噬菌体克服水稻细菌性褐条病菌CRISPR/Cas免疫的anti-CRISPR机制研究

为有效利用自然界广泛存在的噬菌体来生态调控植物细菌病害，迫切需要搞清楚细菌和天敌噬菌体共进化过程中产生的攻防策略。申请者团队前期不仅国内外首次揭示了水稻褐条病菌利用Type Ι-C CRISPR/Cas抵御噬菌体的入侵，还发现与抗性菌株相比，噬菌体敏感菌株基因组的前噬菌体序列存在多个可被CRISPR/Cas识别的self-targets，显示其已进化出能克服CRISPR/Cas免疫以避免自身被降解的anti-CRISPRs。本申请项目拟在前期基础上首先在self-targets附近确定anti-CRISPRs；然后鉴定出与Cas互作的anti-CRISPRs；接着突变和回补分析anti-CRISPRs在噬菌体侵染中的功能；最后过表达和原核表达anti-CRISPRs并评价其对CRISPR/Cas的抑制，以最终阐明噬菌体克服水稻褐条病菌CRISPR/Cas免疫的anti-CRISPR机制。

In order to effectively control plant bacterial diseases using the bacteriophages that widely distributed in nature, it is very necessary to make clear the attack and defense strategies in the coevolution process between bacteria and bacteriophage. The applicants not only firstly revealed the importance of type I-C CRISPR/Cas in bacterial defense against bacteriophage infection, but also found that compared with bacteriophage-resistant strain, there were multiple self-targets that could be recognized by CRISPR/Cas in prophages from genome of bacteriophage-susceptible strain, indicating that anti-CRISPRs have been successfully developed to avoid its own degradation by CRISPR/Cas immunity. The aim of this project is to find the anti-CRISPRs by TXTL analysis of sequences near self-targets; next to identify the anti-CRISPRs interacting with Cas based on analysis of bacterial two-hybrid, BiFC and pull down as well as correlation analysis of gene expression; and then elucidate the role of anti-CRISPRs in bacteriophage infection by comparing the wild type with the constructed mutants and corresponding complements; and finally determinate the anti-CRISPR mechanism of bacteriophage overcome the CRISPR/Cas immune system of Acidovorax oryzae by using overexpression and prokaryotic expression techniques.