Prospects for functional analysis of effectors from cereal rust fungi
- 584 Downloads
With the advancement of several Puccinia genome sequencing projects, along with gene expression data and methods for predicting secreted proteins, it is now possible to predict many effector proteins from the cereal rusts. Biological assays that can be conducted in a relatively high throughput fashion are necessary to assign specific functions, such as avirulence. Biolistic delivery of potential effectors is limited by the need to examine individual cells and delivery by Agrobacterium generally also affects small numbers of cells in grasses. An approach that has had some success in dicots is the use of bacterial systems to deliver proteins by their type III secretion systems (TTSS). Several bacterial systems were thus tested for their suitability in delivering effectors to wheat. Pseudomonas syringae DC3000 caused hypersensitive reactions (HR) when infiltrated into all tested wheat lines but only some barley lines. A variant strain with multiple effectors deleted showed a reduced HR on wheat lines. Pseudomonas fluorescens with an engineered TTSS system showed no HR in wheat lines but was able to deliver bacterial effectors AvrRpm1 and AvrRpt2 and the fungal toxin ToxA. Delivery of the effectors by P. fluorescens could be detected by HR or by staining for presence of hydrogen peroxide or callose deposits. The bacterial systems thus showed good potential for their ability to deliver foreign proteins into wheat cells.
KeywordsType III secretion system Pseudomonas syringae Pseudomonas fluorescens RNAi Wheat Barley
This work was supported in part by National Institute of Food and Agriculture Grant No. 2010-65108-20568. We are grateful to Timothy Friesen for providing seeds of Langdon and the ToxA-insensitive mutant and Xianmin Chen for providing seeds of differential barley cultivars and differential wheat cultivars. Alan Collmer kindly provided P. syringae strains DC3000 and CUCPB5500 and the Pseudomonas fluorescens strains were provided by Jeff Chang. The pEDV6 and pEDV3 vectors were a gift from Jonathan Jones. We are also grateful to Zhaohui Liu, Justin Faris, and Timothy Friesen for supplying a clone of the ToxA gene and consultation on its use.
- Ballance GM, Lamari L, Kowatsch R, and Bernier CC (1996) Cloning, expression and occurrence of the gene encoding the Ptr necrosis toxin from Pyrenophora tritici-repentis. Mol Plant Pathol http://www.bspp.org.uk/mppol/1996/1209ballance/
- Brueggeman R, Druka A, Nirmala J, Cavileer T, Drader T, Rostoks N, Mirlohi A, Bennypaul H, Gill U, Kudrna D, Whitelaw C, Kilian A, Han F, Sun Y, Gill K, Steffenson B, Kleinhofs A (2008) The stem rust resistance gene Rpg5 encodes a novel protein with nucleotide binding site, leucine-rich and protein kinase domains. Proc Natl Acad Sci USA 105:14970–14975PubMedCrossRefGoogle Scholar
- Dodds PN, Lawrence GJ, Catanzariti A-M, Teh T, Ching I, Wang A, Ayliffe MA, Kobe B, Ellis JG (2006) Direct protein interaction underlies gene-for-gene specificity and co-evolution of the flax resistance genes and flax rust avirulence genes. Proc Natl Acad Sci USA 103:8888–8893PubMedCrossRefGoogle Scholar
- Ham JH, Bauer DW, Fouts DE, Collmer A (1998) A cloned Erwinia chrysanthemi Hrp (type III protein secretion) system functions in Escherichia coli to deliver Pseudomonas syringae Avr signals to plant cells and to secrete Avr proteins in culture. Proc Natl Acad Sci USA 95:10206–10211PubMedCrossRefGoogle Scholar
- Webb CA, Richter TE, Collins NC, Nicolas M, Trick HN, Pryor T, Hulbert SH (2003) Genetic and molecular characterizatio of the maize rp3 rust resistance locus. Genetics 169:381–394Google Scholar
- Wellings C, Kandel K (2004) Pathogen dynamics associated with historic stripe (yellow) rust epidemics in Australia in 2002 and 2003. In: Proceedings of 11th In Cereal Rusts and Powdery Mildews Conf, Norwich, England, 22–27 August 2004, Cereal Rusts and Powdery Mildews Bulletin, Abstr A2.74Google Scholar
- Zhou H, Li S, Deng Z, Wang X, Chen T, Zhang J, Chen S, Ling H, Zhang A, Wang D, Zhang X (2007) Molecular analysis of three new receptor-like kinase genes from hexaploid wheat and evidence for their participation in the wheat hypersensitive response to stripe rust fungus infection. Plant J 52:420–434PubMedCrossRefGoogle Scholar