Journal
MOLECULAR PLANT PATHOLOGY
Volume 16, Issue 3, Pages 262-275Publisher
WILEY
DOI: 10.1111/mpp.12179
Keywords
cell wall-degrading enzymes; fruit-pathogen interaction; gene expression; gene knockout; phytopathogenic fungi; proteases
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Funding
- Spanish Ministry of Economy and Innovation [AGL-2008-04828-C03-02, AGL2011-30519-C03-01]
- Generalitat Valenciana [PROMETEO/2010/010, PROMETEOII/2014/027, ACOMP/2011/250]
- Formacion de Personal Investigador scholarship [BES-2006-12983]
- Spanish National Research Council
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The fungus Penicillium digitatum, the causal agent of green mould rot, is the most destructive post-harvest pathogen of citrus fruit in Mediterranean regions. In order to identify P. digitatum genes up-regulated during the infection of oranges that may constitute putative virulence factors, we followed a polymerase chain reaction (PCR)-based suppression subtractive hybridization and cDNA macroarray hybridization approach. The origin of expressed sequence tags (ESTs) was determined by comparison against the available genome sequences of both organisms. Genes coding for fungal proteases and plant cell wall-degrading enzymes represent the largest categories in the subtracted cDNA library. Northern blot analysis of a selection of P. digitatum genes, including those coding for proteases, cell wall-related enzymes, redox homoeostasis and detoxification processes, confirmed their up-regulation at varying time points during the infection process. Agrobacterium tumefaciens-mediated transformation was used to generate knockout mutants for two genes encoding a pectin lyase (Pnl1) and a naphthalene dioxygenase (Ndo1). Two independent P. digitatum Delta ndo1 mutants were as virulent as the wild-type. However, the two Delta pnl1 mutants analysed were less virulent than the parental strain or an ectopic transformant. Together, these results provide a significant advance in our understanding of the putative determinants of the virulence mechanisms of P. digitatum.
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