Plants respond to external stimuli through signal transduction pathways. An external signal binds...
1
Plants respond to external stimuli through signal transduction pathways. An external signal binds the extracellular domain of a transmembrane receptor- like kinase (RLK) protein, and the signal is transmitted inside the cell through the RLK’s kinase domain. Two important plant RLKs are Arabidopsis CLV1 and rice Xa21. CLV1 regulates plant meristem growth and development, and Xa21 provides resistance to the pathogenic bacterium Xanthomonas oryzae. These functions are initiated by a peptide ligand binding a leucine-rich repeat (LRR) region in the extracellular domains of CLV1 and Xa21. Peptide phage display technology will be used to isolate additional peptide ligands that bind CLV1 and Xa21 LRRs. To this end, the DNAs encoding these LRRs are being cloned into vector pSUMO_Kan for soluble Fusion proteins will be isolated from E. coli cells by immobilized metal affinity chromatography. We thank Pamela Ronald (Univ. California, Davis) for Xa21-containing plasmid pCR504 and the Meyerowitz laboratory (California Institute of Technology) for CLV1-containing plasmid pKR123. This work was funded by Iowa-NASA EPSCoR, Drake University, and Iowa State University. 1 Morris, E. and J. Walker. 2003. Receptor-like protein kinases: the keys to response. Curr Opin Plant Biol. 6:339-42. 2 Walker, J. 1994. Structure and function of the receptor-like protein kinases of higher plants. Plant Mol Biol. 26: 1599-609. 3 Ito, Y. et al. 2006. Dodeca-CLE peptides as suppressors of plant stem cell differentiation. Science 313: 842-5. 4 Kondo, T. et al. 2008. Dual assay for MCLV3 activity reveals structure-activity relationship of CLE peptides. Biochem Biophys Res Commun. 377:312-6. 5 Trotochaud, A. et al. 2000. CLAVATA3, a Multimeric Ligand for the CLAVATA1 Receptor-Kinase. Science ABSTRACT ACKNOWLEDGEMENTS & REFERENCES SUMMARY EXPRESSION OF PLANT RECEPTOR-LIKE KINASE LEUCINE RICH REPEAT REGIONS IN E. COLI Andrea Corns, Emily Heffernen, Ara May Hubbard, Paula Macksey, Kelsey Pigg, Andrew Platt, Maria Stieff, Julia Tucci, Guru Rao, and Heidi Sleister. Biology and BCMB, Drake University; BBMB, Iowa State University PROJECT AIM & SIGNIFICANCE PROJECT AIM : To express and purify the leucine rich repeats (LRRs) from plant receptor-like kinases (RLKs) CLV1 and Xa21 as soluble fusions with SUMO protein in E. coli. SIGNIFICANCE : If successful, this project will be the first reported example of soluble expression of a large plant LRR region in E. coli. In the future, soluble LRR protein will be used as the target in peptide phage display to isolate peptides that bind LRR region N-terminal region Flanking paired Cys Flanking paired Cys Transmembrane domain Kinase domain VISLNVSFTPLFGTISPEIGMLTHLVNL TLAANNFTGELPLEMKSLTSLKVL NISNNGNLTGTFPGEILKAMVDLEVL DTYNNNFNGKLPPEMSELKKLKYL