Biology of Maize Kernel Infection by Fusarium verticillioides

Fusarium kernel rot disease starburst symptomatology was characterized fully for the first time. Two maize lines were hand pollinated and inoculated, using a fluorescent protein-expressing transformant of the fungal pathogen Fusarium verticillioides, by introduction of a conidial suspension through the silk channel of intact ears. Microscopy was used to identify the infection court and document initial stages of kernel colonization and subsequent manifestation of macroscopic symptoms. The fungus entered kernels of susceptible line AD38 via an open stylar canal and spread extracellularly and over the kernel through the nucellus region, sporadically entering pericarp and filling the long thick-walled mesocarp cells. Hyphae spread within pericarp from cell to cell via pits, colonizing files of host cells by growing both up and down the kernel in a radial pattern that preceded macroscopic symptom development. The starburst symptom developed subsequently, and mirrored colonization exactly, when there was extensive dissolution of the thick walls of pericarp cells. Line HT1 exhibited a closed stylar canal phenotype and was not susceptible—except when the pericarp surface was breached mechanically. We hypothesize the passive movement of conidia along the surface of silks, perhaps via capillarity, as a possible mechanism for pathogen access to the infection court.

Download Full-text

Multiple knockout mutants reveal a high redundancy of phytotoxic compounds that determine necrotrophic pathogenesis of Botrytis cinerea

10.1101/2021.08.21.457223 ◽

2021 ◽

Author(s):

Thomas Leisen ◽

Janina Werner ◽

Patrick Pattar ◽

Edita Ymeri ◽

Frederik Sommer ◽

...

Keyword(s):

Cell Death ◽

Botrytis Cinerea ◽

Plant Cell ◽

Fungal Pathogen ◽

Host Plants ◽

Host Cells ◽

Necrotic Tissue ◽

Knockout Mutants ◽

Marker Free ◽

First Time

Botrytis cinerea is a major pathogen of more than 1400 plant species. During infection, the kills host cells during infection and spreads through necrotic tissue, which is believed to be supported by induction of programmed plant cell death. To comprehensively evaluate the contributions of most of the currently known plant cell death inducing proteins (CDIPs) and metabolites for necrotrophic infection, an optimized CRISPR/Cas protocol was established which allowed serial marker-free mutagenesis to generate Botrytis mutants lacking up to 12 different CDIPs. Infection analysis revealed a decrease in virulence with increasing numbers of knockouts, and differences in the effects of knockouts on different host plants. The on planta secretomes obtained from these mutants revealed substantial remaining necrotic activity after infiltration into leaves. Our study has addressed for the first time the functional redundancy of virulence factors of a fungal pathogen, and demonstrates that B. cinerea releases a highly redundant cocktail of proteins and metabolites to achieve necrotrophic infection of a wide variety of host plants.

Download Full-text

Interaction of Pasteurella multocida with Free-Living Amoebae

Applied and Environmental Microbiology ◽

10.1128/aem.71.9.5458-5464.2005 ◽

2005 ◽

Vol 71 (9) ◽

pp. 5458-5464 ◽

Cited By ~ 11

Author(s):

Matthew J. Hundt ◽

Carmel G. Ruffolo

Keyword(s):

Pasteurella Multocida ◽

Fluorescent Protein ◽

Host Cells ◽

Free Living ◽

Fowl Cholera ◽

Transmission Electron ◽

Membrane Bound ◽

Green Fluorescent ◽

First Time

ABSTRACT Pasteurella multocida is a highly infectious, facultative intracellular bacterium which causes fowl cholera in birds. This study reports, for the first time, the observed interaction between P. multocida and free-living amoebae. Amoebal trophozoites were coinfected with fowl-cholera-causing P. multocida strain X-73 that expressed the green fluorescent protein (GFP). Using confocal fluorescence microscopy, GFP expressing X-73 was located within the trophozoite. Transmission electron microscopy of coinfection preparations revealed clusters of intact X-73 cells in membrane-bound vacuoles within the trophozoite cytoplasm. A coinfection assay employing gentamicin to kill extracellular bacteria was used to assess the survival and replication of P. multocida within amoebae. In the presence of amoebae, the number of recoverable intracellular X-73 cells increased over a 24-h period; in contrast, X-73 cultured alone in assay medium showed a consistent decline in growth. Cytotoxicity assays and microscopy showed that X-73 was able to lyse and exit the amoebal cells approximately 18 h after coinfection. The observed interaction between P. multocida and amoebae can be considered as an infective process as the bacterium was able to invade, survive, replicate, and lyse the amoebal host. This raises the possibility that similar interactions occur in vivo between P. multocida and host cells. Free-living amoebae are ubiquitous within water and soil environments, and P. multocida has been observed to survive within these same ecosystems. Thus, our findings suggest that the interaction between P. multocida and amoebae may occur within the natural environment.

