Developmental regulators and secreted effector molecules of the fungal pathogen Verticillium spp.
by Miriam Leonard
Date of Examination:2019-09-30
Date of issue:2020-09-29
Advisor:Prof. Dr. Gerhard Braus
Referee:Prof. Dr. Gerhard Braus
Referee:Prof. Dr. Kai Heimel
Referee:Prof. Ph.D. James Kronstad
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EnglishThe haploid Verticillium dahliae and the allodiploid V. longisporum are two filamentous fungal pathogens, which colonize the plant vascular system of a broad host range including important crops. They survive outside of the plant for years in the soil with microsclerotia as resting structures and colonize specific host plants through the roots. In the xylem they form conidia to facilitate transportation within the plant. This life cycle requires a tight control of fungal development and a cautious communication with the host through the secretome without causing extensive plant defense reactions. The focus of this work was (i) to explore the regulatory function of two transcriptional regulators in development, Frq (Frequency) and Sfl1 (Suppressor of flocculation 1), and (ii) to compare fungal secretomes in different environmental conditions. One major result was that Frq and Sfl1 are required for microsclerotia and conidia formation of V. dahliae. The circadian clock component Frq acts as light-dependent microsclerotia formation repressor and has the opposite function for conidia where it operates as activator. Sfl1 functions as well as activator of conidia, but also as activator of microsclerotia formation in V. dahliae. A genetic analysis using deletion mutant strains revealed that SFL1 is epistatic to FRQ because the absence of SFL1 leads to the same severe reduction of microsclerotia formation in single as in double deletion mutant strains. This suggests that SFL1 is genetically located downstream in the regulation of fungal development. This genetic analysis demonstrated as second major result that both genes are required for full virulence of V. dahliae on tomato. The fungal communication with the host plant was examined by the secretome of allodiploid V. longisporum as pathogen of rape seed. Minimal or complete media delivered a similar broad exoprotein pattern. In contrast, cultivation in the plant-related media, a pectin-rich medium or pure xylem sap isolated from Brassica napus, triggered the secretion of another exoproteome pattern that includes similar but also distinct features. This corroborates significant differences in the sensing of the fungus of different environments resulting in different secretion responses. The xylem sap-specific exoproteome included carbohydrate-active enzymes (CAZys), peptidases and proteins with effector domains. Deletion strains of the corresponding homologs of haploid V. dahliae were phenotypically similar to wildtype growth ex planta. Whereas single deletions of CAZy encoding genes, and even a double deletion of the MEP1 and MEP2 genes encoding metallopeptidases or the CP1 and CP2 encoding cerato-platanin proteins did not alter virulence, the single metalloprotease Mep1 and the necrosis and ethylene inducing-like proteins Nlp2 and Nlp3 are necessary for V. dahliae pathogenicity on tomato. Therefore, the exoproteome approach pinpointed Mep1, Nlp2 and Nlp3 as single effectors required for successful V. dahliae colonization whereas other secreted proteins might provide redundant functions. This study shows the flexibility of the Verticillium response throughout the life cycle to adjust to distinct external stimuli. Developmental regulators such as the Frq and Sfl1 proteins are essential for microsclerotia or conidia production and for pathogenicity. The secretome response adapts with a distinct pattern to different nutrient combinations in the environment. En masse, these findings revealed Frq, Sfl1, Mep1 and Nlp2/3 as factors important for V. dahliae virulence, which all display potential targets for new growth control strategies of the fungus.
Keywords: Verticillium longisporum; Verticillium dahliae; plant pathogen; plant- and media-dependent exoproteomes; effectors; developmental regulators