Bacterial rot of lettuce Necrotic leaf spot of eggplant

An aldehyde dehydrogenase gene and a phosphinothricin n-acetyltransferase gene compose of a pathogenicity island with hrp genes of Pseudomonas cichirii

Y. Hikichi, M. Tanaka, U. Md. Wali, H. Mizumoto, K. Ohnishi and A. Kiba

hrp genes of Pseudomonas cichorii strain SPC9018

The consensus hrp-box ( , GGAACC-N15-16-CCANNCA) was identified at putative promoters of hrpA, hrpF, hrpW, avrE, avrF and hrpJ.

Hojo et al. Microbiology, 2008

hrcT mutant

SPC9018

hrpG mutant

hrpS mutant

hrpL mutant

DW

hrcT mutant

SPC9018

hrpG mutant

hrpS mutant

hrpL mutant

DW

eggplant lettuce

Hojo et al. Microbiology, 2008

Virulence of P. cichorii strain SPC9018 on eggplant and lettuce

The hrp mutants of SPC9018 lost their virulence on eggplant 1 days after inoculation

P. syringae type

P. cichorii type

T-PAI S-PAI

Araki et al. PNAS, 2006

Nucleotide sequences of the hrp genes of SPC9018 and their genetic structure werehomologous to those of S-PAI of P. viridiflava BS group strains.

Pathogenicity island and hrp cluster of P. viridiflava

Virulence of the hrpG mutant on eggplant was restored by transformation with ahrpF operon, originating from Pv9504, suggesting that functional conservation of hrpFoperons between SPC9018 and Pv9504 .

hrcT mutant

SPC9018

hrpG mutant

hrpS mutant

hrpL mutant

hrpG mutant (phrpFPc)

DW

hrpG mutant (phrpFPv)

eggplant

Complementation of hrpG mutant with hrpF operon from a BS group member P. viridiflava strain 9504 (Pv9504)

eggplant

Hojo et al. Microbiology, 2008

The hrp is involved in full virulence of P. cichorii on eggplant and lettuce

Hojo et al. Microbiology, 2008

hrcT mutant

hrpG mutant

hrpS mutant

hrcTmutant

hrpL mutant

Transformant ofhrpG mutant with hrpF operon

P. viridiflava strain 9504 (BS group, Pv9504)

P. cichorii strain SPC9018

phosphinorthiricin N-acetyltransferase (pat)

LysR family transcriptional regulator(lysR)

hrpL hrpJ

hrpL hrpJ

phosphinorthiricin N-acetyltransferase (bar)

An aldehyde dehydrogenase gene (aldh) and a phosphinorthiricin N-acetyltransferasegene (pat) are located adjacent to hrpL of SPC9018 and Pv9504.

aldehyde dehydrogenase (aldH)

hrp cluster region A11.3-k bp

J V Q N Q R T UOP SL

LysR-typeTranscription regulator

aldH

Phosphinothricin N-acetyltransferase (pat)

hrpL

Phosphinothricin N-acetyltransferase

(bar)

LysR-typeTranscription regulator

aldH

ISPsy7, transposase

Phosphinothricin N-acetyltransferase (bar)

P. cichorii

P. syringae pv. tomato strain DC3000

The aldH but not the pat in the genome of P. cichorii and the S-PAI of P. viridiflavais located in the same positions of the genome of P. syringae strains and T-PAI of P. viridiflava.

SPC9018

ΔaldHΔpat

SPC9018SPC9018

ΔaldH

Δpat

ΔaldH (Pc-aldH)

Δpat (Pc-pat)

DW DW DW

eggplant lettuce

The aldH and pat was involved in virulence of P. cichorii strain SPC9018 on eggplant but not on lettuce.

Virulence of aldH-deficient mutant (ΔaldH) and pat-deficient mutant (Δpat) on eggplant and lettuce

SPC9018

ΔaldH

ΔaldH (Pc-aldH)

DW

eggplant

ΔaldH (Pv-aldH)

ΔaldH (Pst-aldH)

ΔaldH (Pa-aldH)

Function of aldH is conserved among these Pseudomonad.

Complementation of ΔaldH with aldH from P. viridiflava 9504, P. syringae pv.tomato strain DC3000 and P. aeruginosa strain PAO1

Δpat

SPC9018

Δpat (Pc-pat)

DW

Δpat (Pv-pat)

Complementation of Δpat with pat from P. viridiflava 9504

eggplant

Function of pat is conserved between P. cichorii SPC9018 and P. viridiflava 9504.

Expression analysis on hrpL, aldH and pat in SPC9018, ΔaldH, Δpat and hrpL-mutant (SPC9018-L) in infected leaves of eggplant by RT-PCR analysis

Expression of aldH and pat was not regulated by HrpL. Deletion of aldH and pat did not influence expression of hrpL. These results suggest that expression of aldH and pat is regulated independently of hrp regulo

Conclusion

1. An aldehyde dehydrogenase gene (aldH) and a phosphinothricin N-acetyltransferase gene (pat) are located in the flanking regions of the hrp innot only P. cichorii but also the BS and AS groups of P. viridiflava strainsharboring S-PAI .

2. Both aldH and pat are involved in P. cichorii virulence on eggplant but notlettuce.

3. The aldH is functionally conserved among Pseudomonad.4. The pat is functionally conserved between P. cichorii and the S-PAI.5. Expression of aldH and pat in P. cichorii infecting eggplant is independently

of that of hrpL.6. These results lead us to consider that P. cichorii may implicate not only the

hrp but also aldH and pat in its virulence.

Acknowledgement

Kobe UniversityProf. Shigeyuki MayamaProf. Pyoyun ParkProf. Yukio TosaDr. Nakayashiki

Sun Yat-Sen UniversityDr. Nan Yao

Boyce Thompson Institute for Plant ResearchProf. Gregory B. Martin

NIVTSDr. Takashi Shirakawa

Found

Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, JapanThe Asahi Glass Foundation

Mr. Tanaka

Dr. KibaDr. Mizumoto

Mr.Wali

Prof. Ohnishi