Putative interactor proteins of whitefly effector protein B1
may suppress plant defenses in Arabidopsis
Jaimy Bon Biomedical Sciences Bachelor Thesis Student ID: 11323612 Date of Submission: 01-07-2020 Words: 4.492
Abstract
Whiteflies are mainly known for their economic damage in farming industries, caused by depletion of plant resources and virus transmission. Plant defenses can be circumvented by e.g. effector proteins secreted by the whitefly during phloem-feeding. These whitefly effector proteins interact with plant proteins (interactors), which causes a chain of events that could interrupt plant defenses. Earlier findings showed that expression of B. tabaci effector protein B1 showed an increased whitefly fitness. The aim of current research is to find plant proteins that can interact with effector B1. Therefore, five putative target proteins were selected that could interact with effector B1. These proteins were selected based on interaction with B1 in a Yeast-Two Hybrid assay performed by an external company. The Y2H assay was performed on truncated protein. Hence in our current research we want to verify the Y2H assay with full-length proteins. In addition, a co-localization analysis and split-YFP assay were performed to provide an indication of the localization of the putative target proteins and B1 inside a plant cell. Additionally, the split-YFP assay shows if interaction occurs between the target proteins and B1 within the plant cell. The Y2H assay and split-YFP showed interaction between B1 and the putative targets. The co-localization and split-YFP assay showed the putative targets and B1 to be colocalized in the cytosol and nucleus. Our findings indicate that the putative target proteins are interactors of B1 and could therefore contribute to circumventing plant defenses.
The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) are herbivory insects. They are mainly known for their economic damage in farming industries, caused by plant virus transmission and depleting plant resources by phloem-feeding. Excess phloem-feeding of the whitefly results in honeydew excretion, causing reduced sunlight intake and thereby depleting photosynthesis of the plant (Chen et al., 2004). However, plants do have defense mechanisms against phloem uptake by the whitefly. The whitefly damages the phloem sieve tubes during feeding, resulting in a turgor shift in the plant (Gould et al., 2013). The turgor shift leads to intracellular signaling which results in occlusion of the sieve tubes, preventing further phloem feeding of the whitefly. This is considered a primary defense response of the plant (Knoblauch et al., 1998). Another defensive trait of plants can be their appearance and architecture. Whitefly density on leaves correlates positively with trichome density and is negatively correlated with cuticle thickness (Firdaus et al., 2011). Phloem-feeding by insects induces salicylic acid (SA) accumulation in the plant, which results in activation of
pathogenesis-related (PR) proteins. This activation is even more noticeable if the whitefly is infectious (Mayer et al., 2002). A study by Shi et al. in 2016 has shown in an experiment on tomato plants that SA reduces the fecundity and longevity of whiteflies. However, it is shown that SA is inefficient and inhibits a more effective defense mechanism; jasmonic acid (JA) defense (Alon et al., 2013).
Despite plant defense mechanisms mentioned, whiteflies are still capable of damaging the plant by circumventing the plant defense. This is mainly achieved by whitefly saliva containing effectors, which is excreted during phloem-feeding. Van Kleeff et al., stated in 2016 that effectors are proteins, metabolites or sRNA that could engage in an interaction with a plant protein, metabolite or sRNA. This interaction results in plant defenses being (partially) terminated. An example of plant defense termination by effectors is shown in a study done by Xu et al. in 2019. This study showed a whitefly effector (Bt56) being able to elicit SA-signaling by interacting with plant-protein KNOTTED-1, which resulted in inhibiting the JA-pathway, enabling the whitefly to feed on host plants. Not only whiteflies make use of effectors, also other organisms are able to circumvent the plant defenses with saliva effectors. Spider mites were found to excrete salivary effector proteins on N. benthamiana which resulted in suppression of the plant defenses and increase in mite reproduction (Villarroel et al., 2016).
Earlier research has found two effectors of whitefly B. tabaci: B1 and S1 (van Kleeff, unpublished). It is shown that overexpression of B1 results in increased whitefly fitness. Follow up research identified over 200 possible interactors of B1 and S1 with the Yeast Two-Hybrid technique. One of the interactors found is SPL7 (Squamosa promoter binding protein-like 7); it was found to have a D-interaction with both B1 and S1 screens. A D-D-interaction indicates an D-interaction with moderate confidence in the interaction (van Kleeff, unpublished). A study from Yamasaki et al. in 2009 found that SPL7 is essential in the activation of genes in response to copper deficiency. This could be relevant for current research because copper ions activate the defense response in Arabidopsis (Liu et al., 2015). Because of this, there can be hypothesized that effector B1 and S1 of whitefly could inhibit defense response in plants, resulting in reduction of plant survivability.
