Progress 03/01/24 to 02/28/25
Outputs
Target Audience:- Research community investigating construction of recombinant plasmids, protein expression in transformed bacteria, and purification of fusion protein - Research community in the area of preventing pathogen infection using purified plant extracellular vesicles - Undergraduates and graduates involved with this research Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One PhD student is now familiar with recombinant plasmid construction, protein expression in T7-based bacteria system, and protein purification using affinity chromatography. This study has also learned to grow Arabidopsis thaliana and collect apoplastic washing fluids obtained from Arabidopsis leaves infected with pathogenic bacteria. The PhD student is in the final stage of completing the remaining experimental studies for the dissertation writing. One undergraduate student has been trained to grow A. thaliana, collect AWF, and use AWF to green synthesis of silver nanoparticles revealing anti-bacterial efficacy. How have the results been disseminated to communities of interest?The techniques of constructing, expressing, purifying, and characterizing lactadherin C1C2 fused proteins have been reported. The PEG-based precipitation of plant EVs have been published. One manuscript is under preparation for publishing the research outcomes of EVs separated from A. thaliana infected with P. syringe for 3 days could prevent Arabidopsis leaves from disease symptom (i.e., no yellowing or withering caused by P. syringe infection) over a long period of time. The other manuscript is also under preparation to report the efficient particle-based separation of plant EVs comparing to standard ultracentrifugation method. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Construction and Expression of lactadherin C1C2-streptavidin and pTXB1-lactadherin C1C2 encoding plasmid. The lactC1C2-SA encoding recombinant plasmid (pMA002) was constructed by inserting the gene sequences of lactadherin C1C2 domain into the multiple cloning sites from EcoRV and EcoRI in the established plasmid pMA001 (i.e., pET-30a(+)-coreSA). Moreover, we worked on an additional plasmid construction of pTXB1-CBD (chitin binding domain)-intein-C1C2. the gene encoding lactadherin C1C2 cloned by PCR were verified by DNA gel electrophoresis showing the size of ~0.9 kb on an agarose gel. The cloned lactadherin C1C2 was inserted between NdeI and SapI of pTXB1 plasmid. Collection of apoplastic washing fluid from Arabidopsis thaliana. Arabidopsis thaliana seeds [wild-type Arabidopsis thaliana (L.) Hyenh. Columbia-0 (Col-0)], obtained from Arabidopsis Biological Resource Center (Columbus, OH), planted by sprinkling uniformly over moist soil in plastic pots. The pots were then placed at 4°C for 4 days to allow enough time to properly germinate. On the 5th day, the pots were shifted to the growth chamber maintained at 22°C, and exposed to light (100 µmol/m2/s) with a cycle of 16 h on and 8 h off. A total of fifty leaves (fresh weight around 2 g) of 4-week-old Arabidopsis thaliana were cut at petioles, washed thrice with water, and dried on a paper towel. Dried leaves were then placed inside a needleless syringe and infiltrated with vesicle infiltration buffer.The apoplastic washing fluid (AWF) was collected, filtered with a 0.45-µm Nylon filter, and aliquoted 500 µL into three 1.5-mL microcentrifuge tubes which were stored at 4°C for follow-up experiments and at -80°C for long-term storage. Isolation of EVs from AWF by polyethylene glycol 8000 and sodium chloride. The collected AWFs (500 µL each) were mixed with various amounts of polyethylene glycol 8000 (PEG8000) to obtain 5%, 8% and 12% w/v concentration, respectively. The mixture was further added with NaCl to get 0.5 M salinity and then incubated at 4°C for 2 h and centrifuged at 10,000× g force for 30 min at 4°C using a microcentrifuge. The PEG-EVs agglomerates were sedimented at the bottom of the 1.5-mL microcentrifuge tube. The pellet was re-suspended in 1× phosphate buffered saline for characterization. Immuno dot blot assay. To examine the efficacy of EVs separated from AWF by the PEG-precipitation method, a dot blot assay was used. Briefly, for three different concentrations of PEG8000 used, 5 µL each from the supernatant and resuspended pellets was taken and carefully spotted on the nitrocellulose membrane. After 10 min of drying, the membranes were blocked using blocking buffer. Then, the membranes were incubated with mammalian monoclonal antibodies against tetraspanin proteins (CD9, CD63, and CD81) and PEG, respectively for 1 h at 4°C. After 1 h, the membranes were washed three times with TBST and incubated with secondary antibody (mouse IgG horseradish peroxide (HRP)-conjugated antibodyin the blocking buffer at 1:1000 dilution) for 1 h at room temperature.Our