Colonization and movement of GFP-labeled Clavibacter nebraskensis in maize

Clavibacter nebraskensis (Cn) causes Goss’s bacterial wilt and leaf blight, a major disease of maize. Infected crop residue is the primary inoculum source and infection can occur via wounds or natural openings, such as stomata or hydathodes. The use of resistant hybrids is the primary control method for Goss’s wilt. In this study, colonization and movement patterns of Cn during infection were examined using green fluorescent protein (GFP)-labeled bacterial strains. We successfully introduced a plasmid to Cn via electroporation, which resulted in GFP accumulation. Fluorescence microscopy revealed that in the absence of wounding, bacteria colonize leaf tissue via entry through the hydathodes when guttation droplets are present. Stomatal penetration was not observed under natural conditions. Bacteria initially colonize the xylem and subsequently the mesophyll, which creates the freckles that are characteristic of the disease. Bacteria infiltrated into the mesophyll did not cause disease symptoms, could not enter the vasculature, and did not spread from the initial inoculation point. Bacteria were observed exuding through stomata onto the leaf surface, resulting in the characteristic sheen of diseased leaves. Resistant maize lines exhibited decreased bacterial spread in the vasculature and the mesophyll. These tools to examine Cn movement offer opportunities and new insights into the pathogenesis process and can form the basis for improved Goss’s wilt management through host resistance.

Combined Antibacterial and Anti-Inflammatory Activity of a Cationic Disubstituted Dexamethasone-Spermine Conjugate

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01682-09 ◽

2010 ◽

Vol 54 (6) ◽

pp. 2525-2533 ◽

Cited By ~ 10

Author(s):

Robert Bucki ◽

Katarzyna Leszczyńska ◽

Fitzroy J. Byfield ◽

David E. Fein ◽

Esther Won ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Bacterial Infections ◽

Glucocorticoid Receptors ◽

Fluorescent Protein ◽

Lipoteichoic Acid ◽

Bacterial Strains ◽

Bacterial Killing ◽

Antibiotic Resistant ◽

Anti Inflammatory ◽

ABSTRACT The rising number of antibiotic-resistant bacterial strains represents an emerging health problem that has motivated efforts to develop new antibacterial agents. Endogenous cationic antibacterial peptides (CAPs) that are produced in tissues exposed to the external environment are one model for the design of novel antibacterial compounds. Here, we report evidence that disubstituted dexamethasone-spermine (D2S), a cationic corticosteroid derivative initially identified as a by-product of synthesis of dexamethasone-spermine (DS) for the purpose of improving cellular gene delivery, functions as an antibacterial peptide-mimicking molecule. This moiety exhibits bacterial killing activity against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa present in cystic fibrosis (CF) sputa, and Pseudomonas aeruginosa biofilm. Although compromised in the presence of plasma, D2S antibacterial activity resists the proteolytic activity of pepsin and is maintained in ascites, cerebrospinal fluid, saliva, and bronchoalveolar lavage (BAL) fluid. D2S also enhances S. aureus susceptibility to antibiotics, such as amoxicillin (AMC), tetracycline (T), and amikacin (AN). Inhibition of interleukin-6 (IL-6) and IL-8 release from lipopolysaccharide (LPS)- or lipoteichoic acid (LTA)-treated neutrophils in the presence of D2S suggests that this molecule might also prevent systemic inflammation caused by bacterial wall products. D2S-mediated translocation of green fluorescent protein (GFP)-labeled glucocorticoid receptor (GR) in bovine aorta endothelial cells (BAECs) suggests that some of its anti-inflammatory activities involve engagement of glucocorticoid receptors. The combined antibacterial and anti-inflammatory activities of D2S suggest its potential as an alternative to natural CAPs in the prevention and treatment of some bacterial infections.

