scholarly journals Invasion and Colonization Pattern of Fusarium fujikuroi in Rice

2020 ◽  
Vol 110 (12) ◽  
pp. 1934-1945
Author(s):  
Chieh-Yi Chen ◽  
Szu-Yu Chen ◽  
Chun-Wei Liu ◽  
Dong-Hong Wu ◽  
Chien-Chih Kuo ◽  
...  
Keyword(s):  
Adventitious Roots ◽  
Fluorescent Protein ◽  
Rice Cultivar ◽  
Leaf Angle ◽  
Fusarium Fujikuroi ◽  
Growth Stages ◽  
Vascular Bundles ◽  
Infected Adult ◽  
Green Fluorescent ◽  
Adult Plants

Bakanae disease in rice can cause abnormal elongation of the stem and leaves, development of adventitious roots, a larger leaf angle, and even death. Little is known about the infection, colonization, and distribution of Fusarium fujikuroi in rice plants across different growth stages. In this study, microscopic observation and quantitative real-time PCR were combined to investigate the pathogenesis of bakanae, using artificially inoculated seedlings of a susceptible rice cultivar, Zerawchanica karatals (ZK), a resistant cultivar, Tainung 67 (TNG67), naturally infected adult field plants (cultivars Kaohsiung 139, Taikeng 2, and Tainan 11), and an F. fujikuroi isolate expressing green fluorescent protein. In rice seedlings, F. fujikuroi hyphae were found to directly penetrate the epidermis of basal stems and roots, then extend inter- and intracellularly to invade the vascular bundles. Occlusion of vascular bundles and radial hyphal expansion from vascular bundles to surrounding parenchyma were observed in adult plants. Analysis of consecutive 3-cm segments of the whole plant revealed that F. fujikuroi was largely confined to the embryo, basal stem, and basal roots in seedlings, and distributed unevenly in the lower aerial parts (including nodes and internodes) of adult plants. The elongation and development of adventitious roots did not necessarily correlate with the amount of F. fujikuroi in diseased plants. Treatment of rice seeds with gibberellic acid-3 (GA3) at 0.5 mg/liter resulted in significantly more elongation of ZK than TNG67 seedlings, suggesting that the susceptibility of ZK to bakanae is associated with its higher sensitivity to GA3.

scholarly journals The Colonization Process of Sunflower by a Green Fluorescent Protein-Tagged Isolate of Verticillium dahliae and its Seed Transmission

Plant Disease ◽  
2018 ◽  
Vol 102 (9) ◽  
pp. 1772-1778 ◽  
Author(s):  
Y. Zhang ◽  
J. Zhang ◽  
J. Gao ◽  
G. Zhang ◽  
Y. Yu ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Verticillium Dahliae ◽  
Fluorescent Protein ◽  
Lateral Roots ◽  
Pollen Grains ◽  
Seed Transmission ◽  
Vascular Bundles ◽  
Long Distance ◽  
Vascular Elements ◽  
Green Fluorescent

Sunflower Verticillium wilt is a widespread and destructive disease caused by the soilborne pathogen Verticillium dahliae. To better understand the process of infection and seed transmission of the fungus, sunflower roots were inoculated with a V. dahliae strain (VdBM9-6) labeled with green fluorescent protein (GFP) and monitored microscopically. After 24 to 96 h postinoculation (hpi), conidia germinated and developed into mycelium on root hairs, elongation zones, and caps of lateral roots. Mycelium colonized vascular bundles of lateral roots and taproots at 7 days postinoculation (dpi). At 10 weeks postinoculation (wpi), the epidermal cells, cortical tissues, and vascular elements of stem, petiole, and leaf veins were colonized by mycelium. By 12 wpi, strong GFP signals were detected not only on different tissues of inflorescence but also on testa of seed and a small fraction of pollen grains. A GFP signal was not observed on cotyledon tissues in the seed. Additionally, the colonization of V. dahliae on testa was also confirmed with MNP-10 selection medium, indicating that the testa of seed is the main carrier for the long distance transmission of sunflower yellow wilt.

