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Identification of Pathogenesis-Associated Genes by T-DNA–Mediated Insertional Mutagenesis in Botrytis cinerea: A Type 2A Phosphoprotein Phosphatase and an SPT3 Transcription Factor Have Significant Impact on Virulence

April 2012 , Volume 25 , Number  4
Pages  481 - 495

S. Giesbert,1 J. Schumacher,1 V. Kupas,1 J. Espino,1 N. Segmüller,1 I. Haeuser-Hahn,2 P. H. Schreier,2 and P. Tudzynski1

1Institut für Biologie und Biotechnologie der Pflanzen, Westf. Wilhelms-Universität, Hindenburgplatz 55, D-48143 Münster, Germany; 2Bayer Crop Science AG, Alfred Nobel-Straße 50, D-40789 Monheim, Germany

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Accepted 16 November 2011.

Agrobacterium tumefaciens–mediated transformation (ATMT) was used to generate an insertional mutant library of the gray mold fungus Botrytis cinerea. From a total of 2,367 transformants, 68 mutants showing significant reduction in virulence on tomato and bean plants were analyzed in detail. As reported for other fungal ATMT libraries, integrations were mostly single copy, occurred preferentially in noncoding (regulatory) regions, and were frequently accompanied by small deletions of the target sequences and loss of parts of the border sequence. Two T-DNA integration events that were found to be linked to virulence were characterized in more detail: a catalytic subunit of a PP2A serine/threonine protein phosphatase (BcPP2Ac) and the SPT3 subunit of a Spt-Ada-Gcn5-acetyltransferase (SAGA-like) transcriptional regulator complex. Gene replacement and silencing approaches revealed that both Bcpp2Ac and SPT3 are crucial for virulence, growth, and differentiation as well as for resistance to H2O2 in B. cinerea.

© 2012 The American Phytopathological Society