotrophs (monomertrophs). To investigate fungal pathogenicity, we selected eighteen candidate effector proteins that were previously shown to be expressed during primary disease development. We assessed whether these putative effectors share predicted structural similarity with other characterized fungal effectors and determined whether any suppress plant immune responses. Using AlphaFold2 and Foldseek, we showed one candidate effector, PM02_g1115, adopts a predicted protein structure similar to that of an effector from Verticillium dahlia. Furthermore, transient expression of candidate effector-fluorescent protein fusions in Nicotiana benthamiana revealed that most effector proteins localize to both the nucleus and the cytosol. Importantly, three candidate effectors consistently attenuated chitin-mediated reactive oxygen species production in N. benthamiana. Collectively, these results presented herein provide valuable insights into the predicted trophic lifestyle and putative functions of effectors from P. maydis and will likely stimulate continued research to elucidate the molecular mechanisms used by P. maydis to induce tar spot.

plant biology↗

bioRxiv2023

A Ralstonia solanacearum type III effector alters the actin and microtubule cytoskeleton to promote bacterial virulence in plants

Cellular responses to biotic stress frequently involve signaling pathways that are conserved across eukaryotes. These pathways include the cytoskeleton, a proteinaceous network that senses external cues at the cell surface and signals to interior cellular components. During biotic stress, dynamic cytoskeletal rearrangements serve as a platform from which early immune-associated processes are organized and activated. Bacterial pathogens of plants and animals use proteins called type III effectors (T3Es) to interfere with host immune signaling, thereby promoting virulence. We previously found that RipU, a T3E from the soilborne phytobacterial pathogen Ralstonia solanacearum K60 (Rs K60), co-localizes with the plant cytoskeleton. Here, we show that RipU from Rs K60 (RipUK60) physically associates with both actin and tubulin and disrupts actin and microtubule cytoskeleton organization. We find that pharmacological disruption of the tomato (Solanum lycopersicum) cytoskeleton promotes Rs K60 colonization. RipUK60 suppresses cell surface-triggered immune responses including flg22-mediated reactive oxygen species (ROS) production and callose deposition. Importantly, tomato plants inoculated with Rs K60 lacking RipUK60 ({Delta}ripUK60) had reduced wilting symptoms and significantly reduced root colonization when compared to plants inoculated with wild-type Rs K60. Collectively, our data suggest that Rs K60 uses the type III effector RipUK60 to remodel cytoskeletal organization, thereby promoting pathogen virulence.

plant biology↗

bioRxiv2023

Uncovering the Infection Strategy of Phyllachora maydis during Maize Colonization: A Comprehensive Analysis

Tar spot, a disease caused by the ascomycete fungal pathogen Phyllachora maydis, is considered one of the most significant yield-limiting diseases of maize (Zea mays L.) within the United States. P. maydis may also be found in association with other fungi, forming a disease complex with characteristic fish eye lesions. Understanding how P. maydis colonizes maize leaf cells is essential for developing effective disease control strategies. Here, we used histological approaches to elucidate how P. maydis infects and multiplies within susceptible maize leaves. We collected tar spot-infected maize leaf samples from four different fields in northern Indiana at three different time points during the growing season. Samples were chemically fixed and paraffin-embedded for high-resolution light and scanning electron microscopy. We observed a consistent pattern of disease progression in independent leaf samples collected across different geographical regions. Each stromata contained a central pycnidium that produced asexual spores. Perithecia with sexual spores developed in the stomatal chambers adjacent to the pycnidia, and a cap of spores formed over the stromata. P. maydis reproductive structures formed around but not within the vasculature. In our samples containing fish eye lesions, P. maydis is associated with two additional fungi, one of which is likely a member of the Paraphaeospheria genus; the other is an unknown fungi. Our data provide fundamental insights into how this pathogen colonizes and spreads within maize leaves. This knowledge can inform new approaches to managing tar spot, which could help mitigate the significant economic losses caused by this disease.

plant biology↗

bioRxiv2022

Candidate effector proteins from the maize tar spot pathogen Phyllachora maydis localize to diverse plant cell compartments

Most fungal pathogens secrete effector proteins into host cells to modulate their immune responses, thereby promoting pathogenesis and fungal growth. One such fungal pathogen is the ascomycete Phyllachora maydis, which causes tar spot disease on leaves of maize (Zea mays). Sequencing of the P. maydis genome revealed 462 putatively secreted proteins of which 40 contain expected effector-like sequence characteristics. However, the subcellular compartments targeted by P. maydis effector candidate (PmECs) proteins remain unknown and it will be important to prioritize them for further functional characterization. To test the hypothesis that PmECs target diverse subcellular compartments, cellular locations of super Yellow Fluorescent Protein (sYFP)-tagged P. maydis effector candidate proteins were identified using a Nicotiana benthamiana-based heterologous expression system. Immunoblot analyses showed that most of the PmEC-fluorescent protein fusions accumulated protein in N. benthamiana, indicating the candidate effectors could be expressed in dicot leaf cells. Laser-scanning confocal microscopy of N. benthamiana epidermal cells revealed most of the P. maydis putative effectors localized to the nucleus and cytosol. One candidate effector, PmEC01597, localized to multiple subcellular compartments including the nucleus, nucleolus, and plasma membrane while an additional putative effector, PmEC03792, preferentially labelled both the nucleus and nucleolus. Intriguingly, one candidate effector, PmEC04573, consistently localized to the stroma of chloroplasts as well as stroma-containing tubules (stromules). Collectively, these data suggest effector candidate proteins from P. maydis target diverse cellular organelles and may thus provide valuable insights into their putative functions as well as host processes potentially manipulated by this fungal pathogen.

