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

Abstract

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 H(2)O(2) in B. cinerea.

Developmental and metabolic plasticity of white-skinned grape berries in response to Botrytis cinerea during noble rot

“…The defense mechanisms established by the plant, which could have slowed down the infection, were inadequate to contain the spread of B. cinerea mycelia in the berry. B. cinerea may also rely on virulence factors, such as fungal protein phosphatases (Amselem et al, 2011;Giesbert et al, 2012;Yang et al, 2013), to alter plant signaling cascades (i.e. mitogen-activated protein kinase cascades) that are crucial for berry development and responses to environmental cues.…”

“…) to the positive sides of DIM1 and DIM2 (Supplemental Table S5; Amselem et al, 2011;Giesbert et al, 2012;Yang et al, 2013;Ambrose et al, 2015). These comprised two putative salicylate hydroxylases (BC1T_14568 and BC1T_15387), a protein-Tyr phosphatase (BC1T_07726), a Ser/Thr protein phosphatase type 2A (BC1T_05741), and a nonribosomal peptide synthetase (BC1T_01317).…”

Tools for Botrytis cinerea: New expression vectors make the gray mold fungus more accessible to cell biology approaches

“…RNA-mediated gene silencing approaches were also implemented as an alternative strategy for gene function characterization especially for essential genes (Patel et al, 2008;Schumacher et al, 2008b). In addition, random mutagenesis via Agrobacterium tumefaciens-mediated transformation (ATMT) has been shown to be a useful tool for the identification of new virulence-associated genes (Giesbert et al, 2012).…”

Functional Analysis of BcBem1 and Its Interaction Partners in Botrytis cinerea: Impact on Differentiation and Virulence

“…In parallel to this study, we identified bcsep1 as a virulence-associated gene by performing a random mutagenesis approach via Agrobacterium tumefaciens -mediated transformation (ATMT) [32]. TAIL-PCR analyses revealed that the left border (LB) of the T-DNA was inserted 1,270 bp upstream of bcsep1 in the non-pathogenic transformant PA3135.…”