Current research wants to investigate whether the interaction between SPL7 and B1 and S1 occurs by using three protein-interaction assays; Yeast Two-Hybrid, in planta co-localization analysis and split-YFP.
To investigate interaction between SPL7 and B1 and S1 with the three protein-interaction analyses mentioned above, vectors were constructed following the Gateway cloning system (Katzen et al., 2007). These vectors were constructed using DH5α e. coli cells to maximize the transformation
efficiency (Inoue et al., 1990). cDNA of both tomato variety Money-maker and Arabidopsis thaliana was used to PCR SPL7. The PCR product was subsequently transferred into pJET plasmids to attach attB-sites. The attB-sites are useful to clone SPL7 directionally into pDonR vectors containing attP sites using BP clonase (Katzen et al., 2007). After the final vectors are obtained via the LR clonase Gateway cloning principle, co-localization analysis, split-YFP and Yeast Two-hybrid will be performed.
The expectations based on earlier research is that the Yeast Two-hybrid analysis will demonstrate SPL7 to interact with both B1 and S1 with a D-interaction. If this finding will be proven, it is also expected that the co-localization assay and split-YFP analysis will show colocalization and interaction between SPL7 and effectors B1 and S1. These findings would show B1 and S1 to be possible effectors of B. tabaci and might give new insights for future research to explore if B1 and S1 have any effect on plant defenses.
Methods
Cloning Plasmid vectors
To perform the experiments; Yeast Two-Hybrid (Y2H), co-localization and split-YFP, the required vector plasmids were cloned. The positive control in this experiment are two proteins which are known to interact in the nucleus in Solanum lycopersicum “Money-Maker” plant cells: Myc1 and Jaz2 (van Kleeff, unpublished). A mutated version of Myc1 (E161K) was cloned and used as the negative control. SPL7 isolated from Arabidopsis (AtSPL7) and SPL7 isolated from S. lycopersicum “Money-Maker” (SlSPL7) plants were both cloned into vectors. A list of required vectors in described in the Supplemental data (Table S4) and the vectors are prepared as described below.
PCR
The first step was amplifying the cDNA of AtSPL7, SlSPL7, SlMyc1, SlMyc1-E161K and SlJaz2 with Phusion PCR. Table 1 shows the PCR settings for Phusion PCR, primers (Table S1) and PCR mastermix (Table S2) can be found in supplementary data.
Table 1. Phusion PCR settings used. Temperature (°C) Time (seconds) Cycle s 98 30 98 10 45 10 5X 72 30s per 1kB 98 10 57 10 25X 72 30s per 1kB 72 600 16 - -Gel electrophoresis
After PCR, the samples were run on an agarose gel to check whether the amplification was successful. A 1% agarose gel was prepared by mixing 0,5g agarose mixed with 50ml TAE 1X. Loading dye (6X) was added to every PCR product and loaded onto the gel. The gel electrophoresis settings were set at 400A and 80V for 35 minutes. DNA bands of the correct size, SlJaz2 = 759bp, SlMyc1 = 1893 bp, Sl/AtSPL7 = 2457 bp were cut from the agarose gel and extracted using a GeneJet Gel extraction kit from Thermos Fisher (according to manufacturer’s protocol), followed by measuring the DNA concentration by Nanodrop (Table S5).
pJET Cloning
SlJaz2, SlSPL7 and AtSPL7 were cloned into pJET-plasmid using the Thermo Fisher CloneJET PCR Cloning kit (according to manufacturer’s protocol).
E.coli DH5 alpha heat-shock transformation
DH5α cells were thawed on ice and added to the transformation mixture. The cells were incubated on ice for 20 minutes and heat shocked at 42 °C for 45 seconds. Next, 450 μl of low salt LB was added and incubated in a 37 °C shaker for 45 minutes. Afterwards, the cells were spun down for 2min at 5000 rpm. The supernatant was removed by flicking and the remaining LB was resuspended with the cells and plated on an ampicillin(pJET)/kanamycin(pDONR) plate which was incubated at 37 °C overnight (O/N).