Download Full-text

First Report of Root Rot on Rhodiola sachalinensis Caused by Fusarium verticillioides (Gibberella fujikuroi) in China

Plant Disease ◽

10.1094/pdis-08-10-0582 ◽

2011 ◽

Vol 95 (2) ◽

pp. 222-222

Author(s):

J. F. Liu ◽

Y. Q. Cheng

Keyword(s):

Root Rot ◽

Tap Water ◽

Fusarium Verticillioides ◽

Gibberella Fujikuroi ◽

Herbaceous Plant ◽

Conidial Suspension ◽

First Report ◽

Rhodiola Sachalinensis ◽

Root Rot Disease ◽

Rot Disease

Rhodiola sachalinensis (family Crassulaceae), a perennial herbaceous plant with adaptogenic properties, cardiopulmonary protective effects, and central nervous system activities, is widely used as a traditional Chinese medicine (3). R. sachalinensis naturally exists only above 1,500 m elevation in the mountain area of Changbai Mountain (average July temperature ≤10°C), China. Introduction and cultivation of R. sachalinensis has been carried out in several low-altitude districts of northeast China. From 2007 to 2010, severe root rot disease was observed on R. sachalinensis in Siping districts, Jilin, China. Approximately 75 to 95% of the fields were affected with root rot disease incidence ranging from 85 to 100% under conditions of high temperatures (24 to 30°C) and high humidity. As the disease progressed, brown lesions expanded on lateral and main roots, and aboveground tissues shriveled and died. Over the 4- to 5-year period from culture to harvest, root rot became more serious. Symptomatic plants were collected from Siping districts. Samples were rinsed in tap water, necrotic areas were excised and cut into 2-mm pieces, surface sterilized with 5% NaOCl for 30 s, and rinsed in four successive changes of sterile distilled water. A single fungus was isolated on potato dextrose agar (PDA). The fungus was white, then pink and cottony, with nearly round margins after 8 days (27°C). Hyphae were separate and hyaline but macroconidia were sparse and occurred in a false head. Conidiogenous cells were monophialides. Microconidia in chains were abundant and mostly nonseptate, oval to clavate, and measured 8.1 to 14.5 × 2.0 to 3.4 μm. Morphological characteristics suggested the isolate was Fusarium verticillioides (Gibberella fujikuroi), which differed from the reported root rot pathogen of R. sachalinensis, F. oxysporum by forming microconidia in chains (1). The sexual stage of F. verticillioides was not observed. The internal transcribed spacer (ITS) fragments were amplified using ITS1 and ITS4 as primers and the 351-bp sequence was deposited in GenBank (Accession No. HQ025928). The ITS sequence showed 100% nucleotide sequence identity with F. verticillioides (GenBank Accession No. AY188916.1.). For pathogenicity tests, the isolate was cultured on PDA for 8 days. Inoculations were performed on 15 healthy R. sachalinensis plants by spraying a conidial suspension (2.0 × 105 microconidia ml–1) on roots wounded with a metal needle (6 wounds cm–2) (2). Ten plants were mock inoculated with water. Plants were incubated in a growth chamber (25°C, 70 to 80% relative humidity, 300 μmol·m–2·s–1 light intensity, and a 12-h photoperiod). After 15 days, defoliation and root rot symptoms were similar to the original symptoms observed under field conditions. F. verticillioides was reisolated from the roots of infected plants. Control plants remained asymptomatic. To our knowledge, this is the first report of F. verticillioides on R. sachalinensis in China. References: (1) X. Y. Li et al. J. Northeast For. Univ. 34:12, 2003. (2) M. Ma. Syahit et al. Am. J. Appl. Sci. 6:902, 2009. (3) T. F. Yan et al. Conserv. Genet. 4:213, 2003.

