Intrinsic Disorder in Pathogen Effectors: Protein Flexibility as an Evolutionary Hallmark in a Molecular Arms Race

Effector proteins represent a refined mechanism of bacterial pathogens to overcome plants’ innate immune systems. These modular proteins often manipulate host physiology by directly interfering with immune signaling of plant cells. Even if host cells have developed efficient strategies to perceive the presence of pathogenic microbes and to recognize intracellular effector activity, it remains an open question why only few effectors are recognized directly by plant resistance proteins. Based on in-silico genome-wide surveys and a reevaluation of published structural data, we estimated that bacterial effectors of phytopathogens are highly enriched in long-disordered regions (>50 residues). These structurally flexible segments have no secondary structure under physiological conditions but can fold in a stimulus-dependent manner (e.g., during protein–protein interactions). The high abundance of intrinsic disorder in effectors strongly suggests positive evolutionary selection of this structural feature and highlights the dynamic nature of these proteins. We postulate that such structural flexibility may be essential for (1) effector translocation, (2) evasion of the innate immune system, and (3) host function mimicry. The study of these dynamical regions will greatly complement current structural approaches to understand the molecular mechanisms of these proteins and may help in the prediction of new effectors.Plants and pathogens are entangled in a continual arms race. While host organisms have developed complex and dynamic immune systems able to recognize a wide range of pathogens and to discriminate them from beneficial microbes (Jones and Dangl, 2006; Medzhitov, 2007), bacterial pathogens have evolved refined adaptation strategies to overcome the plant’s innate immune system. Among these ingenious adaptations are effector proteins. Most of these proteins are secreted via the type III secretion system (TTSS) into the host cytoplasm, where they manipulate the immune signaling and the physiology of plant cells and thereby improve bacterial fitness within the host (Dean, 2011).Plant–pathogen interactions are highly dynamic processes, both from the evolutionary and the physiological point of view. Here, we postulate that they are equally dynamic at the protein-structure level. This is based on our finding that numerous effector proteins are predicted to be intrinsically disordered (ID) and that this feature may be essential for (1) effector translocation, (2) evasion of the innate immune system, and (3) host function mimicry. Intrinsic disorder has so far been postulated to preferentially occur in eukaryotic proteins. While on average ∼20% of the eukaryotic proteome harbors long (>50 residues) ID segments, these regions are only predicted at low abundance (8% on average) in bacterial proteomes (Dunker et al., 2000). The most likely reason for this discrepancy is the lack of efficient mechanisms to protect unfolded proteins from degradation (Ward et al., 2004). However, when surveying genomes of pathogenic bacteria with the widely used PONDR VL-XT program (Romero et al., 2001), we observed that not only the average percentage of sequence disorder, but most strikingly long (>50 residues) stretches of intrinsic disorder are highly overrepresented in secreted effectors, with especially high levels in phytopathogenic bacteria (Pseudomonas syringae, ∼39%; Ralstonia solanacearum, ∼70%; Xanthomonas spp, ∼77%) (Supplemental Table 1 online). This striking enrichment of unstructured regions strongly suggests positive evolutionary selection of intrinsic disorder in effector proteins and highlights their dynamic nature.

Table 1.

Predictions of Intrinsic Disorder in Effectors and Whole Proteomes of Different Bacterial Species

Organism	Average Percentage of Disordered Residues		Percentage of Proteins Harboring ID Regions >50 Residues
	All Proteins	TTSS Effectors	All Proteins	TTSS Effectors
P. syringae		38.6		35.6
phaseolicola 1448A	26.1	42.0	10.1	52.4
syringae B728a	26.2	41.4	10.7	57.1
tomato DC3000	26.4	39.7	10.2	34.4
R. solanacearum		42.6		69.6
GMI1000	29.2	43.5	11.9	66.7
Xanthomonas sp		49.2		75.7
X. campestris pv vesicatoria 85-10	29.6	50.9	13.5	69.6
X. oryzae pv oryzae KACC10331	29.7	46.3	12.5	82.3
X. campestris pv campestris ATCC 33913	29.1	44.6	13.3	68.9
S. enterica		22.1		18.5
enterica ser. typhimurium LT2	23.0	21.5	7.0	19.2

Open in a separate windowDisorder parameters of representative effectors (see Supplemental Table 1 online) were calculated per species (highlighted in bold) and were compared to the values calculated for the proteomes from which the majority of the effectors were extracted. For completeness, effectors belonging to protein families absent in these strains were extracted from closely related strains (see Supplemental Table 1 online). Proteomes of P. syringae pv phaseolicola (strain 1448A; 5170 proteins), P. syringae pv syringae (strain B728a; 5088 proteins), P. syringae pv tomato (strain DC3000; 5618 proteins), R. solanacearum (strain GMI1000; 5108 proteins), X. campestris pv vesicatoria (strain 85-10; 4726 proteins), X. oryzae pv oryzae (strain KACC10331; 4065 proteins), X. campestris pv campestris (strain ATCC 33913; 4178 proteins), and S. typhimurium (strain LT2 ; 4555 proteins) were downloaded from the National Center for Biotechnology Information server (http://www.ncbi.nlm.nih.gov/genome/). Additionally, parameters were individually calculated for the different strains. Intrinsic disorder predictions were calculated with the PONDR VL-XT program (Romero et al., 2001). Here, scores below and above 0.5 indicate residues predicted to be ordered and disordered, respectively. The average percentage of sequence disordered was calculated as the mean value of the percentage of disordered residues (PONDR score > 0.5) per protein from all proteins. The percentage of long ID regions was calculated as the percentage of proteins harboring ID regions >50 residues.