Gateway Cloning
The constructs were cloned into pDONR plasmids through BP cloning. The amount of PCR product used was calculated by dividing the needed amount of pDONR-PP (31.2 ng) by the concentration of the purified PCR product.
pDONR221-SlMyc1 attb 1/2 was provided by Pietro to perform a LR reaction. The amount pDONR needed was 25ng. The amount of destination vector (PGADT7) was calculated by dividing 50 by the concentration of the destination vector.
The LR and BP reaction are performed using Thermo Fisher Gateway Gene Cloning kit according to manufacturer’s protocol.
Colony PCR
After DH5 alpha transformation of pJET or pDONR, a colony PCR was performed on three colonies, to verify the presence of the insert in the plasmid. The colonies were streaked on a separate plate with a tip, after which the remaining colonies was used for PCR. Primers used in the colony PCR are the same as for the insert PCR (Table S1). The colony PCR mastermix can be found in Table S3, and the PCR settings in Table 2.
Table 2. Colony PCR settings used.
Temperature (°C) Time (seconds) Cycles
95 180
95 30
57 30
72 60s per 1 kB Back to step 2 X30
72 600
16
-The PCR product was run on a 1% agarose gel (400A, 80V) for 35 minutes. Colonies with the correct size insert were inoculated in 5ml LB + 5 μl Ampicillin (1000X) for pJET or 2.5 μl Kanamycin (2000X) for pDONR221 O/N in a 37 °C shaker. Next, the inoculated colony was miniprepped using Thermo Fisher Plasmid DNA Isolation kit (according to manufacturer’s protocol)(Table S6).
---At this point in time I was not able to continue my lab work due to the corona virus, my supervisors (Marieke Mastop and Paula van Kleeff) and I decided to analyze previous data from similar
experiments. Different target proteins were used against B1. These target proteins are unknown in literature. The results of the experiments were performed by my supervisors and handed to me via email for me to analyze. With this change of my research paper, we decided to leave the introduction for as it was. However, the research question and focus on SPL7 cannot be maintained. Therefore, the research currently aims to find putative target proteins from Arabidopsis that interact with effector B1.
---Experiment 1 – Y2H
Clones of the identified putative B1 interactors have been ordered from Hybrigenics.
NMY51 yeast strain was grown O/N in 3 ml YPDA medium at 30 °C and shaken at 220 rpm while being slightly tilted. The next day, 150 µl of NMY51 has been inoculated in 5 ml YPDA medium and grown O/N in the same conditions as the last incubation step. The following day 500 µl culture has been spun down in an Eppendorf tube for 20 seconds at 10000 rpm at room temperature (RT). The supernatant was removed using a P20 pipette. The yeast cells were resuspended in 2.5 µl of each plasmid (+/- 200 ng/µl) following Table 1. Sonicated salmon sperm has been boiled for 5 minutes at 100˚C, placed on ice and 10 µl was added to the yeast/plasmid mixture. 100 µl LiAc +PEG has been added to every sample and vortexed for 30 seconds. Subsequently the samples were incubated at 30 ˚C for 30 minutes by 220 rpm, then incubated in a waterbath at 42 ˚C for 15 minutes. The cells have been plated on selection media (SD – Leu/ -Trp = DDO) and grown for 8 days at 30 ˚C.
Table 3. Transformation of yeast for Y2H was done according to this table. The numbers 1-11 are indicated to visualize the results in Figure 3. pP6 and pB27 are the plasmids used for the Y2H screen. Numbers 1-5 show the negative control for the pB27 plasmid. Number 1 and 6 show the negative control for the pP6 plasmid. Number 7-10 are samples which could lead to indication of interaction between B1 and target protein. Number 11 is the positive control where S1 interacts with itself.
pB27-empty pB27-B1 pB27-S1 pP6-empty 1 6 pP6-target 1 2 7 pP6-target 2 3 8 pP6-target 3 4 9 pP6-target 4 5 10 pP6-S1 11*
*S1 was found to bind itself in previous Y2H screen.
Experiment 2 - Y2H #2
The protocol for this Y2H assay was the same as the Y2H described in the previous section, except that newly ordered salmon sperm was used. Also, the PJ69-4A yeast strain was selectively grown on yeast medium minus Lysine (SD -K). Therefore, YPDA was replaced for SD -K medium in this Y2H assay. The yeast was transformed according to Table 1 of the first Y2H experiment. Subsequently, five colonies for every sample have been spotted on four different media: DDO, TDO, TDO +5 mM 3-Amino-1,2,4-triazole (3AT) and QDO.
