Directly after, the adults were added to each plate by tapping each tube content in each well and checking if all 5 adults have been transferred to the well. This process was conducted quickly to avoid thrips from gaining consciousness. Once adults were added to all wells in one plate, the wells were quickly closed with the adjusted individual lids. The same was done for the second plate. Next, the two plates were closed with the original rectangular plate-cover and transferred to the climate box for a 24 h infestation.
The thrips-free wells were closed with both the adjusted individual lids and plate cover and further incubated in the climate box for 5 days. On day 8 post-infiltration, the nymphs in each well were counted using a stereo microscope M3, Wild-Heerbrugg. When significant differences with the negative control are encountered, the analysis is followed by comparisons of the predicted means of the treatment factor the over-expressed gene back to the control eGFP group mean using the Least Significant Difference LSD procedure.
Leaf discs were agro-infiltrated as described above using A. The obtained values reflect the difference in GUS activity and thus the level of GUS gene expression among the samples.
A list of all used equipment is provided in Supplementary Material. The use of a synchronized thrips colony makes it easier to distinguish female adult thrips from male adult thrips, as the latter are slightly smaller in size, and thus reduces the variations in nymph counts among treatments.
It is important to maintain this OD to ensure that the cells are in the logarithmic growth phase and thus reduce variability between treatments and increase reproducibility. It is important to avoid causing any damage to the leaf discs during the whole procedure to avoid triggering wound responses.
This is also why the leaf discs should be cut using a cork borer with a sharp edge to minimize tissue damage. The cover of a Petri dish is used as it is shallow and when placed near to the air stream in the down flow, it allows air to circulate around the leaf discs and thus improve water evaporation from the intracellular space.
This rapid recovery step is meant to evaporate the excess of water and remove the water soaking phenotype from the infiltrated leaf discs in a short time but without allowing the them to dry out. The recovery from infiltration is necessary to prevent hypoxia which can otherwise lead to tissue necrosis. Although the leaf discs are not sterile, we use clean or sterilized material to reduce the chance of developing fungal infection on the leaf discs during the course of the experiment.
The use of the lifters allows further recovery of the leaf discs from infiltration during incubation, maintains better humidity around the leaf disc and allow the adult thrips and the nymphs to move freely around the leaf disc.
The cavity of the lifters should be facing the agar surface. This prevents trapping humidity if they were facing the leaf tissue which increases the chance of fungal infection. A shallow contact with the agar surface is important to reduce tissue necrosis at this region of the leaf disc due to hypoxia. It is important to avoid collecting dead thrips to which pollen are usually stuck or collect the pollen themselves which will disturb the starvation process.
Avoid stretching the Parafilm more than one time its length to prevent possible water leakage and drowning the thrips. Depending on the temperature of the surrounding, the incubation time on ice for thrips can be decreased as long it will remain anesthetized. Do not exceed the 1 min as it can lead to mortality.
Compared to pressure infiltration or the infiltration using a conventional vacuum pomp, our infiltration procedure allowed us to obtain complete tissue infiltration where the water soaking phenotype was observed on the whole tissue.
This is despite that we have used large leaf discs which are usually more difficult to infiltrate. Also, the leaf discs did not suffer from wounding as that caused by pressure infiltration. We found that tomato leaf discs of the cultivar Moneymaker recover slightly faster 45 min to 1 h after infiltration from the water-soaking phenotype, caused by the infiltration, than N.
We found it to be essential that the infiltrated leaf discs reach a near-complete recovery before being transferred to the agar plates, otherwise tissue necrosis could appear on them 2—3 days later and then they become more vulnerable to fungal infection.
Although protein expression was often still visible in these non-recovered regions, it was somewhat diffused as observed after the expression of eGFP Figures 3C,D. Notably, the recovery of the leaf discs from the infiltration needed to be continued on agar. Immersing a large part of the leaf disc into the agar or having direct contact of the complete leaf disc surface with the agar often resulted in reoccurrence of the water soaking phenotype and necrosis often developed at these contact regions.
To solve this, the leaf discs were lifted from the agar from one side using clean plastic micro-tube coders see materials while the thick side of the midrib remained slightly inserted into the agar.
Failing to use these settings caused a prolonged presence of water soaked tissue and the development of necrosis at the positions where the leaf disc was contacting the agar. Also, thrips benefit from this as adults, and later, nymphs are often encountered at the abaxial side of the leaf disc which becomes more accessible due to lifting the leaf discs. Figure 3. C,D GFP signal phenotype observed as a result of hypoxia when the leaf discs were not sufficiently recovered from the vacuum infiltration of A.
We used the eGFP control as an indicator for protein expression during the course of the experiment by monitoring GFP fluorescence. The GFP signal was visible at 4 dpi and continued to increase at 5- and 6 dpi after which it started to decline at 8 dpi. At 14 dpi the signal was much weaker and was often confined to the areas adjacent to the veins Figures 3A,B , 4. Figure 4.
