This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (NRF-2018R1A2A1A05019892) to DC and by a grant from Plant Molecular Breeding Center (PMBC) of the Next Generation Biogreen 21 program of the Rural Development Administration (PJ013201) to EP.&#160;We thank the imaging center of the National Instrumentation Center for Environmental Management to provide a confocal microscope for filming.

Note: All steps are performed at room temperature, unless stated otherwise.
1. Preparation of Plant Materials (4 Weeks)
<ol>
	<li>Preparation for the N. benthamiana plants
	<ol>
		<li>Sow 2 seeds of N. benthamiana on the soil surface of each pot, cover the tray with a plastic dome, and allow seeds to germinate in a 25 &#176;C, 60% humidity growth chamber with a 16/8-h light/dark photoperiod cycle.</li>
		<li>After two weeks, pick out and discard the smallest seedling in each pot and continue to grow plants under the same growth conditions as applied for the germination in step 1.1.1. Add 1 L of water per tray every two days. 
		Note: The growth conditions for plants may vary across labs. Therefore, follow the regular watering protocol to grow the plant in a healthy condition.</li>
		<li>In a week, transfer the plants to a new tray and arrange them with adequate space for further growth. Keep growing the plants under the conditions described in step 1.1.1 until they are ready to be infiltrated at 4 weeks of age. 
		Note: Plant growth may differ depending on the growth condition across labs. Usually, we find that the 4-week-old N. benthamiana plants bear about six leaves.</li>
	</ol>
	</li>
	<li>Preparation for A. thaliana transgenic plants
	<ol>
		<li>Refer to the Table of Materials and order the transgenic Arabidopsis seeds.</li>
		<li>Soak ~50 -&#160;100 transgenic Arabidopsis seeds in 1 mL of distilled water and store them at 4 &#176;C for 3 days in the dark to synchronize the onset of germination.</li>
		<li>Sow ~2 - 3 seeds on the soil surface of a plug plant tray and cover the tray with a plastic dome. Allow seeds to germinate at 23&#160;&#176;C, 60% humidity with a 10/14-h light/dark photoperiod cycle. 
		Note: The seeds should be homozygotes. However, we recommend reconfirming the presence of the transgene in the batch. In this case, sterilize the seeds by washing with 70% ethanol for 2 min, 50% bleach (about 2% hypochlorite) containing 0.05% triton X-100 for 5 min. Follow by washing 5 - 6 times with sterile double distilled water (ddH2O). After sterilization, stratify at 4 &#176;C for 3 days and plate them on plant germination media containing 25 &#181;g/L of hygromycin B to select the transgenic plants.</li>
		<li>After a week, leave only one plant per plug and continue growing plants under the same growth conditions used for step 1.2.3. 
		Note: Four-week-old plants were used for the P. syringae infection. Water plants every other day to keep plants healthy.</li>
	</ol>
	</li>
</ol>
2. Preparation of Pseudomonas Culture (~1 Week)
<ol>
	<li>Construction of the plasmid for Pseudomonas transformation
	<ol>
		<li>Refer to the Table of Materials and order the desired vector(s) of the T3SS-based effector delivery system vector25.</li>
		<li>Insert the effector gene of interest into the effector delivery vector using site-specific recombination cloning25. 
		Note: When monitoring subcellular localization of full-length effector protein, put the full-length gene into pBK-GW-1-2 or pBG-GW-1-2. It is also possible to choose pBK-GW-1-4 or pBG-GW-1-4 containing 2x sfGFP11 for increased fluorescence signal. In the case of a partial effector lacking signal peptide, use pBK-GW-2-2 or pBK-GW-2-4. Refer to Park et al. for detailed information about effector delivery vectors25.</li>
	</ol>
	</li>
	<li>Transform the plasmid carrying an effector fused to the sfGFP11 tag to P. syringae pv. Tomato (Pst) CUCPB5500 using standard electroporation29. 
	Note: Other Pseudomonas strains can be used if necessary. The sfGFP11 tag system is constructed for a broad range of vectors30 and the gene expression of the effector is regulated by the AvrRpm1 promoter, which is comparable with, e.g., Pseudomonas fluorescens (EthAn)31.</li>
	<li>Spread the transformed bacterial cells gently over the surface of the King&#8217;s B agar plates containing 100 &#181;g/mL rifampicin and 25 &#181;g/mL kanamycin or 25 &#181;g/mL gentamycin. Incubate at 28 &#176;C for 2 days.</li>
	<li>Inoculate one colony into King&#8217;s B liquid media with antibiotics appropriate for the vector used, and grow the cells overnight at 28 &#176;C with shaking at 200 rpm.</li>
	<li>Make a glycerol stock. Add autoclaved glycerol to a final concentration of 50% and store at -80 &#176;C.</li>
</ol>
3. Transient Expression of Organelle-targeted sfGFP1-10OPT in N. benthamiana (4 Days)
<ol>
	<li>Preparation of Agrobacterium culture
	<ol>
		<li>Order the desired vector(s) of organelle-targeted sfGFP1-10OPT plasmid(s) (refer to the Table of Materials).</li>