Experiment 3 – Co-localization
The construct used for co-expression is pFRETgc-2in1-NN (described in 2015 by Hecker et al.), where B1 is N-terminally fused to mCherry (red channel) and the putative target is N-terminally fused to EGFP (green channel) (see Figure 1). The constructs were transformed by heat-shock into
Agrobacterium tumefaciens GV3101 (pMP90) and grown on LB containing Spectinomycin, Rifampicin and Gentamycin at 28 ℃ for 2-3 days. Subsequently, a colony-PCR was performed. A single positive colony was inoculated in 5 ml LB low salt medium supplemented with Rifampicin (25μg/ml),
Gentamycin (50 μg/ml) and Spectinomycin (50 μg/ml) or Kanamycin for p38 (a silencing suppressor of N. tabacum plants). The agrobacteria were grown for 24 hours at 28 °C while shaking. Next, the
cells were diluted 1:5 with LB low salt medium and the OD600 was measured using a photometer, where the LB low salt medium was used as a blank. To obtain a final agrobacterium suspension of OD 0.1, X μl (depending on OD value) of agro culture was transferred into a 2 ml Eppendorf where the cells were harvested by centrifugation for 15 min at 3000 x g (RT). The supernatant was removed, and 2 ml of infiltration buffer (5g/L MS salt (Murashige & Skoog Basal Salt Mixture without vitamins, Duchefa), 20 g/L Sucrose, 10 mM MES (pH 5.6 with 5M KOH), 200 μM acetosyringone (0.2M)) was added to obtain an OD600 of 0.5. Finally, the cells were infiltrated into the leaves of N. tabacum plants (4-5 weeks, 16 h day/8 h night photoperiod, day 20°C/night 18°C, 60% humidity) using a syringe without a needle.
Three days after infiltration samples were taken for confocal (Nikon Eclipse Ti) analysis. The confocal settings were excitation 488 nm/emission 495 to 530 nm (mEGFP), excitation 561 nm/emission 580 to 630 nm (mCherry).
The confocal microscopy images are analyzed using Image J (version 1.8.0).
Figure 1. The pFRETgc-2in1-NN construct that is used for the co-localisation experiment. Made by Saskia Mulder.
Experiment 4 – Split-YFP
The constructs used for co-expression are the pBiFC-2in1-NN (described in 2012 by Grefen et al.), where B1 is N-terminally fused to cYFP and the putative target is N-terminally fused to nYFP (see Figure 2). The constructs were transformed into Agrobacterium tumefaciens GV3101 (pMP90) according to the same protocol as Experiment 4 Co-localization.
The YFP/RFP ratio was calculated in ImageJ by averaging two images per target, the background was subtracted before the pixel intensity of the image was measured (YFPintensity / RFPintensity (x 100)). RFP is visible if the cell is successfully infiltrated via agro infiltration.
Three days after infiltration samples were taken for confocal (Nikon Eclipse Ti) analysis. The confocal settings were excitation 514 nm/emission 521 to 553 nm (YFP), excitation 543 nm/emission 580 to 615 nm (RFP).
The confocal microscopy images are analyzed using Image J (version 1.8.0).
Figure 2. The pBiFc-2in1-NN construct that was used for the BiFC experiment. Made by Saskia Mulder.
Yeast Two-Hybrid screens are a valuable technique to narrow down the search of putative effector interactors. Previous Y2H screen, performed by Hybrigenics, resulted in a list of putative B1 interactors. We selected four putative interactors to verify their interaction with B1. The yeast has been transformed as shown in Table 3.
No yeast growth was seen (Figure 3).
Figure 3. Yeast Two-Hybrid results. The numbering is indicated in Table 4. None of the yeast transformations has grown.
There can be concluded that none of the yeast has grown in the Y2H experiment, which suggests a technical or biological flaw in the protocol or execution of it.
Yeast-Two Hybrid #2
Because the first Y2H experiment did not show yeast growth, we decided to re-clone the targets to a different GAL4 system and the effectors to the same GAL4 system as the targets. Also, the
transformation was done in the PJ69-4A yeast strain with newly ordered sonicated salmon sperm. Colonies were visible after three days of incubation of the cells plated on media selecting for the presence of the two plasmids.
