The characterization of defense responses to fungal infection in alfalfa

The enzyme activity and transcript level of three enzymes involved in flavonoid biosynthesis, phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (CA4H), and isoflavone reductase (IFR), were monitored in alfalfa seedlings (Medicago sativa) that had been challenged with the fungal pathogen Colletotrichum trifolii. In many legumes, a pathogen-host infection leads to an induced synthesis of fungitoxic phytoalexins produced via these key enzymes. For example, when alfalfa is exposed to an avirulent fungal type, phytoalexins are produced by the plant providing protection from additional exposure to a virulent fungal race. This defensive plant protection response was accompanied by increases in transcript levels of PAL, CA4H and IFR gene expression, by increases in PAL enzyme activity, and by production of the end product phytoalexins, medicarpin and sativan. The expression of these defense genes and PAL enzyme activity were significantly greater in seedlings responding to combined inoculations of avirulent and virulent fungi, compared to plants inoculated with the avirulent fungus alone. Inoculation with the virulent race alone elevated the gene transcripts compared with control plants but these levels were less than found in plants inoculated with the avirulent race or those inoculated with both. Production of gene transcripts reached its peak in treated plants after 49 h. The phytoalexins medicarpin and sativan increased rapidly and reached maximal levels 97 h after inoculation with the avirulent fungal race. The plants challenged with combined inoculations of the avirulent and the virulent fungi showed significantly greater accumulation of medicarpin than in any other treatment. These results suggest that the increased medicarpin accumulation produced in induced resistant tissues, following challenge inoculation with virulent race, is attributable to increased expression of genes of flavonoid biosynthesis.