		<li>Transform the plasmid(s) into Agrobacterium tumefaciens strain GV3101 cells32. Grow the cells on Luria-Bertani (LB) agar medium supplemented with 50 &#181;g/mL kanamycin and 50 &#181;g/mL rifampicin at 28 &#176;C for 2 days.</li>
		<li>From a single colony on the LB agar medium, inoculate the cells into 5 mL of liquid LB media supplemented with 50 &#181;g/mL kanamycin and 50 &#181;g/mL rifampicin. Grow the cells overnight at 28 &#176;C with shaking at 200 rpm.</li>
		<li>Harvest the cells by centrifugation at 3,000 x g for 10 min. Pour off the supernatant media and resuspend the pellet in 1 mL of freshly made infiltration buffer.</li>
		<li>Measure the quantity of Agrobacterium by obtaining the optical density (OD) value at an absorbance of 600 nm (Abs 600 nm). Adjust the OD600 of the bacteria to 0.5 with infiltration buffer. 
		Note: 1 mL of suspension is enough to infiltrate on two spots.</li>
		<li>Leave the culture at room temperature on a gentle rocker for 1 - 5 h before infiltration.</li>
		<li>Poke a hole into the center of the leaves to be infiltrated with a 10 &#181;L tip. Use a 1-mL needleless syringe to infiltrate the Agrobacterium suspensions. Carefully and slowly inject about 500 &#181;L of the suspensions prepared from step 3.1.5 into the leaf adaxial side via the syringe. Repeat the infiltration on at least three different plants for experimental replicates. 
		Note: For health and safety reasons, eye protection should be worn during infiltration.</li>
		<li>Wipe off the remaining bacterial suspension on the leaves and mark the boundary of the infiltrated region.</li>
		<li>Keep the infiltrated plants under the same growth conditions used for step 1.1.1 for 2 days.</li>
	</ol>
	</li>
</ol>
4. Inoculation of Pseudomonas (4 Days)
<ol>
	<li>Streak the transformed Pseudomonas strain from the glycerol stock in step 2.5 on King&#8217;s B agar media with the appropriate antibiotics at 28 &#176;C for 2 days. 
	Note: The health of Pseudomonas is very critical. If colonies do not form well, streak the cells again or propagate the cells in the King&#8217;s liquid media prior to proceeding.</li>
	<li>Inoculate a loopful of Pseudomonas cells in Mannitol-Glutamate (MG) liquid media at 28 &#176;C with shaking at 200 rpm for overnight.</li>
	<li>Harvest the cells by centrifugation at 3,000 x g for 10 min. Pour off the supernatant media, resuspend the pellet in 10 mM MgCl2, and adjust the OD600 to 0.02 (1 x 107 cfu/mL) for N. benthamiana leaves and to 0.002 (1 x 106 cfu/mL) for Arabidopsis leaves.</li>
	<li>For the N. benthamiana, infiltrate the Pseudomonas suspension into the area of the leaves where the Agrobacterium carrying sfGFP1-10OPT construct was infiltrated 2 days previously (as in step 3.1.7). For the sfGFP1-10OPT transgenic Arabidopsis, infiltrate the Pseudomonas suspension into two 4-week-old short day-grown leaves. 
	Note: At least three plants are needed for experimental replicates.</li>
</ol>
5. Observation of sfGFP Signal via Confocal Microscopy (1 Day)
<ol>
	<li>Cut out the leaf disc from the Pseudomonas-inoculated leaves. At specific time points after infiltration of Pseudomonas, image two 2-cm2 leaf discs from the single plant using a laser scanning confocal system with 40X/1.2 NA C-Apochromat water immersion objective or 63X/0.8 NA C-Apochromat oil immersion objective. To avoid dead cells killed by wounding, observe the cells away from the infiltration hole.</li>
	<li>Start the observation at a low power setting of the 488-nm argon laser. Increase the laser power to detect sfGFP. 
	Note: We usually use 2 - 15% of the laser intensity to detect the fluorescence signal. However, the laser power and detection setting should be adjusted based upon the user&#8217;s microscopy system. Here, the emission filters were set to 520 - 550 nm. The dead cells often emit auto-fluorescence under the 488-nm laser excitation. Therefore, as a negative control, the same effector without the sfGFP11 tag should be infiltrated and observed under the same observation conditions. In addition, high laser excitation can induce chlorophyll autofluorescence. Therefore, adjust the laser intensity using the control plant cells so as&#160;not to induce chlorophyll autofluorescence.
	<ol>
		<li>For a counterstaining of the cell wall, infiltrate 20 mM propidium iodide (PI) into the leaf disc at 5 - 10 min prior to observation.</li>
		<li>For the nucleus staining, submerge the leaf discs into 0.1% paraformaldehyde for 5 min followed by washing with water. Then, infiltrate the 10 mM PI into the leaf disc at 5 - 10 min before microscopic observation. Repeat the experiments at least three times. 
		Note: This step is not critical but helpful to define the effector localization at the plasma membrane or the nucleus. If the effectors of interest showed their localization in a specific organelle, use a marker for the given organelle to confirm the localization of the effector.</li>
	</ol>
	</li>
</ol>