scholarly journals A Simplified Protocol for Reversing Phenotypic Conversion of Ralstonia solanacearum during Experimentation

2020 ◽  
Vol 17 (12) ◽  
pp. 4274
Author(s):  
Pramod Kumar Sahu ◽  
Shailendra Singh ◽  
Amrita Gupta ◽  
Udai B. Singh ◽  
Surinder Paul ◽  
...  
Keyword(s):  
Ralstonia Solanacearum ◽  
Fluorescent Protein ◽  
Synergistic Effects ◽  
Dead Cell ◽  
Vascular Bundles ◽  
Virulent Type ◽  
Confocal Scanning Laser ◽  
The Stability ◽  
Green Fluorescent ◽  
Cell Fractions

Background: Ralstonia solanacearum has the problem of losing the virulence in laboratory conditions, during prolonged experimentation. Since pure colonies of R. solanacearum contain cell fractions differing in virulence, it was considered worthwhile to find a way of selecting the cells with lower attenuation. Therefore, a methodology for inducing virulent-type colonies occurrence in Ralstonia solanacearum was developed. Methods: Nutrient gradient was created by swabbing R. solanacearum culture in a slanted KMTTC medium, and Phyllanthus emblica extract was given by well diffusion. Live–dead cell imaging using BacLight, effects of ascorbic acid on cell viability, and production of virulence factors (exopolysaccharides, cellulase, and pectinase) supported this hypothesis. The tagging of R. solanacearum with green fluorescent protein and further confocal scanning laser microscopic visualization confirmed the colonization in vascular bundles of tomato. Results: P. emblica extract suppressed R. solanacearum initially in well diffusion, but further developed virulent-type colonies around the wells. Nutrient deprivation was found to have synergistic effects with P. emblica extract. The converted fluidal (virulent type) colonies could be able to colonize vascular bundles and cause wilting symptoms. Conclusion: This method will be useful in the laboratories working on biocontrol of R. solanacearum for maintaining virulent-type colonies. Moreover, it could form the basis for studies on the stability of phenotypic conversion and cell fractions in R. solanacearum.

scholarly journals Seed Transmission of Fusarium verticillioides in Maize Plants Grown Under Three Different Temperature Regimes

Plant Disease ◽  
2007 ◽  
Vol 91 (9) ◽  
pp. 1109-1115 ◽  
Author(s):  
A. L. Wilke ◽  
C. R. Bronson ◽  
A. Tomas ◽  
G. P. Munkvold
Keyword(s):  
Temperature Regime ◽  
Fluorescent Protein ◽  
Fusarium Verticillioides ◽  
Systemic Infection ◽  
Seed Transmission ◽  
Infection Process ◽  
Effect Of Temperature ◽  
Growth Stages ◽  
Temperature Regimes ◽  
Green Fluorescent

Fusarium verticillioides can be seed transmitted and cause systemic infection of maize; however, the frequency of these phenomena has varied widely among and within individual studies. In order to better understand this variability, we evaluated the effect of temperature on the first step in the systemic infection process, the transmission of F. verticillioides from seed to seedling. Seed of a commercial maize hybrid were inoculated with a strain of F. verticillioides that had been transformed with a gene for green fluorescent protein (GFP). The seed were planted in a greenhouse potting mix and incubated in growth chambers. Plants were incubated at one of three temperature regimes designed to simulate average and extreme temperatures occurring in Iowa during the weeks following planting. Root, mesocotyl, and stem tissues were sampled at growth stages V2 and V6, surface disinfested, and cultured on a semiselective medium. At V2, >90% of root and mesocotyl tissues was infected by the GFP-expressing strain at all three temperature regimes. Also at V2, infection was detected in 68 to 75% of stems. At V6, infection of root and mesocotyl tissues persisted and was detected in 97 to 100% of plants at all three temperature regimes. Plants also had symptomless systemic infection of belowground and aboveground internodes at V6. Infection of the three basal aboveground internodes was 24, 6, and 3% for the low-temperature regime; 35, 9, and 0% for the average-temperature regime; and 46, 24, and 9% for the high-temperature regime. Seed transmission and systemic infection occurred at all temperatures and did not differ significantly among treatments. These results indicate that, if maize seed is infected with F. verticillioides, seed transmission is common and symptomless systemic infection can be initiated under a broad range of temperature conditions.