SFGGNFFSGEIPESYGDIQSLEYL GLNGAGLSGKSPAFLSRLKNLRE MYIGYYNSYTGGVPPEFGGLTKLEIL DMASCTLTGEIPTSLSNLKHLHTL FLHINNLTGHIPPELSGLVSLKSL DLSINQLTGEIPQSFINLGNITLI NLFRNNLYGQIPEAIGELPKLEVF EVWENNFTLQLPANLGRNGNLIKL DVSDNHLTGLIPKDLCRGEKLEML ILSNNFFFGPIPEELGKCKSLTKI RIVKNLLNGTVPAGLFNLPLVTII ELTDNFFSGELPVTMSGDVLDQI YLSNNWFSGEIPPAIGNFPNLQTL FLDRNRFRGNIPREIFELKHLSRI NTSANNITGGIPDSISRCSTLISV DLSRNRINGEIPKGINNVKNLGTL NISGNQLTGSIPTGIGNMTSLTTL DLSFNDLSGRVPLGGQFL CLV1 is important for shoot apical meristem development. CLV’s natural ligand is a 13-amino acid arabinosylated peptide derived from 96-amino acid CLV3 (RTVPSGP DPLHHH). VVKLLLRSSNLSGIISPS LGNLSFLRELDLGDNYLSGEIPPE LSRLSRLQLLELSDNSIQGSIPAA IGACTKLTSLDLSHNQLRGMIPREI GASLKHLSNLYLYKNGLSGEIPSA LGNLTSLQEFDLSFNRLSGAIPSS LGQLSSLLTMNLGQNNLSGMIPNS IWNLSSLRAFSVRENKLGGMIPTNA FKTLHLLEVIDMGTNRFHGKIPAS VANASHLTVIQIYGNLFSGIITSG FGRLRNLTELYLWRNLFQTREQDDWGFISD LTNCSKLQTLNLGENNLGGVLPNSF SNLSTSLSFLALELNKITGSIPKD IGNLIGLQHLYLCNNNFRGSLPSS LGRLKNLGILLAYENNLSGSIPLA IGNLTELNILLLGTNKFSGWIPYT LSNLTNLLSLGLSTNNLSGPIPSE LFNIQTLSIMINVSKNNLEGSIPQE IGHLKNLVEFHAESNRLSGKIPNT LGDCQLLRYLYLQNNLLSGSIPSA LGQLKGLETLDLSSNNLSGQIPTS LADITMLHSLNLSFNSFVGEVPT IGAFAAASG ISIQGNAKLCGGIP Xa21 is important for resistance to Xanthomonas oryzae which causes rice bacterial blight disease. A 17-amino acid sulfated peptide derived from 194-amino acid Ax21 (AENLSY NFVEGDYVRTP) functions as a ligand for Xa21. LRR region N-terminal region Transmembrane domain Kinase domain C-terminal domain RECEPTOR-LIKE KINASES CLV1 AND Xa21 EXPERIMENTAL METHODS A library of phage, each displaying a different peptide, is allowed to bind LRR- HaloTag fusion protein immobilized on HaloLink magnetic beads. phage magnetic bead HaloTag LRR peptide Specifically-bound phage are eluted, amplified, and used for the next round of panning. After ~4 rounds of panning, individual clones are isolated and sequenced. Panning process is repeated ~3 times. Unbound phage are washed away. Panning Phage Display Library for Peptides that Bind CLV1 / Xa21 LRR Regions Cut PCR product with EcoRV, gel purify Linearize vector pSUMO_Kan with BsaI, polish ends with Pfu DNA Polymerase, dephosphorylate, gel purify Ligate prepared vector + prepared CLV1/Xa21 LRR Transform ligation mixture into E. coli competent cells Screen transformants by colony PCR, restriction digests, and sequence analysis Express LRR region fused with SUMO in bacteria PCR amplify CLV1 and Xa21 LRR regions to incorporate protein tags (Halo or FLAG) and 5’ and 3’ EcoRV restriction sites EcoRV CLV1 / Xa21 LRR region EcoRV Isolate soluble SUMO-LRR fusion protein by immobilized metal affinity chromatography (IMAC) FUTURE WORK RESULTS Figure 1. Structures of CLV1 and Xa21 proteins. The leucine rich repeat (LRR) region is the extracellular portion of the protein that binds a ligand to initiate signal transduction. Figure 2. Methods to clone and express CLV1 and Xa21 LRRs as fusions with SUMO protein. Table 1. PCR primers and conditions CLV1(LRR) Halo CLV1(LRR) FLAG Xa21(LRR) Halo Xa21(LRR) FLAG Figure 5. Protein expression constructs. 