Hypersensitive response (HR) is an efficient defense strategy leading to restriction of evoked by Psm. In contrast, there was a distinct time lapse between the first signs of tissue pathogen growth. It can be activated during host as well as nonhost interactions. This process collapse (9 hpi) and the occurrence of plasma membrane discontinuity (12 hpi) after Pst involves programmed cell death (PCD) and manifests itself in tissue collapse at the site of treatment. Detailed ultrastructural analysis of dying cells in bacteria infiltrated areas revealed pathogen attack. The detailed mechanisms underlying plant PCD are still poorly elucidated. that, as expected, cytological events distinctly preceded macroscopic necrotization. The both Apparently there are various forms of plant PCD. The role of HR as a direct defense pathosystems varied, however, in the order in which alterations in morphology of organelles mechanism has been recently questioned. HR can act, however, as a signal of infection for the occurred. Moreover, we failed to detect any abnormalities in nuclei of Psm treated tissue, whole plant and is usually followed by enhanced resistance to secondary infection. whereas different forms of nucleus degeneration were observed in leaf zones infiltrated with Tobacco plants (Nicotiana tabacum cv. Xanthi-nc) infiltrated with either one of two pathovars of Pst Additionally, synthetic caspase inhibitors abolished HR induced by Pst but not Psm.Pseudomonas syringae, avirulent strain of pv. tabaci (Pst) or nonhost pathogen pv. maculicola Interestingly, only preinoculation with Pst led to the mounting of long distance acquired M2 (Psm), developed a hypersensitive response (HR). There were, however, considerable resistance (LDAR), although locally a typical set of defense responses including acquired differences in HR phenotype, timing and sequence of cell dismantling between the two resistance was activated in response to Psm. It seems likely that lack of LDAR is due to rapid pathosystems. Following Psm infiltration the first macroscopic signs were visible at 4.5 hours degeneration of vascular bundle cells after Psm infection. Proteomic approach has been post infiltration (hpi). Simultaneously, an increased plasma membrane permeability was applied in order to identify specific bacterial factors which may contribute to observed distinct observed, suggesting that the loss of cell membrane integrity commences macroscopic HR morphotypes of host cell death.

Infection of tobacco with Pseudomonas syringae leads to distinct morphotypes of programmed cell death

1 1 1 2 1M. Krzymowska , D. Konopka-Postupolska , I. Goszczyñska-Legat , M. Sobczak and J. Hennig1Institute of Biochemistry and Biophysics, PAS, Pawinskiego 5a, 02-106 Warsaw, Poland

2Department of Botany, Agricultural University, Nowoursynowska 159, 02-776 Warsaw, Poland

Tobacco leaves were infiltrated with bacteria (at 8concentration 10 cfu/ml) mixed respectively with

synthetic caspase inhibitors (200µM in DMSO), DMSO (negative control) or bacteria alone (positive control). The picture was taken 48h after infiltration.

Ac-DEVD-CHO synthetic caspase-3 inhibitor

Ac-YVAD-CMK s y n t h e t i c c a s p a s e - 1 inhibitorVacuolar processing enzyme (VPE) has been recently shown as a protease essential for vacuole-mediated cell death during TMV induced HR. Although structurally unrelated to caspases, VPE possesses caspase-1 acitivity which could be blocked by caspase-1 inhibitor.