Evaluation of Olive as a Host of Xylella fastidiosa and Associated Sharpshooter Vectors

Plant Disease ◽

10.1094/pdis-01-14-0014-re ◽

2014 ◽

Vol 98 (9) ◽

pp. 1186-1193 ◽

Cited By ~ 44

Author(s):

Rodrigo Krugner ◽

Mark S. Sisterson ◽

Jianchi Chen ◽

Drake C. Stenger ◽

Marshall W. Johnson

Keyword(s):

Control Method ◽

Xylella Fastidiosa ◽

Primary Control ◽

Homalodisca Vitripennis ◽

Insecticide Treatment ◽

Disease Symptoms ◽

Leaf Scorch ◽

Polymerase Chain ◽

Low Efficiency ◽

Branch Dieback

Olive (Olea europaea) trees exhibiting leaf scorch or branch dieback symptoms in California were surveyed for the xylem-limited, fastidious bacterium Xylella fastidiosa. Only approximately 17% of diseased trees tested positive for X. fastidiosa by polymerase chain reaction, and disease symptoms could not be attributed to X. fastidiosa infection of olive in greenhouse pathogenicity assays. Six strains of X. fastidiosa were isolated from olive in Southern California. Molecular assays identified strains recovered from olive as belonging to X. fastidiosa subsp. multiplex. Pathogenicity testing of olive strains on grapevine and almond confirmed that X. fastidiosa strains isolated from olive yield disease phenotypes on almond and grapevine typical of those expected for subsp. multiplex. Mechanical inoculation of X. fastidiosa olive strains to olive resulted in infection at low efficiency but infections remained asymptomatic and tended to be self-limiting. Vector transmission assays demonstrated that glassy-winged sharpshooter (Homalodisca vitripennis) could transmit strains of both subspp. multiplex and fastidiosa to olive at low efficiency. Insect trapping data indicated that two vectors of X. fastidiosa, glassy-winged sharpshooter and green sharpshooter (Draeculacephala minerva), were active in olive orchards. Collectively, the data indicate that X. fastidiosa did not cause olive leaf scorch or branch dieback but olive may contribute to the epidemiology of X. fastidiosa-elicited diseases in California. Olive may serve as an alternative, albeit suboptimal, host of X. fastidiosa. Olive also may be a refuge where sharpshooter vectors evade intensive areawide insecticide treatment of citrus, the primary control method used in California to limit glassy-winged sharpshooter populations and, indirectly, epidemics of Pierce's disease of grapevine.

Proof that Burkholderia Strains Form Effective Symbioses with Legumes: a Study of Novel Mimosa-Nodulating Strains from South America

10.1128/aem.71.11.7461-7471.2005 ◽

2005 ◽

Vol 71 (11) ◽

pp. 7461-7471 ◽

Cited By ~ 124

Author(s):

Wen-Ming Chen ◽

Sergio M. de Faria ◽

Rosângela Straliotto ◽

Rosa M. Pitard ◽

Jean L. Simões-Araùjo ◽

...

Keyword(s):

