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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.

Powdery mildew is a widespread plant disease caused by obligate biotrophic fungal pathogens involving species-specific interactions between host and parasite. To gain genomic insights into the underlying obligate biotrophic mechanisms, we analyzed 15 microbial genomes covering powdery and downy mildews and rusts. We observed a genome-wide, massive contraction of multiple gene families in powdery mildews, such as enzymes in the carbohydrate metabolism pathway, when compared with ascomycete phytopathogens, while the fatty acid metabolism pathway maintained its integrity. We also observed significant differences in candidate secreted effector protein (CSEP) families between monocot and dicot powdery mildews, perhaps due to different selection forces. While CSEPs in monocot mildews are likely subject to positive selection causing rapid expansion, CSEP families in dicot mildews are shrinking under strong purifying selection. Our results not only illustrate obligate biotrophic mechanisms of powdery mildews driven by gene family evolution in nutrient metabolism, but also demonstrate how the divergence of CSEPs between monocot and dicot lineages might contribute to species-specific adaption.

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