Navarrete, F. | Search bioRxiv

bioRxiv2019

The Pleiades cluster of fungal effector genes inhibit 1 host defenses

SummaryBiotrophic plant pathogens secrete effector proteins to manipulate the host physiology. Effectors suppress defenses and induce an environment favorable to disease development. Sequence-based prediction of effector function is difficulted by their rapid evolution rate. In the maize pathogen Ustilago maydis, effector-coding genes frequently organize in clusters. Here we describe the functional characterization of the pleiades, a cluster of ten symplastic effectors. Deletion of the pleiades leads to strongly impaired virulence and accumulation of reactive oxygen species (ROS) in infected tissue. Eight of the Pleiades suppress the production of ROS upon perception of pathogen associated molecular patterns (PAMPs). Although genetically redundant, the Pleiades target different host components. The paralogs Taygeta1 and Merope1 suppress ROS production in either the cytoplasm or nucleus, respectively. Merope1 targets and promotes the autoubiquitination activity of RFI2, a conserved family of E3 ligases that regulates the production of PAMP-triggered ROS burst and influences flowering time in plants.

microbiology↗

bioRxiv2019

A High-throughput Screening Method to Identify Proteins Involved in Unfolded Protein Response Signaling in Plants

BackgroundThe unfolded protein response (UPR) is a highly conserved process in eukaryotic organisms that plays a crucial role in adaptation and development. While the most ubiquitous components of this pathway have been characterized, current efforts are focused on identifying and characterizing other UPR factors that play a role in specific conditions, such as developmental changes, abiotic cues, and biotic interactions. Considering the central role of protein secretion in plant pathogen interactions, there has also been a recent focus on understanding how pathogens manipulate their hosts UPR to facilitate infection.\n\nResultsWe developed a high-throughput screening assay to identify proteins that interfere with UPR signaling in planta. A set of 35 genes from a library of secreted proteins from the maize pathogen Ustilago maydis were transiently co-expressed with a reporter construct that upregulates enhanced yellow fluorescent protein (eYFP) expression upon UPR stress in Nicotiana benthamiana plants. After UPR stress induction, leaf discs were placed in 96 well plates and eYFP expression was measured. This allowed us to identify a previously undescribed fungal protein that inhibits plant UPR signaling, which was then confirmed using the classical but more laborious qRT-PCR method.\n\nConclusionsWe have established a rapid and reliable fluorescence-based method to identify heterologously expressed proteins involved in UPR stress in plants. This system can be used for initial screens with libraries of proteins and potentially other molecules to identify candidates for further validation and characterization.

plant biology↗