Download Full-text

A Cowpea Seed Rot Disease Caused by Fusarium equiseti Identified in Nigeria

Plant Disease ◽

10.1094/pdis.1999.83.10.964a ◽

1999 ◽

Vol 83 (10) ◽

pp. 964-964 ◽

Cited By ~ 3

Author(s):

S. O. Aigbe ◽

B. Fawole ◽

D. K. Berner

Keyword(s):

Fusarium Species ◽

Conidial Suspension ◽

Basal Cells ◽

Fusarium Equiseti ◽

Cowpea Seeds ◽

Seed Rot ◽

Cowpea Seed ◽

Rot Disease ◽

Dark Incubator ◽

First Time

Fusarium equiseti (Corda) Sacc., reported on cowpea (Vigna unguiculata (L.) Walp.) seeds in India (2), was isolated for the first time in Nigeria from naturally infected cowpea seeds. Cowpea, cv. IT90K-76, seeds (400) from plants grown in Nigeria were surface-disinfested in 0.05% NaOCl and placed on moist filter paper in petri dishes (10 seeds per dish) and then in a dark incubator for 4 days at 27°C. After incubation, some seeds had fungal mycelia growing on their surfaces. When cultured on potato dextrose (PDA) and Spezieller Nährstoffarmer (SNA) agars, the fungi produced macroconidia characteristic of F. equiseti (1). Septate macroconidia were three to six celled with extended apical and distinctive foot-shaped basal cells. F. equiseti was recovered from 4.25% of seeds, and incidence correlated positively with development of seed rot symptoms. To confirm pathogenicity, 80 cowpea seeds were surface-disinfested with NaOCl, and 40 were soaked for 6 h in a suspension of 3 × 105 conidia of F. equiseti per ml of water. The remaining seeds were soaked in sterile distilled water. After incubation, white mycelia developed on 87.5% of seeds soaked in the conidial suspension and rotted without germinating. Only 5% of seeds soaked in sterile water developed seed rot symptoms. When cultured on PDA and SNA, fungi isolated from artificially infested seeds with rot symptoms again were identified as F. equiseti. References: (1) P. E. Nelson et al. 1983. Fusarium species: An illustrated Manual for Identification. Pennsylvania University Press, University Park. (2) O. K. Sinha and M. N. Khare. Seed Sci. Technol. 5:721, 1977.

Download Full-text

Resveratrol Inhibits Venezuelan Equine Encephalitis Virus Infection by Interfering with the AKT/GSK Pathway

Plants ◽

10.3390/plants10020346 ◽

2021 ◽

Vol 10 (2) ◽

pp. 346

Author(s):

Caitlin W. Lehman ◽

Kylene Kehn-Hall ◽

Megha Aggarwal ◽

Nicole R. Bracci ◽

Han-Chi Pan ◽

...

Keyword(s):

Antiviral Activity ◽

Fluorescent Protein ◽

Glycogen Synthase ◽

Venezuelan Equine Encephalitis Virus ◽

Encephalitis Virus ◽

Host Cells ◽

Dynamics Simulation ◽

Luciferase Reporter ◽

Venezuelan Equine Encephalitis ◽

Equine Encephalitis Virus

The host proteins Protein Kinase B (AKT) and glycogen synthase kinase-3 (GSK-3) are associated with multiple neurodegenerative disorders. They are also important for the replication of Venezuelan equine encephalitis virus (VEEV), thereby making the AKT/GSK-3 pathway an attractive target for developing anti-VEEV therapeutics. Resveratrol, a natural phytochemical, has been shown to substantially inhibit the AKT pathway. Therefore, we attempted to explore whether it exerts any antiviral activity against VEEV. In this study, we utilized green fluorescent protein (GFP)- and luciferase-encoding recombinant VEEV to determine the cytotoxicity and antiviral efficacy via luciferase reporter assays, flow cytometry, and immunofluorescent assays. Our results indicate that resveratrol treatment is capable of inhibiting VEEV replication, resulting in increased viability of Vero and U87MG cells as well as reduced virion production and viral RNA contents within host cells for at least 48 h with a single treatment. Furthermore, the suppression of apoptotic signaling adaptors, caspase-3, caspase-7, and annexin V may also be implicated in resveratrol-mediated antiviral activity. We found that decreased phosphorylation of the AKT/GSK-3 pathway, mediated by resveratrol, can be triggered during the early stages of VEEV infection, suggesting that resveratrol disrupts the viral replication cycle and consequently promotes cell survival. Finally, molecular docking and dynamics simulation studies revealed that resveratrol can directly bind to VEEV glycoproteins, which may interfere with virus attachment and entry. In conclusion, our results suggest that resveratrol exerts inhibitory activity against VEEV infection and upon further modification could be a useful compound to study in neuroprotective research and veterinary sciences.

