Mutations Affecting Virulence Induced By Biolistics In Puccinia Striiformis

Wheat strip rust,caused by Puccinia striiformis West. f.sp tritici,is an air-borne disease and one of the most important wheat diseases in wheat growth region in our country and all over the world. The studies and practices in China and the other countries showed that growing rust-resistant cultivars was the most economical, effective and feasible environment-friendly measure to manage wheat strip rust integrally. However, the virulence of wheat strip rust varied frequently and produced new virulent races which led to the breakdown of resistance genes and the periodical prevalence of wheat strip rust. Therefore, releating the reasons and rules on virulence variation of wheat strip rust and putting forward effective countermeasures to prolong service life of resistant cultivars, which is the key to control wheat strip rust and wheat breeding. Mutation and heterokaryosis are the two main ways of Puccinia striiformis virulence variation, the construction of pathogens mutation library is an important means for molecular genetic analysis as well as a short cut to clone pathogenicity-related genes. This study explored the ways to produce P. striiformis mutation and establish a set of effective genetic transformation system in P. striiformis1. Established the transformation system of P. striiformis by biolistics: there were many factors to affect the transformation rate including the delivery pressure, the speed and gun shot of microprojectiles, the dosage and the diameter of gold powder, the concentration and hydrate time of urediospores of P. striiformis, He pressure in bombardment, and so on. Established the optimized transformation system of P. striiformis by biolistics: Urediospores of P. striiformis (0.2mg) were sprayed onto plates. Plates were maintained at 4℃for 2h prior to bombardment initiate spore hydration. Gold particles (500μg) of 0.6μm diameter were coated with plasmid DNA(1μg). The plates of hydrated P. striiformis urediospores were bombarded at delivery pressures of 1100 and 1350psi and placed 6cm from the blocked net. The distance between the membrance-cracking and membrance-bearing was 2.5cm, the distance between membrance-bearing and blocked net was 0.8cm, and the He pressure was 2.7×104Pa.2. Transient expression of reporter gene in P. striiformis transformants using biolistics: urediospores of Puccinia striiformis is transformed with the plasmid pGUS6L20 carrying the GUS reporter gene. The GUS in transformed urediospores are able to express temporarily under the delivery pressure of 1100, 1350, 1550psi. The transformation efficiency is high up to 0.2% and 0.34% under the delivery pressure of 1350psi and 1550psi respectively. However, the germination rates of these urediospores are as low as 25%. After stained with X-Gluc, some of the spores are deep blue and some are light blue which shows that there are difference in the copies and expression quantity of GUS gene within the transformed urediospores. When transformed with the pKLHyg14 which contains the hygromycin B resistance gene hpt, under the delivery pressure of 650, 900, 1100psi, the urediospores have higher germination rates on media containing 50μg/mL hygromycin B than the control. This indicates that hpt gene has expressed in those urediospores. Therefore, reporter gene GUS and hygromycin B resistance gene hpt can both be used as selection marker for P. striiformis transformants.3. Screened of virulence mutants of P. striiformis using biolistics: Single spores of albino strain-5 are used as the recipients transformed by biolistics with vectors carrying GUS gene and hygromycin B resistance gene hpt. When the urediospores of P. striiformis is transformed with the plasmid pGUS6L20 carrying GUS gene under the delivery pressure of 1350psi and 1550psi, the GUS gene expression in progenies is detected. The expression quantity of GUS gene is decreased with increased of the progenies, and GUS gene expression in the third generation of transformants isn't detected. When the urediospores of P. striiformis is transformed with the plasmid pKLHyg14 carrying the hpt gene under delivery pressure of 1100psi, two stable virulence mutants M1100-4-2 and M1100-4-3 are screened. Mutant M1100-4-2 showed reduced virulence on Mentana with reaction type changing from 4 (wild-type) to 2. Reaction type of M1100-4-3 on Mentana decreased from 4 (wild-type) to 2 and 3 which shows the virulence differentiation. Infection assay of mutants on Chinese differential host shows that virulence of all the mutants changes significantly in contrast with wild-type isolates. PCR amplification in the two mutants yields the hpt gene fragment and this indicates that the mutation in the two mutants results from the insertion of hpt gene.The foreign gene markers are introduced by particle bombardment and pathogenicity mutants of P. striiformis are screened in the study. Therefore, a novel technical system for studying inheritance of P. striiformis pathogenicity is established, which lays a strong foundation for cloning virulence genes in P. striiformis and elucidating the mechanism of virulence variation in P. striiformis.