Fluorescent Protein ◽

Restriction Analysis ◽

Rrna Gene ◽

Bacterial Strains ◽

Consensus Sequences ◽

Mimosa Pigra ◽

Green Fluorescent ◽

Gene Restriction ◽

Single Cluster ◽

The 16S Rrna Gene

ABSTRACT Twenty Mimosa-nodulating bacterial strains from Brazil and Venezuela, together with eight reference Mimosa-nodulating rhizobial strains and two other β-rhizobial strains, were examined by amplified rRNA gene restriction analysis. They fell into 16 patterns and formed a single cluster together with the known β-rhizobia, Burkholderia caribensis, Burkholderia phymatum, and Burkholderia tuberum. The 16S rRNA gene sequences of 15 of the 20 strains were determined, and all were shown to belong to the genus Burkholderia; four distinct clusters could be discerned, with strains isolated from the same host species usually clustering very closely. Five of the strains (MAP3-5, Br3407, Br3454, Br3461, and Br3469) were selected for further studies of the symbiosis-related genes nodA, the NodD-dependent regulatory consensus sequences (nod box), and nifH. The nodA and nifH sequences were very close to each other and to those of B. phymatum STM815, B. caribensis TJ182, and Cupriavidus taiwanensis LMG19424 but were relatively distant from those of B. tuberum STM678. In addition to nodulating their original hosts, all five strains could also nodulate other Mimosa spp., and all produced nodules on Mimosa pudica that had nitrogenase (acetylene reduction) activities and structures typical of effective N2-fixing symbioses. Finally, both wild-type and green fluorescent protein-expressing transconjugant strains of Br3461 and MAP3-5 produced N2-fixing nodules on their original hosts, Mimosa bimucronata (Br3461) and Mimosa pigra (MAP3-5), and hence this confirms strongly that Burkholderia strains can form effective symbioses with legumes.

Cloning and Expression Analysis of the BocMBF1c Gene Involved in Heat Tolerance in Chinese Kale

International Journal of Molecular Sciences ◽

10.3390/ijms20225637 ◽

2019 ◽

Vol 20 (22) ◽

pp. 5637 ◽

Cited By ~ 1

Author(s):

Lifang Zou ◽

Bingwei Yu ◽

Xing-Liang Ma ◽

Bihao Cao ◽

Guoju Chen ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Heat Stress ◽

Green Fluorescent Protein ◽

Stress Response ◽

Nicotiana Benthamiana ◽

Thermal Tolerance ◽

Fluorescent Protein ◽

Leaf Tissue ◽

Chinese Kale ◽

Chinese kale (Brassica oleracea var. chinensis Lei) is an important vegetable crop in South China, valued for its nutritional content and taste. Nonetheless, the thermal tolerance of Chinese kale still needs improvement. Molecular characterization of Chinese kale’s heat stress response could provide a timely solution for developing a thermally tolerant Chinese kale variety. Here, we report the cloning of multi-protein bridging factor (MBF) 1c from Chinese kale (BocMBF1c), an ortholog to the key heat stress responsive gene MBF1c. Phylogenetic analysis showed that BocMBF1c is highly similar to the stress-response transcriptional coactivator MBF1c from Arabidopsis thaliana (AtMBF1c), and the BocMBF1c coding region conserves MBF1 and helix-turn-helix (HTH) domains. Moreover, the promoter region of BocMBF1c contains three heat shock elements (HSEs) and, thus, is highly responsive to heat treatment. This was verified in Nicotiana benthamiana leaf tissue using a green fluorescent protein (GFP) reporter. In addition, the expression of BocMBF1c can be induced by various abiotic stresses in Chinese kale which indicates the involvement of stress responses. The BocMBF1c-eGFP (enhanced green fluorescent protein) chimeric protein quickly translocated into the nucleus under high temperature treatment in Nicotiana benthamiana leaf tissue. Overexpression of BocMBF1c in Arabidopsis thaliana results in a larger size and enhanced thermal tolerance compared with the wild type. Our results provide valuable insight for the role of BocMBF1c during heat stress in Chinese kale.

Interactions between Food-Borne Pathogens and Protozoa Isolated from Lettuce and Spinach

10.1128/aem.02709-07 ◽

2008 ◽

Vol 74 (8) ◽

pp. 2518-2525 ◽

Cited By ~ 72

Author(s):

Poornima Gourabathini ◽

Maria T. Brandl ◽

Katherine S. Redding ◽

John H. Gunderson ◽

Sharon G. Berk

Keyword(s):