Download Full-text

Systemic Infection by Fusarium verticillioides in Maize Plants Grown Under Three Temperature Regimes

Plant Disease ◽

10.1094/pdis-92-12-1695 ◽

2008 ◽

Vol 92 (12) ◽

pp. 1695-1700 ◽

Cited By ~ 36

Author(s):

A. Murillo-Williams ◽

G. P. Munkvold

Keyword(s):

High Temperature ◽

Environmental Factors ◽

Fusarium Verticillioides ◽

Systemic Infection ◽

Temperature Treatment ◽

High Temperature Treatment ◽

Kernel Infection ◽

Temperature Regimes ◽

Maize Plants ◽

Systemic Development

Fusarium verticillioides causes seedling decay, stalk rot, ear rot, and mycotoxin contamination (primarily fumonisins) in maize. Systemic infection of maize plants by F. verticillioides can lead to kernel infection, but the frequency of this phenomenon has varied widely among experiments. Variation in the incidence of systemic infection has been attributed to environmental factors. In order to better understand the influence of environment, we investigated the effect of temperature on systemic development of F. verticillioides during vegetative and reproductive stages of plant development. Maize seeds were inoculated with a green fluorescent protein-expressing strain of F. verticillioides, and grown in growth chambers under three different temperature regimes. In the vegetative-stage and reproductive-stage experiments, plants were evaluated at tasseling (VT stage), and at physiological maturity (R6 stage), respectively. Independently of the temperature treatment, F. verticillioides was reisolated from nearly 100% of belowground plant tissues. Frequency of reisolation of the inoculated strain declined acropetally in aboveground internodes at all temperature regimes. At VT, the high-temperature treatment had the highest systemic development of F. verticillioides in aboveground tissues. At R6, incidence of systemic infection was greater at both the high- and low-temperature regimes than at the average-temperature regime. F. verticillioides was isolated from higher internodes in plants at R6, compared to stage VT. The seed-inoculated strain was recovered from kernels of mature plants, although incidence of kernel infection did not differ significantly among treatments. During the vegetative growth stages, temperature had a significant effect on systemic development of F. verticillioides in stalks. At R6, the fungus reached higher internodes in the high-temperature treatment, but temperature did not have an effect on the incidence of kernels (either symptomatic or asymptomatic) or ear peduncles infected with the inoculated strain. These results support the role of high temperatures in promoting systemic infection of maize by F. verticillioides, but plant-to-seed transmission may be limited by other environmental factors that interact with temperature during the reproductive stages.

Download Full-text

Effectors of biotrophic fungal plant pathogens

Functional Plant Biology ◽

10.1071/fp10072 ◽

2010 ◽

Vol 37 (10) ◽

pp. 913 ◽

Cited By ~ 12

Author(s):

Pamela H. P. Gan ◽

Maryam Rafiqi ◽

Adrienne R. Hardham ◽

Peter N. Dodds

Keyword(s):

Plant Tissue ◽

Fungal Pathogen ◽

Plant Pathogens ◽

Plant Cells ◽

Host Cells ◽

Plant Proteins ◽

Living Plant ◽

Host Cytoplasm ◽

Fungal Plant Pathogens ◽

Biotrophic Fungi

Plant pathogenic biotrophic fungi are able to grow within living plant tissue due to the action of secreted pathogen proteins known as effectors that alter the response of plant cells to pathogens. The discovery and identification of these proteins has greatly expanded with the sequencing and annotation of fungal pathogen genomes. Studies to characterise effector function have revealed that a subset of these secreted pathogen proteins interact with plant proteins within the host cytoplasm. This review focuses on the effectors of intracellular biotrophic and hemibiotrophic fungal plant pathogens and summarises advances in understanding the roles of these proteins in disease and in elucidating the mechanism of fungal effector uptake into host cells.

Download Full-text

Optical reconstruction of murine colorectal mucosa at cellular resolution

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00310.2014 ◽

2015 ◽

Vol 308 (9) ◽

pp. G721-G735 ◽

Cited By ~ 10

Author(s):

Cambrian Y. Liu ◽

Philip E. Dubé ◽

Nandini Girish ◽

Ajay T. Reddy ◽

D. Brent Polk

Keyword(s):

