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

Abstract

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 PiGPAI, 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 data-bases 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.