A
B
Figure 3. Results of the Y2H assay on different media; DDO, TDO, TDO +3AT & QDO. (A) The DDO plate, indicating presence of both plasmids, shows grown yeast for every sample. The TDO plate shows yeast growth in every sample for 2, 8, 9 and 11. Sample 7 shows yeast growth in 4 out of 5 cases. The TDO + 5 mM 3AT plate shows yeast growth in sample 2, 7 and 9. The QDO plate shows yeast growth in every sample for 2, 8, 9 and 11. Sample 7 shows yeast growth in 4 out of 5 cases. (B) The legend is shown to show which sample number corresponds with which target.
From the second Y2H assay can be concluded that both plasmids have been successfully transformed into the yeast. Target protein 2 and 3 showed to interact with B1 in every plate (TDO, TDO+3AT, QDO). Also, the negative control for target 1 showed interaction (TDO, TDO+3AT, QDO). Hence target 1 interacting on the plates is not reliable. Target protein 2 does not show interaction with B1 in the TDO+3AT plate. Target protein 4 showed no interaction with B1 in any of the plates.
Co-localization analysis
Even though a Y2H assay shows interaction, the proteins need to be in the same subcellular space within the plant to be able to interact in the plant tissue. The aim of this analysis is to determine the localization of the putative target proteins and B1, to ultimately discover if they colocalize. For this experiment, a fifth putative target protein has been analyzed.
Figure 5. Confocal microscopy images of fluorescent proteins in N. tabacum cells, analyzed using Image J. The first column (GFP-Target) shows images of the fluorescent target proteins (1-5) attached to GFP. The second column (mCherry-B1) shows images of fluorescent B1 attached to mCherry. In the
third column (Merged) the GFP and mCherry images are merged to visualize colocalization in the cell. The GFP channel also shows the line which was used to visualize the plot profiles in Figure 6.
A – Target 1 B – Target 2
C – Target 3 D – Target 4
E – Target 5
Figure 6. Plot profiles of putative target proteins and B1. The red line shows B1 (mCherry) and the green line shows the putative target protein (GFP). The x-axis shows the distance of measurements, which was done horizontally throughout the entire cell though the nucleus.The y-axis shows the pixel intensety of the image i.e. the fluoresence intensity.
From the co-localization experiment can be concluded that every target protein does colocalize with B1 in the cytosol and nucleus, the plot-profiles strengthens this finding. Target 1, 2 and 5 also show to co-localize with B1 in the cytoskeleton.
Split-YFP assay
The Y2H assay showed if interaction occurs, while the co-localization experiment showed where the proteins of interest localize in the plant cell. The Split-YFP assay shows if and where interaction between the target proteins and B1 occurs within the plant. Thereby, the Split-YFP assay can strengthen the results from the previous experiments.
YFP-channel RFP-channel Merged YFP-gray
Ta rg et 1 Ta rg et 2 Ta rg et 3 Ta rg et 4 Ta rg et 5
Figure 8. Split-YFP confocal microscopy images. This figure shows if and where interactions occurs between the putative targets and B1 (green fluorescence – YFP) in N. tabacum plant cells. The RFP channel which is used to quantify interaction is also shown. The fluorescence by interaction is shown by merging the YFP-fluorescence images with the visible cells.
Table 4. YFP/RFP ratios in percentages. The following ratios were calculated using an average for two images per target
Target YFP/RFP ratio (in %)
1 9,8%
2 18,2%
3 9,7%
4 13,3%
5 197,7%
In conclusion, the Split-YFP experiment showed interaction between B1 and every target protein (1-5). Also shown is the target proteins and B1 colocalize in the nucleus and cytosol.
Discussion
The primary purpose of this study was to research putative target proteins of whitefly effector B1. Earlier research suggested that effectors can circumvent plant defenses and thereby impact plant, and whitefly survivability (Xu et al., 2018). Determining plant interactors could give new insights on how to oppose plant damage inflicted by whiteflies. In this study we showed that target 2 and 3 showed interaction in a Y2H assay. In planta, a co-localization assay showed that B1 and the target proteins both localized in the cytoplasm and nucleus. The split-YFP confirmed the findings of the co-localization assay by visualizing interaction in the cytoplasm and nucleus. However, the split-YFP showed B1 to interact with every target protein, while the Y2H assay showed only target protein 2 and 3 to interact with B1.
