Transposon insertion libraries for the characterization of mutants from the kiwifruit pathogen Pseudomonas syringae pv. actinidiae

Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit canker, is one of the most devastating plant diseases of recent times. We have generated two mini-Tn5-based random insertion libraries of Psa ICMP 18884. The first, a ‘phenotype of interest’ (POI) library, consists of 10,368 independent mutants gridded into 96-well plates. By replica plating onto selective media, the POI library was successfully screened for auxotrophic and motility mutants. Lipopolysaccharide (LPS) biosynthesis mutants with ‘Fuzzy-Spreader’-like morphologies were also identified through a visual screen. The second, a ‘mutant of interest’ (MOI) library, comprises around 96,000 independent mutants, also stored in 96-well plates, with approximately 200 individuals per well. The MOI library was sequenced on the Illumina MiSeq platform using Transposon-Directed Insertion site Sequencing (TraDIS) to map insertion sites onto the Psa genome. A grid-based PCR method was developed to recover individual mutants, and using this strategy, the MOI library was successfully screened for a putative LPS mutant not identified in the visual screen. The Psa chromosome and plasmid had 24,031 and 1,236 independent insertion events respectively, giving insertion frequencies of 3.65 and 16.6 per kb respectively. These data suggest that the MOI library is near saturation, with the theoretical probability of finding an insert in any one chromosomal gene estimated to be 97.5%. However, only 47% of chromosomal genes had insertions. This surprisingly low rate cannot be solely explained by the lack of insertions in essential genes, which would be expected to be around 5%. Strikingly, many accessory genes, including most of those encoding type III effectors, lacked insertions. In contrast, 94% of genes on the Psa plasmid had insertions, including for example, the type III effector HopAU1. These results suggest that some chromosomal sites are rendered inaccessible to transposon insertion, either by DNA-binding proteins or by the architecture of the nucleoid.

丁香假单胞菌猕猴桃致病变种（Pseudomonas syringae pv. actinidiae，Psa）是猕猴桃溃疡病的病原菌，也是近年来极具破坏性的植物病害之一。本研究构建了两株基于mini-Tn5转座子的Psa ICMP 18884随机插入突变体库。第一株为"目标表型（phenotype of interest，POI）"突变体库，包含10368株独立突变体，以网格化形式接种于96孔板中。通过影印平板法接种至选择性培养基，本研究成功对该POI突变体库进行了营养缺陷型和运动能力缺陷型突变体的筛选；同时通过目视筛选，还鉴定出具有"模糊扩散"样形态的脂多糖（Lipopolysaccharide，LPS）生物合成突变体。第二株为"目标突变体（mutant of interest，MOI）"突变体库，包含约96000株独立突变体，同样保存于96孔板中，每孔约含200株突变体。本研究采用转座子定向插入位点测序（Transposon-Directed Insertion site Sequencing，TraDIS）技术，在Illumina MiSeq测序平台上对该MOI突变体库进行测序，以将插入位点定位至Psa基因组中。此外，本研究开发了一种基于网格化的PCR方法以回收单株突变体，利用该策略成功从MOI突变体库中筛选出一株在目视筛选中未被鉴定到的潜在LPS突变体。Psa的染色体和质粒分别具有24031和1236次独立插入事件，对应的插入频率分别为每千碱基3.65和16.6次。上述数据表明该MOI突变体库已接近饱和，理论上在任意一条染色体基因中检测到插入的概率可达97.5%；但实际仅有47%的染色体基因检测到插入。这一偏低的插入覆盖率无法仅用必需基因缺乏插入来解释——通常必需基因的插入缺失率约为5%。值得注意的是，大量附属基因（包括多数编码III型效应蛋白的基因）均未检测到插入。与之相反，Psa质粒上94%的基因均检测到插入，例如III型效应蛋白HopAU1的编码基因。上述结果表明，部分染色体位点因DNA结合蛋白或类核结构的影响，无法发生转座子插入。