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 The Use of Green Fluorescent Protein-Tagged Recombinant Viruses to Test Lettuce mosaic virus Resistance in Lettuce

2002 ◽  
Vol 92 (2) ◽  
pp. 169-176 ◽  
Author(s):  
T. Candresse ◽  
O. Le Gall ◽  
B. Maisonneuve ◽  
S. German-Retana ◽  
E. Redondo
Keyword(s):  
Green Fluorescent Protein ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Lettuce Mosaic Virus ◽  
Recombinant Viruses ◽  
Systemic Movement ◽  
Resistance Breaking ◽  
Recessive Genes ◽  
Lettuce Breeding ◽  
Green Fluorescent

Seed certification and the use of cultivars containing one of two, probably allelic, recessive genes, mo11 and mo12, are the principal control methods for Lettuce mosaic virus (LMV) in lettuce. Although for a few LMV isolates, mo12 confers resistance with most isolates, the genes mo11 or mo12 confer a tolerance, and virus accumulation is readily detected in mo1-carrying plants. This phenotype complicates evaluation of the resistance status, in particular for mo11, for which there are no viral strains against which a true resistance is expressed. Two green fluorescent protein (GFP)-tagged viruses were constructed, derived from a non-resistance breaking isolate (LMV-0) and from a resistance-breaking isolate (LMV-E). An evaluation of 101 cultivars of known status was carried out with these recombinant viruses. Using the LMV-0-derived recombinant, identification of mo1-carrying cultivars was simple because, contrary to its wild-type parent, systemic movement of LMV-0-GFP was abolished in resistant plants. This assay detected four cases of misidentification of resistance status. In all these cases, further tests confirmed that the prior resistance status information was incorrect, so that a 100% correlation was observed between LMV-0-GFP behavior and the mo1 resistance status. Similarly, the LMV-E-derived recombinant allowed the identification of mo12 lettuce lines because its systemic movement was restricted in mo12 lines but not in susceptible or in mo11 lines. The tagged viruses were able to systemically invade another host, pea, irrespective of its resistance status against another member of the genus Potyvirus, Pea seed-borne mosaic virus. The use of these recombinant viruses could therefore greatly facilitate LMV resistance evaluation and speed up lettuce breeding programs.

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 Cell-to-Cell and Systemic Movement of Recombinant Green Fluorescent Protein-Tagged Turnip Crinkle Viruses

Virology ◽  
2000 ◽  
Vol 273 (2) ◽  
pp. 258-266 ◽  
Author(s):  
Yuval Cohen ◽  
Andreas Gisel ◽  
Patricia C. Zambryski
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Systemic Movement ◽  
Green Fluorescent

scholarly journals Lettuce chlorosis virus P23 Suppresses RNA Silencing and Induces Local Necrosis with Increased Severity at Raised Temperatures

2016 ◽  
Vol 106 (6) ◽  
pp. 653-662 ◽  
Author(s):  
Kenji Kubota ◽  
James C. K. Ng
Keyword(s):  
Rna Silencing ◽  
Elevated Temperatures ◽  
Fluorescent Protein ◽  
Plant Viruses ◽  
Suppressor Activity ◽  
Systemic Movement ◽  
Symptom Modulation ◽  
Green Fluorescent ◽  
Rna Accumulation ◽  
Local Necrosis

RNA silencing functions as an antivirus defense strategy in plants, one that plant viruses counter by producing viral suppressors of RNA silencing (VSRs). VSRs have been identified in three members of the genus Crinivirus but they do not all share identical suppression mechanisms. Here, we used Agrobacterium co-infiltration assays to investigate the suppressor activity of proteins encoded by Lettuce chlorosis virus (LCV). Of 7 LCV proteins (1b, P23, HSP70 homolog, P60, CP, CPm, and P27) tested for the suppression of silencing of green fluorescent protein (GFP) expression in wild-type Nicotiana benthamiana plants, only P23 suppressed the onset of local silencing. Small-interfering (si)RNA accumulation was reduced in leaves co-infiltrated with P23, suggesting that P23 inhibited the accumulation or enhanced the degradation of siRNA. P23 also inhibited the cell-to-cell and systemic movement of RNA silencing in GFP-expressing transgenic N. benthamiana plants. Expression of P23 via agroinfiltration of N. benthamiana leaves induced local necrosis that increased in severity at elevated temperatures, a novelty given that a direct temperature effect on necrosis severity has not been reported for the other crinivirus VSRs. These results further affirm the sophistication of crinivirus VSRs in mediating the evasion of host’s antiviral defenses and in symptom modulation.

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 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 A Cysteine-Rich Plant Protein Potentiates Potyvirus Movement through an Interaction with the Virus Genome-Linked Protein VPg

2004 ◽  
Vol 78 (5) ◽  
pp. 2301-2309 ◽  
Author(s):  
P. Dunoyer ◽  
C. Thomas ◽  
S. Harrison ◽  
F. Revers ◽  
A. Maule
Keyword(s):  
Transcriptional Control ◽  
Fluorescent Protein ◽  
Virus Genome ◽  
Specific Protein ◽  
Interacting Protein ◽  
Diverse Range ◽  
Phd Finger ◽  
Systemic Movement ◽  
Systemic Symptoms ◽  
Green Fluorescent

ABSTRACT We have identified a cellular factor that interacts with the virus genome-linked proteins (VPgs) of a diverse range of potyviruses. The factor, called Potyvirus VPg-interacting protein (PVIP), is a plant-specific protein with homologues in all the species examined, i.e., pea, Arabidopsis thaliana, and Nicotiana benthamiana. The sequence of PVIP does not identify a specific function, although the existence of a “PHD finger” domain may implicate the protein in transcriptional control through chromatin remodeling. Deletion analysis using the yeast two-hybrid system showed that the determinants of the interaction lay close to the N terminus of VPg; indeed, the N-terminal 16 amino acids were shown to be both necessary and sufficient for the interaction with at least one PVIP protein. From a sequence comparison of different potyvirus VPg proteins, a specific amino acid at position 12 was directly implicated in the interaction. This part of VPg is distinct from regions associated with other functional roles of VPg. Through mutation of Turnip mosaic virus (TuMV) at VPg position 12, we showed that the interaction with PVIP affected systemic symptoms in infected plants. This resulted from reduced cell-to-cell and systemic movement more than reduced virus replication, as visualized by comparing green fluorescent protein-tagged wild-type and mutant viruses. Furthermore, by using RNA interference of PVIP in Arabidopsis, we showed that reduced expression of PVIP genes reduced susceptibility to TuMV infection. We conclude that PVIP functions as an ancillary factor to support potyvirus movement in plants.

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>

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