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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
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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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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Cellular aggregation vs Biofilm formation
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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Cellular aggregation vs Biofilm formation
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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