Fluorescent Protein ◽

Tetrahymena Pyriformis ◽

Enteric Bacteria ◽

Enteric Pathogens ◽

Leafy Vegetables ◽

Human Pathogens ◽

Bacterial Strains ◽

E Coli ◽

ABSTRACT The survival of Salmonella enterica was recently shown to increase when the bacteria were sequestered in expelled food vacuoles (vesicles) of Tetrahymena. Because fresh produce is increasingly linked to outbreaks of enteric illness, the present investigation aimed to determine the prevalence of protozoa on spinach and lettuce and to examine their interactions with S. enterica, Escherichia coli O157:H7, and Listeria monocytogenes. Glaucoma sp., Colpoda steinii, and Acanthamoeba palestinensis were cultured from store-bought spinach and lettuce and used in our study. A strain of Tetrahymena pyriformis previously isolated from spinach and a soil-borne Tetrahymena sp. were also used. Washed protozoa were allowed to graze on green fluorescent protein- or red fluorescent protein-labeled enteric pathogens. Significant differences in interactions among the various protist-enteric pathogen combinations were observed. Vesicles were produced by Glaucoma with all of the bacterial strains, although L. monocytogenes resulted in the smallest number per ciliate. Vesicle production was observed also during grazing of Tetrahymena on E. coli O157:H7 and S. enterica but not during grazing on L. monocytogenes, in vitro and on leaves. All vesicles contained intact fluorescing bacteria. In contrast, C. steinii and the amoeba did not produce vesicles from any of the enteric pathogens, nor were pathogens trapped within their cysts. Studies of the fate of E. coli O157:H7 in expelled vesicles revealed that by 4 h after addition of spinach extract, the bacteria multiplied and escaped the vesicles. The presence of protozoa on leafy vegetables and their sequestration of enteric bacteria in vesicles indicate that they may play an important role in the ecology of human pathogens on produce.

Use of Green Fluorescent Protein-Transgenic Strains to Study Pathogenic and Nonpathogenic Lifestyles in Colletotrichum acutatum

Phytopathology ◽

10.1094/phyto.2002.92.7.743 ◽

2002 ◽

Vol 92 (7) ◽

pp. 743-749 ◽

Cited By ~ 47

Author(s):

Sigal Horowitz ◽

Stanley Freeman ◽

Amir Sharon

Keyword(s):

Green Fluorescent Protein ◽

Plant Species ◽

Fluorescent Protein ◽

Leaf Tissue ◽

Colletotrichum Acutatum ◽

Signal Molecules ◽

Primary Host ◽

Large Numbers ◽

Cell Layers ◽

Colletotrichum acutatum, which causes anthracnose disease on strawberry, can also persist on several other plant species without causing disease symptoms. The genetic and molecular bases that determine pathogenic and nonpathogenic lifestyles in C. acutatum are unclear. We developed a transformation system for C. acutatum by electroporation of germinating conidia, and transgenic isolates that express the green fluorescent protein (GFP) were produced. Details of the pathogenic and nonpathogenic lifestyles of C. acutatum were determined by using GFP-transgenic isolates. Major differences between colonization-mediating processes of strawberry and of other plants were observed. On the main host, strawberry, the germinating conidia formed branched, thick hyphae, and large numbers of appressoria were produced that were essential for plant penetration. In strawberry, the fungus developed rapidly, filling the mesophyll with dense mycelium that invaded the cells and caused necrosis of the tissue. In nonpathogenic interactions on pepper, eggplant, and tomato, the conidia germinated, producing thin, straight germ tubes. Appressoria were produced but failed to germinate and penetrate leaf tissue, resulting in epiphytic growth without invasion of the plant. Penetration of the plant occurred only several days after inoculation and was restricted to the intercellular spaces of the first cell layers of infected tissue without causing any visible damage. Much of the new fungal biomass continued to develop on the surface of inoculated organs in the nonpathogenic interaction. The differences in fungal development on strawberry compared with the other plant species suggest that signal molecules, which may be present only in strawberry, trigger appressorial germination and penetration of the primary host.

