K. Norihito1, S. Takehisa1, L. Sam2, S. Satoshi1, M. Hiroki1, M. Yukihiro1, H. Marcelle2,
'Dept Global Agri. Sci., U. Tokyo, Tokyo, Japan
2Dept Plantengenetica, VIB, U. Gent, Gent, Belgium
Sesbania rostrata is an annual, fast-growing legume from the Sahel region of West Africa and parts of Angora, Mozambique and Madagascar with tropical climates and flooded soils, where it engages in symbiotic nitrogen fixation with Azorhizobium caulinodans. Nitrogen-fixing nodules are formed at lateral root bases and at the bases of adventitious rootlets that are located on the stem. We want to examine the potential overlap between symbiosis- and pathogenesis-related plant responses and collected a few genes commonly implicated in defense responses for use as molecular markers.
Chalcone synthase (CHS) and phenylalanine ammonia-lyase (PAL) are two key enzymes in the biosynthesis of isoflavonoid phytoalexins. In general, these antimicrobial plant compounds accumulate strongly upon pathogen attack. Other important plant proteins in the response to pathogen attack are hydrolytic enzymes such as (3-1,3-glucanase (GLU), that inhibit growth of fungal infection.
To isolate homologs from S. rostrata, DNA sequences for these genes were aligned and degenerate primers were designed corresponding with conserved regions. DNA fragments were amplified on a cDNA template prepared from a mixture of S. rostrata root primordial samples harvested at different time points after inoculation with Ralstonia solanacearum. This wide host range pathogen causes a nonhost defense response upon infection of the adventitious rootlets. Sequencing resulted in the identification of an S. rostrata clone for each of the three defense genes. Subsequently gene specific primers were designed for RT-PCR detection of the transcripts corresponding to SrPAL, SrGLU and SrCHS.
To check whether these genes are expressed in S. rostrata following pathogen attack, RT-PCR analysis was applied to Botrytis cinerea infected leaf material. Uninfected leaves were compared with leaves harvested at 4, 8, 17, 30 and 48 hours after inoculation with the fungus. PAL, CHS and GLU genes were induced already at 4 hours after infection (Lievens 2001). Strongest induction was observed for (3-1,3-glucanase. To isolate the full length p-l,3-glucanase sequence, 3' and 5' RACE were performed. The deduced amino acid sequence contained an ORF of 372 amino acids with 84% identity with Phaseolus vulgaris (5-1,3-glucanase and 83% identity with Medicago sativa acidic glucanase. In wounded leaves, SrGLU transcripts were expressed from 2 h to 2 days. At present the expression of these genes upon Ralstonia infection and during nodule development is being investigated.
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