1. Lambda-HindIII marker 2. Xa21(LRR)-Halo PCR product 3. Xa21(LRR)-FLAG PCR product 4. CLV1(LRR)-Halo PCR product 5. CLV1(LRR)-FLAG PCR product 6. Uncut plasmid template for CLV1 PCR reaction 7. Uncut plasmid template for Xa21 PCR reaction 8. Lambda-HindIII marker Figure 3. 0.8% agarose gel of CLV1(LRR) and Xa21(LRR) PCR products. The size of each PCR product (lanes 2-5, marked with arrows) is correct. 1 2 3 4 5 6 7 8 2.3 kb 2.0 kb 1 2 3 1. pSUMO vector cut with BsaI 2. Uncut pSUMO vector 3. Lambda-HindIII marker Figure 4. Preparation of pSUMO vector. 0.8% agarose gel of BsaI- cut and uncut pSUMO. • Construction of CLV1 and Xa21 LRRs as fusions with SUMO is in progress. The vector and LRR inserts were successfully isolated and ligated. Transformants are being screened for presence of the insert. • Validation of a phage display combinatorial approach to isolate peptide ligands for plant RLKs CLV1 and Xa21 may lead to general utility of this system for signal transduction in plants and animals. PC R Prim erNam e Prim erSequence PrimerLength (N ucleotides) PC R C onditions PC R Product Size (B asepairs) SU M O -C LV1-F2 AGGTGATATCGACGACGATGCTCGTGTTATC 31 SU M O -C LV1-H alo-R 2 C TA G G A TA TC TTA A C C G G A A A TC TC C A G A G 30 SU M O -C LV1-F2 AGGTGATATCGACGACGATGCTCGTGTTATC 31 SU M O -C LV1-Flag-R 2 C TA G G A TA TC TTA C TTA TC G TC G TC A TC C TTG TA A TC G G A A G TTTC G TTG A A C 53 SUM O -Xa21-F2 AGGTGATATCCGCCGGCACCCACACAG 27 SU M O -Xa21-Halo-R 2 CTAGGATATCTTAACCGGAAATCTCCAGAGTAG 33 SUM O -Xa21-F2 AGGTGATATCCGCCGGCACCCACACAG 27 SUM O -Xa21-Flag-R2 CTAGGATATCTTACTTATCGTCGTCATCCTTGTAATCTCGAGGCAGATGTAGATC 55 2582 bp 1664 bp 2663 bp 1745 bp 2 m in 94 o C ;35 cycles [45 sec 94 o C, 45 sec 50 o C ,3 m in 72 o C ];10 m in 72 o C 2 m in 94 o C ;35 cycles [45 sec 94 o C, 45 sec 50 o C ,3 m in 72 o C ];10 m in 72 o C 2 m in 94 o C ;35 cycles [45 sec 94 o C, 45 sec 52 o C ,3 m in 72 o C ];10 m in 72 o C 2 m in 94 o C ;35 cycles [45 sec 94 o C, 45 sec 52 o C ,3 m in 72 o C ];10 m in 72 o C http://faculty.washington.edu/ktorii/ERECTA04.html http://www.google.com/imgres?imgurl=http://www.carto.net/neumann/travelling/ japan_2004_09/04_kyoto_2004_09_12/29_rice_plant.jpg&imgrefurl=http://www.carto.net/neumann/ travelling/japan_2004_09/04_kyoto_2004_09_12/ &h=614&w=819&sz=65&tbnid=6awJqYynLZFAMM:&tbnh=108&tbnw=144&prev=/search%3Fq%3Drice%2Bplant %26tbm%3Disch%26tbo%3Du&zoom=1&q=rice+plant&usg=__of- NUAjqtzK9TJpvVmYzMzpNd_M=&sa=X&ei=azibTbOnCoz2swO94ImbBA&ved=0CDEQ9QEwAA
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Transcript of Plants respond to external stimuli through signal transduction pathways. An external signal binds...