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Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+Agrobacterium-delivered+VirE2+in+recipient+cells.">Direct visualization of Agrobacterium-delivered VirE2 in recipient cells.</a> Plant J. 77 (3), 487-495 (2014).</li><li>Tanaka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+approaches+to+investigate+effector+translocation+into+host+cells+in+the+Ustilago+maydis/maize+pathosystem.">Experimental approaches to investigate effector translocation into host cells in the Ustilago maydis/maize pathosystem.</a> Eur J Cell Biol. 94 (7-9), 349-358 (2015).</li></ol>

The authors have no conflicts of interest to disclose.

The protocol described here is used for monitoring the accurate localization of the effector proteins injected by the bacterial T3SS into the host plant cell upon infection. Previously, the split GFP system was used as a tool to study the subcellular localization of mammalian proteins23,36, Salmonella T3E localization, and Agrobacterium VirE2 delivery through the T4SS into the plant cells37. To apply this system in the pl...

Plants are sessile organisms that encounter numerous invading pathogens including bacteria, fungi, viruses, insects, and nematodes throughout their life cycle. Among the phytopathogens, the gram-negative bacterial pathogens such as Pseudomonas spp. and Ralstonia spp., infect their host plants by entering through wounds or natural openings, such as the stomata and hydathode1. To successfully colonize host plants, bacterial pathogens have evolved to develop a variety of virulence factors2. When bacteria invade a host plant, they inject a series of virulence proteins &#8212; known as effectors &#8212; directly into the plant cells to promote their pathogenicity. These effectors suppress or modulate the plant innate immunity, and manipulate the host cellular processes to result in bacterial survival3.
Pathogenic bacteria mainly use a T3SS to deliver effector proteins directly into host cells4. The T3SS resembles a molecular syringe with a needle-like channel connecting from a scaffold protein structure across the inner and the outer bacterial membranes to the injection site of the host cell5. This T3SS-mediated effector (T3E) secretion mechanism is well-conserved in various gram-negative bacterial pathogens of the plant as well as human. One of the representative plant pathogens, the P. syringae pv. Tomato DC3000 hrcC mutant which typically has a defective T3SS, has restricted growth in plants likely due to the inability of this mutant to fully suppress the plant immunity (by injecting effector proteins)6. Upon translocation into the host cells, effectors target various host proteins that are important for the host cell system, including plant defense responses, gene transcription, cell death, proteasome, vesicle trafficking, and hormone pathways7,8,9,10. Therefore, tracking of the cellular localization of the effector proteins in the host cells is an attractive target to understand their functions with respect to modulation of the plant immunity.
Most of the localization studies of the T3Es have employed Agrobacterium-mediated overexpression with a large fluorescence protein in the host plant9. However, the heterologous expression method for genes that are introduced in other species has been shown to be mis-localized or occasionally non-functional11,12,13. In addition, several studies revealed that bacterial effectors undergo modification for proper targeting in the host cells14,15,16,17. Therefore, transiently expressed effectors in the cytosol of the plant cells may not be functionally or quantitatively identical to the effectors which are delivered by the T3SS upon pathogen infection18. Moreover, the fusion of large fluorescent tags to effector proteins may disrupt the proper effector delivery and visualization18,19. Therefore, these approaches to assay the T3E function may not fully reflect the native localization of the T3SS-secreted effectors.
A green fluorescent protein (GFP) is composed of an 11-stranded &#946;-barrel enclosing a central strand that includes a chromophore20. Waldo et al. reported a novel split-GFP system that consists of a small component (GFP &#946; strand 11; GFP11) and a large complementary fragment (GFP &#946; strand 1-10; GFP1-10)21. The fragments do not fluoresce by themselves but fluoresce upon their self-association when both fragments are in close proximity with each other. For the optimization of the protein folding efficiency, robust folding variants of the GFP, i.e., sfGFP and sfGFPOPT, were subsequently developed for the split GFP system20,21,22. Recently, single amino acid mutated variants of sfGFP1-10OPT- sfYFP1-10OPT and sfCFP1-10OPT- that can reconstitute with a sfGFP11 fragment, and show yellow and cyan fluorescence respectively, were generated23. Moreover, sfCherry, a derivative of mCherry, can be split into sfCherry1-10 and sfCherry11 fragments in the same manner as sfGFP23.
This system has been adapted to label and track the T3SS effectors in HeLa cells during infection using the effectors from Salmonella24. However, it was previously not optimized for the host plant-bacterial pathogen system. Recently, we optimized the split GFP system based on the improved sfGFP1-10OPT to monitor the subcellular localization of the T3Es delivered from P. syringae into plant cells25. To facilitate the localization studies of the T3Es to different subcellular compartments in the plant cells, a set of transgenic Arabidopsis thaliana plants were generated to express sfGFP1-10OPT in the various subcellular compartments25. Moreover, the plasmids carrying a variety of organelle-targeted sfGFP1-10OPT for the Agrobacterium-mediated transient overexpression and the sfGFP11-tagged vectors for the T3SS-based effector delivery were also generated. The seeds of various transgenic Arabidopsis lines and the plasmids to express the T3Es of interest can be obtained from sources mentioned in the Table of Materials26,27.
In the following protocol, we describe an optimized system to monitor the dynamics of effectors delivered by bacteria in the host cells using the split sfGFP system. Infection of plants expressing sfGFP1-10OPT with transgenic Pseudomonas carrying recombinant sfGFP11 plasmid results in a delivery of the sfGFP11-tagged effector from Pseudomonas into the host cell. Consequently, these proteins are reconstituted and translocate to the specific effector target compartment(s). The Pseudomonas syringae pv. tomato CUCPB5500 strain in which 18 effectors are deleted, was used because this strain showed low or no cell death in both A. thaliana and N. benthamianas28. However, all of the materials and steps described here can be replaced or modified to adapt the split sfGFP system for investigation of other biological questions or optimization in the given laboratory conditions.

