軟腐細菌harpin所誘導之免疫反應可影響其對文心蘭之毒力

Virulence of soft rot bacteria is associated with harpin-mediated immunity in Oncidium orchid

謝宛婷(Wan-Ting Xie)

指導教授 ： 林宜賢

國立屏東科技大學/農學院/植物醫學系所/碩士(2016年)

摘要

文心蘭細菌性軟腐病主要由Pectobacterium chrysanthemi (Pch)所引起，可引起嚴重之軟腐病徵。在分類是與其十分接近之P. carotovorum subsp. carotovorum (Pcc)雖對文心蘭具有致病性，但對文心蘭致腐能力弱。報導顯示此種在毒力上之差異性可能與文心蘭辨識Pcc而引起病原相關小分子 (pathogen-associated molecular patterns, PAMPs) 所誘導之植物免疫有關。因此，本研究擬針對Pcc及Pch在其所誘導之植物免疫反應的差異上探討其在毒力上所扮演角色。首先將Pcc菌株與Pch菌株接種於文心蘭，可顯示Pch菌株相較Pcc菌株對文心蘭致腐能力較強。進一步將不同Pcc菌株接種於文心蘭，也顯示出不同Pcc菌株間致腐能力亦具有差異，並可區分成SRI、SRII及SRIII三群。此三群Pcc菌株在文心蘭除可誘導激激活化氧 (reactive oxygen species, ROS)的快速產生外，亦可誘導癒傷葡聚醣(callose)的累積。同時，分別於抑制鈣離子通道或NADPH 氧化酵素活性下，三群菌株均會增強對文心蘭之致腐能力且族群建立亦顯著提高。為進一步分析Pcc中之PAMPs是否與此致腐能力有關，選擇軟腐細菌表現harpin之hrpN基因在選殖定序後利用胺基酸進行親緣性分析的結果顯示，Pcc與Pch之HrpN於胺基酸序列上在N端具有極大差異性。有趣的是，Pcc菌株間亦有差異性，致腐能力較強的SRI與SRII可被歸類至同一群，最弱的SRIII則為獨立的一群，推測Pcc之致腐能力可能與HrpN有關。為確認文心蘭可能辨識Pcc之HrpN而誘發植物免疫反應而影響其對文心蘭之致腐能力，以大腸桿菌表現不同軟腐細菌之HrpN重組蛋白接種菸草與文心蘭後，均能誘發菸草產生過敏性反應，且可誘導文心蘭激活化氧的快速產生及癒傷葡聚醣的累積。其中，在文心蘭的防禦訊號中又以Pcc之HrpN所誘導之反應較強。進一步將不同軟腐菌之HrpN混合Pch菌株接種於文心蘭，可顯示出Pcc之HrpN具有減弱Pch對文心蘭的致腐能力，而Pch之HrpN卻能增強Pch對文心蘭之致腐能力。綜合上述結果，可推測不同軟腐細菌之HrpN在文心蘭辨識之免疫反應具有差異性，此差異性為影響軟腐細菌對文心蘭之毒力的重要因子。

關鍵字

Pectobacterium carotovorum subsp. carotovorum ； Pectobacterium chrysanthemi ； HrpN ； 文心蘭細菌性軟腐病

並列摘要

Pectobacterium chrysanthemi (Pch) is the causal agent of bacterial soft rot in Oncidium orchid. Pch is a destructive pathogen on ornamental flowers that causes strong plant tissue maceration. However, the symptoms caused by another common soft rot pathogen, P. carotovorum subsp. carotovorum (Pcc), in Oncidium orchid is weak. Our preliminary study indicates that this weak virulence is associated with the immune response triggered by pathogen-associated molecular patterns (PAMPs) from Pcc. To gain more insights into the role of PAMPs-triggered immunity on the virulence of Pcc and Pch in Oncidium orchid, its effects on virulence and immune responses need to be evaluated. First, the inoculation assay showed all Pch strains were more virulent in Oncidium orchid than all Pcc strains based on the length of soft rot symptoms and bacterial populations. We also found variations of Pcc strains on their weak virulence and could be further classified into three SR groups. The SRI strains were the most virulent in Pcc, and SRIII strains were the lowest in virulence. In addition, results revealed that the virulence of Pcc strains could be increased by applying calcium channel blocker or the inhibitor of NADPH oxidase. Moreover, the levels on virulence of Pcc exhibited strong associations with plant immune responses. The SRIII strains induced stronger intensities on rapid ROS (reactive oxygen species) generation and callose deposition than those induced by SRI and SRII strains. Thus, we suggested that the virulence of soft rot bacteria was associated with the immune response triggered by the PAMPs from Pcc or Pch. Among the PAMPs, amino acid sequences of harpin encoded by hrpN in soft rot bacteria were analyzed. Phylogenetic tree revealed that the HrpN in Pcc is different from that in Pch. More interestingly, the HrpN in Pcc was divided into two groups. One group included the SRI and SRII strains, the other group included the SRIII strains. To further evaluate the differences of HrpN on virulence of soft rot bacteria, distinct HrpNs expressed by Escherichia coli were used as an elicitor on plants. All of the HrpNs induced the hypersensitive response in tobacco leaves rather than that in Oncidium orchid. The HrpN from Pcc exhibited stronger intensification on rapid ROS generation and callose deposition than that from Pch. Most importantly, the inoculation assay carried out with Pch showed the virulence in Oncidium orchid was reduced by the HrpN from Pcc but increased by the HrpN from Pch. We concluded that the recognition of cells on HrpN from soft rot bacteria was different, and these differences were important factors to affect the virulence in Oncidium orchid.

並列關鍵字

Pectobacterium carotovorum subsp. carotovorum ； Pectobacterium chrysanthemi ； HrpN ； bacterial soft rot ； Oncidium orchi

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