mcpQ13 與 cheZ 對 Pseudomonas kribbensis XP1-6 化學趨向性及番茄根部定殖能力影響之研究

Abstract

Pseudomonas kribbensis XP1-6 為一種能定殖於番茄根部的促進植物生長根棲細菌能促進番茄生長，提高番茄對青枯病之抗性並降低高溫、乾旱、鹽害及淹水等非生物逆境對番茄造成之危害，因此具有發展為生物肥料或生物農藥的潛力。雖然已知 P. kribbensis XP1-6 對番茄植株生長有諸多助益，但對其定殖於番茄宿主根部之機制仍所知甚少。根棲菌定殖於植物根部之過程中，化學趨向性與泳動能力對於根部之初期附著至關重要，亦會影響到後續定殖之生物膜形成。本研究探討 P. kribbensis XP1-6 中，cheA操作子中蛋白質產物預測為趨化受體的 mcpQ13 以及趨化作用相關基因 cheA 與 cheZ ，對 P. kribbensis XP1-6 移動能力、化學趨向性及番茄根部定殖之影響。試驗結果顯示，野生型 P. kribbensis XP1-6 不僅會受到種子及根部滲出液之吸引，亦會對番茄根部滲出液中琥珀酸、蘋果酸及檸檬酸等三種最主要的有機酸有趨化反應，但對滲出液中葡萄糖、麥芽糖和果糖等三種最主要的糖僅有微弱的趨化性。在營養充足之 KBM 培養基上，cheA2、cheZ 及 mcpQ13之突變株表面移行能力與泳動能力和野生型菌株皆無顯著差異，但 cheA1 突變株雖然表面移行能力沒有受影響，泳動能力卻顯著下降。個別剔除 cheZ 與 mcpQ13 基因後，相比於野生型，突變株對種子及根部滲出液的趨性指數顯著下降；而以cheA2、cheZ 及 mcpQ13突變株進行上述三種有機酸之趨化試驗，僅 cheZ 及 mcpQ13 突變株之趨化反應顯著下降。後續聚焦於 cheZ 及 mcpQ13兩基因，在低營養且僅以檸檬酸作為碳源之情況下，剔除 cheZ 及 mcpQ13 兩基因並不會影響 P. kribbensis XP1-6 對檸檬酸的趨化性。而在生物膜形成能力試驗中，雖然刪除 cheZ 或 mcpQ13 並未影響 P. kribbensis XP1-6 生物膜初期附著的能力，但會顯著降低後期成熟生物膜的形成。最後以 cheZ 及 mcpQ13 兩基因之突變株及互補株進行番茄根部定殖試驗，mcpQ13 回補株相比於帶有空質體之突變株，其定殖於番茄根部之菌量有顯著提高，而 cheZ 基因之回補株亦有較高之趨勢。此結果有助於了解 P. kribbensis XP1-6 化學趨向性的機制和根部定殖之關聯性，未來可透過番茄植株生長及逆境試驗，來探討化學趨向性對 P. kribbensis XP1-6 促進番茄植株生長能力之影響。

Pseudomonas kribbensis XP1-6 is a plant growth-promoting rhizobacterium (PGPR) that can colonize tomato roots. In addition to promoting tomato vegetative growth, it can increase resistance to bacterial wilt and mitigate the damage caused by abiotic stresses such as heat, drought, salinity, and flooding. These traits make P. kribbensis XP1-6 hold its potential for development as a biofertilizer or biopesticide. While the various benefits of P. kribbensis XP1-6 on tomato plant growth have been reported, the mechanisms underlying its colonization of tomato roots remain poorly understood. Effective root colonization by rhizobacteria depends on robust chemotactic responses and bacterial motility, which are essential for early root attachment and can influence subsequent biofilm formation, a key step for persistent root colonization. This study aimed to investigate the effects of the chemotaxis receptor gene mcpQ13 and the chemotaxis-related genes cheA and cheZ, which are located within two cheA operons in P. kribbensis XP1-6, on its motility, chemotaxis, and tomato root colonization. The results showed that P. kribbensis XP1-6 demonstrated strong chemotaxis toward tomato seed and root exudates and succinic acid, malic acid, and citric acid, three major organic acids in tomato root exudates. However, glucose, maltose, and fructose, the three major sugars in root exudates, elicited only weak chemotaxis. On nutrient-rich KBM medium, the swarming and swimming motility of cheA2, cheZ, and mcpQ13 mutants showed no significant difference compared to the wild-type strain. However, while the swarming motility of the cheA1 mutant was unaffected, its swimming motility significantly decreased. After individual deletion of the cheZ and mcpQ13, the chemotaxis indices to seed and root exudates of the mutants were significantly reduced compared to the wild-type strain. Chemotaxis assays with the three aforementioned organic acids using cheA2, cheZ, and mcpQ13 mutants revealed that only the cheZ and mcpQ13 mutants showed a significant decrease in chemotactic response. This study further focused on the cheZ and mcpQ13 genes, and under low-nutrient conditions with citric acid as the sole carbon source, the deletion of either cheZ or mcpQ13 had no significant effect on the chemotaxis towards citric acid of the wild-type strain. In the biofilm formation assay, mcpQ13 and cheZ mutations did not impact early root attachment but significantly impaired the ability to form mature biofilms. Finally, tomato root colonization assays were conducted using mutants and complementation strains of the cheZ and mcpQ13 genes. The bacterial population of the mcpQ13 complementation strain on tomato roots was significantly higher than that of the mutant carrying an empty plasmid, and the cheZ complementation strain also showed a higher trend. These results contribute to understanding the chemotaxis mechanisms in Pseudomonas kribbensis XP1-6 and its correlation with root colonization. Future studies will incorporate tomato plant growth and abiotic stress experiments to investigate the impact of chemotaxis on the ability of P. kribbensis XP1-6 to promote tomato plant growth.