A systematic analysis of T-DNA insertion events in Magnaporthe oryzae.
We describe here the analysis of random T-DNA insertions that were generated as part of a large-scale insertional mutagenesis project for Magnaporthe oryzae. Chromosomal regions flanking T-DNA insertions were rescued by inverse PCR, sequenced and used to search the M. oryzae genome assembly. Among the 175 insertions for which at least one flank was rescued, 137 had integrated in single-copy regions of the genome, 17 were in repeated sequences, one had no match to the genome, and the remainder were unassigned due to illegitimate T-DNA integration events. These included in order of abundance: head-to-tail tandem insertions, right border excision failures, left border excision failures and insertion of one T-DNA into another. The left borders of the T-DNA were frequently truncated and inserted in sequences with micro-homology to the left terminus. By contrast the right borders were less prone to degradation and appeared to have been integrated in a homology-independent manner. Gross genome rearrangements rarely occurred when the T-DNAs integrated in single-copy regions, although most insertions did cause small deletions at the target site. Significant insertion bias was detected, with promoters receiving two times more T-DNA hits than expected, and open reading frames receiving three times fewer. In addition, we found that the distribution of T-DNA inserts among the M. oryzae chromosomes was not random. The implications of these findings with regard to saturation mutagenesis of the M. oryzae genome are discussed.
Meng
Yan
Y
Department of Plant Pathology, University of Kentucky, Lexington, KY 40546, USA.
Patel
Gayatri
G
Heist
Melanie
M
Betts
Melania F
MF
Tucker
Sara L
SL
Galadima
Natalia
N
Donofrio
Nicole M
NM
Brown
Doug
D
Mitchell
Thomas K
TK
Li
Lei
L
Xu
Jin-Rong
JR
Orbach
Marc
M
Thon
Michael
M
Dean
Ralph A
RA
Farman
Mark L
ML
D024509:3' Flanking Region; D024506:5' Flanking Region; D016960:Agrobacterium tumefaciens / Q000235:genetics; D015825:Chromosomes, Fungal; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D016681:Genome, Fungal; D020082:Magnaporthe / Q000235:genetics*; D016254:Mutagenesis, Insertional; D012091:Repetitive Sequences, Nucleic Acid; D014170:Transformation, Genetic
D004269:DNA, Bacterial; C036483:T-DNA