plant biology↗

bioRxiv2022

The NIN-LIKE PROTEIN 7 (NLP7) transcription factor modulates auxin pathways to regulate root cap development

The root cap surrounds the root tip and promotes root growth by protecting the root apical meristem, influencing root branching, and sensing environmental signals like nitrate. The root cap maintains a constant size through the coordination of cell production in the root meristem with cell release at the tip of the root, a process that requires an auxin minima in the last layer of the root cap. To perform its functions, the root cap must maintain a constant size and synchronize external cues with development, but mechanisms underlying such coordination are not well understood. Mutations in the NIN LIKE PROTEIN 7 (NLP7) transcription factor, a master regulator of nitrate signaling, lead to defects in root cap cell release and cell production. Nitrate impacts root development through crosstalk with auxin. Therefore, we hypothesized that NLP7 regulates root cap cell release and cell production by modulating auxin pathways. Here we show that mutations in NLP7 abolish the auxin minima required for root cap cell release and alter root cap expression levels of the auxin carriers PIN-LIKES 3 (PILS3) and PIN-FORMED 7 (PIN7). We find that NLP7 is required for proper root cap cell production and differentiation and for expression of transcription factors that regulate these processes. Nitrate deficiency impacts auxin pathways in the last layer of the root cap, and this is mediated in part by NLP7. Together, our data suggest that NLP7 integrates nitrate signaling with auxin pathways to optimize root cap development in response to external nitrate cues. One sentence summaryThe nitrate master regulator NLP7 controls root cap development through auxin pathways.

plant biology↗

bioRxiv2021

Trehalose induces tomato defenses and increases drought tolerance and bacterial wilt disease resistance

Ralstonia solanacearum causes bacterial wilt disease, leading to severe crop losses. Xylem sap from R. solanacearum-infected tomato is enriched in the disaccharide trehalose. Water-stressed plants also accumulate trehalose, which increases drought tolerance via abscisic acid (ABA) signaling. Because R. solanacearum-infected plants suffer reduced water flow, we hypothesized that bacterial wilt physiologically mimics drought stress, which trehalose could mitigate. We found that R. solanacearum-infected plants differentially expressed drought-associated genes, including those involved in ABA and trehalose metabolism, and had more ABA in xylem sap. Consistent with this, treating tomato roots with ABA reduced both stomatal conductance and stem colonization by R. solanacearum. Treating roots with trehalose increased xylem sap ABA and reduced plant water use by lowering stomatal conductance and temporarily improving water use efficiency. Trehalose treatment also upregulated expression of salicylic acid (SA)-dependent tomato defense genes; increased xylem sap levels of SA and other antimicrobial compounds; and increased bacterial wilt resistance of SA-insensitive NahG tomato plants. Additionally, trehalose treatment increased xylem concentrations of jasmonic acid and related oxylipins. Finally, trehalose-treated plants were substantially more resistant to bacterial wilt disease. Together, these data show that exogenous trehalose reduced both water stress and bacterial wilt disease and triggered systemic disease resistance, possibly through a Damage Associated Molecular Pattern (DAMP) response pathway. This suite of responses revealed unexpected linkages between plant responses to biotic and abiotic stress and suggested that R. solanacearum-infected plants increase trehalose to improve water use efficiency and increase wilt disease resistance. The pathogen may degrade trehalose to counter these efforts. Together, these results suggest that treating tomatoes with exogenous trehalose could be a practical strategy for bacterial wilt management.

plant biology↗

bioRxiv2021

Image-based assessment of plant disease progression identifies new genetic loci for resistance

A major challenge in global crop production is mitigating yield loss due to plant diseases. One of the best means of disease control is plant resistance, but the identification of genes that promote resistance has been limited by the subjective quantification of disease, which is typically scored by the human eye. We hypothesized that image-based, non-destructive quantification of disease phenotypes would enable the rapid identification of new disease resistance loci. We tested this using the interaction between tomato and Ralstonia solanacearum, a soilborne pathogen that causes bacterial wilt disease. We acquired over 40,000 time-series images of disease progression in a tomato recombinant inbred line population, and developed an image analysis pipeline providing a suite of ten traits to quantify wilt disease based on plant shape and size. Quantitative trait loci (QTL) analyses using image-based phenotyping identified QTL that were both unique and shared compared with those identified by human assessment of wilting. When shared loci were identified, image-based phenotyping could detect some QTL several days earlier than human assessment. Thus, expanding the phenotypic space of disease with image-based, non- destructive phenotyping allowed both earlier detection and identified new genetic components of resistance.

plant biology↗

bioRxiv2021

Revisiting the source of wilt symptoms: X-ray microcomputed tomography provides direct evidence that Ralstonia biomass clogs xylem vessels.

