Effects of grape xylem sap and cell wall constituents on in vitro growth, biofilm formation and cellular aggregation of Xylella fastidiosa

7/31/2019 Effects of grape xylem sap and cell wall constituents on in vitro growth, biofilm formation and cellular aggregation of Xylella fastidiosa

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Presented by :

Kusuma Darma (A362090031/FIT)

Advanced Plant Pathogenic Bacteria

Davis W. Cheng, Hong Lin, M. Andrew Walker, Drake C. Stenger & Edwin L. Civerolo

Eur J Plant Pathol (2009) 125:213222


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Xylella fastidiosa (Xf)

Gram negative; xylem limited bacterium

Tansmitted by xylem-feeding insects (ex. Homalodisca

vitripennis)

Cause of Pierces Disease (PD) of grapevine : wilt/waterstress due to xylem blockage by EPS, pectins, tyloses, and

gum

Bacterial movement and multiplication occurs more rapidlyin xylem of susceptible grapevines


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Xylem of grapevines

Xylem pit membranes in grapevine do not allow passage of

bacteria between adjacent vessels as the pore size (5-20 nm) is

much smaller than the diameter ofXfcells (0.3-0.5 um)

Xfproduces CWDEs (ex. PG) for overcome physical barrier Cell wall degradation product effectXfcell growth aggregation,

biofilm formation, and movement within xylem vessels either

directly as a source of nutrients and/or indirectly by induction or

repression ofXfgenes Xylem sap contain various amino acids, organic acids,

inorganic ions and proteins


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The objective of study

to compareXfgrowth, biofilm formation, cell aggregation in

vitro in response to the amendment of media with xylem sap

from different sources (PD-resistant and PD-susceptible

grapes) or amendment of media with a variety of cell wallconstituents.


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Procedure

Plant growth and collection of xylem sap

Bacterial growth and treatments

Measurements of bacterial planktonic growth,cellular aggregation and biofilm formation

Statistical analysis


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Grapevines cultivars/lines

Plant propagated in green house

V. rupestrisA. de Serres x V.

arizonica/girdana b42-26

D8909-15V. rupestrisA. de Serres x V.

arizonica/candicans b42-26

F8909-17

9621

9621-67PD-resistant 9621-67PD-susceptible


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Inoculation and xylem sap collecting

Inoculation : 3 weeks old plant

Xylem sap collecting : 3 months post inoculation

Using pressure chamber (PMS Instrument Co., USA)

From actively growing shoots

First few drops of xylem were discarded

Volume of xylem sap : 0.5-2.0 ml for @ sampel

Collected sap was transferred to15 ml tubes andimmediately stored at -80C


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Bacterial growth and treatment

XF strain temecula-I

Solid PW medium

28C; 2 weeks

Liquid PW medium24C; rotary shaking

95 rpm; until 1x108

Bacterial cells

Centrifuge : 2000g, 4C

Bacterial cells

Washing with liquid PW (-BSA)

Bacterial cells

Resuspended in 15 ml liquid PW

Precondition bacterial culture

Xylem sap

Filter-sterilized; mix with 1/10 Vol

of 10X PW (-BSA) liquid medium

100 l cell wall

constituent solution*

Added to 15 ml of 1X PW (-BSA)

liquid medium

Bacterial culture Bacterial culture

Incubation : 24C, rotary

shaking 95 rpm

Incubation : 24C, rotary

shaking 95 rpm

Bacterial assay in 1, 3 &

7 dai for :

Bacterial growth (cell

density)

Biofilm formation

Cellular aggregation

Bacterial assay in 1, 3 &

7 dai for :

Bacterial growth (cell

density)

Biofilm formation

Cellular aggregation


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Table 1. Cell wall constituents used in study


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Measurements of bacterial planktonic growth,

cellular aggregation and biofilm formation

Bacterial culture

Cell suspension

Centrifuge : 2000g, 4C, 10

Washed with 0.8 NaCl in PBS

Centrifuge : 2000g, 4C, 10

Resuspended in 500 l 0.8

NaCl in PBS

Spectrophotometry

= 600 nm

Platting

Bacterial tube culture

Bacterial biofilm

3x rinse with sterilized water

Stain with 0.5 ml of 1% crystal violet; 15

3x rinse with sterilized water

Dried completely

Spectrophotometry

= 600 nm

dissolved in 2 ml of absolute ethanol

vortexing at the highest setting for 3

Cell suspenatant

Stand 20

Cell aggregate

Bacterial culture

1 dispersed with

tissue homogenizer

Spectrophotometry

= 540 nmODt

Spectrophotometry

= 540 nmODs

% cell aggregation = [(ODtODs)/Odt] x 100

Bacterial Growth Biofilm FormationCell Aggregation


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Effects of xylem sap onXfgrowth in vitro In 1 DAI culture : no significant different inXfgrowth

cultured in liquid PW (-BSA) medium amended with xylem

sap of resistant or susceptible plant

In 3 and 7 DAI :Xfgrowth cultured in liquid PW (-BSA)medium amended with xylem sap of susceptible plant was

significantly greater


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Effects of xylem sap onXfgrowth in vitro