scholarly journals Characterization of the Dual Subcellular Localization of Lilium LsGRP1, a Plant Class II Glycine-Rich Protein

2014 ◽  
Vol 104 (10) ◽  
pp. 1012-1020 ◽  
Author(s):  
Chia-Hua Lin ◽  
Chao-Ying Chen
Keyword(s):  
Subcellular Localization ◽  
Time Course ◽  
Fluorescent Protein ◽  
Protein Extraction ◽  
Protein A ◽  
Class Ii ◽  
Growth Stages ◽  
Weakly Bound ◽  
Green Fluorescent ◽  
Glycine Rich Protein

The defense-related gene LsGRP1 exhibits an increased level of expression in Lilium spp. after being infected by Botrytis elliptica, the fungal pathogen of lily leaf blight. In this study, the expression profile of the LsGRP1 protein (a plant class II glycine-rich protein) was characterized biochemically and its subcellular localization in lily leaves was evaluated using immunohistochemistry, enhanced green fluorescent protein (EGFP) imaging, and protein extraction analysis. Using an LsGRP1-specific antibody, LsGRP1 was found to be most abundant in epidermal cells and phloem tissues. Leaves from lily plants at different growth stages demonstrated similar levels of 14- and 16-kDa LsGRP1 and a decreased amount of 23-kDa LsGRP1 at the senescence stage. LsGRP1-EGFP imaging and protein extraction assays revealed that 14-kDa LsGRP1 was located in the plasma membrane whereas 16- and 23-kDa LsGRP1 was weakly bound to the cell wall. The time course analyses of LsGRP1 expression in response to salicylic acid treatment or B. elliptica infection showed an increased accumulation of 14- and 23-kDa LsGRP1 over time. Because 23-kDa LsGRP1 could be detected by an ubiquitin antibody, conversion of 14-kDa to 23-kDa LsGRP1 via mono-ubiquitination was presumed, which is a phenomenon that has not been reported for a plant class II glycine-rich protein.

scholarly journals Colonization and Movement of Xanthomonas fragariae in Strawberry Tissues

2018 ◽  
Vol 108 (6) ◽  
pp. 681-690 ◽  
Author(s):  
Hehe Wang ◽  
Christine McTavish ◽  
William W. Turechek
Keyword(s):  
Fluorescent Protein ◽  
Vascular Bundles ◽  
Leaf Petiole ◽  
Initial Inoculum ◽  
Systemic Movement ◽  
Xanthomonas Fragariae ◽  
Crown Tissue ◽  
Direct Infiltration ◽  
Taqman Pcr ◽  
Green Fluorescent

Xanthomonas fragariae causes angular leaf spot of strawberry, an important disease in strawberry growing regions worldwide. To better understand how X. fragariae multiplies and moves in strawberry plants, a green fluorescent protein (GFP)-labeled strain was constructed and used to monitor the pathogen’s presence in leaf, petiole, and crown tissue with fluorescence microscopy following natural and wound inoculation in three strawberry cultivars. Taqman PCR was used to quantify bacterial densities in these same tissues regardless of the presence of GFP signal. Results showed X. fragariae colonized leaf mesophyll, the top 1 cm portion of the petiole adjacent to the leaf blade, and was occasionally found colonizing xylem vessels down to the middle of the petioles. The colonization of vascular bundles and the limited systemic movement that was observed appeared to be a passive process, of which the frequency increased with wounding and direct infiltration of bacteria into leaf veins. X. fragariae was able to directly enter petioles and colonize the space under the epidermis. Systemic movement of the bacteria into crown and other uninoculated tissues was not detected visually by GFP. However, X. fragariae was occasionally detected in these tissues by qPCR, but at quantities very near the qPCR detection limit. Petiole tissue harboring bacteria introduced either by direct entry through natural openings or wounds, or by systemic movement from infected foliar tissue, likely serves as a main source of initial inoculum in field plantings.

