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FACOLTÀ DI SCIENZE MATEMATICHE FISICHE E NATURALI. Corso di Laurea Magistrale in Biotecnologie Industriali ed Ambientali. Characterization of the Arabidopsis thaliana LysM -CONTAINING RECEPTOR-LIKE KINASE 3 gene. Candidate : Lukasz Pawel Tarkowski.

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FACOLTÀ DI SCIENZE MATEMATICHE FISICHE E NATURALI

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Facolt di scienze matematiche fisiche e naturali

FACOLTÀ DI SCIENZE MATEMATICHE FISICHE

E NATURALI

Corso di Laurea Magistrale in Biotecnologie Industriali ed Ambientali

Characterization of the Arabidopsis thalianaLysM-CONTAINING RECEPTOR-LIKE KINASE 3 gene

Candidate: LukaszPawelTarkowski

Mentor: Dott. Simone Ferrari

Department of Biology and Biotechnologies "Charles Darwin"

AcademicYear2011/2012


Facolt di scienze matematiche fisiche e naturali

Plant-pathogeninteraction


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Crop losses due to pathogens represents a large percentage of the total potential production of all the world's major food crops

59% of the attainable production of $ 580 billion are obtainedonly, whereas 41% are lost(Oenke et al., 2006)


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  • Plants are able to recognize microbe-associated molecular patterns (MAMPs)

FUNGI

BACTERIA

flagellinEF-Tu PGN

ElicitinsChitin


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Plants are alsoable to perceiveendogenoussignalsreleasedduringinfection:

DAMPs (Damage-AssociatedMolecularPatterns)

  • A well-characterizedexample of DAMP are the oligogalacturonides (OGs):

  • pecticfragmentsreleased from homogalacturonan’sdegradation


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  • Recognition of MAMPs/DAMPs:

  • PRRs (pattern-recognition receptors)

Adapted from Brohm et al., 2014


The lysin motif lysm

The lysinmotif (LysM)

  • 42-48 amino acids long motif, found in all living organisms except in Archaea

  • Topology βααβ (two α-helices on one side of an antiparallel β sheet)

  • in Eukaryotes it was first identified in proteins that degrade peptidoglycan (e.g. lysozyme)

LysM domain of the E. coli membrane-bound lytic mureintransglycosylase (Bateman and Bycroft, 2000)


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LysM-containingproteins: mediators of symbiosis and defenceresponses

Nurnberger et al., 2012


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LysM-containingkinasesin Arabidopsisthaliana

Tanaka et al., 2013


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PreviousfindingsaboutAtLYK3…

  • AtLYK3 nullmutant (atlyk3-1) showedenhancedresistanceagainstplantpathogenslikeB. cinerea and P. carotovorum

  • …Thisfeatureisdependent on PAD3, a gene thatencodesa P450 cytocromeoxidaseinvolved in the last step of camalexinbiosynthesis

  • PAD3isa major determinant of the resistanceagainstB.cinerea(Ferrari et al. 2003)


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Abscisic acid negativelyregulatesdefenceresponses

againstB. cinerea

  • Arabidopsismutantsdefective in ABA biosynthetis show enhancedresistanceagainstB. cinerea(Audenaert et al., 2002; Asselbergh et al., 2007; L’Haridon et al., 2011)

ABA is involved in several physiological processes in plants (Barrero et al., 2006)


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AtLYK3expressionispositivelyregulated by ABA

  • Low-dose ABA treatmentsinduces AtLYK3 expression

  • AtLYK3isrequired for late responses to ABA, suchasprimaryrootelongation and germinationinhibition(Paparella et al., 2014)

Germinationinhibitionassay with 5 µM ABA


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AIM OF THE THESIS

All the data obtainedaboutAtLYK3 came from the studies on onemutant: in order to corroborate previousfindings, I isolate and characterize a new insertionalmutant, atlyk3-3.