results showed that there is an increase in pellet size with increasing concentration of PEG8000 from 5% to 12%. The precipitation of EVs was further verified by dot blot assay of both the pellet and supernatant. The dot blot analysis that was performed using the monoclonal antibodies against mammalian tetraspanin proteins CD9, CD63, and CD81, confirmed the presence of EVs in the pellet. Characterization of PEG-precipitated EVs. The measurements for the size distribution of EVs were performed with dynamic light scattering (DLS) instrument. EVs, resuspended in 1× PBS, were appropriately diluted with 0.2-µm membrane filtered distilled water and loaded into a 750-µL cuvette for the detection. Zeta potential of EVs was measured by the same instrument. The particle size distribution and concentration of isolated EVs were further determined by nanoparticle tracking analysis (NTA). Briefly, each sample was diluted 1:1000 in PBS and loaded into a 1-mL cuvette and placed into the instrument. Twenty-five videos with 3 frames per sec and 300 frames per video were recorded at 22°C. Software version 2.12/1.3.5 was used for the analysis. To characterize size and morphology through transmission electron microscopy (TEM), 3 µL of EVs purified by 12% PEG8000 and 0.5 M NaCl was loaded onto the 200-mesh size Formvar-carbon coated electron microscopy grids.Our results based on the DLS analysis revealing a normal distribution with mean EVs size of 266 nm and zeta potential measured to be -48 mV. Nanoparticle tracking analysis was conducted to confirm the size distribution and concentration of the isolated EVs. The average size of EVs separated by 12% PEG8000 was determined as 265 nm and the concentration of the diluted sample was calculated to be 1×107 particles/mL. Furthermore, TEM image exhibits the typical cup-shaped morphology of exosome-like EVs and large EVs, as well as some protein aggregates which has been reported in the literature. Western Blot Analysis. Our results indicated the EVs acquired by PEG-based precipitation approach reveal the surface protein expression of mammalian exosome associated tetraspanin family including CD9 (24 kDa), CD63 (60 kDa), and CD81 (26 kDa). The bands for endosomal sorting complex required for transport (ESCRT) dependent biogenic proteins TSG101 (50 kDa) and ALIX (95 kDa) were also clearly displayed. Additionally, no band was detected using Calnexin as a negative marker of exosomes. Infection of Arabidopsis thaliana. Pseudomonas syringe strain DC3000 colonies scraped from the agar plate were suspended in culture medium and grown to OD600 = 0.2. A. thaliana leaves were sprayed with the bacterial solution or a control solution without bacteria. Three days after the initial infection, apoplastic washing fluid (AWF) was collected from infected and mock plant leaves, respectively. The images of Arabidopsis thaliana post-infection with P. syringe for 3 days. Separation of PS-exposing EVs by Functionalized Chitin nanoparticles (NPs). The procedures of separating PS-exposing EVs by chitin NPs functionalized with lactadherin C1C2 were illustrated using 50 mM dithiothreitol (DTT) and 500 mM NaCl. The isolated EVs in the supernatant were analyzed for size, polydispersity, and zeta potential by dynamic light scattering device. The average size of collected EVs is around 117 nm with negatively charged. The EVs-chitin NP, EVs, and chitin NP were further analyzed by transmission electron microscopy (TEM). Infection of A. thaliana pre-treated with EVs from infected plant. A. thaliana leaves were sprayed with EVs purified from plant leaves infected with P. syringe for 3 days, and then infected with the bacterial solution for 3 days. Images of A. thaliana 3 days post-infection were taken under various conditions: (A) pre-treated with EVs collected from mock plant; (B) pre-treated with EVs purified from plant infected with P. syringe for 3 days; (C) no pre-treatment of EVs; (D) control (i.e., not infected). Our results showed that EVs separated from A. thaliana infected with P. syringe for 3 days could silence its virulence genes.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Jokhio S, Peng, I, Peng CA. Extracellular vesicles isolated from Arabidopsis thaliana leaves reveal characteristics of mammalian exosomes. Protoplasma 261:1025-1033, 2024.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Peng, CA. Nanoparticle-based separation of plant extracellular vesicles for silencing virulence genes. 2024 GRC Nanoscale Science and Engineering for Agriculture and Food Systems. Manchester, NH (June 23-28).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Jokhio S, Peng CA. Apoptosis of Myrosinase Gene Expressed Lung Cancer Cells Treated with Glucosinolate-Containing Extracellular Vesicles Isolated from Arabidopsis thaliana. 2024 AIChE Annual Meeting, San Diego, CA (Oct 27-31).