Plants respond to external stimuli through signal transduction pathways. An external signal binds the extracellular domain of a transmembrane receptor-like kinase (RLK) protein, and the signal is transmitted inside the cell through the RLK’s kinase domain. Two important plant RLKs are Arabidopsis CLV1 and rice Xa21. CLV1 regulates plant meristem growth and development, and Xa21 provides resistance to the pathogenic bacterium Xanthomonas oryzae. These functions are initiated by a peptide ligand binding a leucine-rich repeat (LRR) region in the extracellular domains of CLV1 and Xa21. Peptide phage display technology will be used to isolate additional peptide ligands that bind CLV1 and Xa21 LRRs. To this end, the DNAs encoding these LRRs are being cloned into vector pSUMO_Kan for soluble expression in E. coli as a fusion with SUMO protein. Fusion proteins will be isolated from E. coli cells by immobilized metal affinity chromatography.
We thank Pamela Ronald (Univ. California, Davis) for Xa21-containing plasmid pCR504 and the Meyerowitz laboratory (California Institute of Technology) for CLV1-containing plasmid pKR123. This work was funded by Iowa-NASA EPSCoR, Drake University, and Iowa State University.
1 Morris, E. and J. Walker. 2003. Receptor-like protein kinases: the keys to response. Curr Opin Plant Biol. 6:339-42.2 Walker, J. 1994. Structure and function of the receptor-like protein kinases of higher plants. Plant Mol Biol. 26: 1599-609.3 Ito, Y. et al. 2006. Dodeca-CLE peptides as suppressors of plant stem cell differentiation. Science 313: 842-5.4 Kondo, T. et al. 2008. Dual assay for MCLV3 activity reveals structure-activity relationship of CLE peptides. Biochem Biophys Res Commun. 377:312-6.5 Trotochaud, A. et al. 2000. CLAVATA3, a Multimeric Ligand for the CLAVATA1 Receptor-Kinase. Science 289: 613-7.6 Lee, et al. 2009. A Type I–Secreted, Sulfated PeptideTriggers XA21-Mediated Innate Immunity. Science 326: 850-853.
ABSTRACT
ACKNOWLEDGEMENTS & REFERENCES
SUMMARY
EXPRESSION OF PLANT RECEPTOR-LIKE KINASE LEUCINE RICH REPEAT REGIONS IN E. COLI
Andrea Corns, Emily Heffernen, Ara May Hubbard, Paula Macksey, Kelsey Pigg, Andrew Platt, Maria Stieff, Julia Tucci, Guru Rao, and Heidi Sleister. Biology and BCMB, Drake University; BBMB, Iowa State University
PROJECT AIM & SIGNIFICANCEPROJECT AIM: To express and purify the leucine rich repeats (LRRs) from plant receptor-like kinases (RLKs) CLV1 and Xa21 as soluble fusions with SUMO protein in E. coli.
SIGNIFICANCE: If successful, this project will be the first reported example of soluble expression of a large plant LRR region in E. coli. In the future, soluble LRR protein will be used as the target in peptide phage display to isolate peptides that bind the LRR.
LRR region
N-terminal regionFlanking paired Cys
Flanking paired CysTransmembrane domain
Kinase domain
VISLNVSFTPLFGTISPEIGMLTHLVNL TLAANNFTGELPLEMKSLTSLKVL NISNNGNLTGTFPGEILKAMVDLEVL DTYNNNFNGKLPPEMSELKKLKYL SFGGNFFSGEIPESYGDIQSLEYL GLNGAGLSGKSPAFLSRLKNLRE MYIGYYNSYTGGVPPEFGGLTKLEIL DMASCTLTGEIPTSLSNLKHLHTL FLHINNLTGHIPPELSGLVSLKSL DLSINQLTGEIPQSFINLGNITLI NLFRNNLYGQIPEAIGELPKLEVF EVWENNFTLQLPANLGRNGNLIKL DVSDNHLTGLIPKDLCRGEKLEML ILSNNFFFGPIPEELGKCKSLTKI RIVKNLLNGTVPAGLFNLPLVTII ELTDNFFSGELPVTMSGDVLDQI YLSNNWFSGEIPPAIGNFPNLQTL FLDRNRFRGNIPREIFELKHLSRI NTSANNITGGIPDSISRCSTLISV DLSRNRINGEIPKGINNVKNLGTL NISGNQLTGSIPTGIGNMTSLTTL DLSFNDLSGRVPLGGQFL
CLV1 is important for shoot apical meristem development. CLV’s natural ligand is a 13-amino acid arabinosylated peptide derived from 96-amino acid CLV3 (RTVPSGPDPLHHH).
