Optimization Of Genetic Transformation System And Preliminary Investigation On The Infection Mechanism Of Ustilago Hordei

Ustilago hordei is a kind of basidiomycete fungi that causes covered smut of barley(Hordeum vulgare L.), which can bring about significant yield loss to global barley production. In this paper, we studied the physiological and infection properties of Ustilago hordei. To establish a gene transformation system of Ustilago hordei, ATMT(Agrobacterium tumefaciens mediated transformation) method was chosen, and the factors might influence transformation efficency were optimized. In addition, how Ustilago hordei mates and infects plant was also observed. Main results obtained were shown as follows:1. To generate 3×pgpdA-1301 and 3×phsp70-1301 vector, Ustilago hordei specific promoters gpdA and hsp70 were cloned into pCAMBIA1301 plasmid by replacing the 35S promoter, respectively. For construction of 3XpgpdA-1301-GFP and 3×phsp70-1301-GFP, the GFP coding sequence was inserted into the two vector. All the four vectors were used to transform Ustilago hordei, and transformants were selected by hygromycin resistance gene and GFP gene, which were driven by gpdA and hsp70 promoters.2. Through analysis the effect of culture condition on Ustilago hordei growth, temperature, pH and composition of mediun were optimized. The optimal condition was 23℃and pH 7.0. Medium composition might have little effect on fungi growth. Low concentration of carbon and nitrogen were enough, and salt free would not affect fungi growth.3. In order to develop an efficient method for Ustilago hordei transformation, PEG-CaCl2, eletroporation and ATMT method were performed and the efficiency of four methods were compared. The efficiency of ATMT method was notable higher than two other methods. Then central factors that influence ATMT efficiency were optimized, the optimal condition was 200-400mM acetosyringone concentration,12-18h pre-culture time and 48-72h co-culture time.4. Mating and infection process of Ustilago hordei 364/365 on Arabidopsis thaliana and barley seedlings were observed. It indicated that the infection hypha formation of Ustilago hordei was the same with Ustilago maydis:conjugation tube emerged from one end of the sporidial, then elongated to the conjugation tube of opposite mating type of sporidial in response to pheromone. Conjugaton tubes of opposite mating type fused, often at their tips, and infection hypha emerged from conjugation bridge. Ustilago hordei infection would hurt meristem region and central cylinder of plant root, and inhibited development of plant root. At the same time, the development of root hair was stimulated.