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Progress 03/01/22 to 02/28/25
Outputs
Target Audience:- Research community investigating construction of recombinant plasmids, protein expression in transformed bacteria, and purification of fusion protein - Research community in the area of preventing pathogen infection using purified plant extracellular vesicles - Undergraduates and graduates involved with this research Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One PhD student is now familiar with recombinant plasmid construction, protein expression in T7-based bacteria system, and protein purification using affinity chromatography. This study has also learned to grow Arabidopsis thaliana and collect apoplastic washing fluids obtained from Arabidopsis leaves infected with pathogenic bacteria. The PhD student is in the final stage of completing the remaining experimental studies for the dissertation writing. One undergraduate student has been trained to grow A. thaliana, collect AWF, and use AWF to green synthesis of silver nanoparticles revealing anti-bacterial efficacy. How have the results been disseminated to communities of interest? The techniques of constructing, expressing, purifying, and characterizing lactadherin C1C2 fused proteins have been reported. The PEG-based precipitation of plant EVs have been published. One manuscript is under preparation for publishing the research outcomes of EVs separated from A. thaliana infected with P. syringe for 3 days could prevent Arabidopsis leaves from disease symptom (i.e., no yellowing or withering caused by P. syringe infection) over a long period of time. The other manuscript is also under preparation to report the efficient particle-based separation of plant EVs comparing to standard ultracentrifugation method. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Construction and Expression of lactadherin C1C2-streptavidin and pTXB1-lactadherin C1C2 encoding plasmid. The lactC1C2-SA encoding recombinant plasmid (pMA002) was constructed by inserting the gene sequences of lactadherin C1C2 domain into the multiple cloning sites from EcoRV and EcoRI in the established plasmid pMA001 (i.e., pET-30a(+)-coreSA). Moreover, we worked on an additional plasmid construction of pTXB1-CBD (chitin binding domain)-intein-C1C2. the gene encoding lactadherin C1C2 cloned by PCR were verified by DNA gel electrophoresis showing the size of ~0.9 kb on an agarose gel. The cloned lactadherin C1C2 was inserted between NdeI and SapI of pTXB1 plasmid. Collection of apoplastic washing fluid from Arabidopsis thaliana. Arabidopsis thaliana seeds [wild-type Arabidopsis thaliana (L.) Hyenh. Columbia-0 (Col-0)], obtained from Arabidopsis Biological Resource Center (Columbus, OH), planted by sprinkling uniformly over moist soil in plastic pots. The pots were then placed at 4°C for 4 days to allow enough time to properly germinate. On the 5th day, the pots were shifted to the growth chamber maintained at 22°C, and exposed to light (100 μmol/m2/s) with a cycle of 16 h on and 8 h off. A total of fifty leaves (fresh weight around 2 g) of 4-week-old Arabidopsis thaliana were cut at petioles, washed thrice with water, and dried on a paper towel. Dried leaves were then placed inside a needleless syringe and infiltrated with vesicle infiltration buffer. The apoplastic washing fluid (AWF) was collected, filtered with a 0.45-μm Nylon filter, and aliquoted 500 μL into three 1.5-mL microcentrifuge tubes which were stored at 4°C for follow-up experiments and at -80°C for long-term storage. Isolation of EVs from AWF by polyethylene glycol 8000 and sodium chloride. The collected AWFs (500 μL each) were mixed with various amounts of polyethylene glycol 8000 (PEG8000) to obtain 5%, 8% and 12% w/v concentration, respectively. The mixture was further added with NaCl to get 0.5 M salinity and then incubated at 4°C for 2 h and centrifuged at 10,000× g force for 30 min at 4°C using a microcentrifuge. The PEG-EVs agglomerates were sedimented at the bottom of the 1.5-mL microcentrifuge tube. The pellet was re-suspended in 1× phosphate buffered saline for characterization. Immuno dot blot assay. To examine the efficacy of EVs separated from AWF by the PEG-precipitation method, a dot blot assay was used. Briefly, for three different concentrations of PEG8000 used, 5 μL each from the supernatant and resuspended pellets was taken and carefully spotted on the nitrocellulose membrane. After 10 min of drying, the membranes were blocked using blocking buffer. Then, the membranes were incubated with mammalian monoclonal antibodies against tetraspanin proteins (CD9, CD63, and CD81) and PEG, respectively for 1 h at 4°C. After 1 h, the membranes were washed three times with TBST and incubated with secondary antibody (mouse IgG horseradish peroxide (HRP)-conjugated antibody in the blocking buffer at 1:1000 dilution) for 