<table>
	<tbody>
		<tr>
			<td>Arabidopsis transgenic lines</td>
			<td></td>
			<td></td>
			<td>Park, E., Lee, H. Y., Woo, J., Choi, D. &#38; Dinesh-Kumar, S. P. Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. Plant Cell. 29 (7), 1571-1584 (2017)</td>
		</tr>
		<tr>
			<td>CYTO-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69831</td>
			<td></td>
		</tr>
		<tr>
			<td>NU-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69832</td>
			<td></td>
		</tr>
		<tr>
			<td>PT-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69833</td>
			<td></td>
		</tr>
		<tr>
			<td>MT-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69834</td>
			<td></td>
		</tr>
		<tr>
			<td>PX-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69835</td>
			<td></td>
		</tr>
		<tr>
			<td>ER-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69836</td>
			<td></td>
		</tr>
		<tr>
			<td>GO-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69837</td>
			<td></td>
		</tr>
		<tr>
			<td>PM-sfGFP1-10</td>
			<td>ABRC</td>
			<td>CS69838</td>
			<td></td>
		</tr>
		<tr>
			<td>Organelle-targeted sfGFP1-10OPT plasmid</td>
			<td></td>
			<td></td>
			<td>Park, E., Lee, H. Y., Woo, J., Choi, D. &#38; Dinesh-Kumar, S. P. Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. Plant Cell. 29 (7), 1571-1584 (2017)</td>
		</tr>
		<tr>
			<td>CYTO-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97387</td>
			<td></td>
		</tr>
		<tr>
			<td>NU-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97388</td>
			<td></td>
		</tr>
		<tr>
			<td>PT-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97389</td>
			<td></td>
		</tr>
		<tr>
			<td>MT-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97390</td>
			<td></td>
		</tr>
		<tr>
			<td>PX-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97391</td>
			<td></td>
		</tr>
		<tr>
			<td>ER-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97392</td>
			<td></td>
		</tr>
		<tr>
			<td>GO-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97393</td>
			<td></td>
		</tr>
		<tr>
			<td>PM-sfGFP1-10</td>
			<td>Addgene</td>
			<td>97394</td>
			<td></td>
		</tr>
		<tr>
			<td>ER-sfCherry1-10</td>
			<td>Addgene</td>
			<td>97403</td>
			<td></td>
		</tr>
		<tr>
			<td>ER-sfYFP1-10</td>
			<td>Addgene</td>
			<td>97404</td>
			<td></td>
		</tr>
		<tr>
			<td>CYTO-sfCFP1-10</td>
			<td>Addgene</td>
			<td>97405</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">sfGFP11-tagged Gateway compatible vector for T3SS-based effector delivery system</td>
			<td>Park, E., Lee, H. Y., Woo, J., Choi, D. &#38; Dinesh-Kumar, S. P. Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. Plant Cell. 29 (7), 1571-1584 (2017)</td>
		</tr>
		<tr>
			<td>pBK-GW-1-2</td>
			<td>Addgene</td>
			<td>98250</td>
			<td>pAvrRpm1:GW:HA-sfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBK-GW-1-4</td>
			<td>Addgene</td>
			<td>98251</td>
			<td>pAvrRpm1:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBK-GW-2-2</td>
			<td>Addgene</td>
			<td>98252</td>
			<td>pAvrRpm1:AvrRPM1sp:GW:HA-sfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBK-GW-2-4</td>
			<td>Addgene</td>
			<td>98253</td>
			<td>pAvrRpm1:AvrRPM1sp:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Kanamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBG-GW-1-2</td>
			<td>Addgene</td>
			<td>98254</td>
			<td>pAvrRpm1:GW:HA-sfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBG-GW-1-4</td>
			<td>Addgene</td>
			<td>98255</td>
			<td>pAvrRpm1:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBG-GW-2-2</td>
			<td>Addgene</td>
			<td>98256</td>
			<td>pAvrRpm1:AvrRPM1sp:GW:HA-sfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>pBG-GW-2-4</td>
			<td>Addgene</td>
			<td>98257</td>
			<td>pAvrRpm1:AvrRPM1sp:GW:HA-2xsfGFP11:AvrRpm1t; Resistant to Gentamycin (25 ug/ml)</td>
		</tr>
		<tr>
			<td>Bacterial strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agrobacterium tumefaciens GV3101</td>
