Effector prediction in Pseudomonas syringae pv. tomato str. DC3000: a case study illustrating interpretation guidelines

This case study illustrates the interpretation of Effectidor's results from a pangenome analysis of Pseudomonas syringae genomospecies 3. This analysis incorporated secretion signal prediction, regulatory elements (hrp-box), and protein sequences from phylogenetically related strains lacking a type 3 secretion system (T3SS). We focus on Pseudomonas syringae pv. tomato str. DC3000 (Pto DC3000), a well-studied bacterium possessing T3SS and numerous known type 3 effectors (T3Es). Details on input data and accession numbers are provided in the 'Detailed input' section below.

Results:

Effectidor II identified 54 orthologous groups (OGs) containing Pto DC3000 genes and pseudogenes as effectors (scores 0.661–0.999), all exhibiting high homology to known effectors in our database. However, three predicted effector OGs appear to contain Pto DC3000 false positives: locus tags PSPTO_RS14865, PSPTO_RS07255, and PSPTO_RS22510, annotated as “putative virulence factor”, “Hrp pilus protein”, and “SpvB/TcaC N-terminal domain-containing protein”, respectively.
To identify false positives, we examined annotations, feature values, and feature importance, consulting external databases and publications. Key features (Table 1) were homology to known T3Es, similarity to effectors versus non-effectors, presence of hrp-box in the promoters, and proximity to known effectors.

Table 1: Feature importance as ranked in Effectidor II analysis for Pto DC3000. Full feature importance table is available in the feature importance file

Feature	Importance
homology_to_T3Es_(bit_score)_mean	0.323
T3_signal_max	0.186
similarity_to_effectors_vs_non_effectors	0.086
hrp_box_max	0.073
distance_from_closest_effector_median	0.026

PSPTO_RS14865, encoding a putative virulence factor was predicted to be an effector with a score of 0.676. While exhibiting effector-like characteristics (homology to T3Es, proximity to effectors in some genomes, and lack of homology to most representative taxa lacking a T3SS), it also displays features inconsistent with effectors: similarity to a non-T3 protein from Azotobacter vinelandii, absence of a hrp-box, and a low T3 signal score. Although some effectors lack hrp-boxes or have weak T3 signals, the ambiguous nature of this OG necessitated search for experimental validation. We found that the locus encodes the protein HopL1, which was previously considered a potential effector due to its coiled-coil domain and other effector-like features but was subsequently shown to be a non-effector [1,2].
PSPTO_RS07255 encodes the HrpA protein, a major structural component of the Hrp pilus. Although exhibiting high homology to the known effector HopY1 (present in the Effectidor II database), PSPTO_RS07255 is considerably shorter and was thus excluded from our positive training set due to the 50% coverage threshold. This annotation suggests a potential false positive, as structural T3SS proteins often share characteristics with effectors. The high variability of these proteins across bacterial species and T3SS subtypes may lead to misclassification. An analysis of the OG features shows high scores for homology to T3Es, T3 signal, hrp box presence, and proximity to other effectors, resulting in a high overall score (0.662). However, this score is lower than those of all true effectors but one, despite the shared characteristics with the positive training set.
PSPTO_RS22510 encodes a SpvB/TcaC N-terminal domain-containing protein. Although its annotation did not clearly indicate a false positive, its relatively low score (0.572) – the lowest among the known and predicted effectors, warranted further investigation. Similarly to PSPTO_RS14865, it shows some effector-like characteristics (homology to T3Es, proximity to effectors in some genomes), but is deficient in key features, with a low T3 signal score and the absence of a hrp-box. Therefore, we searched for experimental validation. Testing showed that the first 100 amino acids fused to AvrRpt2 (without a signal peptide) failed to deliver it to plant cells, indicating this is not an effector [2].
Three additional OGs containing Pto DC3000 genes were predicted to be effectors, scoring between 0.581 and 0.745, but little information on the proteins was available, as they are currently poorly characterized (see full results in the results and OG features files).

Detailed input:

The pangenome analysis included the Pseudomonas syringae species pv. tomato str. DC3000 (GCF_000007805.1), pv. tomato str. B13-200 (GCF_002966555.1), pv. tomato DAPP-PG 215 (GCF_949769235.1), pv. maculicola str. ES4326 (GCF_000145845.2), pv. tomato str. Delta X (GCF_009800225.1), pv. maculicola str. MAFF 302723 (GCF_016599655.1), Pseudomonas syringae pv. antirrhini str. 126 (GCF_030252405.1), pv. tomato str. GM 113 (GCF_041379965.2), and pv. morsprunorum CFBP 6411 (GCF_900235905.1). The input consisted of ORFs, full genome and GFF files for each pangenome species. The closely related T3SS-lacking bacteria included Acinetobacter baumannii str. ATCC 19606 (GCF_009035845.1), Azotobacter vinelandii DJ (GCF_000021045.1), and Ectopseudomonas khazarica (GCF_017915135.1). The input consisted of protein sequences of each T3SS-lacking bacteria. All input files used are available in NCBI database.