Wake of the flood: ascribing functions to the wave of type III effector proteins of phytopathogenic bacteria
Introduction
Many plant pathogenic bacteria rely on a type III secretion system (TTSS; encoded by hrp/hrc genes) to cause disease on their hosts (Figure 1; [1]). The protein effectors traversing the TTSS are delivered into the host cell, where they function as the virulence factors that modulate susceptible hosts to benefit the pathogen. Type III effector proteins of plant pathogens are characterized by some common traits (Figure 1). It is perhaps no surprise that the plant surveillance system in resistant plants (R proteins) relies on recognition of these same type III effector proteins. This ‘recognition’ results in the induction of a suite of defense responses, including hypersensitive cell death (HR), which renders the pathogen avirulent [2]. For more in-depth reviews on plant resistance, please refer to 2., 3., 4.. As a consequence, the type III effectors identified via their ability to trigger the specific function of a given R protein were historically referred to as avirulence proteins (Avr). The genetic analysis of R-mediated disease resistance suggested that the R protein would function as a receptor binding a ligand encoded by the Avr protein [5]. Though a couple of examples do fit this idea, most do not. This led to a proposal suggesting that the type III effector proteins (and by extension, virulence factors from other pathogens) are indirectly recognized by resistant hosts, and that R proteins monitor the homeostasis of a particular host cellular machine targeted by the pathogen 2., 6., 7.. Unless otherwise stated, we use ‘recognition’ to imply either direct or indirect molecular interaction between R protein and type III effectors acting as Avr proteins.
Three general virulence functions have been proposed for type III effectors: release of bacteria to the organ surface, nutrient acquisition and suppression of basal or induced host defenses. The basal host defenses are those that operate, even in susceptible plants, to limit the extent of disease in the absence of R-mediated recognition. Basal defense is genetically defined [8], and steps required for basal defense can overlap with genetically defined steps in R-mediated defense. This leads to the suggestion that the R proteins act to accelerate the overall defense response 2., 8.. Experimental evidence suggests that some type III effectors are required for optimal release of bacteria to the leaf surface 9., 10.. No reports have been published demonstrating a role for effectors in acquiring nutrients. By contrast, much of the data presented to date strongly suggests that type III effectors can function to suppress the host defense response. Generally, it is difficult to determine the function of a particular type III effector. This is owing to a combination of functional overlap or redundancy between the suite of type III effectors in a given strain, the probable subtle effects they may exert to increase virulence, or roles that are possibly specific to certain stages of the pathogens’ life cycle or colonization of particular hosts.
Whole genome sequencing of strains Pseudomonas syringae pv. tomato (Pto) DC3000 [11], Pseudomonas syringae pv. syringae (Psy) B728a (http://genome.jgi-psf.org/draft_microbes/psesy/psesy.home.html), Xanthomonas axonopodis pv. citri (Xac) [12], Xanthomonas campestris pv. campestris (Xcc) [12], Ralstonia solanacearum [13] and that of Pseudomonas syringae pv. phaseolicola (Pph) race 6 (currently in progress; RC Buell, personal communication), functional screens, and several bioinformatics approaches have forced the floodgates open, leading to the discovery of many more proven and putative type III effectors 14., 15., 16., 17., 18.; reviewed in 19., 20.•. The next hurdle to overcome is unveiling their functions and determining how they benefit the pathogen. Several recent publications provide inklings as to how pathogenic bacteria use type III effectors (Table 1; reviewed in [21]). These findings also highlight the many strategies that can be used by flummoxed researchers determined to crack the secret function of type III effectors from plant pathogenic bacteria.
Section snippets
Tsunamis look like tidal waves: homology-based approaches to type III effector function
There is nothing simpler, nothing more satisfying than clicking ‘BLAST!’ and instantaneously having symbols appear that could potentially dictate how to characterize a protein. It is frustrating to wait over ten minutes for a response, but even more exasperating to see, ‘No hits found’. Yet, this has been the standard response to simple database searches using type III effector gene and deduced protein sequences. As the microbial genome projects progress, however, one can expect to be rewarded
Surf’s up! Don’t forget the family
Many type III effectors are conserved between bacterial strains, and even between species. This is presumably owing to horizontal transmission as supported by the observation that many type III effector genes are associated with mobile genetic elements, and most display some features of pathogenicity islands 30., 31., 32.. Comparing members of these gene families can reveal conserved amino acid residues and domains. Conservation of domains should reflect conserved function, presumably in
Look closer to see the capillary waves
Genetic knockout of type III effector genes in plant pathogens, and subsequent assay of the mutants for differences in growth relative to wild-type strains is a standard approach towards understanding function. However, several attributes of type III effectors make this approach akin to searching for a pilus in a haystack. Badel et al. 35., 36. created strains of Pto DC3000 lacking either HopPtoM or both of the homologous genes, HopPtoA1 and HopPtoA2. Neither mutant exhibited significant
Standing waves: interference at the nodes
Recognition of a particular type III effector by a host R protein induces a multitude of responses that suppress pathogen growth (see above). Pathogenic strains can express type III effectors that interfere with the R-mediated recognition of another type III effector 43., 44., 45., 46.••. In one case, the mechanism by which the type III effector AvrRpt2 interferes with the ability of another type III effector, AvrRpm1, to trigger the RPM1 R protein has been deciphered [46••]. Expression of
The next wave: inferring effector function from structure
Determining protein structure and comparing the primary amino acid sequence with other members of the protein family can aid in identifying critical residues required for function. The AvrB protein structure was solved to 2.2 Å resolution with the intention of gaining insight into its molecular function (C Lee et al. unpublished data). The AvrB structure consists of a novel bi-lobal fold separated by a deep interlobal cleft. This cleft is highly conserved among the four AvrB family members,
Waving goodbye: conclusions and outlook
There will be a veritable flood of data regarding type III effector protein function from plant pathogens. Plant pathology, as a discipline, has provided a broad wealth of host and pathogen genotypes from which to sample interaction phenotypes. High throughput screens will be used to further our understanding of effector function. The transcriptional profile of a given host can be determined in response to effectors. Their roles in virulence might then be predicted on the basis of commonly
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
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of special interest
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of outstanding interest
Acknowledgements
We thank Chris Lee, Fumi Katagiri and Robin Buell for permission to cite their unpublished work and Laurence Rohmer for providing Figure 2. We would also like to thank Sheng Yang He and members of the Dangl/Grant laboratory for their many fruitful discussions. We apologize for not being able to cover all advances in the field owing to strict space constraints. Work on P. syringae type III effector in our group is supported by grants from the United States Department of Energy
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