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Downstream targets of the Phytophthora infestans Gα subunit PiGPA1 revealed by cDNA-AFLP

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In many plant pathogens heterotrimeric G-proteins are essential signalling components involved in development and pathogenicity. In the late blight oomycete pathogen Phytophthora infestans the G-protein α subunit PiGPA1 controls zoospore motility and is required for virulence. To identify G-protein targets and signalling pathways downstream of PiGPA1, we used an optimized cDNA-AFLP protocol for analysing gene expression profiles in hypovirulent P.¬†infestans strains that were previously generated by silencing the Pigpa1 gene. First, expression profiles in sporangia and mycelium of the wild-type strain were compared, and this revealed a substantial number of mycelium- or sporangia-specific transcript derived fragments (TDFs). Subsequently, profiles in sporangia of wild-type, Pigpa1-silenced mutants and of a strain expressing a constitutively active form of PiGPA1 were compared. From a total of 2860 TDFs, 92 were down- and 19 up-regulated in the Pigpa1-silenced mutants. A subset of the differential TDFs was cloned and sequenced, and homology searches were carried out against Phytophthora EST and genomic databases and the NCBI database. cDNA-AFLP expression profiles were verified by Northern blot analysis or RT-PCR. The power of cDNA-AFLP for the identification of target genes in knock-down or gain-of-function mutants is discussed.

Document Type: Research Article


Affiliations: Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, NL-6709 PD Wageningen and Graduate School Experimental Plant Sciences, The Netherlands

Publication date: 2004-09-01

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