			<td></td>
			<td></td>
			<td>Csaba Koncz and Jeff Schell, The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet. 204,383-396 (1986); Resistant to gentamycin (50 ug/ml) and rifampicin (50 ug/ml)</td>
		</tr>
		<tr>
			<td>Pseudomonas syringae pv. Tomato CUCPB5500</td>
			<td></td>
			<td></td>
			<td>Kvitko, B. H. et al. Deletions in the repertoire of Pseudomonas syringae pv. tomato DC3000 type III secretion effector genes reveal functional overlap among effectors. PLoS Pathog. 5 (4) (2009).; Resistant to rifampicin (100 ug/ml)</td>
		</tr>
		<tr>
			<td>Media components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plant germination media</td>
			<td></td>
			<td></td>
			<td>Add 2.165g/L Murashige &#38; Skoog powder, 10 g/L sucrose to water. Adjust to pH 5.8 and add 2.2 g/L phytagel. Autocalve.</td>
		</tr>
		<tr>
			<td>Murashige &#38; Skoog medium including vitamins</td>
			<td>Duchefa Biochemie</td>
			<td>M0222</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Duchefa Biochemie</td>
			<td>S0809</td>
			<td></td>
		</tr>
		<tr>
			<td>Phytagel</td>
			<td>Sigma-Aldrich</td>
			<td>P8169</td>
			<td></td>
		</tr>
		<tr>
			<td>LB media</td>
			<td></td>
			<td></td>
			<td>Add 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl to water. For solid media, add 15 g/L micro agar. Autoclave.&#160; Allow solution to cool to 55 &#176;C, and add antibiotic if needed.</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>BD Bioscience</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD Bioscience</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Duchefa Biochemie</td>
			<td>S0520</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro agar</td>
			<td>Duchefa Biochemie</td>
			<td>M1002</td>
			<td></td>
		</tr>
		<tr>
			<td>King&#39;s B media</td>
			<td></td>
			<td></td>
			<td>10 g/L protease peptone #2, 1.5 g/L anhydrous K2HPO4, 15 g/L of agar to water. Autoclave. Cool down to 55 &#176;C and add sterile 15 ml/L glycerol, 5 ml/L MgSO4 to the medium. Add antibiotics if needed.</td>
		</tr>
		<tr>
			<td>Proteose peptone</td>
			<td>BD Bioscience</td>
			<td>212120</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous K2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>1551128 USP</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Duchefa Biochemie</td>
			<td>G1345</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD Bioscience</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol-Glutamate (MG) liquid media </td>
			<td></td>
			<td></td>
			<td>Add 10 g/L of mannitol, 2 g/L of L-glutamic acid, 0.5 g/L of KH2PO4, 0.2 g/L of NaCl, and 0.2 g/L of MgSO4 to water. Adjust to pH 7</td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Duchefa Biochemie</td>
			<td>M0803</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamic acid</td>
			<td>Duchefa Biochemie</td>
			<td>G0707</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>NIST200B</td>
			<td></td>
		</tr>
		<tr>
			<td>Infiltration buffer</td>
			<td></td>
			<td></td>
			<td>10 mM MES (2-(N-morpholino)-ethane sulfonic acid), 10 mM MgCl2, 150 &#181;M acetosyringone. pH 5.6; Prepare a fresh buffer before use.</td>
		</tr>
		<tr>
			<td>MES</td>
			<td>Duchefa Biochemie</td>
			<td>M1503</td>
			<td>Prepare 100 mM (pH 5.6) stock in water. Filter sterilize.</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>Prepare 100 mM stock in water. Autoclave.</td>
		</tr>
		<tr>
			<td>Acetosyringone</td>
			<td>Sigma-Aldrich</td>
			<td>D134406</td>
			<td>Prepare 150 mM stock in DMSO.</td>
		</tr>
		<tr>
			<td>Confocal microscope equipments/materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>710 laser scanning confocal system</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axio observer Z1 inverted microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>ThermoFisher</td>
			<td>P1304MP</td>
			<td></td>
		</tr>
	</tbody>
</table>