VVKLLLRSSNLSGIISPS LGNLSFLRELDLGDNYLSGEIPPE LSRLSRLQLLELSDNSIQGSIPAA IGACTKLTSLDLSHNQLRGMIPREI GASLKHLSNLYLYKNGLSGEIPSA LGNLTSLQEFDLSFNRLSGAIPSS LGQLSSLLTMNLGQNNLSGMIPNS IWNLSSLRAFSVRENKLGGMIPTNA FKTLHLLEVIDMGTNRFHGKIPAS VANASHLTVIQIYGNLFSGIITSG FGRLRNLTELYLWRNLFQTREQDDWGFISD LTNCSKLQTLNLGENNLGGVLPNSF SNLSTSLSFLALELNKITGSIPKD IGNLIGLQHLYLCNNNFRGSLPSS LGRLKNLGILLAYENNLSGSIPLA IGNLTELNILLLGTNKFSGWIPYT LSNLTNLLSLGLSTNNLSGPIPSE LFNIQTLSIMINVSKNNLEGSIPQE IGHLKNLVEFHAESNRLSGKIPNT LGDCQLLRYLYLQNNLLSGSIPSA LGQLKGLETLDLSSNNLSGQIPTS LADITMLHSLNLSFNSFVGEVPT IGAFAAASG ISIQGNAKLCGGIP
Xa21 is important for resistance to Xanthomonas oryzae which causes rice bacterial blight disease. A 17-amino acid sulfated peptide derived from 194-amino acid Ax21 (AENLSYNFVEGDYVRTP) functions as a ligand for Xa21.
LRR region
N-terminal region
Transmembrane domain
Kinase domain
C-terminal domain
RECEPTOR-LIKE KINASES CLV1 AND Xa21
EXPERIMENTAL METHODS
A library of phage, each displaying a different peptide, is allowed to bind LRR-HaloTag fusion protein immobilized on HaloLink magnetic beads.
pha
ge
magnetic beadHaloTag
LRR
peptide
Specifically-bound phage are eluted, amplified, and used for the next round of panning.
After ~4 rounds of panning, individual clones are isolated and sequenced.
Pa
nn
ing
pro
ces
s is
rep
eat
ed
~3
tim
es.
Unbound phage are washed away.
Panning Phage Display Library for Peptides that Bind CLV1 / Xa21 LRR Regions
Cut PCR product with EcoRV, gel purify
Linearize vector pSUMO_Kan with BsaI, polish ends with Pfu DNA Polymerase,
dephosphorylate, gel purify
Ligate prepared vector + prepared CLV1/Xa21 LRR
Transform ligation mixture into E. coli competent cells
Screen transformants by colony PCR, restriction digests, and sequence analysis
Express LRR region fused with SUMO in bacteria
PCR amplify CLV1 and Xa21 LRR regions to incorporate protein tags (Halo or FLAG) and 5’ and 3’ EcoRV
restriction sites
EcoRV
CLV1 / Xa21 LRR region
EcoRV
Isolate soluble SUMO-LRR fusion protein by immobilized metal affinity chromatography (IMAC)
FUTURE WORK
RESULTS
Figure 1. Structures of CLV1 and Xa21 proteins. The leucine rich repeat (LRR) region is the extracellular portion of the protein that binds a ligand to initiate signal transduction.
Figure 2. Methods to clone and express CLV1 and Xa21 LRRs as fusions with SUMO protein.