Successful expression of GFP in tomato leaf discs after vacuum infiltration with Agrobacterium tumefaciens and recovery from infiltration. Leaf discs prepared from 3. Leaf discs were allowed to recover from the infiltration for 1 h under an air stream and then placed on water agar in 6-well plates.
Pictures were taken using a wide-field fluorescence microscope at the indicated time points and magnification. Counting the number of the adult female thrips after infestation and oviposition showed that no escapes occurred, which reflected efficient containment of thrips in the utilized experimental setup.
This is different from the mortality rate that we observed in treated N. After several trials, we found that it is important to optimize the number of adult female thrips used for infestation and oviposition, the contact time with the plant leaf discs and the incubation time of the leaf discs before counting the nymphs. This was particularly important to reduce sample to sample variation and increase the reproducibility of the results.
This ensures obtaining a reasonable number of nymphs that can be counted concurrently in 1 day for a single screen containing 15 treatments, including the controls. The infestation and oviposition was allowed for 24 h in the climate box. We found this to be important for generating a synchronized emergence of the progeny so that most eggs would have hatched and could be counted in the scoring day.
Under these experimental settings, the optimum incubation time that allowed most eggs to hatch was between 7 in the summer and 8 in the winter days post-oviposition. This incubation time might differ slightly depending on the tested plant species as thrips reproductive performance is known to vary among plant species Baez et al. The plotted nymph counts showed variations within the treatments, but also between some treatments and the negative control eGFP.
Thus, Foc is a putative effector that can now be further tested with increased sample sizes. Foc is predicted to contain a complete open reading frame, which encodes a small protein AA with a signal peptide and two repeat domains. It is highly expressed in the salivary glands of thrips and has no significant similarity with known sequences from other organisms.
We are currently using this screening method to study a large set of putative effector candidates from thrips in a medium throughput setup to find those that have a stronger effect on the reproductive performance of the thrips. Figure 5. Effect of transient expression of thrips effector-candidate genes on thrips reproductive performance.
The box plot represents the results from an effector screen, showing the number of nymphs scored at 8 days post-agro-infiltration to transiently express eGFP control or thrips effector candidate genes in tomato leaf discs. Oviposition by adult female thrips was started at 2 dpi and continued for 24 h. Mock represents leaf discs infiltrated with only infiltration medium. The box center-lines represent the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.
The tremendous increase in sequence-based genetic information from plants and their herbivores and the quest for plant genetic resistance requires rapid genetic screening procedures to identify resistance-related traits. Our screening procedure addresses this by eliminating the need for generating stable transgenic plants and to be able to screen with herbivorous insects for up to, at least, 8 days.
It also allows conducting the screen under strictly controlled conditions and in a relatively small space which increases the reproducibility of the results. Another benefit is the cost reduction due to omitting the occupation of a large greenhouse space equipped to accommodate the combination of genetic screens with genetically modified organisms and insect bioassays.
Furthermore, the procedure allows a necrosis-free, transient protein expression in tomato leaf tissue. This is accomplished by the use of A. Although infiltration of the tomato leaf discs with the A. The observed chlorosis was similar to that formed when infiltrating leaves attached to whole plants with this strain data not shown.
However, this did not prevent protein expression until the end of the thrips bioassay as observed in the eGFP control treatments Figure 4 and on the whole leaf-disc level after expression of the GUS gene Figure S1. The utilized vacuum infiltration method, followed by a recovery-from-infiltration procedure, increases the homogeneity of protein expression in the tissue. This is important to reduce the score variations among samples of the same treatment and increases the reproducibility of the results.
The use of an age-synchronized thrips colony and the efficient containment of adult thrips and their progeny during the course of the experiment also contributes to reducing variations. It is possible to use this method for other plant species to screen either single effectors or plant genes or to screen wild accessions to identify their significance to thrips reproductive performance.
In the latter, the screen becomes even faster as the vacuum infiltration and recovery steps are not required. Similarly, rapid genetic functional analysis techniques like virus-induced gene silencing can also be combined with this screen, where silenced plants are used to generate leaf discs that are directly used without the need for agro-infiltration and recovery.
Despite that one of the aims of this procedure is to reduce the within-treatment variations, we still see them. This may have to do with the complexity of the involved biological system which includes plant, bacteria and thrips. Therefore, treatments that cause small effect on thrips performance might not directly show statistically significant difference.
A possible solution for this could be to increase the sample size, although this will limit the number of genes that can be tested in one screen. Thrips effectors that have positive or negative effect on thrips reproductive performance are very useful to include in the screen. However, there are no thrips effectors known to date. However, the expression of none of these genes affected thrips reproductive performance in our assay.
Finally, this method can be used to identify genes that play a role in the plant interaction with other small pests or microbes as long as the screen can be conducted within the time frame of expression. However, we think that in the future there is room for further optimization of this method by using more potent expression vectors and A.