1 h at room temperature. Our results showed that there is an increase in pellet size with increasing concentration of PEG8000 from 5% to 12%. The precipitation of EVs was further verified by dot blot assay of both the pellet and supernatant. The dot blot analysis that was performed using the monoclonal antibodies against mammalian tetraspanin proteins CD9, CD63, and CD81, confirmed the presence of EVs in the pellet. Characterization of PEG-precipitated EVs. The measurements for the size distribution of EVs were performed with dynamic light scattering (DLS) instrument. EVs, resuspended in 1× PBS, were appropriately diluted with 0.2-μm membrane filtered distilled water and loaded into a 750-μL cuvette for the detection. Zeta potential of EVs was measured by the same instrument. The particle size distribution and concentration of isolated EVs were further determined by nanoparticle tracking analysis (NTA). Briefly, each sample was diluted 1:1000 in PBS and loaded into a 1-mL cuvette and placed into the instrument. Twenty-five videos with 3 frames per sec and 300 frames per video were recorded at 22°C. Software version 2.12/1.3.5 was used for the analysis. To characterize size and morphology through transmission electron microscopy (TEM), 3 μL of EVs purified by 12% PEG8000 and 0.5 M NaCl was loaded onto the 200-mesh size Formvar-carbon coated electron microscopy grids. Our results based on the DLS analysis revealing a normal distribution with mean EVs size of 266 nm and zeta potential measured to be -48 mV. Nanoparticle tracking analysis was conducted to confirm the size distribution and concentration of the isolated EVs. The average size of EVs separated by 12% PEG8000 was determined as 265 nm and the concentration of the diluted sample was calculated to be 1×107 particles/mL. Furthermore, TEM image exhibits the typical cup-shaped morphology of exosome-like EVs and large EVs, as well as some protein aggregates which has been reported in the literature. Western Blot Analysis. Our results indicated the EVs acquired by PEG-based precipitation approach reveal the surface protein expression of mammalian exosome associated tetraspanin family including CD9 (24 kDa), CD63 (60 kDa), and CD81 (26 kDa). The bands for endosomal sorting complex required for transport (ESCRT) dependent biogenic proteins TSG101 (50 kDa) and ALIX (95 kDa) were also clearly displayed. Additionally, no band was detected using Calnexin as a negative marker of exosomes. Infection of Arabidopsis thaliana. Pseudomonas syringe strain DC3000 colonies scraped from the agar plate were suspended in culture medium and grown to OD600 = 0.2. A. thaliana leaves were sprayed with the bacterial solution or a control solution without bacteria. Three days after the initial infection, apoplastic washing fluid (AWF) was collected from infected and mock plant leaves, respectively. The images of Arabidopsis thaliana post-infection with P. syringe for 3 days. Separation of PS-exposing EVs by Functionalized Chitin nanoparticles (NPs). The procedures of separating PS- exposing EVs by chitin NPs functionalized with lactadherin C1C2 were illustrated using 50 mM dithiothreitol (DTT) and 500 mM NaCl. The isolated EVs in the supernatant were analyzed for size, polydispersity, and zeta potential by dynamic light scattering device. The average size of collected EVs is around 117 nm with negatively charged. The EVs-chitin NP, EVs, and chitin NP were further analyzed by transmission electron microscopy (TEM). Infection of A. thaliana pre-treated with EVs from infected plant. A. thaliana leaves were sprayed with EVs purified from plant leaves infected with P. syringe for 3 days, and then infected with the bacterial solution for 3 days. Images of A. thaliana 3 days post-infection were taken under various conditions: (A) pre-treated with EVs collected from mock plant; (B) pre-treated with EVs purified from plant infected with P. syringe for 3 days; (C) no pre-treatment of EVs; (D) control (i.e., not infected). Our results showed that EVs separated from A. thaliana infected with P. syringe for 3 days could silence its virulence genes.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Jokhio S, Peng, I, Peng CA. Extracellular vesicles isolated from Arabidopsis thaliana leaves reveal characteristics of mammalian exosomes. Protoplasma 261:1025-1033, 2024.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Jokhio S, Peng CA. Apoptosis of Myrosinase Gene Expressed Lung Cancer Cells Treated with Glucosinolate-Containing Extracellular Vesicles Isolated from Arabidopsis thaliana. 2024 AIChE Annual Meeting, San Diego, CA (Oct 27-31).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Peng, CA. Nanoparticle-based separation of plant extracellular vesicles for silencing virulence genes. 2024 GRC Nanoscale Science and Engineering for Agriculture and Food Systems. Manchester, NH (June 23-28).
