十字花科黑腐病病菌第三型分泌系統之 effectors 的功能分析

Abstract

Xanthomonas campestris pv. campestris (Xcc) 與 X. campestris pv. raphani (Xcr) 為造成台灣十字花科蔬菜細菌性病害之 X. campestris 的兩個病原小種，分別可在寄主植物的葉片上造成系統性之黑腐病與非系統性的斑點病，此外，Xcr 可感染茄科植物之蕃茄、甜椒，但不能感染十字花科之辣根，故比較不同植物之感染能力已成為Xcc 與 Xcr 的鑑別依據之一，而根據寄主範圍與病徵型態分析，已將前人分離之辣根細菌性葉斑病菌 X. campestris pv. armoraciae 756C 重新歸類為 Xcr 756C (Fargier and Manceau, 2007, Plant Pathol. 56: 805 - 818)。Xanthomonas 屬病原細菌感染植物過程中常以黏附素 (adhesin) 附著於植物表面、伺機從自然開口入侵至細胞間隙，再藉由第三型分泌系統 (type III secretion system，T3SS) 將 effector 蛋白 (T3Es) 送入植物細胞中干擾植物的防禦反應以利其侵染寄主。比對 X. campestris pv. armoraciae 756C 與 X. campestris pv. campestris ATCC33913 全基因體序列後得知兩者具有不同的 t3es 組合，推測 Xcr 與 Xcc 之寄主範圍與病徵型態差異可能與其帶有的 t3es 有關。由於前人以 loss-of-function 的策略分析 T3Es 功能時，常無法分辨 t3e 缺損之突變菌株與野生型菌株在致病能力上的差異，因此本研究採用 gain-of-function 的策略，於台灣分離之 Xcr 菌株 cxsp3 中分別表現 Xcc 之 avrBs2Xcc17、avrXccE1Xcc100、avrXccBXcc17 及 xopQXcc17 等t3es 並分析其 in vitro 與 in planta 表現型。研究中發現液態培養於 NYG broth 中之 Xcr cxsp3 (pCHUB247) (avrXccE1+)、Xcr cxsp3 (pCHUB248) (avrXccB+) 及 Xcr cxsp3 (pCHUB249) (xopQ+) 均可產生明顯的細胞凝聚現象。以穿透式電子顯微鏡觀察上述三株轉型菌株後發現菌體兩極著生許多長絲狀 pili，菌體間之 pili 會相互纏繞、集結成束而形成細菌群集，且其生物膜生合成能力與 twitching motility 亦較對照組 Xcr cxsp3 (pBBR1MCS-5) 或異源表現 avrBs2Xcc17 之 Xcr cxsp3 轉型菌株低。根據定量 RT-PCR 之結果，推測 Xcr cxsp3 大量表現 avrXccE1、avrXccB 及 xopQ 後可能降低 pilT 轉錄而使菌體呈現 hyperpiliation，但 Xcr cxsp3 (pCHUB246) (avrBs2+) 則因 pilA 的表現量提高而出現細菌群集能力低、生物膜生合成能力與 twitching motility 高於其它菌株的表現型；定量 RT-PCR 之結果顯示 Xcr cxsp3 表現 avrBs2、avrXccE1、avrXccB 及 xopQ 後可能會影響細菌第四型纖毛基因的表現並進而改變 in vitro 培養時轉型菌株的細胞型態，但此 4 個 effectors 與細菌調控第四型纖毛基因表現之關聯性仍有待證實。分析轉型菌株之致病能力時，發現將上述 4 轉型菌株接種至甘藍葉片二天後即可誘發病徵產生，且 Xcr cxsp3 表現 avrBs2、avrXccE1 及 xopQ 之轉型菌株在甘藍葉片中的生長量與野生型菌株無明顯差異，僅有表現 avrXccBXcc17 之Xcr cxsp3 (pCHUB248) 在甘藍葉片中的生長量高於對照組及其它轉型菌株，顯示 Xcc AvrXccB 可促進 Xcr cxsp3在甘藍葉片中之生長；此外，將上述 4 個 Xcc t3es 以 Agrobacterium-mediated transient expression 表現在番茄 (Solanum lycopersicum cv. Farmers 301) 葉肉細胞中，2 天後測量接種葉的導電度，發現番茄接種葉短暫表現 xopQ 後之導電度明顯高於對照組與短暫表現 avrXccB 之接種葉，顯示 Xcc XopQ 可誘發番茄葉肉細胞的凋亡路徑 (programmed cell death)。本研究以異源表現策略分析 4 個 Xcc T3Es 後得知 Xcc AvrXccB 可促進 Xcr cxsp3 在甘藍葉片中之生長，而 Xcc XopQ 可誘發番茄葉肉細胞凋亡，故此方法應可用於分析其他功能未知的 T3Es 在病原細菌致病過程中對植物的影響。

