Two linked genes encoding a secreted effector and a membrane protein are essential for Ustilago maydis-induced tumour formation

Ustilago maydis is a biotrophic fungal pathogen that colonizes living tissue of its host plant maize. Based on transcriptional upregulation during biotrophic development we identified the pit (proteins important for tumours) cluster, a novel gene cluster comprising four genes of which two are predicted to encode secreted effectors. Disruption of the gene cluster abolishes U. maydis-induced tumour formation and this phenotype can be caused by deleting either pit1 encoding a transmembrane protein or pit2 encoding a secreted protein. Pit1 localizes to the fungal plasma membrane at hyphal tips, endosomes and vacuoles while Pit2 is secreted to the biotrophic interface. Both Delta pit1 and Delta pit2 mutants are able to penetrate maize epidermis and grow intracellularly at sites of infection but fail to spread in the infected leaf. Microarray analysis shows an indistinguishable response of the plant to infection by Delta pit1 and Delta pit2 mutant strains. Transcriptional activation of maize defence genes in infections with Delta pit1/2 mutant strains indicates that the mutants have a defect in suppressing plant immune responses. Our results suggest that the activity of Pit1 and Pit2 during tumour formation might be functionally linked and we discuss possibilities for a putative functional connection of the two proteins.