Identification of novel rate-limiting components in Arabidopsis cell entry control against non-adapted fungal pathogens
by Josephine Mittendorf
Date of Examination:2024-09-02
Date of issue:2024-09-27
Advisor:Prof. Dr. Volker Lipka
Referee:Prof. Dr. Volker Lipka
Referee:Prof. Dr. Ivo Feußner
Persistent Address:
http://dx.doi.org/10.53846/goediss-10756
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Abstract
English
Cell entry into host cells represents a crucial step during pathogenesis of fungal phytopathogens. However, plants have evolved an effective cell entry control mechanism to block a majority of intruders at the front line. Upon penetration attempts, the earliest cellular response is the rearrangement of the cytoskeleton towards host-pathogen contact sites, which is accompanied with organelle translocation and secretion processes to form cell wall appositions called papillae. In the past, forward genetic screens uncovered components called PENETRATION (PEN) that are involved in Arabidopsis cell entry control against non-adapted powdery mildew fungi. The plasma membrane (PM)-resident SOLUBLE N-ETHYLMALEIMIDE-SENSITIVE-FACTOR ATTACHMENT RECEPTOR (SNARE) protein PEN1 contributes to vesicle fusion events to ensure papilla formation. Another PEN1-independent pathway deals with the production of toxic indole glucosinolate-derived compounds by the atypical myrosinase PEN2, which are secreted by the ATP binding cassette (ABC) transporter PEN3 into the apoplastic battlefield. Upon pathogen attack, PEN1 and PEN3 were reported to be recycled from the PM to the trans-Golgi-network (TGN), followed by recruitment within extracellular papillae to terminate fungal invasion attempts. However, previous studies suggested that this pathogen-induced recruitment of PEN1 and PEN3 is facilitated by distinct trafficking pathways and depends on yet unknown fine-tuned selection processes at the TGN. In this study, a forward genetic screen uncovered functions of INOSITOL PHOSPHORYLCERAMIDE SYNTHASE 2 (IPCS2) in Arabidopsis cell entry control against non-adapted powdery mildew fungi. IPCS2 was recently described as a TGN-resident enzyme involved in the biosynthesis of complex sphingolipids. Further genetic and biochemical approaches in this study provided evidence that IPCS2-derived glycosyl inositol phosphorylceramides (GIPCs) are required for penetration resistance. Confocal laser scanning microscopy (CLSM) analyses revealed reduced focal accumulation of PEN3-GFP at sites of attempted fungal invasion in the absence of IPCS2, whereas genetic analyses suggested that IPCS2 is not involved in the PEN1-dependent pathway. Thus, a model was proposed in which IPCS2-derived complex sphingolipids facilitate sorting processes at the TGN, regulating polar secretion of PEN3 and not PEN1 to sites of attempted fungal invasion. In contrast to PEN1 and PEN3, PEN2 was previously observed to tag a mitochondria subpopulation that is recruited to and become immobilized at sites of fungal penetration attempts in single epidermal cells. This is coordinated with PEN2 substrate production via the cytochrome P450 monooxygenase CYP81F2 on the surface of the recruited endoplasmic reticulum (ER). As the molecular basis for these pathogen-induced polarization events is not fully understood, this study provides an innovative tool to unravel components involved in the concerted recruitment of PEN2 and CYP81F2 towards host-pathogen contact sites. A novel mutant population derived from a double transgenic line stably co-expressing GFP-tagged PEN2 and CYP81F2-mKate2 was generated, allowing a CLSM-based forward genetic screen to isolate mutants with an aberrant localization pattern of these proteins upon pathogen attack. Furthermore, transgenic Arabidopsis plant lines were designed to isolate and analyze epidermis-specific and pathogen-activated mitochondria to gain more information about the function of mitochondria (subpopulations) in cell entry control.
Keywords: disease resistance; non-host resistance; powdery mildew; sphingolipids; forward genetic screen; endomembrane trafficking; plant-microbe interaction
