Virulence Mutants Of Puccinia Striiformis F.SP. Tritici Gained By Particle Bombardment With GUS Gene

Wheat strip rust, caused by Puccinia striiformis f.sp. tritici, is one of the most important wheat diseases in wheat growth region all over the world. Growing rust-resistant cultivars was the most economical and effective measure to manage wheat strip rust integrally. However, continuous occurrence of new virulent races was the main cause to led to the breakdown of resistance genes and the periodical prevalence of wheat strip rust. It had been proved that mutation and heterokaryosis were the two main ways of Puccinia striiformis virulence variation and gene mutation was the main approach to produce new virulence genes. Therefore, pathogenicity-related genes were researched for the key to control wheat strip rust and wheat breeding. To obtain virulence mutants and study their biological characteristics and optimize the transformation system of P. striiformis by gene gun, plasmid pGUS6L20 containing the GUS gene was transformed into urediniospores of P. striiformis f. sp. tritici via particle bombardment.1.Optimized the transformation system of P. striiformis by biolistics: Plates sprayed urediospores (0.7mg) of P. striiformis were maintained at 4℃for 2h to 2.5h to initiate spore hydration before bombardment. Gold particles (350μg) of 0.6μm diameter were coated with plasmid DNA (1μg). The plates of hydrated P. striiformis urediospores were bombarded at delivery pressures of 900psi, 1100 psi and 1350psi. He pressure was 2.7×104Pa. The distance between the membrance-cracking and membrance-bearing was 2.5cm, the distance between membrance-bearing and blocked net was 0.8cm and placed 6cm from the blocked net.2.The separation method of the transformants with GUS Gene in wheat stripe rust was studied:The result showed that to separate transformants from screening cultivars while separating transformants from breeding cultivar Huixianhong as an assistant way can increase the detectable rate efficiently. A new way which can separate the transformants with GUS gene through pasting and dyeing of urediospores of P. striiformis by transparent tape was established.3.Transient expression of reporter gene and screened of virulence mutants of P. striiformis using biolistics: Plasmid pGUS6L20 containing the GUS gene was transformed into urediniospores of P. striiformis f. sp. Tritici(CYR29)via particle bombardment. The transient expression of GUS was identified in the transformed urediniospores. The GUS-carrying spores were cultured and increased consecutively. The GUS gene was expressed in the uredia and urediniospores of the T1 and T2 generation of the stripe rust fungus. Two stable insertion mutants of increased virulence were identified on wheat cultivars JubilejinaⅡ(MJ-4 and MJ-7)and Kangyin 655(MK-2 and MK-3), respectively. Mutant MK-2 showed virulence on Mentana with reaction type changing from 0 (wild-type) to 3. Reaction types of MJ-4, MJ-7 and MK-3 on JubilejinaⅡand Kangyin 655 is 4, respectively and the wild-type is 0. The presence of the GUS gene was detected by the polymerase chain reaction (PCR) and Southern blot in these four virulence mutants. The pathogenicity of four mutants cultures on the differentials and Near-isogenic lines has changed greatly. The result shows that the particle bombardment is an effective way to obtain the virulence strains of Puccinia striiformis.The foreign gene markers are introduced by particle bombardment, the optimized transformation system and pathogenicity mutants of P. striiformis gained in the study were useful for cloning virulence genes in P. striiformis and elucidating the mechanism of virulence variation in P. striiformis.