Genomic and mutational analysis of Pseudomonas syringae pv. tagetis EB037 pathogenicity on sunflower

Abstract

Background: Pseudomonas syringae pv. tagetis (Pstag) causes apical chlorosis on sunflower and various other plants of the Asteraceae family. Whole genome sequencing of Pstag strain EB037 and transposon-mutant derivatives, no longer capable of causing apical chlorosis, was conducted to improve understanding of the molecular basis of disease caused by this pathogen.

Results: A tripartite pathogenicity island (TPI) for a Type III secretion system (T3SS) with the complete hrp-hrc gene cluster and conserved effector locus was detected in the Pstag genome. The exchange effector region of the TPI contained genes potentially functioning in detoxification of the environment as well as two integrases, but no previously described T3SS effector homologues. In all, the Pstag EB037 genome contained homologues for at least 44 T3SS effectors with 30 having known functions. Plasmids similar with pTagA and pTagB of P. syringae pv. tagetis ICMP 4091 were also identified in the Pstag genome. The pTagA-like plasmid contained a complete Type IV secretion system (T4SS) with associated putative killer protein. Mutational analysis using transposon insertions within genes functioning in the T3SS and T4SS confirmed the role of both secretion systems and these plasmids in apical chlorosis. Transposon mutagenesis identified an additional 22 genes in loci, including two more plasmid-bound loci, involved in apical chlorosis on sunflower; some with known importance in other plant or animal pathosystems.

Conclusions: Apical chlorosis disease caused by Pstag EB037 is the result of a complex set of mechanisms. This study identified a TPI and homologues for at least 44 T3SS effectors, 30 of which with known functions in disease, and another 20 genes in loci correlated with apical chlorosis on sunflower. Two plasmids were detected that were correlated with apical chlorosis disease, one of which contained a complete T4SS that was correlated with disease. To our knowledge, we provide the first direct evidence for a T4SS functioning in disease by a pathogenic P. syringae strain.

Keywords: Pseudomonas syringae pv. tagetis; Apical chlorosis; Disease; Effectors; Sunflower; Type III secretion system; Type IV secretion system.

Fig. 1

Pseudomonas syringae pv. tagetis EB037 tirpartite pathogenicity island. Arrows indicate direction of transcription of individual genes. hopAA-1, Type III secretion system (T3SS) effector protein HopAA-1; hrpW1, harpin HrpW1; hopM1, T3SS effector protein HopM1; cesT, chaperone protein CesT; avrE, Type III secretion transcription activator, regulates HrpL; hrpS, NtrC-like transcription activator, regulates HrpL; hrpA, needle protein HrpA; hrpZ, helper protein HrpZ; hrpB, inner rod protein HrpB; hrcJ, inner membrane ring protein HrcJ; hrpE, ATPase complex protein HrpE; hrpD, ATPase complex factor HrpD; hrpF, negative regulator protein HrpF, stabilizes HrpA; hrpG, regulatory protein, suppresses negative regulation by HrpV; hrcC, component of Type III secretion machinery; hrpT, secretin pilotin protein HrpT; hrpV, HrpV protein, negatively regulates HrpR and HrpS; hrcU, autoprotease HrcU; hrcT, inner membrane component protein HrcT; hrcS, inner membrane component protein HrcS; hrcR, inner membrane component protein HrcR; hrcQb, cytoplasmic ring protein HrcQb; hrcQa, cytoplasmic ring protein HrcQa; hrpP, needle-length regulator protein HrpP; hrpO, ATPase complex protein HrpO; hrcN, ATPase complex protein HrcN; hrpQ, inner membrane ring protein HrpQ; hrcV, export gate protein HrcV; hrpJ, switch regulator protein HrpJ; hrpL, sigma factor, master regulator of majority of hrp, hrc, hop,and avr genes; hrpK, translocation pore protein HrpK; lysR, LysR-type transcriptional regulator; SDR oxidoreductase; glutathione transferase; tetR, TetR family regulatory protein; trxB, thioredoxin reductase; ubiE1, ubiquinone/menaquinone biosynthesis C-methyltransferase; hyp, hypothetical protein; Int, integrase; IntS, integrase; tRNA^leu, encodes tRNA.^Leu; queA, S-adenosylmethionine:tRNA ribosyltransferase-isomerase [19, 25, 122, 126]

Fig. 2

Pseudomonas syringae pv. tagetis EB037 Type IV secretion system locus. Arrows indicate direction of transcription of individual genes. rfa3A, replication factor A protein; virB1, peptidoglycan hydrolase; virB2, pilin protein; virB3, inner membrane complex protein; virB4, inner membrane complex ATPase; virB5, inner membrane complex pilus-tip protein; hyp4-vir, hypothetical virulence protein; virB6, polytopic inner membrane core protein; virB7, outer membrane complex lipoprotein; virB8, inner membrane complex bitopic transmembrane protein; virB9, outermembrane complex protein; virB10, inner membrane complex/outer membrane complex bitopic transmembrane protein; virB11, cytoplasmic/inner membrane complex ATPase; hyp3-vir, hypothetical virulence protein; hyp2-vir, hypothetical virulence protein; virD4, Type IV coupling protein, ATPase; killer protein, predicted function in ubiquination and cell apoptosis; topB5, DNA topoisomerase B protein; hyp1-vir, hypothetical virulence protein; ssb2, replication factor A protein 2 [108]

Fig. 3

Sunflower bioassay for apical chlorosis. In each subfigure, sunflower plants were inoculated with cells of Pseudomonas syringae pv. syringae EB037 derivative strains just below the hypocotyle using a hypodermic needle. Plants were evaluated for apical chlorosis, indicative of tagetitoxin production, at 5 to 7 days after inoculation. A The leftmost plant was inoculated with EB037 containing the empty cloning vector pME6031, the center plant was inoculated with Tox-17, and rightmost plant was inoculated with Tox-17(pJLCP-11) which contains wild-type gacS in pME6031. B The leftmost plant was inoculated with EB037 containing the empty cloning vector pME6032, the center plant was inoculated with Tox-18, and rightmost plant was inoculated with Tox-18(pJLCP-5) which contains wild-type gacA in pME6032