Characterization of T-DNA insertion patterns in the genome of rice blast fungus Magnaporthe oryzae

Abstract

Agrobacterium tumefaciens-mediated transformation (ATMT) has been proven to be a powerful strategy for gene disruption in plants and fungi. Patterns associated with transferred DNA (T-DNA) integration in plants and yeast have been studied comprehensively, whereas no detailed analysis of T-DNA integration has been reported yet in filamentous fungi. Here, we reported the T-DNA insertion patterns in the genome of filamentous fungus Magnaporthe oryzae. Using ATMT, a T-DNA tagged population consisting of 6,179 transformants of M. oryzae was constructed. With thermal asymmetric interlaced-PCR (TAIL-PCR), 623 right border (RB) flanking sequences and 124 left border (LB) flanking sequences were generated. Analysis of these flanking sequences indicated a significant integration bias toward non-coding sequences, suggesting distribution of T-DNAs was not random. Comparing to T-DNA RB, LB was nicked inaccurately and truncated frequently during integration. Chromosomal rearrangements, such as deletion, inversion, and translocation, were associated with T-DNA integration in some transformants. Our data suggest that, comparing with plant cells, T-DNA integrates into this filamentous fungus with more precise and simpler patterns. Some phenotypic mutants were observed in our T-DNA tagged population, and these transformants will be very useful for functional genomics research of M. oryzae.