split green fluorescent protein system to visualize effectors delivered from bacteria during infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

The &#946;-barrel structure of GFP is composed of eleven &#946; strands and can be divided into two fragments, the 1 - 10th strand (GFP1-10OPT) and the 11th (GFP11) strand. Although neither of two fragments fluorescent by themselves, self-assembled sfGFP can emit the fluorescence when the two fragments exist in close proximity (Figure 1A). In this system, sfGFP1-10OPT-expressing Arabidopsis or N. bentha...

Watch this Scientific Journal Video about Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection at JoVE.com

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

split-green-fluorescent-protein-system-to-visualize-effectors

Research

JoVE Journal

Biology

10.3K Views.  Seoul National University. Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

Video: Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia.
Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer.
DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia.
We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.

Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)&#x2013;dgn fusion protein (GFP&#x2013;dgn, unstable) and a variant carrying a frameshift mutation (GFP&#x2013;dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP&#x2013;dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian countries, with a reported frequency of 1 in 5,000 in Taiwan. Although much is known about the management of biliary atresia, its pathogenesis is still elusive. The sea lamprey (Petromyzon marinus) provides a unique opportunity to examine the mechanism and progression of biliary degeneration. Sea lamprey develop through three distinct life stages: larval, parasitic, and adult. During the transition from larvae to parasitic juvenile, sea lamprey undergo metamorphosis with dramatic reorganization and remodeling in external morphology and internal organs. In the liver, the entire biliary system is lost, including the gall bladder and the biliary tree. A newly-developed method called &#8220;CLARITY&#8221; was modified to clarify the entire liver and the junction with the intestine in metamorphic sea lamprey. The process of biliary degeneration was visualized and discerned during sea lamprey metamorphosis by using laser scanning confocal microscopy. This method provides a powerful tool to study biliary atresia in a unique animal model.

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus

Sea lamprey lose the gall bladder and bile ducts during metamorphosis, a process similar to human biliary atresia. A new fixation and clarification method (CLARITY) was modified to visualize the entire biliary tree using laser scanning confocal microscopy. This method provides a powerful tool to study biliary degeneration.

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian ...