A Novel Function for the Highly Conserved Signal Peptidase in the Regulation of Biotrophy in a Plant Pathogenic Fungus

Plant pathogens, most of them fungi, destroy at least 10% of the global food supply annually and represent an increasing threat to world food security. Fungal pathogens secrete diverse arrays of "effector" proteins into living host tissues, where they induce accessibility of the plant cells by suppressing resistance and promoting susceptibility. There is clear evidence for specific temporal and spatial expression of these effectors, suggesting a variety of important roles in multiple aspects of pathogenesis. Although we have made significant progress on identifying the modes of action of individual effectors, and have catalogued the effector repertoires of many pathogenic fungi, we still know very little about the underlying mechanisms that regulate the production and secretion of effectors in planta. And yet, given that individual effectors are often functionally redundant and poorly conserved across taxa, understanding the secretion mechanism of effectors offers our best hope for broad-spectrum control of these devastating fungal pathogens. We propose to characterize a novel phenomenon that may reveal a previously unknown mechanism regulating effector secretion in the maize anthracnose fungus, Colletotrichum graminicola. Anthracnose causes losses of approximately 5% of the potential maize yield in the U.S. annually (28). This would have been worth about 2.5 billion dollars in 2015. Using a random mutagenesis approach, we discovered that a highly conserved subunit of the endoplasmic reticulum (ER)-localized signal peptidase complex (SPC) controls the biotrophic establishment of C. graminicola in living maize tissues, in a process that is separate from its normal "housekeeping" function. We named this SPC component CPR1. Our hypothesis is that CPR1 undergoes post-transcriptional and/or post-translational modifications in response to plant signals and fungal stress, and acts as a developmental switch controlling the secretion of effector proteins that are necessary for the induction of host cell accessibility and biotrophic colonization. Our first objective will be to evaluate in detail the role of CPR1 in the secretion of fungal effectors by tracking a selected group of fluorescently-tagged proteins in the CPR1-mutant (MT) and wild type (WT) strains in planta and in vitro in the presence and absence of stress. Our second objective will be to characterize and compare post-transcriptional and post-translational modifications of CPR1 in the MT versus WT strains during biotrophic establishment and in response to stress. If our hypothesis is supported, this work will result in: (1) an association between the CPR1 mutation and loss of effector secretion; (2) an understanding of the role of post-transcriptional/post-translational modifications in the regulation of CPR1; and (3) a mechanistic insight into the specific secretion of effectors via a previously unknown CPR1- regulated pathway. The proposed research will address how a housekeeping function (processing of pre-proteins during protein transport) may have evolved to acquire specific new roles in conditioning virulence in plant-pathogenic fungi. CPR1 is highly conserved among fungi, thus it may provide a conserved target for the development of broad-spectrum control of fungal pathogens.