| Verticillium wilt is a soil-borne fungal disease giving serious wilt phenotype for various plant species and resulting in severe loss of crop production. Most Verticillium diseases are mainly caused by either Verticillium dahliae or V. albo-atrum. The Verticillium species can produce extracellular toxins that are the cause of most of the symptoms associated with Verticillium wilt disease. Although the great efforts have been made to investigate molecular mechanisms of the pathogen-host interaction, little is known about chemical property of the pathogen-secreted toxin as an effective elicitor as well as of the perception site (a receptor) responding to the elicitor. In this dissertation work, the pathogenic fungi Verticillium. dahliae and Arabidopsis thaliana are used as a plant-microb interaction model system to identify and isolate the effective wilting factor from the Verticillium. dahliae secreted toxins.The previous work in this laboratory has analyzed the susceptibility of Arabidopsis thaliana (ecotype Columbia) to V. dahliae (strain V229). The inoculation of plants with cultured pathogens resulted in typical disease symptoms (wilt, chlorosis) as well as significant growth inhibition of the seedlings, and the the injection of the extracted fungal toxins into Arabidopsis rosette leaves caused hypersensitive reaction around the acupunctured spots.The methods of liquid chromatography, including FPLC, HPLC and IC, combined with the mass spectrometry were applied to identify and purify the effective wilting factor. The pathogenic activity of the fractions was detected using the leaf puncture tests and/or the seedling culture tests. The raw toxin extracted from the liquid culture of pathogens was fractioned through a gel column chromatography and 5 fractions. The P2 fraction was identified as an effective fraction to cause the hypersensitive reaction (necrosis symptom) by injection of P2 and to result in the inhibition of seedling growth after addition of P2 in the medium. In addition, the transcriptional expression of the pathogen-related protein 1 (PR1) was dramatically induced by P2 fraction. The results indicate that P2 fraction contains the effective wilting factor that triggers signaling process for host hypersensitive reaction. Using Ion Exchange (IEX) Chromatography, the P2 fraction was further separated into 5 fractions, named from Dl to D5. The Dl fraction was identified as the most effective one for induction of hypersensitive reaction and inhibition of seedling growth. By application of HPLC, the Dl was fractioned and H112 was further identified as the fraction containing effective factor to induce hypersensitive reaction of host plants. The H112 was identified as a carbohydrate compound. The fraction H112 was further analyzed using ion chromatography (IC) and 6 sub-fractions were obtained. These sub-fractions were subsequently analyzed by application of Reverse Phase-HPLC, and the effective fractions with similar retention times were collected. The fraction named IC4-1 was identified by using IT-MS to mainly contain fragment of m/z 520.7.In conclusion, IC4-1, a carbohydrate-group containing compound with molecular weight of 521.7 dalton, was identified as the effective wilting factor in the Verticillium. dahliae secreted toxins. Thissimple compound is effective to induce the hypersensitive reaction of host plants. It is expected that this study may provide important information for further investigation of the pathogen-plant interaction. |
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