Differences between Pseudomonas syringae pv. syringae B728a and Pantoea agglomerans BRT98 in Epiphytic and Endophytic Colonization of Leaves

10.1128/aem.69.2.1220-1228.2003 ◽

2003 ◽

Vol 69 (2) ◽

pp. 1220-1228 ◽

Cited By ~ 35

Author(s):

Siva Sabaratnam ◽

Gwyn A. Beattie

Keyword(s):

Plant Species ◽

Pseudomonas Syringae ◽

Rapid Growth ◽

Fluorescent Protein ◽

Pantoea Agglomerans ◽

Bacterial Strains ◽

Surface Sites ◽

Maize Leaves ◽

Green Fluorescent ◽

Favorable Conditions

ABSTRACT The leaf colonization strategies of two bacterial strains were investigated. The foliar pathogen Pseudomonas syringae pv. syringae strain B728a and the nonpathogen Pantoea agglomerans strain BRT98 were marked with a green fluorescent protein, and surface (epiphytic) and subsurface (endophytic) sites of bean and maize leaves in the laboratory and the field were monitored to see if populations of these strains developed. The populations were monitored using both fluorescence microscopy and counts of culturable cells recovered from nonsterilized and surface-sterilized leaves. The P. agglomerans strain exclusively colonized epiphytic sites on the two plant species. Under favorable conditions, the P. agglomerans strain formed aggregates that often extended over multiple epidermal cells. The P. syringae pv. syringae strain established epiphytic and endophytic populations on asymptomatic leaves of the two plant species in the field, with most of the P. syringae pv. syringae B728a cells remaining in epiphytic sites of the maize leaves and an increasing number occupying endophytic sites of the bean leaves in the 15-day monitoring period. The epiphytic P. syringae pv. syringae B728a populations appeared to originate primarily from multiplication in surface sites rather than from the movement of cells from subsurface to surface sites. The endophytic P. syringae pv. syringae B728a populations appeared to originate primarily from inward movement through the stomata, with higher levels of multiplication occurring in bean than in maize. A rainstorm involving a high raindrop momentum was associated with rapid growth of the P. agglomerans strain on both plant species and with rapid growth of both the epiphytic and endophytic populations of the P. syringae pv. syringae strain on bean but not with growth of the P. syringae pv. syringae strain on maize. These results demonstrate that the two bacterial strains employed distinct colonization strategies and that the epiphytic and endophytic population dynamics of the pathogenic P. syringae pv. syringae strain were dependent on the plant species, whereas those of the nonpathogenic P. agglomerans strain were not.

Early Events in the Fusarium verticillioides-Maize Interaction Characterized by Using a Green Fluorescent Protein-Expressing Transgenic Isolate

10.1128/aem.69.3.1695-1701.2003 ◽

2003 ◽

Vol 69 (3) ◽

pp. 1695-1701 ◽

Cited By ~ 103

Author(s):

Liat Oren ◽

Smadar Ezrati ◽

David Cohen ◽

Amir Sharon

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Lateral Roots ◽

Fusarium Verticillioides ◽

Systemic Infection ◽

Fungal Mycelium ◽

Infested Soil ◽

Disease Symptoms ◽

Fungal Hyphae ◽

ABSTRACT The infection of maize by Fusarium verticillioides can result in highly variable disease symptoms ranging from asymptomatic plants to severe rotting and wilting. We produced F. verticillioides green fluorescent protein-expressing transgenic isolates and used them to characterize early events in the F. verticillioides-maize interaction that may affect later symptom appearance. Plants grown in F. verticillioides-infested soil were smaller and chlorotic. The fungus colonized all of the underground parts of a plant but was found primarily in lateral roots and mesocotyl tissue. In some mesocotyl cells, conidia were produced within 14 to 21 days after infection. Intercellular mycelium was detected, but additional cells were not infected until 21 days after planting. At 25 to 30 days after planting, the mesocotyl and main roots were heavily infected, and rotting developed in these tissues. Other tissues, including the adventitious roots and the stem, appeared to be healthy and contained only a small number of hyphae. These results imply that asymptomatic systemic infection is characterized by a mode of fungal development that includes infection of certain tissues, intercellular growth of a limited number of fungal hyphae, and reproduction of the fungus in a few cells without invasion of other cells. Development of visibly rotted tissue is associated with massive production of fungal mycelium and much less organized growth.