Estimated based on spectrophotometric measurements

PD-resistant (962167) or PD-susceptible (962194) grapevines

(*) indicates ANOVA-test results of significance at P


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Effects of xylem sap onXfgrowth in vitro

Xfgrowth on normal PW medium-agar plates

after preculture for 7 days in PW medium

containing 1/10X BSA and amended with

xylem sap from PD-resistant (962167) or PD-

susceptible (962194) grapevines

The number of viable cells recovered from

growth medium amended with xylem sap from

PD-susceptible plants averaged 2.24-foldmore than the number of viable cells

recovered from medium supplemented with

xylem sap from PD-resistant plants (431 : 192)


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Effects of cell wall constituent onXfgrowth in vitro

The comparison was taken statistically between control and each treatment in a column,where asterisks * means significant level at P


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Effects of cell wall constituent onXfgrowth in vitro

Most cell wall constituents had positive effects on bacterial

growth in vitro especially after 7 days of culture.

CM-cellulose had the most promoting effect for all time-

points examined, followed by xylan, cello-oligosaccharide,-Dglucan, k-carrageenan, and laminarin.

In contrast, lichenan inhibitedXfbacterial growth.

Laminarin and k-carrageenan also had negative effects onXfgrowth initially after 1 day of culture. Xylan inhibitedXf

growth after 3 days of culture.


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Effects of xylem sap on bacterial biofilm formation

xylem sap from PD-susceptible grapevinesignificantly increasedXfbiofilm formation invitro (1.48 times higher than the unamendedcontrol, 1.51 times higher than resistantxylem sap from the PD-resistant)

The biofilm formation / growth ratio analysis

indicated that xylem sap from the PD-resistant grapevine significantly decreasedXfbiofilm formation / growth ratio in vitro (-1.38times lower than the unamended control,; -1.43 times lower than xylem sap from thePD-susceptible grapevine

Xylem sap from the PD-susceptiblegrapevine did not show any significantdifference from the unamended control inbiofilm formation /growth ratio in vitro (1.04times higher) than the unamended control,but it significantly promoted Xf biofilmformation compared with xylem sap from the

Pdresistant grapevines (1.44 times higher)


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Effects of cell wall constituent on biofilm formation

b

Spectrophotometric absorbance value at 600 nm


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Effects of cell wall constituent on biofilm formation

Laminarin, xylan and k-carrageenan significantly enhancedXfbiofilm formation

Lichenan had the most significant effect on increasing theXfbiofim formation/growth ratio (6.04 times higher than control),

followed by laminarin and k-carrageenan (1.92 and 1.54 timeshigher than control respectively)

CM-cellulose and cello-oligosaccharide significantly decreasedthe biofilm formation/growth ratio (-3.25 and -1.30 times lowerthan the unamended control respectively).

Xylan and -D-glucan did not have significant effects onXfbiofilm formation/growth ratios when compared with theunamended medium controls


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Effects of xylem sap on bacterial aggregation

Effects of xylem sap on bacterial aggregationdue to partial degradation of xylem cell wall

Xylem sap from both PD-susceptible and PD-resistant grapevines significantly decreasedXfcellular aggregation in vitro (-3.28 and 2.20 timeslower than the unamended control respectively)

Xylem sap from PD-susceptible grapevinesdecreasedXfcellular aggregation more than PD-resistant grapevines in vitro (-1.49 times lower)

Similarly, even though the xylem sap from bothPD-susceptible and PD-resistant grapevinessignificantly decreasedXfcellular aggregation /

growth ratios in vitro (-4.66 and -2.84 times lowerthan the unamended control respectively)

Xylem sap from PD-susceptible grapevinesdecreasedXfcellular aggregation / growth ratiosmore than sap from PD-resistant grapevines invitro (-1.64 times lower)


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Effects of cell wall constituents onXfaggregates after

7-day culture in vitro

b

Spectrophotometric absorbance value at 540 nm


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Effects of cell wall constituents onXfcell aggregates

Lichenan, cello-oligosaccharide, k-carrageenan, laminarin,xylan and -D-glucan each decreasedXfcellular aggregationsignificantly (3 to 21 times lower than the unamended control

Interestingly, effects of these cell wall constituents onXfcellular

aggregation were similar to that observed when the growthmedium was amended with xylem sap from PD-susceptibleplants

CM cellulose, k-carrageenan, oligosaccharide, laminarin, -D-

glucan, and xylan all decreasedXfcellular aggregation /growthratios significantly (-2.29 to -16 times lower than theunamended control)


