RESUMEN
In a search for novel plant-derived antimicrobial proteins, we screened extracts from salicylic acid (SA)-treated lettuce and sunflower leaves. These extracts displayed very potent antimicrobial activity against a set of phytopathogens. Characterisation of these extracts revealed that in both extracts, proteins of approximately 60 kDa were responsible for the antimicrobial activity. Further characterisation of these proteins and cloning of the respective cDNAs revealed close homology to a range of (plant) oxidases. Dissection of the enzymatic activity of both proteins revealed them to be carbohydrate oxidases (Helianthus annuus carbohydrate oxidase (Ha-CHOX) and Lactuca sativa carbohydrate oxidase (Ls-CHOX)) with broad substrate specificity and with hydrogen peroxide (H(2)O(2)) as one of the reaction products. The sunflower transcript, in addition to being SA inducible, was also inducible by fungal pathogens but not by ethylene and jasmonate. To determine whether Ha-CHOX plays a role in pathogen defence, it was transformed into tobacco and the effect of resistance to Pectobacterium carotovorum ssp. carotovorum was examined. Transgenic plants overexpressing Ha-CHOX displayed enhanced resistance to infection by this pathogen, and the resistance level was proportional to enzyme expression.
Asunto(s)
Helianthus/genética , Lactuca/genética , Proteínas de Plantas/aislamiento & purificación , Secuencia de Aminoácidos , Escherichia coli/genética , Regulación de la Expresión Génica de las Plantas , Helianthus/metabolismo , Helianthus/microbiología , Peróxido de Hidrógeno/metabolismo , Lactuca/metabolismo , Lactuca/microbiología , Datos de Secuencia Molecular , Pectobacterium carotovorum/patogenicidad , Filogenia , Hojas de la Planta/química , Proteínas de Plantas/química , Plantas Modificadas Genéticamente , Ácido Salicílico/farmacología , Especificidad por Sustrato , Nicotiana/genética , Nicotiana/microbiología , Transformación GenéticaRESUMEN
Summary Many events associated with the plant defence responses are regulated on the transcriptional level. Here we report the results of a promoter tagging approach to identify promoters that are induced upon pathogen attack in Arabidopsis thaliana. A line was identified in a T-DNA UidA tagged Arabidopsis library with induced GUS expression after Botrytis cinerea infection around the site of fungal infection. The upstream sequence was isolated and fused to the UidA gene and tested in transgenic Arabidopsis thaliana and Brassica napus plants. Promoter function was very similar to the expression pattern found in the original promoter tagged line. We found that the promoter sequence was located on Arabidopsis chromosome III and linked to a predicted open reading frame in the reverse orientation. The predicted gene codes for a putative receptor serine threonine protein kinase of 383 amino acids in size. The clone contains a protein kinase ATP binding region, a protein kinase active site, a region with similarity to motifs found in Alpha Isopropylmalate/homocitrate synthase enzymes and a putative leucine zipper motif. Analysis of the expression pattern of the gene using RT-PCR demonstrated that the putative receptor serine threonine protein kinase is up-regulated after Salicylic acid treatment and Botrytis infection.