Infection of the Native California Grass, Bromus carinatus, by Fusarium circinatum, the Cause of Pitch Canker in Pines

Plant Disease ◽

10.1094/pdis-04-19-0800-re ◽

2020 ◽

Vol 104 (1) ◽

pp. 194-197

Author(s):

Jason W. Carter ◽

Thomas R. Gordon

Keyword(s):

Fluorescent Protein ◽

Leaf Tissue ◽

Pitch Canker ◽

Fusarium Circinatum ◽

Holcus Lanatus ◽

Native Grass ◽

Point Reyes National Seashore ◽

Bromus Carinatus ◽

Point Reyes ◽

At Point Reyes National Seashore in California, Fusarium circinatum, the causal agent of pitch canker in pines, was isolated from Pinus muricata, the California native grass, Bromus carinatus, and the introduced grass, Holcus lanatus. All grass plants from which F. circinatum was isolated were symptomless. Pathogenicity of grass isolates was confirmed by inoculation of P. radiata trees, which developed symptoms similar to trees inoculated with a pine isolate of F. circinatum. Isolates from grasses were somatically compatible with isolates recovered from symptomatic pines. B. carinatus grown in a growth chamber was inoculated with a green fluorescent protein-expressing strain of F. circinatum. Segments of inoculated leaves were incubated in moist chambers; after 1 to 2 days, sporulating hyphae were observed growing from leaf tissue. Spores of F. circinatum removed from B. carinatus leaves were confirmed to be fluorescent when illuminated with ultraviolet light. These results raise the possibility that B. carinatus cryptically infected by F. circinatum may be a source of propagules capable of infecting pines.

Diverse Microhabitats Experienced by Halomonas variabilis on Salt-Secreting Leaves

10.1128/aem.02791-12 ◽

2012 ◽

Vol 79 (3) ◽

pp. 845-852 ◽

Cited By ~ 11

Author(s):

Adrien Y. Burch ◽

Omri M. Finkel ◽

Juliana K. Cho ◽

Shimshon Belkin ◽

Steven E. Lindow

Keyword(s):

Salt Concentration ◽

Fluorescent Protein ◽

Leaf Surface ◽

Green Fluorescent Protein Fluorescence ◽

Protein Fluorescence ◽

Content Type ◽

Osmotic Tolerance ◽

Leaf Surfaces ◽

High Concentrations ◽

ABSTRACTThe leaf surfaces of the salt-excreting treeTamarix aphyllaharbor a wide diversity of halophilic microorganisms, includingHalomonassp., but little is known of the factors that shape community composition in this extreme habitat. We isolated a strain ofHalomonas variabilisfrom the leaf surface ofT. aphyllaand used it to determine the heterogeneity of salt concentrations experienced by bacteria in this environment. This halophilic strain was transformed with aproU::gfpreporter gene fusion, the fluorescence of which was responsive to NaCl concentrations up to 200 g liter−1. These bioreporting cells were applied toT. aphyllaleaves and were subsequently recovered from dew droplets adhering to the leaf surface. Although cells from within a given dew droplet exhibited similar green fluorescent protein fluorescence, the fluorescence intensity varied between droplets and was correlated with the salt concentration measured in each drop. Growth ofH. variabiliswas observed in all droplets, regardless of the salt concentration. However, cells found in desiccated microniches between dew drops were low in abundance and generally dead. Other bacteria recovered fromT. aphylladisplayed higher desiccation tolerance thanH. variabilis, both in culture and on inoculated plants, despite having lower osmotic tolerance. Thus, theTamarixleaf surface can be described as a salty desert with occasional oases where water droplets form under humid conditions. While halotolerant bacteria such asHalomonasgrow in high concentrations of salt in such wet microniches, other organisms are better suited to survive desiccation in sites that are not wetted.