Effectomics - Exploring the Toolbox of Plant Pathogens
Effector functions of biotrophic pathogens in maize and Brachypodium
Parasitic biotrophic interactions are molecularly based on a battery of effector-molecules, which are used by the invader to manipulate the host without killing it. Effector molecules are known to fulfill a broad variety of functions including camouflaging the pathogen against recognition, suppression of host-defense responses by detoxifying or sequestering harmful plant compounds, redirecting host developmental processes or manipulating the host metabolism for the reproductive success of the pathogen.
An effector can be any molecule that is released by the biotroph and that interferes directly or indirectly with the host cell, thereby affecting the interaction between the two organisms.Thus, effectors can be secreted proteins but also non-proteinous secreted molecules. Effectors can promote infection but they can also be specifically recognized by the host and thereby trigger defense responses. Thus, the term “effector” is neutral and can imply a positive or negative impact on virulence. The identification of effectors and a comprehensive knowledge about effector target molecules will not only provide a deep understanding of the needs of the pathogen but also identify critical pathogen or plant targets for disease intervention and pest control. In our group we are developing a systematic and generalizable workflow to identify and functionally categorize effectors. We are establishing this pipeline for the plant-pathogen model system Ustilago maydis-Zea mays and a currently newly explored pathosystem Ustilago bromivora - Brachypodium. In the future we intend to expand our research to include effectors from closely and distantly related biotrophic fungi.
U. maydis and U. bromivora are basidiomycetes that cause smut disease on their host plants maize or the model grass Brachypodium, respectively. The most prominent feature of U. maydis is its ability to cause tumor formation on all aerial parts of the plant. The yet largely unstudied head smut U. bromivora induces symptoms only in the floral part of its host Brachypodium.
After mating and formation of a dikaryotic filament, U. maydis forms a penetration structure on the leaf surface, a so-called appressorium. Previously it has been shown that hydrophobicity and hydroxyl- fattyacids are the two cues that induce this developmental program. Microarray-studies and mass-spectrometric analyses of different developmental stages of U. maydis indicate that filament formation and appressoria formation on the host plant maize induce expression of a number of genes encoding putative secreted protein effectors. Other secreted effector genes are either expressed during penetration into the plant tissue, or later in a maize-organ-specific expression profile as recently reported by the Walbot group (Skibbe et al. 2010).
Advantages of Brachypodium as a plant model
- Small plant size
- Modest growth requirements
- Short generation time
- Easily and efficiently transformed
- Genomic information available
- Growing toolbox available
This indicates that U. maydis uses different mixtures of secreted factors depending on the conditions present in its host. Deletions of effector genes lead in several cases to severe virulence defects or even to a loss of pathogenicity, whereas in other cases no virulence phenotype could be observed. Whereas functional effector studies in the model U. maydis are well feasible, on the plant side the research on maize is hindered by several disadvantageous properties including plant size, generation time and tedious transformation techniques. Therefore we are currently elucidating the model grass Brachypodium for plant sided effector studies.
Linking effectors to functions
The functional characterization of most effectors is difficult as many of them show no sequence homology to known proteins. In our recent work we could for the first time ascribe a biological function to an U. maydis effector. Cmu1 is a secreted chorismate mutase which interferes with the salicylic acid (SA) biosynthesis pathway of the host plant through a rechanneling of chorismate, the precursor for SA. Our lab is interested in establishing and developing high throughput screens to functionally categorize the complete effectome of Ustilago maydis.
For this we are adopting established screens as well as developing new functional screens based on metabolomic and transcriptomic datasets which are available for various stages of the U.maydis – maize interaction.
For a proof of principle experiment we generated a Gateway®-compatible effector library, which currently comprises about 300 U. maydis genes encoding small secreted proteins. This library represents the starting point for the functional, robotic-based high throughput-screening.
We imagine the secreted effectors to be involved in the following processes:
- Avoidance of fungal recognition by the plant defense system
- Suppression of the multilayered plant defense responses
- Anti-apoptotic functions
- Detoxification of host-derived metabolites
- Suppression of microbial competitors
- Sensing and communication
- Manipulation of the host-cell metabolism
Respective screens to functionally categorize effectors are ongoing or being set up. Once proof of concept is achieved, this pipeline will be extended to effectors from less accessible but economically important biotrophic pathogens.
Selected effectors with distinct functions will be analyzed in detail; mutants will be generated by reverse genetics. The knowledge of functional properties of the tested effector proteins may allow detection of even subtle virulence defects of the corresponding deletion strain. Host-Interaction partners will be identified by yeast two hybrid screens, co-immunoprecipitations followed by mass spectrometry, and new genetic screens for extracellular interactions.
As a future perspective the accumulation of functional effector-data will allow the modeling of a synthetic core effectome of biotrophs and the corresponding core-targets on the host side. The understanding of the effector toolbox of biotrophs will also enable the use of effectors as genetic tools to manipulate the metabolism of important crop plants.