移動性對 Pseudomonas sp. XP1-6 生物膜形成與定殖於番茄根部之影響

Abstract

Pseudomonas sp. XP1-6 為一種能在番茄根部定殖 (colonization) 的促進植物生長根棲細菌 (plant growth-promoting rhizobacteria, PGPR)，其能促進番茄生長、提高番茄對茄科青枯病之抗性以及降低高溫、乾旱、淹水及鹽害等非生物逆境對番茄造成之傷害，因此具有發展為生物肥料 (biofertilizer)、生物農藥 (biopesticide) 或植物生物刺激素 (plant biostimulant) 的潛力。雖然已知 Pseudomonas sp. XP1-6 對番茄有諸多益處，然而其與宿主之間交互作用的機制仍所知甚少。過去的研究發現，促進植物生長根棲細菌的移動能力會影響其定殖在植物根部的效率，推測 Pseudomonas sp. XP1-6 能定殖於番茄根部可能也與此特性有關。因此本研究想尋找 Pseudomonas sp. XP1-6 的移動性相關基因，並測試選定基因之突變株是否有鞭毛型態、生物膜形成能力、細胞外多醣 (Exopolysaccharide, EPS) 合成能力，及趨化性等與鞭毛有關特性上的變化，進而以計算 Pseudomonas sp. XP1-6 根內菌數和即時聚合酶連鎖反應 (Real-time polymerase chain reaction) 定量法等兩種方法驗證候選基因是否會影響 Pseudomonas sp. XP1-6定殖在番茄根部的能力。從跳躍子 (transposon) 突變株庫中篩選移動性降低的突變株，以質體拯救 (plasmid rescue) 方式定序找出被跳躍子插入之位置，得知因而突變的基因為何後，共篩選出 11 個具有潛力的基因。除了建構這 11 個基因的全基因刪除突變株之外，亦刪除鞭毛蛋白基因 fliC，以進行後續驗證。結果顯示，aceE 基因和 tolB 基因刪除後，Pseudomonas sp. XP1-6 移動能力降低的程度最為顯著；後續實驗中也發現 fliC 如預期一般無鞭毛產生，而 aceE 和 tolB 的鞭毛則是容易斷裂和散開。fliC、aceE 和 tolB 不僅趨化性受到影響，其生物膜和細胞外多醣生成量均明顯低於野生型。此外，tolB 突變株中第六型分泌系統表現量下降，且毒殺 E. coli MG1655 的能力也下降；而 aceE 和 tolB的螯鐵能力也和野生型有差異，即 aceE 幾乎無螯鐵能力，但 tolB 的螯鐵能力較野生型高。最後，以兩種方式評估根部定殖效率均發現 fliC、aceE 和 tolB 在番茄根部定殖的效率下降。本研究發現會影響移動性的 tolB 基因和 aceE 基因，與鞭毛蛋白基因 fliC 均會影響 Pseudomonas sp. XP1-6的定殖效率，此結果有助於了解 Pseudomonas sp. XP1-6 定殖的機制，並可能增加其未來應用於農業時的成效。

Pseudomonas sp. XP1-6 is a plant growth-promoting rhizobacterium (PGPR) that can colonize in tomato roots. It can also promote tomato growth, and enhance tolerance to Ralstonia solanacearum and abiotic stresses, including heat, drought, flooding, and salinity. Therefore, Pseudomonas sp. XP1-6 has the potential to be developed into a biofertilizer, pesticide, or plant biostimulant. Although Pseudomonas sp. XP1-6 is known to have numerous benefits to tomato, the mechanism underlying the interactions between Pseudomonas sp. XP1-6 and its host remains poorly understood. Previous studies reported that the motility of PGPR can affect the colonization efficiency of plant roots, suggesting that the tomato root colonization by Pseudomonas sp. XP1-6 may also be associated with its motility. In this study, genes involved in motility of Pseudomonas sp. XP1-6 were screened, and whether changes in flagellar morphology, biofilm formation, exopolysaccharide (EPS) production, and chemotaxis can be detected in the mutants of the candidate genes was investigated. Finally, the effect of the candidate genes on tomato root colonization of Pseudomonas sp. XP1-6 was assessed using direct bacterial counts and estimation of DNA copies of Pseudomonas sp. XP1-6 in tomato roots by qRT-PCR. Eleven candidate genes were identified by screening the transposon mutant library followed by insertion site determination using plasmid rescue and sequencing. In-frame deletion mutants of the candidate genes as well as fliC, the gene coding for flagellin were generated for further study. Deletion of aceE and tolB dramatically decreased the motility of Pseudomonas sp. XP1-6. As predicted, fliC did not produce flagella, and aceE and tolB produced fragile or tumbling flagella. fliC, aceE and tolB altered their chemotaxis activity. In addition, the amounts of biofilm and extracellular polysaccharide (EPS) produced by aceE and tolB were both lower when compared to the wild type. Deletion of tolB reduced the expression of genes related to type VI secretion system as well as the ability of Pseudomonas sp. XP1-6 to kill E. coli MG1655. The iron-chelating ability of aceE and tolB was also different from the wild type as aceE almost had no activity while tolB possessed higher activity. At last, using both methods, lower tomato root colonization efficiency was observed for fliC, aceE and tolB. In this study, we found that two motility-related genes tolB and aceE, and the flagellin gene fliC, can affect the colonization efficiency of Pseudomonas sp. XP1-6. These results help us understand the mechanisms of colonization of Pseudomonas sp. XP1-6 in planta, perhaps increasing the effectiveness while applying Pseudomonas sp. XP1-6 in the field in the future.