The desired improvements would be extending the duration of maintaining plant tissue in a healthier state while obtaining sufficient levels of protein expression.
This will allow using this approach for studying other organisms for which the screen requires a longer time frame.
AA designed the experiments. AA and SH conducted the experiments. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We acknowledge Dr. Saioa Legarrea Imizcoz and Dr.
Arne Janssen for providing starting material for the thrips colony, Prof. Jorunn Bos for providing the aphid effectors, Dr. Patrick Smith for providing the A. Michel Haring is acknowledged for the critical reading of this manuscript. Baez, I. Variation within and between Frankliniella thrips species in host plant utilization. Insect Sci. Bate, S. Cambridge: Cambridge University Press. Bos, J. A functional genomics approach identifies candidate effectors from the aphid species Myzus persicae green peach aphid.
PLoS Genet. Douglas, A. Strategies for enhanced crop resistance to insect pests. Plant Biol. Fery, R. Resistance in pepper Capsicum annuum L. HortScience 26, — Google Scholar. Hopp, T. A short polypeptide marker sequence useful for recombinant protein identification and purification.
Horton, P. Nucleic Acids Res. Hunter, W. When all the disks have floated, try putting the cup in a dark cabinet or room, or cover the cup with aluminum foil. Check the cup after about fifteen minutes. What happens to the disks? Plants occupy a fundamental part of the food chain and the carbon cycle due to their ability to carry out photosynthesis, the biochemical process of capturing and storing energy from the sun and matter from the air.
At any given point in this experiment, the number of floating leaf disks is an indirect measurement of the net rate of photosynthesis.
In photosynthesis, plants use energy from the sun, water, and carbon dioxide CO 2 from the air to store carbon and energy in the form of glucose molecules. Oxygen gas O 2 is a byproduct of this reaction. Oxygen production by photosynthetic organisms explains why earth has an oxygen-rich atmosphere. In the leaf-disk assay, all of the components necessary for photosynthesis are present. The light source provides light energy, the solution provides water, and sodium bicarbonate provides dissolved CO 2.
Plant material will generally float in water. This is because leaves have air in the spaces between cells, which helps them collect CO 2 gas from their environment to use in photosynthesis.
When you apply a gentle vacuum to the leaf disks in solution, this air is forced out and replaced with solution, causing the leaves to sink. Accumulation of O 2 on the disks causes them to float. The rate of production of O 2 can be affected by the intensity of the light source, but there is a maximum rate after which more light energy will not increase photosynthesis.
To use the energy stored by photosynthesis, plants like all other organisms with mitochondria use the process of respiration, which is basically the reverse of photosynthesis.
In respiration, glucose is broken down to produce energy that can be used by the cell, a reaction that uses O 2 and produces CO 2 as a byproduct. Because the leaf disks are living plant material that still require energy, they are simultaneously using O 2 gas during respiration and producing O 2 gas during photosynthesis.
Therefore, the bubbles of O 2 that you see represent the net products of photosynthesis, minus the O 2 used by respiration. When you put floating leaf disks in the dark, they will eventually sink.
Without light energy, no photosynthesis will occur, so no more O 2 gas will be produced. However, respiration continues in the dark, so the disks will use the accumulated O 2 gas. Try changing other factors that might affect photosynthesis and see what happens. How long does it take for the disks to float under different conditions?
For example, you can compare the effects of different types of light sources—lower- or higher-wattage incandescent, fluorescent, or LED bulbs. You can change the temperature of the solution by placing the beaker in an ice bath or a larger container of hot water. You can increase or decrease the concentration of sodium bicarbonate in the solution, or eliminate it entirely.
You can try to identify the range of wavelengths of light used in photosynthesis by wrapping and covering the beaker with colored gel filters that remove certain wavelengths. This experiment is extremely amenable to manipulations, making it possible for students to design investigations that will quantify the effects of different variables on the rate of photosynthesis.
It is helpful to have students familiar with the basic protocol prior to changing the experimental conditions. Ask your students to think carefully about how to isolate one variable at a time. It is important to hold certain parts of the experimental setup constant—for example, the distance from the light source to the beaker, the type of light bulb used, the temperature of the solution, the height of the solution, and so on.
Certain treatments may eliminate photosynthesis altogether—water with no bicarbonate, very low temperature, and total darkness. A typical way to collect data in this assay is to record the number of disks floating at regular one-minute time intervals. This is easily graphed, with time on the x-axis and number of floaters on the y-axis. To make comparisons between treatments, the number traditionally used is the time point at which half of the disks in the sample were floating, also known as the E This experiment was originally described in Steucek, Guy L.
Compare the brightness of two light sources with an oil spot on a white card. Attribution: Exploratorium Teacher Institute. Connect with us! Get at-home activities and learning tools delivered straight to your inbox. The Exploratorium is a c 3 nonprofit organization. Photosynthetic Floatation Light leaves leaves light.
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