Plant perception of pathogen-associated molecular patterns (PAMPs) triggers a phosphorylation relay leading to PAMP-triggered immunity (PTI). Despite increasing knowledge of PTI signaling, how immune homeostasis is maintained remains largely unknown. Here we describe a forward-genetic screen to identify loci involved in PTI and characterize the Arabidopsis calcium-dependent protein kinase CPK28 as a negative regulator of immune signaling. Genetic analyses demonstrate that CPK28 attenuates PAMP-triggered immune responses and antibacterial immunity. CPK28 interacts with and phosphorylates the plasma-membrane-associated cytoplasmic kinase BIK1, an important convergent substrate of multiple pattern recognition receptor (PRR) complexes. We find that BIK1 is rate limiting in PTI signaling and that it is continuously turned over to maintain cellular homeostasis. We further show that CPK28 contributes to BIK1 turnover. Our results suggest a negative regulatory mechanism that continually buffers immune signaling by controlling the turnover of this key signaling kinase.

With the exception of Ustilago maydis, smut fungi infecting monocotyledonous hosts systemically colonize infected plants and cause symptoms exclusively in the inflorescences. Ustilago may disinfects primordia of all aerial organs of maize (Zea mays L.) and results in the formation of large plant tumours. Previously, we have found that U. maydis infection of seedling leaves, adult leaves and tassels causes organ-specific transcriptional changes in both the pathogen and the host. Of particular interest, U. may disgenes encoding secreted proteins are differentially expressed depending on the colonized maize organ. Therefore, we hypothesized that the fungus secretes virulence-related proteins (effectors)that act in an organ-specific manner. Here, we present the identification and functional characterization of 20 presumptive organ-specific U. maydis effector genes. Ustilago maydis deletion strains for these genes were generated and tested for infectivity of maize seedling leaves and tassels. This approach identified 11 effector genes required for the full virulence of U. maydis. In nine cases, virulence was only affected in one of the tested plant organs. These results demonstrate that individual fungal effector proteins contribute to fungal virulence in an organ-specific manner.

The biotrophic fungus Ustilago maydis causes smut disease on maize (Zea mays), which is characterized by immense plant tumours. To establish disease and reprogram organ primordia to tumours, U. maydis deploys effector proteins in an organ-specific manner. However, the cellular contribution to leaf tumours remains unknown. We investigated leaf tumour formation at the tissue- and cell type-specific levels. Cytology and metabolite analysis were deployed to understand the cellular basis for tumourigenesis. Laser-capture microdissection was performed to gain a cell type-specific transcriptome of U. maydis during tumour formation. In vivo visualization of plant DNA synthesis identified bundle sheath cells as the origin of hyperplasic tumour cells, while mesophyll cells become hypertrophic tumour cells. Cell type-specific transcriptome profiling of U. maydis revealed tailored expression of fungal effector genes. Moreover, U. maydis See1 was identified as the first cell type-specific fungal effector, being required for induction of cell cycle reactivation in bundle sheath cells. Identification of distinct cellular mechanisms in two different leaf cell types and of See1 as an effector for induction of proliferation of bundle sheath cells are major steps in understanding U. maydis-induced tumour formation. Moreover, the cell type-specific U. maydis transcriptome data are a valuable resource to the scientific community.