Xanthomonas campestris pv. campestris (Xcc) and X. campestris pv. raphani (Xcr) are the causal agents of black rot and bacterial spot diseases on cruciferous plants. Xcc infects plants belonging to the Brassicaceae family, whereas Xcr infects plants in Brassicaceae and tomato and pepper of the Solanaceae family. Xcr is not a pathogen of cruciferous horseradish (Armoracia rusticana), which serves as a diagnostic index for differentiating Xcr from X. campestris pv. armoraciae (Xca) and reclassifies Xca 756C to Xcr 756C (Fargier and Manceau, 2007, Plant Pathol. 56: 805 - 818). Plant-pathogenic xanthomonads attach to the aerial surfaces of plants with extracellular adhesins for subsequent invasion. Once residing in the apoplast, the bacteria synthesize the type III secretion system (T3SS) to deliver the type III system effectors (T3Es) into plant cells to interfere with plant defense signaling pathways and to obtain nutrients from host cells. Whole-genome comparisons of Xcc ATCC33913 and Xcr 756C reveal that the 2 bacteria harbor different repertoires of T3Es, which might be collectively involved in plant-microbe interactions to elicit various plant responses in different plants. To address the functions of T3Es in bacterial pathogenesis, the approaches of loss-of-function by mutation and gain-of-function by overexpression are frequently applied. Previous researchers characterized Xcc T3Es by genetic disruption and showed that the mutant strains exhibited wild type-like phenotypes in symptom production and bacterial multiplication in planta. In this study, the gain-of-function approach is employed to examine the functions of Xcc T3Es. Four of the Xcc T3Es, i.e. avrBs2Xcc17, avrXccE1Xcc100, avrXccBXcc17, and xopQXcc17, that are not found in the genome of Xcr 756C are selected and heterologously expressed in Xcr cxsp3. In comparison with Xcr harboring vector pBBR1MCS-5, Xcr transformants expressing avrXccE1, avrXccB, and xopQ show reduced biofilm formation and twitching motility, whereas avrBs2-expressing Xcr exhibits the opposite phenotypes, including increased biofilm formation and enhanced twitching motility. In contrast to the few clustered cells of the avrBs2-expressing transformant, the 3 Xcr transformants expressing avrXccE1, avrXccB, and xopQ grow and form large clusters in NYG broth. The cellular aggregation is likely due to the synthesis of long, tangled pili that are readily seen on the bacterial surfaces under transmission electron microscope. Results of quantitative RT-PCR reveal that the transcription of pilT is reduced in the hyperpiliated transformants expressing avrXccE1, avrXccB, and xopQ, and the transcription of pilA in avrBs2-expressing Xcr is greatly increased, indicating the expression of the type IV pili biogenesis genes in Xcr is affected by the ectopic expression of the 4 Xcc T3S effectors. The question of how the 4 Xcc T3Es modulate the type IV pili biogenesis in Xcr remains to be elucidated. To test if avrBs2, avrXccE1, avrXccB, and xopQ have virulent functions, the 4 T3Es-expressing Xcr transformants are infiltrated to cabbage leaves for scoring symptom development and bacterial multiplication. Our results show that wild-type Xcr harboring vector pBBR1-MCS5 and the 4 transformants elicit necrotic symptom at 2 days post inoculation. Meanwhile, the population of avrXccB-expressing Xcr increases 3.5 logs, and the rest of the strains have an increment of 2 logs in 2 days, suggesting that Xcc AvrXccB may promote bacterial growth in cabbage through an unknown mechanism. In addition, Agrobacterium- mediated transient expression of the 4 Xcc T3Es in tomato (Solanum lycopersicum cv. Farmers 301) leaves reveals that the expression of xopQ in tomato mesophyll cells causes ion leakage. The high conductivity of leaf disks harvested from the xopQ-infiltrated leaves reveals that Xcc XopQ could induce programmed cell death in tomato cells. Taken together, the results of ectopic-expressing Xcc T3Es show that AvrXccB promotes Xcr growth in cabbage, and XopQ acts as an avirulent protein in tomato. The application of heterologous expression in planta and in closely related recipient strains has a great potential to identify the roles of functionally unknown T3Es in the pathogenesis of bacterial diseases.