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
Corso di Laurea Magistrale in Biotecnologie Industriali ed Ambientali
Characterization of the Arabidopsis thalianaLysM-CONTAINING RECEPTOR-LIKE KINASE 3 gene
Mentor: Dott. Simone Ferrari
Department of Biology and Biotechnologies "Charles Darwin"
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)
Plants are alsoable to perceiveendogenoussignalsreleasedduringinfection:
Adapted from Brohm et al., 2014
LysM domain of the E. coli membrane-bound lytic mureintransglycosylase (Bateman and Bycroft, 2000)
LysM-containingproteins: mediators of symbiosis and defenceresponses
Nurnberger et al., 2012
Tanaka et al., 2013
Abscisic acid negativelyregulatesdefenceresponses
ABA is involved in several physiological processes in plants (Barrero et al., 2006)
AtLYK3expressionispositivelyregulated by ABA
Germinationinhibitionassay with 5 µM ABA
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
The new insertionalmutantatlyk3-3ishypomorphic
Schematicrepresentation of the AtLYK3 locus
days post infection
atlyk3-3 shows increased resistance to pathogen infection similar to atlyk3-1, and increased expression of PAD3
B. cinerea , 2days post infection
P. carotovorum , 1day post infection
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
Furthercharacterization of AtLYK3
2) Determine the subcellularlocalizationof AtLYK3 by generatingtransgenicplantsexpressing the CaMV 35S::AtLYK3-GFP construct
Resistance to pathogens in atlyk mutants is not due to enhanced MAMP/DAMP-triggered callosedeposition
Adult rosette leaves were infiltrated with water, 100 μg ml-1 OG or 100 nMflg22. Leaves were harvested after 24 h.
IncreasedPAD3expression in atlyk3mutantsisnot due to enhancedresponse to elicitors
PAD3expressionisenhancedatsimiliarlevels in WT and atlykmutantstreated with OGs and flg22
Takentogether, callose and PAD3expression data indicate thatatlyk3mutantsenhancedresistanceisnot due to altered MAMP/DAMP perception
Generation of plants expressing the
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
AtLYK3 islocalized in the plasma membrane
GFP FM-64 Merge
epidermal cells seen with confocal laser scanning microscopy
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.
Late ABA responses