CHO

CMK

Caspase inhibitorsFig 2: Pst

Pst+DMSO

Pst+CHO

Pst+CMK

DMSO

Psm

Psm+DMSO

Psm+CHO

Psm+CMK

DMSO

8 representative discs were photographed under dissection Tobacco plants were infiltrated with bacterial suspension 10 scope. cfu/ml and analyzed at given time points. [A] Macroscopic [C] Plasma membrane permeability was analyzed by ion symptoms evoked by bacteria. [B] In order to stain dead cells leakage assay. Eight leaf discs were punched out and floated several leaf discs were punched out and incubated in 2% (w/v) abaxial side up on 5ml of the sterile MiliQ water for 10 min with Evans blue solution for 10 min with gentle shaking (50rpm), at

°° gentle shaking (50rpm), at 18 C and then the conductivity of the 18 C. Afterwards the discs were rinsed with water and solution was measured .chlorophyll was removed with ethanol washes. The

Time [h]

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Response to avirulent strain P.syringae pv. tabaci

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start of necrotizati

on

Evans blue staining to

detect dead cells

Course of tissue damage during HR induced by two pathovars of P. syringaeFig 1:

mock

mock

Psm

Psm

Pst

Pst

0 1.5 3 4.5 6 12 hpi

Expression of hsr203 gene in the infected leaves was tested using Northern analysis. Psm or Pst infiltrated leaves were collected at indicated time points post inoculation. Total RNA was isolated and analyzed with probes corresponding to hsr203 or rDNA as a loading control.I n t e res t i ng l y, t empo ra l mRNA accumulation for HSR203 was very similar in these two systems, the expression was however much stronger in response to Pst infection.

Induction of hsr203 gene - a molecular hallmark for HR-related cell death

loading control (rRNA)

hsr203 mRNA accumulation

1.5 3 4.5 6

Time [h]

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]

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Response to non-host strainP.syringae pv. maculicola

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Acquired resistanceFig 4:

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Long distance

Short distance

0.51.0

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To directly test the level of resistance we compared the diameter of the TMV-induced necroses in plants previously infected with bacteria. Tobacco plats Xanthi-nc were infiltrated either with: 10 mM

8 8MgCl as a negative control, Psm (10 cfu/ml) or Pst (10 cfu/ml) as a 2

positive control. Six days after primary inoculation, non-infected a r e a s o f infected leaves (for short distance acquired resistance, SDAR) or after 7 days non-infected upper leaves (for long distance acquired resistance, LDAR) were challenged with TMV. Diameters of secondary necroses (10 per leaf) were measured 7 days later. Reduction of secondary necrosis size was a direct measure of acquired resistance.

Mascot Search Results

Significant hits: type III effector HrpK1 [Pseudomonas syringae pv. phaseolicola 1448A] HrpA [Pseudomonas syringae pv. phaseolicola] HrpZ [Pseudomonas syringae pv. phaseolicola]

Chain B, Porcine E-Trypsin (E.C.3.4.21.4) type III effector HopAK1 [Pseudomonas syringae pv. phaseolicola 1448A] type III effector HopD1 [Pseudomonas syringae pv. phaseolicola 1448A] HrpZ [Pseudomonas syringae pv. maculicola] harpin [Pseudomonas syringae pv. phaseolicola] type III effector HopAE1 [Pseudomonas syringae pv. phaseolicola 1448A] hypothetical protein PSPPH_2509 [Pseudomonas syringae pv. phaseolicola 1448A] sugar ABC transporter, periplasmic sugar-binding protein [Pseudomonas syringae pv. phaseolicola 1448A] Protein of unknown function DUF796 [Pseudomonas syringae pv. syringae B728a] type III effector HopAU1 [Pseudomonas syringae pv. phaseolicola 1448A] polygalacturonase [Pseudomonas syringae pv. phaseolicola 1448A] type III effector HopI1 [Pseudomonas syringae pv. phaseolicola 1448A] OprF [Pseudomonas syringae pv. phaseolicola 1448A]

