Continual green-fluorescent protein monitoring of cauliflower mosaic virus 35S promoter activity in nematode-induced feeding cells in Arabidopsis thaliana

被引:77
作者
Urwin, PE [1 ]
Moller, SG [1 ]
Lilley, CJ [1 ]
McPherson, MJ [1 ]
Atkinson, HJ [1 ]
机构
[1] UNIV LEEDS, CTR PLANT BIOCHEM & BIOTECHNOL, LEEDS LS2 9JT, W YORKSHIRE, ENGLAND
关键词
regulation; transgenic; GENE-EXPRESSION; TRANSCRIPTS; ROOTS; HOST;
D O I
10.1094/MPMI.1997.10.3.394
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The responsiveness of the cauliflower mosaic virus 35S promoter in feeding sites developed by both sexes of Heterodera schachtii and female Meloidogyne incognito has been studied, The objective was to establish the value of green-fluorescent protein (GFP) as a nondestructive reporter gene system for characterizing promoter activity at nematode feeding sites in vivo. Growth units were devised that allowed individual feeding sites in roots of Arabidopsis thaliana to be observed by both bright-field and epifluorescent illumination. Changes in GFP expression were visually observed under experimental conditions that resulted in chloroplast formation in syncytia but not other root cells, Changes in GFP levels altered the extent of quenching, by this protein, of red light emitted by chlorophyll within the chloroplasts under violet excitation. Image analysis provided a semiquantitative basis for simultaneous measurement of changes in GFP fluorescence and the unquenched emission by chlorophyll, GFP levels were constant in cells surrounding the syncytium induced by H. schachtii, but they fell progressively from 10 to 35 days postinfection within this structure, Significant reduction in GFP levels was not limited to the early part of the time course but also occurred between 27 and 35 days postinfection, GFP was detected by immunoblotting in females of M. incognita but not in H. schachtii parasitizing similar GFP-expressing roots.
引用
收藏
页码:394 / 400
页数:7
相关论文
共 26 条
[11]   CHANGES IN ABUNDANCE OF TRANSLATABLE MESSENGER-RNA SPECIES IN POTATO ROOTS AND LEAVES FOLLOWING ROOT INVASION BY CYST-NEMATODE G-ROSTOCHIENSIS PATHOTYPES [J].
HAMMONDKOSACK, KE ;
ATKINSON, HJ ;
BOWLES, DJ .
PHYSIOLOGICAL AND MOLECULAR PLANT PATHOLOGY, 1990, 37 (05) :339-354
[12]   Differential expression patterns of the wound-inducible transgene wun1-uidA in potato roots following infection with either cyst or root knot nematodes [J].
Hansen, E ;
Harper, G ;
McPherson, MJ ;
Atkinson, HJ .
PHYSIOLOGICAL AND MOLECULAR PLANT PATHOLOGY, 1996, 48 (03) :161-170
[13]   GFP IN PLANTS [J].
HASELOFF, J ;
AMOS, B .
TRENDS IN GENETICS, 1995, 11 (08) :328-329
[14]   CDNA LIBRARY CONSTRUCTION FROM SMALL AMOUNTS OF RNA USING PARAMAGNETIC BEADS AND PCR [J].
LAMBERT, KN ;
WILLIAMSON, VM .
NUCLEIC ACIDS RESEARCH, 1993, 21 (03) :775-776
[15]   Characterization of intestinally active proteinases of cyst-nematodes [J].
Lilley, CJ ;
Urwin, PE ;
McPherson, MJ ;
Atkinson, HJ .
PARASITOLOGY, 1996, 113 :415-424
[16]  
LILLEY CJ, 1997, Patent No. 95243952
[17]   ROOT-KNOT NEMATODE-DIRECTED EXPRESSION OF A PLANT ROOT-SPECIFIC GENE [J].
OPPERMAN, CH ;
TAYLOR, CG ;
CONKLING, MA .
SCIENCE, 1994, 263 (5144) :221-223
[18]   GREEN-FLUORESCENT PROTEIN AS A NEW VITAL MARKER IN PLANT-CELLS [J].
SHEEN, J ;
HWANG, SB ;
NIWA, Y ;
KOBAYASHI, H ;
GALBRAITH, DW .
PLANT JOURNAL, 1995, 8 (05) :777-784
[19]   EFFICIENT TRANSFORMATION OF AGROBACTERIUM SPP BY HIGH-VOLTAGE ELECTROPORATION [J].
SHEN, WJ ;
FORDE, BG .
NUCLEIC ACIDS RESEARCH, 1989, 17 (20) :8385-8385
[20]   PARASITIC STRATEGIES OF ROOT NEMATODES AND ASSOCIATED HOST-CELL RESPONSES [J].
SIJMONS, PC ;
ATKINSON, HJ ;
WYSS, U .
ANNUAL REVIEW OF PHYTOPATHOLOGY, 1994, 32 :235-259