|
Progress 03/01/23 to 02/29/24
Outputs
Target Audience:- Research community in the area of preventing pathogen infection using isolated extracellular vesicles from plant treated with pathogens - Graduates involved with this research Changes/Problems:We will use Pseudomonas syringaerather than B. cinerea as the pathogen to infect Arabidopsis thaliana leaves. What opportunities for training and professional development has the project provided?One PhD student is now familiar with the expression and urification of lactC1C2-streptavidin fusion protein, thepreparation of functionalized nanoparticles for rapid isolation of plant EVs, collection of AWF from Arabidopsis thaliana leaves infected by pathogens. How have the results been disseminated to communities of interest?Some of the results have been reported in the A1511 PD meeting held at Universtiy of Tennessee Conference Center (from August 10 to 11). What do you plan to do during the next reporting period to accomplish the goals?We will examine the efficacy of EVs isolated from infected Arabidopsis thaliana leaves revealing silencing virulence genes of pathogens.
Impacts
What was accomplished under these goals?
The purified lactC1C2-streptavidin fusion protein was bound with biotinylated nanoparticles. The functionalized nanoparticles were able to rapidly isolate extracellular vesicles from apoplastic washing fluid (AWF) obtained bt infiltratingArabidopsis thaliana leaves pre-infected wtih pathogens.The mean size of purified Arabidopsis thaliana EVs determined by dynamic light scattering was 266 nm, which is consistent with nanoparticle tracking analysis. The size was also confirmed via transmission electron microscopy with morphology of a cup-shaped appearance which is the typical mammalian exosome's morphology. Additionally, Western blotting of the purified Arabidopsis thaliana EVs, using commercially available mammalian exosomal kits, displayed surface marker tetraspanin proteins (CD9, CD63, and CD81), and endosomal sorting complexes required for transport (ESCRT)-associated proteins (TSG101 and ALIX). This demonstrates that the purified Arabidopsis thaliana EVs reveal the typical proteins reported in mammalian exosomes.
Publications
|
Progress 03/01/22 to 02/28/23
Outputs
Target Audience:- Research community investigating construction of recombinant plasmids, protein expression in transformed bacteria, and purification of fusion protein - Research community in the area of preventing pathogen infection using purifiedplant extracellular vesicles - Undergraduates and graduates involved with this research Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One PhD student is now familiar with recominant plasmid construction, protein expression in T7-based bacteria system, and protein purification using affinity chromatography. This study has also learned to grow Arabidopsis thaliana and collect apoplastic washing fluids obtained from Arabidopsis leaves infected with pathogenic bacteria. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We will construct and characterize biotinylated nanoparticles for tethering the lactC1C2-SA fusion protein purified from this report period. The purified plant extracellular vesicles will be further used to spray onArabidopsis thaliana leaves to investigate the potency of preventing infection.
Impacts
What was accomplished under these goals?
ThelactC1C2-coreSA encoding recombinant plasmid (pMA002) wasconstructed by inserting the gene sequences of lactadherin C1C2 domain into the multiple cloning sites from EcoRV and EcoRI in the established plasmid pMA001 (i.e., pET-30a(+)-coreSA). The lactC1C2 domain gene was cloned from the plasmid encoding C1C2 domain, acquired from Addgene (plasmid Cat # 52987), by polymerase chain reaction (PCR) with forward primer 5'- ATTAAGATATCTGTTCTACACAGCTGGGC-3' and reverse primer 5'-ATTAAGAATTCACAGCCCAGC AGCTCCAG-3'. LactC1C2 PCR reactions were performed with previous reportedDNA polymerase, times, temperatures, and cycles. The PCR products of lactC1C2 were purified through the PCR purification kit (NEB) and analyzed by 1% agarose gel electrophoresis. The pMA001 plasmid and lactC1C2 cDNA (insert) were cut with restriction enzymes EcoRV and EcoRI. The digested pMA001 and lactC1C2 products were ligased to get the pMA002 plasmid. The pMA002 ligation product was amplified by transformation with subcloning efficiency DH5α competent cells and purified by QIAGEN Plasmid Maxi Kit. The gene sequences encoding lactC1C2 and coreSA from pMA002 cloned by PCR were verified by DNA gel electrophoresis. The constructed pMA002 plasmid was transformed into T7 Express Lemo21(DE3) competent E. coli cells and had lactC1C2-coreSA fusion protein expressed using the protocols reported in public accounts. Expressed lactC1C2-coreSA fusion protein with 6×His tag was purified by IMAC Co-NTA affinity chromatography and characterized by SDS?PAGE and Western blotting analysis.The purified lactC1C2-coreSA fusion protein can be easily bound onto biotinylated chitosan nanoparticles.
Publications
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