Homologous Expression and T3SS-Dependent Secretion of TAP-Tagged Xo2276 in Xanthomonas oryzae pv. oryzae Induced by Rice Leaf Extract and Its Direct In Vitro Recognition of Putative Target DNA Sequence원문보기
Kim, Seunghwan
(Department of Advanced Technology Fusion, Konkuk University)
,
Nguyen, Thi-Dieu-Hanh
(Department of Bioscience and Biotechnology, Konkuk University)
,
Lee, Joohee
(Department of Green Life Science, College of Convergence, Sangmyung University)
,
Hong, Myoung-Ki
(Department of Advanced Technology Fusion, Konkuk University)
,
Pham, Tan-Viet
(Department of Advanced Technology Fusion, Konkuk University)
,
Ahn, Yeh-Jin
(Department of Green Life Science, College of Convergence, Sangmyung University)
,
Lee, Byoung-Moo
(Genomics Division, National Academy of Agricultural Science (NAAS), Rural Development Administration (RDA))
,
Han, Ye Sun
(Department of Advanced Technology Fusion, Konkuk University)
,
Kim, Dong-Eun
(Department of Bioscience and Biotechnology, Konkuk University)
,
Kim, Jeong-Gu
(Genomics Division, National Academy of Agricultural Science (NAAS), Rural Development Administration (RDA))
,
Kang, Lin-Woo
(Department of Biological Sciences, Konkuk University)
Xo2276 is a putative transcription activator-like effector (TALE) in Xanthomonas oryzae pv. oryzae (Xoo). Xo2276 was expressed with a TAP-tag at the C-terminus in Xoo cells to enable quantitative analysis of protein expression and secretion. Nearly all TAP-tagged Xo2276 existed in an insoluble form;...
Xo2276 is a putative transcription activator-like effector (TALE) in Xanthomonas oryzae pv. oryzae (Xoo). Xo2276 was expressed with a TAP-tag at the C-terminus in Xoo cells to enable quantitative analysis of protein expression and secretion. Nearly all TAP-tagged Xo2276 existed in an insoluble form; addition of rice leaf extracts from a Xoosusceptible rice cultivar, Milyang23, significantly stimulated secretion of TAP-tagged Xo2276 into the medium. In a T3SS-defective Xoo mutant strain, secretion of TAPtagged Xo2276 was blocked. Xo2276 is a Xoo ortholog of Xanthomonas campestris pv. vesicatoria (Xcv) AvrBs3 and contains a conserved DNA-binding domain (DBD), which includes 19.5 tandem repeats of 34 amino acids. Xo2276- DBD was expressed in E. coli and purified. Direct in vitro recognition of Xo2276-DBD on a putative target DNA sequence was confirmed using an electrophoretic mobility shift assay. This is the first study measuring the homologous expression and secretion of Xo2276 in vitro using rice leaf extract and its direct in vitro binding to the specific target DNA sequence.
Xo2276 is a putative transcription activator-like effector (TALE) in Xanthomonas oryzae pv. oryzae (Xoo). Xo2276 was expressed with a TAP-tag at the C-terminus in Xoo cells to enable quantitative analysis of protein expression and secretion. Nearly all TAP-tagged Xo2276 existed in an insoluble form; addition of rice leaf extracts from a Xoosusceptible rice cultivar, Milyang23, significantly stimulated secretion of TAP-tagged Xo2276 into the medium. In a T3SS-defective Xoo mutant strain, secretion of TAPtagged Xo2276 was blocked. Xo2276 is a Xoo ortholog of Xanthomonas campestris pv. vesicatoria (Xcv) AvrBs3 and contains a conserved DNA-binding domain (DBD), which includes 19.5 tandem repeats of 34 amino acids. Xo2276- DBD was expressed in E. coli and purified. Direct in vitro recognition of Xo2276-DBD on a putative target DNA sequence was confirmed using an electrophoretic mobility shift assay. This is the first study measuring the homologous expression and secretion of Xo2276 in vitro using rice leaf extract and its direct in vitro binding to the specific target DNA sequence.
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제안 방법
(A) Induced secretion of Xo2276-TAP by IRBB-3 RLX and Milyang 23 RLX in 20 h. A dilution series (1:5) of the soluble medium from Xoo transformant cultures with IRBB-3 RLX and Milyang 23 RLX was prepared and blotted as described in Materials and Methods. Total cell extracts from Xo2276-TAP Xoo transformants and wild-type Xoo cells were used as positive and negative controls for a TAP-tag detection, respectively.
대상 데이터
oryzae (Xoo) strain KACC10331, Xoo strain KACC10859, pVFT6S vector, and pHM1 vector were stored by our laboratory. The Xoo strain KACC13129 (∆hrcC) was provided by KACC (Korean Agricultural Culture Collection, Gyunggi Province, Republic of Korea). Escherichia coli strain BL21-Gold (DE3) and Rpb3-TAP-tagged yeast strain ysc1177 were purchased from Stratagene (La Jolla, CA, USA) and OPEN Biosystem (Huntsville, AL, USA), respectively.
이론/모형
5A). Specific recognition of the putative target DNA sequence was examined using EMSA. Recently, the specific DNA sequence recognition mechanism of TALE was elucidated [3].
성능/효과
The purified Xo2276-DBD specifically recognized the target DNA sequence in the EMSA experiment. Our results showed that Xo2276 is a putative Xoo effector, which could be secreted by the pathogenic interaction with rice, and Xo2276-DBD specifically recognized the target DNA sequence in vitro.
참고문헌 (17)
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