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Host response to Xfinfection

Differential host responses toXfinfection : 72% of observed phenotypic variation controlled by a single

major locus (the dominant resistance allele is PdR1)

23% controlled by several modifying loci with minor effects 5% of phenotypic variance is due to environmental conditions

Host plant responses toXfinfection differ between resistant

and susceptible genotypes at molecular and physiological

levels and also vary with plant organ, as stem and leaftissues of the same plant


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Contribution of xylem sap toXfinfection

Xylem cell wall properties and chemical composition ofxylem sap may significantly affectXfpathogenesis

The most common proteins in xylem sap are peroxidase,

lectin-like protein, protease, glycinerich protein, chitinase,lipid transfer protein-like peptides, and putative apoplasticsecretion proteins

Secretions from metabolically active cells into xylem sap

may differ among PD-resistant and PD-susceptiblegrapevines, and are likely to contribute to host response toXfinfection


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Cell wall constituents as nutrition of pathogen

Xylem is poor nutritive environment

Cell wall-degrading enzymes contribute to nutrient release

and thus mediate interactions between host and pathogen

Xfutilises polysaccharide-degrading enzymes to digest cellwall polymers of the xylem pit membranes

Upon degradation of xylem cell walls, xylem fluid in PD-

resistant and PD-susceptible grapevines is likely to differboth qualitatively and quantitatively with respect to

chemical composition of cell wall-degradation products


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Roles of plant cell wall constituents in bacterial

growth, biofilm formation and cell aggregation in vitro

most cell wall constituents had positive effects on bacterial

growth in vitro especially after 7 days of culture

the effective order is CM-cellulose > xylan > -D-glucan >

carrageenan > cello-oligosaccharide > laminarin In contrast, lichenan inhibitedXfgrowth after 3 days or 7 days

of culture.

Laminarin and k-carrageenan had negative effects onXfgrowth

initially. Both are found in the cell walls of marine algae. Theyare analogues to xyloglucan and pectin respectively, the main

constituents of dicotyledonous woody plant cell walls.


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Plant cell wall constituents have important role in

bacterial pathogenesis

Pathogenicity ofXflikely requires biofilm formation leading toxylem vessel blockage and subsequent water stress

Early release of nutrients upon degradation byXfcell wall-degrading enzymes may be a key step for successful

colonisation and subsequent formation of biofilms in xylemvessels

Bacterial growth, biofilm formation and cell aggregation can bemanipulated by altering xylem chemistry. Artificial manipulationof specific cell wall degradation pathways may lead to a

disruption ofXfbiofilm formation, or blocking ofXfgrowth Degradation and utilisation of cell wall constituents byXfin

xylem vessels of host grapevines would be an importantmechanism forXfpathogenicity upon infection


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Cellular aggregation vs Biofilm formation

Cellular aggregation may result from clumping of cellsfacilitated by extracellular polysaccharides and may be theinitial step of biofilm formation

BothXfcellular aggregation and relative cellular aggregation /

growth ratio were decreased upon treatment with xylem sapfrom a PD-susceptible grapevine compared to that from a PD-resistant grapevine

Negative correlation existed betweenXfcellular aggregationand biofilm formation, i.e. less cellular aggregation, more biofilm

formation Xylan, laminarin and k-carrageenan inhibitedXfcellular

aggregation but promoted biofilm formation


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Media rich in amino acids, rather than carbohydrates, may

stimulateXfaggregation and biofim formation

Stimuli for cellular aggregation and biofilm formation may

involve specific plant-bacterium interactions and thenutrient status of xylem sap

it is not clear whether the cellular aggregation process in

vitro is the same as that in planta and whether such a

relationship betweenXfcellular aggregation and biofilm

formation exists in planta


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A number of genes responsible for metabolic functions, celldivision, host cell wall degradation, membrane attachmentand virulence-related adaptations are highly expressedduring biofilm formation in vitro

Xfrequires polygalacturonase for colonisation andpathogenicity in grapevines potentially through thedigestion of cell wall polymers of xylem pit membranes

It remains unclear what cell wall constituents andregulatory genes are involved inXfgrowth, biofilmformation and aggregation in planta


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Conclusion

Xylem vessels serve as a confined space for host plants to

recognise and interact withXf

Xf-plant host pathogen interactions are mediated by specific

constituents of xylem sap, as opposed to direct interactionbetween bacteria and metabolically active host cells

Xylem sap from different grape cultivars, which are differentially

susceptible and resistant to PD, was affectedXfgrowth, biofilm

formation, and cellular aggregation differently in vitro Xylem sap composition analysis may be useful for screening

grapevines for PD resistance


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Documents

Effects of grape xylem sap and cell wall constituents on in vitro growth, biofilm formation and cellular aggregation of Xylella fastidiosa