Generation and characterization of transgenic plants expressing an AtLYK3-GFP fusion protein


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The new insertionalmutantatlyk3-3ishypomorphic

Schematicrepresentation of the AtLYK3 locus

days post infection

Semiquantitative RT-PCR


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atlyk3-3 shows increased resistance to pathogen infection similar to atlyk3-1, and increased expression of PAD3

  • Lesionsizes of atlyk3-3mutants are comparable to thoseseen on atlyk3-1

  • Expression of PAD3 is enhanced in both atlyk3-1 and atlyk3-3 mutants inoculated with B.cinerea

B. cinerea , 2days post infection

P. carotovorum , 1day post infection


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atlyk3-3 and atlyk3-1 have similar alterations of ABA responses

atlyk3-3mutants display similarresults to thoseobtained with atlyk3-1 in the ABA responsebioassays

- percentage of reduction of root elongation, with respect to the controls, is indicated above bars

- 4-day-old seedlings grown on solid medium with 2.5 µ ABA or 0.05% MeOH (control) for two days

- Seeds grown on solid medium with 0.05% MeOH (control) or 5 μMABA

- Germination rate was determined after three days


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Furthercharacterization of AtLYK3

  • 1) Test iflack of functionalAtLYK3increasesresponses to MAMPs or DAMPs , by using callose deposition assay for well-known characterized examples of MAMPs/DAMPs, such as flg22 and OGs; and the analysis of PAD3 gene expression

  • Callosedeposition is a common-used marker response in the induction of PAMP/DAMP –triggered immunity (Luna et al., 2011)

2) Determine the subcellularlocalizationof AtLYK3 by generatingtransgenicplantsexpressing the CaMV 35S::AtLYK3-GFP construct

  • All Arabidopsis LYK proteins so far characterized are localized in the plasma membrane (Tanaka et al., 2013)


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Resistance to pathogens in atlyk mutants is not due to enhanced MAMP/DAMP-triggered callosedeposition

WATER

WT

Adult rosette leaves were infiltrated with water, 100 μg ml-1 OG or 100 nMflg22. Leaves were harvested after 24 h.


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IncreasedPAD3expression in atlyk3mutantsisnot due to enhancedresponse to elicitors

flg22

OGs

y

PAD3expressionisenhancedatsimiliarlevels in WT and atlykmutantstreated with OGs and flg22

Takentogether, callose and PAD3expression data indicate thatatlyk3mutantsenhancedresistanceisnot due to altered MAMP/DAMP perception


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Generation of plants expressing the

CaMV35S::AtLYK3-GFPconstruct

1) Generation of a expression vector through the Gateway recombination system

2) Trasformationof E. coli, plasmid amplification and purification, trasformation of A. tumefaciens

3) Trasformationof plants by floral dip (Clough and Bent, 1998) , and subsequent selection of F1 resistant plants


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AtLYK3 islocalized in the plasma membrane

GFP FM-64 Merge

epidermal cells seen with confocal laser scanning microscopy

  • The GFP-associatedaccumulation of fluorescence in correspondence to the plasma membrane wasalsoobserved in plasmolysisexperiments


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CONCLUSIONS

AtLYK3 plays a role of negative regulator of defenceresponses against microbial infections.

Among the geneswhichexpressionisnegativelyregulated by AtLYK3, the mostimportantseems to be PAD3, crucial for the resistance to the necrotrophfungusBotrytis cinerea

AtLYK3is required for several late responses to ABA : this gene may have a important role in the negative cross-talk between the plant innate immunity and ABA-mediated processes.

AtLYK3 kinase domain seems to be necessary for the protein function.

AtLYK3 islocalized in the plasma membrane.


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PHYSIOLOGICAL STATE

PATHOLOGICAL STATE

PATHOGEN INFECTION

ABA

MAMPs/

DAMPs

PLANT IMMUNE

RESPONSE

AtLYK3

Camalexin

Late ABA responses

PAD3

PAD3

ABA

AtLYK3


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