HrpK [Pseudomonas syringae pv. tomato] flagellin [Pseudomonas syringae pv. glycinea] virPphAPsv [Pseudomonas syringae pv. savastanoi]

major outer membrane lipoprotein I [Pseudomonas mendocina] type III effector AvrB4-2 [Pseudomonas syringae pv. phaseolicola 1448A]

pancreatic trypsin 1 [Rattus norvegicus] HrpA [Pseudomonas syringae pv. tomato]

porin D [Pseudomonas syringae pv. phaseolicola 1448A] translation initiation factor IF-3 [Pseudomonas syringae pv. tomato str. DC3000] flagellar hook-associated protein FliD [Pseudomonas syringae pv. phaseolicola 1448A] outer membrane protein [Pseudomonas syringae pv. tomato str. DC3000] amino acid ABC transporter, periplasmic amino acid-binding protein [Pseudomonas syringae pv. tomato str. DC3000] type III effector HopAK1 [Pseudomonas syringae pv. tomato str. DC3000] lipoprotein, putative [Pseudomonas syringae pv. phaseolicola 1448A] acetyl-CoA carboxylase [Pseudomonas syringae pv. syringae B728a] a,a-phosphotrehalase [Chromobacterium violaceum ATCC 12472] HrpF [Pseudomonas savastanoi] putative hydrolase [Corynebacterium efficiens YS-314] Extracellular ligand-binding receptor [Pseudomonas syringae pv. syringae B728a] autotransporting lipase, GDSL family [Pseudomonas syringae pv. tomato str. DC3000] sugar ABC transporter, periplasmic sugar-binding protein [Pseudomonas syringae pv. phaseolicola 1448A] hypothetical protein PSPTO3599 [Pseudomonas syringae pv. tomato str. DC3000 autotransporting lipase, GDSL family [Pseudomonas syringae pv. phaseolicola 1448A]

hrpJ

gi|71554439gi|42562016gi|42562015gi|999627gi|71557868gi|71558760gi|33521621gi|28883191gi|71557222gi|71553947gi|71557519gi|66048189gi|71558795gi|71558819gi|71554131gi|71557267gi|8037778gi|40645052gi|20145844gi|3201826gi|71556129gi|6981420gi|790907gi|71558459gi|28869576gi|71557845gi|28868926gi|28871313gi|28871243gi|71557286gi|66047627gi|34498754gi|37931589gi|23494780gi|66047072gi|28867797gi|71555318gi|28870759gi|71555414gi|571517

P. syringae

KB, 28°C

P. syringae

HIM, 18°C concentration 40-60x

SDS-PAGE analysis

Identification of virulence factorsFig 6:

Changes in nucleus morphology were analyzed in Psm or Pst infiltrated leaves at the time of tissue collapse. Sections were prepared from leaf tissue at the infection site. Following fixation and embedding, nucleus structure was studied under the fluorescence microscopy.

Crescent-shaped nucleus

Chromatin locatedalong nuclear envelope

Pst

Granularchromatin

Nuclear blebbing

Psm

Control

Different stages of nucleus degeneration

/

/

Infiltration of tobacco with two pathovars of P. syringae results in different morphotypes of HR.

In contrast to Pst, after Psm infection long distance acquired resistance is not established. The reason for that is probably rapid degeneration of vascular bundle cells.

/

/

Conclusions:

Changes in ultrastructure of organelles in response to infection

Fig 3:

Nu M

Ch

Ch

Ch

M

Ch Ch

Nu

MM Ch

NuM

Ch

Ch

Nu

MM

M

MM

Ch

Ch

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M

M

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M

ChCh

NuM

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M

ChCh

Ch

Ch

mock

Psm 1.5 hpi

Psm 4.5 hpi

Psm 6 hpi

mock

Pst 3.0 hpi

Pst 9.0 hpi

Pst 12.0 hpi

Bars represent 2µmCh-chloroplast,Nu-nucleus, M-mitochondrion

After fixation and embedding material was subjected to TEM analysis. Bars represent 5mm.De-dead cell, Se-sieve element,X-xylem.

Differences in degradation of cellssurrounding conductive elements

Fig 5:

De

De

DeDe

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X

Se

Se

De

De

De

SeSe

Se

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X

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Psm 3.0hpi

Psm 6.0hpi

Pst 9.0hpi

Pst 12.0hpi