| 摘要: | 細菌之第六型分泌系統（Type VI secretion system, T6SS）廣泛存在於變型菌門（Proteobacteria）之細菌中，這些細菌透過第六型分泌系統可將其效應子蛋白直接分泌至體外或與其接觸的真核或原核細胞內，因此，第六型分泌系統常與細菌之致病性或細菌間競爭有關。本實驗室前人實驗顯示，於多種雙子葉植物上造成冠癭病（crown gall disease）之土棲性與土傳性植物病原細菌——農桿菌（Agrobacterium tumefaciens），可經由其T6SS於生體外（in vitro）或植物體內（in planta）攻擊種內或種外之細菌，從而提升其在特定生態棲位之競爭力。此外，前人亦利用直接接種於植物(例如番茄)莖上傷口或馬鈴薯塊腫瘤定量分析之實驗顯示，失去T6SS並不會影響農桿菌之致病能力。然而，我們對農桿菌之T6SS是否對自土壤入侵植物傷口從而造成冠癭的過程、或對冠癭內之植物內生微生物相（microbiota）造成影響等所知甚少。因此，本研究以農桿菌之模式菌株A. tumefaciens C58為材料，建立土壤接種番茄幼苗實驗，觀察A. tumefaciens C58野生株及T6SS突變株ΔtssB及ΔtssL之致病能力是否有不同；同時，由土壤接種實驗所造成的番茄冠癭組織萃取DNA，經由16S核醣體核酸基因擴增子定序（16S rRNA gene amplicon sequencing），調查及比較不同菌株造成的番茄冠癭內生細菌菌相，以得知農桿菌之T6SS是否對冠癭內生菌菌相造成影響。七次土壤接種實驗之結果表明，接種A. tumefaciens C58 T6SS突變株ΔtssB及ΔtssL，使番茄產生冠癭之罹病率比野生株顯著較低，說明農桿菌之T6SS可能經由間接的方式對其致病能力造成影響。由初步16S核醣體核酸基因擴增子定序實驗發現，番茄冠癭DNA的定序資料中含有大量來自葉綠體及粒線體之16S核醣體核酸基因序列，因而大幅度降低對內生細菌菌相之定序深度及解析度。為解決該問題，本研究針對16S核醣體核酸基因之不同高度變異區域（variable regions）設計共四對引子對，並同時針對番茄之葉綠體及粒線體16S核糖核酸基因設計相應之聚合?連鎖反應阻斷性引子（polymerase-chains-reaction blocker），以增加對細菌族群之16S核糖核酸基因的增幅。結果顯示藉由阻斷性引子的運用，成功提高對冠癭內細菌16S核糖核酸基因之定序深度，而最佳化之16S核糖核酸基因增幅條件便可被應用於大規模調查冠癭內生菌相實驗的分析比較。對土壤接種實驗產生的共53個冠癭之內生細菌菌相進行之主座標分析（principle coordinate analysis）後得知A. tumefaciens C58野生株及T6SS突變株ΔtssB及ΔtssL造成之冠癭內生細菌菌相並無顯著差異，但不同月份進行土壤接種實驗造成之冠癭菌相則有顯著的差異。此外，我們發現冠癭之重量與A. tumefaciens C58在內生菌相中的相對豐度有正相關。綜上所述，本研究經由土壤接種番茄幼苗之實驗顯示，農桿菌T6SS突變株之致病力比野生株顯著較低，但對造成之番茄冠癭內之內生菌相則無顯著影響，此結果可能表示農桿菌的T6SS並不是造成冠癭內內生菌相的主因。未來方向可探索農桿菌A. tumefaciens C58之T6SS是否對其於根部的纏據（colonization）或根圈微生物菌相（rhizospheric microbiota）造成影響，以解釋為何T6SS突變株於經由土壤接種番茄幼苗時有較低的致病率。;The type VI secretion system (T6SS) is a widespread nanomachine deployed by many proteobacteria to secrete effector proteins into prokaryotic or eukaryotic cells. This system engages in pathogenesis or interbacterial competition, which increases the fitness of the T6SS-containing bacteria. Previous studies demonstrated that Agrobacterium tumefacien, a soil-inhabiting and soil-borne phytopathogen causing crown gall disease on various plant species, deploys T6SS as an antibacterial weapon to attack closely- and distantly-related bacterial species in vitro and in planta. Additionally, tumorigenesis was not affected by the loss of T6SS when A. tumefaciens was inoculated in sterile condition using potato disc tumor assays or directly inoculated on the stems of various plant species including tomato plant. However, it remains unknown whether T6SS influences tumorigenesis in natural infection process, or affects the composition of bacterial community inside crown galls. Here, we established a soil inoculation method on wounded tomato seedlings and performed 16S rRNA gene amplicon sequencing to address these questions through the comparison of the A. tumefaciens C58 wild-type strain and two T6SS deficient mutants (i.e., ΔtssL and ΔtssB). Based on seven inoculation trials, all three strains could induce tumors through this method. Although the mutants have significantly lower disease incidences, for those successful infections there was no significant difference in crown gall weights. For the 16S amplicon sequencing, the first attempt based on a commonly used protocol for plant-associated microbiota suffered high levels of host DNA contaminations. Hence, we evaluated four newly designed primer pairs and their cognate PCR blockers against tomato chloroplasts and mitochondria. The optimized protocol targeting the V5-7 regions was utilized to survey 53 crown gall samples. Based on the principal coordinate analysis (PCoA) of bacterial composition, a functional T6SS did not significantly affect the bacterial compositions of crown gall, but the season of inoculation time did. A positive correlation was found between the relative abundance of A. tumefacien and the crown gall weight. In summary, the T6SS-dependant interbacterial competitions affected the success of A. tumefaciens infection of host plants through invasion from soil, but may not be an important factor in shaping the crown gall microbiota. Future works are necessary to investigate whether the T6SS enhances Agrobacterium colonization in rhizosphere or on wounded site, and consequently influences the initial stages of infection. |
