The role of natural products in plant-microbe interactions

Danish title: Cyanogene glucosiders betydning for plante-mikrobe interaktioner.

 

The aim of this project is to analyze the variation in cyanogenic glucoside content in different barley cultivars and to investigate whether cyanogenic glucosides are important determinants for disease and pest resistance.
Barley (Hordeum vulgare) seedlings were found to contain five cyano glucosides derived from the amino acid L-leucine (Leu). The chemical structure and the relative abundance of the cyano glucosides were investigated by liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses using spring barley cultivars with high, medium, and low cyanide potential. The barley cultivars showed a 10-fold difference in their cyanogenic glucoside and cyanoalkyl glucoside content, but the relative content among the individual cyano glucosides remained constant. Epiheterodendrin, the only cyanogenic glucoside present, comprised 12% to 18% of the total content of cyano glucosides. It is proposed that the aglycones of all five cyano glucosides are formed by the initial action of a cytochrome P450 enzyme of the CYP79 family converting L-Leu into Z-3-methylbutanal oxime and subsequent action of a less specific CYP71E enzyme converting the oxime into 3-methylbutyro nitrile and mediating subsequent hydroxylations at the a-, b-and g-carbon atoms. Presence of cyano glucosides in the barley seedlings was restricted to leaf tissue, with 99% confined to the epidermis cell layers of the leaf blade. Microsomal preparations from epidermal cells were not able to convert l-[14C]Leu into the biosynthetic intermediate, Z-3-methylbutanal-oxime. This was only achieved using microsomal preparations from other cell types in the basal leaf segment, demonstrating translocation of the cyano glucosides to the epidermal - cell layers after biosynthesis. A b-glucosidase able to degrade epiheterodendrin was detected exclusively in yet a third compartment, the endosperm of the germinating seed. Therefore, in barley, a putative function of cyano glucosides in plant defense is not linked to cyanide release.

Cyanogenesis was reconstituted in barley leaf epidermal cells through single cell expression of a cDNA encoding dhurrinase-2, a cyanogenic β-D-glucosidase from sorghum. This resulted in a 35-60% reduction in colonization rate by an obligate parasite Blumeria graminis f. sp. hordei, the causal agent of barley powdery mildew. A database search for barley homologues of dhurrinase-2 identified a (1,4)-β-D-glucan exohydrolase isozyme βII that is located in the starchy endosperm of barley grain. The purified (1,4)-β-D-glucan exohydrolase isozyme βII was found to hydrolyze the cyanogenic β-D-glucosides epiheterodendrin and dhurrin. Molecular modeling of its active site based on the crystal structure of linamarase from white clover, demonstrated that the disposition of the catalytic active amino acid residues was structurally conserved. Epiheterodendrin stimulated appressoria and appressorial hook formation of B. graminis in vitro, suggesting that loss of cyanogenesis in barley leaves has enabled the fungus to utilize the presence of epiheterodendrin to facilitate host recognition and to establish infection. The aim of future studies will be to elucidate the chemical communication between the powdery mildew spore and the barley epidermal cell layer to more precisely delineate the function of epiheterodendrin and the different cyanoalk(en)yl glucosides in disease resistance.

 

Researchers involved: Kirsten Annette Nielsen, Janni Nyvang Nielsen, Birger Lindberg Møller,

Foreign collaborators: Maria Hrmova, Geoffrey B. Fincher, both School of Agriculture and Wine, The Australian Centre for Plant Functional Genomics, University of Adelaide, Waite Campus, Australia.

 

Financial support: Danish national research Foundation.