A Yeast Two-Hybrid was performed, which initially showed no yeast growth. The implications with the first Y2H experiment could be due to a low concentration of salmon sperm. Salmon sperm is used in Yeast Two-Hybrid transformations as carrier DNA and binds the yeast cell-wall resulting in the plasmid entering the yeast cell (Gietz et al., 2001). When the concentration of salmon sperm is low or not single-stranded, the transformation will be unsuccessful resulting in empty plates. Additionally, the GAL4/LexA screening was used, which is known for false negatives because of their difference in localization (Xing et al., 2016). However, for the second Y2H newly ordered salmon sperm was used and we decided to re-clone the targets to a different GAL4 system and the effectors to the same GAL4 system as the targets. This resulted in yeast growth and the DDO-plate to show that both plasmids had been successfully transformed into the yeast.
In the Y2H experiment, every plate (TDO, TDO +3AT, QDO) showed interaction in one of the negative controls (pGADT7-target 1 –pGBKT7-empty). The reason for this could be that target 1 engages in non-specific interactions (auto-activation) in yeast (Serebriiskii et al., 2000). Target protein 4 did not interact with B1 in the Y2H, which possibly is a false negative after seeing that the Split-YFP did show target protein 4 to interact with B1. False negatives could occur in Y2H assays due to a different or lacking post-translational protein modification system in yeast (Brückner et al., 2009). Another reason could be usage of full-length proteins that might not fold correctly in yeast, resulting in no interaction with B1 (Galletta et al., 2015). Another issue observed was with the TDO +3AT plate, where probably too much 3-AT was added to the plate which caused our positive control not to interact. This resulted in the strongest interactions to show on the plate (Caufield et al., 2012). Possibly, the positive control (S1-S1) is not a strong enough interaction to interact in these conditions. Target protein 3 showed interaction on the TDO +3AT plate, suggesting target 3 to have a strong interaction with B1. The co-localization experiment visualized the possible interaction sites within the cell. The
experiment shows that B1 and target 1 and 2 have nuclear and cytosolic localization with a higher fluorescent intensity in the nucleus. Target 3 shows localization within nucleolus while B1 is excluded from the nucleolus. Target 4 appears in spots within the cytosol, interestingly B1 is also found in these spots. Target 5 seems to be depleted from the nucleus while B1 is present. There can also be argued that B1 and the target proteins are localized in the cytoskeleton for target proteins 1, 2 and 5. To determine this, the cytoskeleton structures shown in the images can be researched with the use of compatible markers described in 2014 by Rocchetti et al.
To investigate whether interaction occurs between B1 and the targets, a split-YFP assay was
performed, which shows interaction between B1 and every putative target protein in the cytoplasm and nucleus, except for target 1 which does not interact in the nucleus.
From the split-YFP experiment, the YFP/RFP ratio was calculated to get an indication on the binding affinity of the putative target proteins with B1. However, it is challenging to consider the spatial particle distribution on the confocal microscopy images while making these calculations, which could result in inaccurate measurements (Dinsmore et al., 2001). Additionally, variation in fluorescence intensity between cells and between different constructs is a problem with confocal microscopy imaging. This issue can cause the YFP/RFP-ratio to exceed a ratio of 1.0, as happened with target protein 5. Also, there were no control samples for the YFP/RFP ratios, which difficulted interpreting the results.
There are also some generic improvements that could have been made for this research. The Y2H technique carries some issues in validity due to the high false positive and false negative rates (Huang et al., 2007). Therefore, repetition and the use of good control samples would have improved this research. However, this research gives a strong indication about which putative target proteins interact with whitefly effector B1 but fails prove if this interaction influenced plant defenses.
Future research could research how and if the interaction between the target proteins and B1 affects the defense response in plants. This could be investigated by knocking down the target plant proteins of whitefly effector B1 with interference RNA. A clip cage can be used to restrict several whiteflies to a leave and thereby measure the effect of knockdown by researching the fitness and survivability of the whiteflies. If whiteflies fitness and survivability were found to be reduced, CRISPR-Cas9 could be used to knock-out the interactor proteins in plants and thereby avoid whitefly infestation. This could have great meaning for the farming industry in disputing plant depletion by whiteflies.
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