scholarly journals Breaking dogmas: the plant vascular pathogen Xanthomonas albilineans is able to invade non-vascular tissues despite its reduced genome

Open Biology ◽  
2014 ◽  
Vol 4 (2) ◽  
pp. 130116 ◽  
Author(s):  
Imène Mensi ◽  
Marie-Stéphanie Vernerey ◽  
Daniel Gargani ◽  
Michel Nicole ◽  
Philippe Rott
Keyword(s):  
Confocal Microscopy ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Vascular Plant ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Vascular Bundles ◽  
Parenchyma Cells ◽  
Green Fluorescent ◽  
Xanthomonas Albilineans

Xanthomonas albilineans , the causal agent of sugarcane leaf scald, is missing the Hrp type III secretion system that is used by many Gram-negative bacteria to colonize their host. Until now, this pathogen was considered as strictly limited to the xylem of sugarcane. We used confocal laser scanning microscopy, immunocytochemistry and transmission electron microscopy (TEM) to investigate the localization of X. albilineans in diseased sugarcane. Sugarcane plants were inoculated with strains of the pathogen labelled with a green fluorescent protein. Confocal microscopy observations of symptomatic leaves confirmed the presence of the pathogen in the protoxylem and metaxylem; however, X. albilineans was also observed in phloem, parenchyma and bulliform cells of the infected leaves. Similarly, vascular bundles of infected sugarcane stalks were invaded by X. albilineans . Surprisingly, the pathogen was also observed in apparently intact storage cells of the stalk and in intercellular spaces between these cells. Most of these observations made by confocal microscopy were confirmed by TEM. The pathogen exits the xylem following cell wall and middle lamellae degradation, thus creating openings to reach parenchyma cells. This is the first description of a plant pathogenic vascular bacterium invading apparently intact non-vascular plant tissues and multiplying in parenchyma cells.

A Unified Model for Photophysical and Electro-Optical Properties of Green Fluorescent Proteins

2019 ◽  
Author(s):  
Chi-Yun Lin ◽  
Matthew Romei ◽  
Luke Oltrogge ◽  
Irimpan Mathews ◽  
Steven Boxer
Keyword(s):  
Driving Force ◽  
Fluorescent Protein ◽  
Charge Transfer Band ◽  
Photophysical Properties ◽  
Polymethine Dyes ◽  
Emission Rates ◽  
Unified Framework ◽  
Underlying Factor ◽  
Green Fluorescent ◽  
Protein Environment

Green fluorescent protein (GFPs) have become indispensable imaging and optogenetic tools. Their absorption and emission properties can be optimized for specific applications. Currently, no unified framework exists to comprehensively describe these photophysical properties, namely the absorption maxima, emission maxima, Stokes shifts, vibronic progressions, extinction coefficients, Stark tuning rates, and spontaneous emission rates, especially one that includes the effects of the protein environment. In this work, we study the correlations among these properties from systematically tuned GFP environmental mutants and chromophore variants. Correlation plots reveal monotonic trends, suggesting all these properties are governed by one underlying factor dependent on the chromophore's environment. By treating the anionic GFP chromophore as a mixed-valence compound existing as a superposition of two resonance forms, we argue that this underlying factor is defined as the difference in energy between the two forms, or the driving force, which is tuned by the environment. We then introduce a Marcus-Hush model with the bond length alternation vibrational mode, treating the GFP absorption band as an intervalence charge transfer band. This model explains all the observed strong correlations among photophysical properties; related subtopics are extensively discussed in Supporting Information. Finally, we demonstrate the model's predictive power by utilizing the additivity of the driving force. The model described here elucidates the role of the protein environment in modulating photophysical properties of the chromophore, providing insights and limitations for designing new GFPs with desired phenotypes. We argue this model should also be generally applicable to both biological and non-biological polymethine dyes.<br>

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

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