Cell Function ◽

Fluorescent Protein ◽

Confocal Imaging ◽

Host Cells ◽

Cellular Mechanisms ◽

Colorectal Mucosa ◽

Cellular Resolution ◽

Volume Imaging ◽

Mucosal Layer ◽

Optical Reconstruction

The mucosal layer of the colon is a unique and dynamic site where host cells interface with one another and the microbiome, with major implications for physiology and disease. However, the cellular mechanisms mediating colonic regeneration, inflammation, dysplasia, and dysbiosis remain undercharacterized, partly because the use of thin tissue sections in many studies removes important volumetric context. To address these challenges in visualization, we have developed the deep mucosal imaging (DMI) method to reconstruct continuous extended volumes of mouse colorectal mucosa at cellular resolution. Use of ScaleA2 and SeeDB clearing agents enabled full visualization of the colonic crypt, the fundamental unit of adult colon. Confocal imaging of large colorectal expanses revealed epithelial structures involved in repair, inflammation, tumorigenesis, and stem cell function, in fluorescent protein-labeled, immunostained, paraffin-embedded, or human biopsy samples. We provide freely available software to reconstruct and explore on computers with standard memory allocations the large DMI datasets containing in toto representations of distal colonic mucosal volume. Extended-volume imaging of colonic mucosa through the novel, extensible, and readily adopted DMI approach will expedite mechanistic investigations of intestinal physiology and pathophysiology at intracrypt to multicrypt length scales.

Download Full-text

Defining the early stages of intestinal colonisation by whipworms

10.1101/2020.08.21.261586 ◽

2020 ◽

Author(s):

María A. Duque-Correa ◽

David Goulding ◽

Claire Cormie ◽

Catherine Sharpe ◽

Judit Gali Moya ◽

...

Keyword(s):

Intestinal Epithelium ◽

Early Stage ◽

Proximal Colon ◽

Host Cells ◽

Parasitic Nematodes ◽

Metazoan Parasites ◽

Multiple Cells ◽

First Time

ABSTRACTHundreds of millions of people are infected with whipworms (Trichuris trichiura), large metazoan parasites that live in the caecum and proximal colon. Whipworms inhabit distinct multi-intracellular epithelial burrows that have been described as syncytial tunnels. However, the interactions between first-stage (L1) larvae and the host epithelia that determine parasite invasion and establishment in the syncytium remain unclear. In vivo experiments investigating these events have been severely hampered by the limited in situ accessibility to intracellular infective larvae at the bottom of the crypts of Lieberkühn, and the lack of genetic tools such as fluorescent organisms that are readily available for other pathogens but not parasitic nematodes. Moreover, cell lines, which do not mimic the complexity of the intestinal epithelium, have been unsuccessful in supporting infection by whipworm larvae. Here, we show that caecaloids grown in an open crypt-like conformation recapitulate the caecal epithelium. Using this system, we establish in vitro infections with T. muris L1 larvae for the first-time, and provide clear evidence that syncytial tunnels are formed at this early stage. We show that larval whipworms are completely intracellular but woven through multiple cells. Using the caecaloids, we are able to visualise the pathways taken by the larvae as they burrow through the epithelial cells. We also demonstrate that larvae degrade the mucus layers overlaying the epithelium, enabling them to access the cells below. We show that early syncytial tunnels are composed of enterocytes and goblet cells that are alive and actively interacting with the larvae during the first 24 h of the infection. Progression of infection results in damage to host cells and by 72 h post-infection, we show that desmosomes of cells from infected epithelium widen and some host cells appear to become liquified. Collectively, our work unravels processes mediating the intestinal epithelium invasion by whipworms and reveals new specific interactions between the host and the parasite that allow the whipworm to establish on its multi-intracellular niche. Our study demonstrates that caecaloids can be used as a relevant in vitro model to investigate the infection biology of T. muris during the early colonisation of its host.

Download Full-text

Molecular architecture of theLegionellaDot/Icm type IV secretion system

10.1101/312009 ◽

2018 ◽

Cited By ~ 3

Author(s):

Debnath Ghosal ◽

Yi-Wei Chang ◽

Kwang Cheol Jeong ◽

Joseph P. Vogel ◽

Grant J. Jensen

Keyword(s):

Legionella Pneumophila ◽

Cell Envelope ◽

Secretion System ◽

Host Cells ◽

Molecular Architecture ◽

Intact Cells ◽

Type Iv ◽

Type Ivb Secretion ◽

Electron Cryotomography ◽

First Time

AbstractLegionella pneumophilasurvives and replicates inside host cells by secreting ~300 effectors through the Dot/Icm type IVB secretion system (T4BSS). Understanding this machine’s structure is challenging because of its large number of components (27) and integration into all layers of the cell envelope. Previously we overcame this obstacle by imaging the Dot/Icm T4BSS in its native state within intact cells through electron cryotomography. Here we extend our observations by imaging a stabilized mutant that yielded a higher resolution map. We describe for the first time the presence of a well-ordered central channel that opens up into a windowed large (~32 nm wide) secretion chamber with an unusual 13-fold symmetry. We then dissect the complex by matching proteins to densities for many components, including all those with periplasmic domains. The placement of known and predicted structures of individual proteins into the map reveals the architecture of the T4BSS and provides a roadmap for further investigation of this amazing specialized secretion system.

Download Full-text