Table 1. PCR primers and conditions
CLV1(LRR) Halo
CLV1(LRR) FLAG
Xa21(LRR) Halo
Xa21(LRR) FLAG
Figure 5. Protein expression constructs.
1. Lambda-HindIII marker2. Xa21(LRR)-Halo PCR product3. Xa21(LRR)-FLAG PCR product4. CLV1(LRR)-Halo PCR product5. CLV1(LRR)-FLAG PCR product6. Uncut plasmid template for CLV1 PCR reaction7. Uncut plasmid template for Xa21 PCR reaction8. Lambda-HindIII marker
Figure 3. 0.8% agarose gel of CLV1(LRR) and Xa21(LRR) PCR products. The size of each PCR product (lanes 2-5, marked with arrows) is correct.
1 2 3 4 5 6 7 8
2.3 kb
2.0 kb
1 2 3
1. pSUMO vector cut with BsaI2. Uncut pSUMO vector3. Lambda-HindIII marker
Figure 4. Preparation of pSUMO vector. 0.8% agarose gel of BsaI-cut and uncut pSUMO.
• Construction of CLV1 and Xa21 LRRs as fusions with SUMO is in progress. The vector and LRR inserts were successfully isolated and ligated. Transformants are being screened for presence of the insert.
• Validation of a phage display combinatorial approach to isolate peptide ligands for plant RLKs CLV1 and Xa21 may lead to general utility of this system for signal transduction in plants and animals.
PCR Primer Name Primer SequencePrimer Length (Nucleotides)
PCR ConditionsPCR Product
Size (Basepairs)
SUMO-CLV1-F2 AGGTGATATCGACGACGATGCTCGTGTTATC 31
SUMO-CLV1-Halo-R2 CTAGGATATCTTAACCGGAAATCTCCAGAG 30
SUMO-CLV1-F2 AGGTGATATCGACGACGATGCTCGTGTTATC 31
SUMO-CLV1-Flag-R2 CTAGGATATCTTACTTATCGTCGTCATCCTTGTAATCGGAAGTTTCGTTGAAC 53
SUMO-Xa21-F2 AGGTGATATCCGCCGGCACCCACACAG 27
SUMO-Xa21-Halo-R2 CTAGGATATCTTAACCGGAAATCTCCAGAGTAG 33
SUMO-Xa21-F2 AGGTGATATCCGCCGGCACCCACACAG 27
SUMO-Xa21-Flag-R2 CTAGGATATCTTACTTATCGTCGTCATCCTTGTAATCTCGAGGCAGATGTAGATC 55
2582 bp
1664 bp
2663 bp
1745 bp
2 min 94oC; 35 cycles [45
sec 94oC, 45 sec 50oC, 3
min 72oC]; 10 min 72oC
2 min 94oC; 35 cycles [45
sec 94oC, 45 sec 50oC, 3
min 72oC]; 10 min 72oC
2 min 94oC; 35 cycles [45
sec 94oC, 45 sec 52oC, 3
min 72oC]; 10 min 72oC
2 min 94oC; 35 cycles [45
sec 94oC, 45 sec 52oC, 3
min 72oC]; 10 min 72oC
http://faculty.washington.edu/ktorii/ERECTA04.htmlhttp://www.google.com/imgres?imgurl=http://www.carto.net/neumann/travelling/japan_2004_09/04_kyoto_2004_09_12/29_rice_plant.jpg&imgrefurl=http://www.carto.net/neumann/travelling/japan_2004_09/04_kyoto_2004_09_12/&h=614&w=819&sz=65&tbnid=6awJqYynLZFAMM:&tbnh=108&tbnw=144&prev=/search%3Fq%3Drice%2Bplant%26tbm%3Disch%26tbo%3Du&zoom=1&q=rice+plant&usg=__of-NUAjqtzK9TJpvVmYzMzpNd_M=&sa=X&ei=azibTbOnCoz2swO94ImbBA&ved=0CDEQ9QEwAA