Plant pathogenic Ralstonia cause wilt diseases by colonizing xylem vessels and disrupting water transport. Due to the abundance of Ralstonia cells in vessels, the dogma is that bacterial biomass clogs vessels and reduces the flow of xylem sap. However, the physiological mechanism of xylem disruption during bacterial wilt disease is untested. Using a tomato and Ralstonia pseudosolanacearum GMI1000 model, we visualized and quantified the spatiotemporal dynamics of xylem disruption during bacterial wilt disease. First, we measured stomatal conductance of leaflets on mock-inoculated and wilt-symptomatic plants. Wilted leaflets had reduced stomatal conductance, as did turgid leaflets located on the same petiole as wilted leaflets. Next, we used X-ray microcomputed tomography (X-ray microCT) and light microscopy to differentiate between mechanisms of xylem disruption: blockage by bacterial biomass, blockage by vascular tyloses, or sap displacement by gas embolisms. We imaged stems on plants with intact roots and leaves to quantify embolized vessels. Embolized vessels were rare, but there was a slight trend of increased vessel embolisms in infected plants with low bacterial population sizes. To test the hypothesis that vessels are clogged during bacterial wilt, we imaged excised stems after allowing the sap to evaporate during a brief dehydration. Most xylem vessels in mock-infected plants emptied their contents after excision, but non-conductive clogged vessels were abundant in infected plants by 2 days post infection. At wilt onset when bacterial populations exceeded 5x108 cfu/g stem tissue, approximately half of the xylem vessels were clogged with electron-dense bacterial biomass. We found no evidence of tyloses in the X-ray microCT reconstructions or light microscopy on the preserved stems. Bacterial blockage of vessels appears to be the principal cause of vascular disruption during Ralstonia wilt.

microbiology↗

bioRxiv2019

Defense hormones modulate root microbiome diversity and composition in tomato

Plant roots live closely associated with soil microbes. Understanding how roots defend against pathogenic microbes while associating with non-pathogenic microbes is critical to using root microbiomes for successful outcomes. The aim of this study was to determine the contribution of the plant defense hormones ethylene (ET), jasmonic acid (JA), and salicylic acid (SA) to the tomato root microbiome. We used 16S rRNA amplicon sequencing to examine the root microbiome of four tomato genotypes defective in three hormone pathways. We found that ET and SA pathways, but not JA, were critical for root endosphere microbial alpha ()-diversity. Root endosphere communities of ACD, deficient in a precursor of ET, and the SA-deficient NahG transgenic, had significantly less -diversity than wild-type tomatoes. NahG and ACD root endosphere enrichment profiles were similar and driven by two taxa. In NahG, ACD, and in a panel of 24 wild-type tomatoes, the abundance of these two taxa was correlated with lower root endosphere -diversity. The abundance of these taxa was also moderately negatively correlated with the level of SA in roots. Our results suggest that SA and ET pathways contribute to tomato root endosphere diversity by modulating the abundance of specific bacterial taxa.

plant biology↗

bioRxiv2017

Whole Root Transcriptomic Analysis Reveals a Role for Auxin Pathways in Resistance to Ralstonia solanacearum in Tomato

The soilborne pathogen Ralstonia solanacearum is the causal agent of bacterial wilt, and causes significant crop loss in the Solanaceae family. The pathogen first infects roots, which are a critical source of resistance in tomato (Solanum lycopersicum L.). Roots of both resistant and susceptible plants are colonized by the pathogen, yet rootstocks can provide significant levels of resistance. Currently, mechanisms of this root-mediated resistance remain largely unknown. To identify the molecular basis of this resistance, we analyzed the genome-wide transcriptional response of roots of resistant (Hawaii 7996) and susceptible (West Virginia700) tomatoes at multiple time points after inoculation with R. solanacearum. We found that defense pathways in roots of the resistant Hawaii7996 are activated earlier and more strongly than roots of susceptible West Virginia700. Further, auxin signaling and transport pathways are suppressed in roots of the resistant variety. Functional analysis of an auxin transport mutant in tomato confirmed a role for auxin pathways in bacterial wilt. Together, our results suggest that roots mediate resistance to R. solanacearum through genome-wide transcriptomic changes that result in strong activation of defense genes and alteration of auxin pathways.

plant biology↗