Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.

Guillaume P Robin,Jochen Kleemann,Jean-Félix Dallery,Nicolas Lapalu,Richard J O’Connell,Ulla Neumann,Lisa Cabre

doi:10.3389/fpls.2018.00562

Abstract

The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium-mediated transformation to transiently express them as N-terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana, and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C-terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.

Highlights

Filamentous plant pathogens such as oomycetes and fungi establish disease by secreting an array of effector proteins that manipulate plant processes and mitigate plant immune responses to create a favorable environment for pathogen growth (Dodds and Rathjen, 2010)
Based on the fungal genome annotation, we identified 365 Candidate Secreted Effector Proteins (CSEPs) defined as extracellular proteins (WoLF PSORT prediction) without homology to proteins from organisms outside the genus Colletotrichum (O’Connell et al, 2012)
We analyzed the subcellular localization of 61 biotrophy-expressed effector candidates from C. higginsianum using a medium-throughput screen based on their heterologous expression as fluorescent proteins (FPs) fusions in N. benthamiana leaves

Summary

Introduction

Filamentous plant pathogens such as oomycetes and fungi establish disease by secreting an array of effector proteins that manipulate plant processes and mitigate plant immune responses to create a favorable environment for pathogen growth (Dodds and Rathjen, 2010). Following their secretion from infection structures such as appressoria, hyphae, and haustoria, effector proteins exert their biological activity either outside plant cells (in the plant apoplast and/or plant–pathogen interface) or inside the plant cytoplasm after translocation across the plant plasma membrane (Giraldo and Valent, 2013; Lo Presti et al, 2015). The plant compartments targeted by effectors from these four pathogens included nuclei, chloroplasts, ER, tonoplast, and plasma membranes

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Results

Conclusion