Structure, function and pathogenicity mechanism of a potato late blight effector that localizes to the host cell nucleus
Final Report Abstract
The causal agent of potato late blight, the oomycete Phytophthora infestans is an economically important pathogen of potato and tomato causing significant yield losses worldwide. Currently, potato late blight is controlled by repeated chemical sprayings. Engineering genetic resistance towards P. infestans requires knowledge on how it manages to successfully conquer its host plants. A hallmark of P. infestans’ aggressiveness is its large repertoire of effector proteins which are translocated into host plant cells via pathogen-host cellular interfaces, called haustoria. Effectors are generally assumed to facilitate infection and to suppress immunity. From the genome of P. infestans several cytoplasmic effector candidates that act inside plant cells were identified. However, their subcellular sites of function in plants are unknown. Studying localization and function of these effectors can highlight mechanisms that are employed by the pathogen to tweak the host cell processes to successfully infect the plant. The major goal of this project was to study effectors that target the host nucleus. To identify nuclear localized effector candidates we applied a bioinformatics pipeline which was based on P. infestans genome sequence. The identified set of nuclear localized effector candidates comprises of 9 RXLR effectors, 5 CRN effectors and 44 proteins without known translocation motif. We experimentally confirmed nuclear localization for 3 RXLR effector candidates including the previously known NUK10. Our studies using CRN proteins with harbor a predicted NLS in the C-terminus identified strong and exclusive nuclear localization. We centered our further work around CRNs as they constituted a less well characterized effector family and had exclusive nuclear localization. In translocation experiments we could show that CRN N termini are functionally equivalent to the N termini of RXLR effector proteins and that they can mediate the delivery of effector proteins inside host cells. Mutations in the conserved LFLAK motif further showed that it is required for effector targeting and translocation. This finding suggests that genome-wide searches for LFLAK-type motifs can be used to identify cytoplasmic effectors in oomycetes as routinely performed for RXLR effectors. Genomic surveys revealed that unlike the RXLR effectors, CRN proteins are ubiquitous in plant pathogenic oomycetes and thus form an ancient effector family that arose early in oomycete evolution and before the emergence of haustoria-forming species. Our localization studies with unrelated CRN effectors showed that they all exclusively target the host nuclear compartment. Furthermore, CRN8 and most likely all other tested CRN proteins rely on nuclear localization for their activities, for example their cell death inducing capabilities. N. benthamiana is emerging as an excellent system for microscopic studies of infections caused by Phytophthora. To further develop this system we compared different Phytophthora species and isolates of P. infestans in their ability to colonize N. benthamiana leaves. We found varying degrees of infection phenotypes ranging from complete HR-based resistance (P. mirabilis) to very susceptible (P. capsici). Confocal fluorescence microscopy and electron microscopy revealed haustorial structures between 2–5 dpi upon infection with P. infestans isolate 88069. As an initial test, we used the N. benthamiana system for functional analysis of genes. We addressed whether basal immunity conferred by membrane receptors of the BAK1/SERK3 type contribute to P. infestans infection in N. benthamiana. We cloned two N. benthamiana SERK3 paralogs and showed that their knockdown significantly enhances P. infestans infection. Our experiments also suggest that recognition of P. infestans INF1 protein requires the SERK3 paralogs and contributes to the infection outcome. The established system will now be employed to study localization and function of effectors during infection. In summary, I shifted my priorities from studying a single nuclear effector candidate mainly towards identification of previously uncharacterized nuclear CRN proteins. Together with colleagues I revealed that CRN effectors are exclusively acting in the host nucleus and facilitate the host nuclear import machinery. I established a system which allows efficient and time-saving analysis of gene functions and protein localizations in the context of infection and applied it to identify SERK3 proteins as elements of plant immunity towards P. infestans in N. benthamiana.
Publications
- (2009): Interactions Between Phytophthora infestans And Solanum. Chapter in: Oomycete Genetics and Genomics: Diversity, Interactions and Research Tools
Van Damme, M., Schornack, S., Cano L. M., Huitema, E. and Kamoun, S.
- (2009): Protein mislocalization in plant cells using a GFP-binding chromobody. Plant J. 60(4)
Schornack S., Fuchs R., Huitema E., Rothbauer U., Lipka V., Kamoun S.
- (2009): Ten things to know about oomycete effectors. Mol Plant Pathol. 10(6)
Schornack S., Huitema E., Cano L.M., Bozkurt T.O., Oliva R., Van Damme M., Schwizer S., Raffaele S., Chaparro- Garcia A., Farrer R., Segretin M.E., Bos J., Haas B.J., Zody M.C., Nusbaum C., Win J., Thines M., Kamoun S.
- (2010): An effector-targeted protease contributes to defense against Phytophthora infestans and is under diversifying selection in natural hosts. Plant Physiol. 154(4)
Kaschani F., Shabab M., Bozkurt T., Shindo T., Schornack S., Gu C., Ilyas M., Win J., Kamoun S., van der Hoorn R.A.
- (2010): Ancient class of translocated oomycete effectors targets the host nucleus. Proc Natl Acad Sci USA 107(40)
Schornack S., van Damme M., Bozkurt T.O., Cano L.M., Smoker M., Thines M., Gaulin E., Kamoun S., Huitema E.
- (2010): Recent developments in effector biology of filamentous plant pathogens. Cell Microbiol. 12(6)
Oliva R., Win J., Raffaele S., Boutemy L., Bozkurt T.O., Chaparro-Garcia A., Segretin M.E., Stam R., Schornack S., Cano L.M., van Damme M., Huitema E., Thines M., Banfield M.J., Kamoun S.
- (2011) The Receptor-Like Kinase SERK3/BAK1 Is Required for Basal Resistance against the Late Blight Pathogen Phytophthora infestans in Nicotiana benthamiana. PLoS ONE 6(1)
Chaparro-Garcia A., Wilkinson R.C., Gimenez-Ibanez S., Findlay K., Coffey M.D., Zipfel C., Rathjen J.P., Kamoun S., Schornack S.
