應用AtGSL5-GFP轉殖阿拉伯芥篩選可提升植物免疫之微生物

植物受到病原菌攻擊時會啟動防禦反應，其中當植物細胞表面受器辨識植物病原菌小分子後，所觸發之免疫反應稱為PAMPs-triggered immunity (PTI)。目前已有文獻利用甜椒上發現之 Plant-Ferredoxin Like Protein (PFLP)蛋白提升植物免疫達到抗病的效果研究。在相關的機制上，已證明PFLP可提升flg22pst所誘導的癒傷葡聚醣累積，及其生合成基因的表現。進一步的研究發現，flg22Pst 所誘導的癒傷葡聚醣累積及其基因之表現也可被Bacillus amyloliquefaciens PMB05 菌株所提升。此菌株目前亦已證明能提高植物對軟腐病與西瓜果斑病之抗病性。因此，在建立提升植物免疫微生物來增加抗病性的菌株篩選上，可利用癒傷葡聚醣生合成基因誘導作為指標。在植物上，與抗病較相關的癒傷葡聚醣生合成基因為GSL5，所以首先選殖阿拉伯芥上AtGSL5 (NM116593) 啟動子區域並使其融合gfp基因。隨後構築於植物轉殖載體上，再進行阿拉伯芥之轉殖，可獲得AtGSL5-GFP轉殖植物。此植株可利用於分析其螢光變化來監測植物免疫之啟動狀態。植物免疫之誘導分別是利用不同PAMP處理後於不同時間點觀察葉片綠色螢光之累積，結果顯示flg22pst與PopW分別在接種後12小時及8小時即有螢光訊號表現。接著利用PopW與已確認可提升 PTI 免疫反應之 PFLP進行分析，結果顯示在接種後4小時PFLP即可增強螢光反應。另外，以 B. amyloliquefaciens PMB05 做為模式菌株評估亦有相同的趨勢。同時，利用此系統評估其他11株Bacillus spp. 菌株，結果亦僅有PMB05菌株顯著提升螢光強度以及癒傷葡聚醣的累積。其他菌株除了PMB04菌株之外，皆顯著降低螢光反應，但對癒傷葡聚醣的累積則無顯著差異。有趣的是，將上述 Bacillus spp. 菌株進行青枯病的接種，結果在接種後二週僅有PMB05 菌株顯著降低青枯病之罹病度，而其餘之Bacillus spp.菌株除了PMB04菌株之外罹病度皆顯著高於對照組，使病害快速發生。以上結果說明，微生物菌株在此平台表現出的螢光訊號越強，對病害防治效果較佳，而表現出的螢光訊號越弱，則會加速病害的發生，因此本平台表現出的螢光訊號確實與青枯病之發生有關。另一方面，將上述11株菌株進行拮抗測試之結果顯示PMB05為供試菌株中對Ralstonia solanacearum拮抗能力最弱的菌株。由此說明，利用植物免疫平台篩選具病害防治能力之微生物確實可行。綜合上述，本研究所建立之AtGSL5-GFP轉殖阿拉伯芥，可應用其螢光訊號作為篩選提升植物免疫反應之微生物於青枯病害防治。

關鍵字

植物免疫；癒傷葡聚醣

並列摘要

PAMPs-triggered immunity (PTI) is a defense response triggered by pathogen-associated molecular patterns (PAMPs) which is recognized on plasma membrane of plant. Recently, application of Plant-Ferredoxin-Like Protein (PFLP) to enhance flg22pst-triggered PTI and disease resistance has been demonstrated. Similarly, a rihzobacterium, Bacillus amyloliquefaciens strain PMB05, also intensifies flg22Pst-triggered PTI responses and disease resistance against bacterial soft rot on Arabidopsis plants and bacterial fruit blotch on watermelon. In both cases of PFLP and PMB05, the flg22Pst-triggered callose deposition and gene expression of AtGSL5, a gene that can enhanced resistance to fungal pathogens, were intensified. Therefore, gene induction of AtGSL5 can be used as a model to establish a screening system for PTI intensifying microbial strains. Firstly, the promoter region of AtGSL5 from Arabidopsis thaliana ecotype Columbia (Col-0) was cloned and fused to the coding sequence of gfp gene which encodes the green fluorescent protein (GFP). Then the DNA fragement was further constructed into pBI121 and generated AtGSL5-GFP transgenic lines of Arabidopsis thaliana. After the homozygous line of AtGSL5-GFP was obtained, fluorescent intensity of GFP was monitored during the activation of PTI. Results showed flg22pst and PopW treatment started to exhibit fluorescence signal at 12 hours and 8 hours post-infiltration, respectively. Thus, the PopW-induced fluorescent was further use to assay the effects carried out with PFLP and PMB05. Results exhibited that the fluorescence signals both intensified by PFLP and PMB05 enhanced at 4 hour post-infiltration. In addition, the intensification of PopW-induced fluorescent signals in AtGSL5-GFP and callose deposition in Col-0 by 11 Bacillus spp. strains was used to assay. Results revealed that the fluorescence intensity and callose deposition was significant intensified by PMB05 rather than other 10 strains. Moreover, these 10 strains, except PMB04, reduced the fluorescence signals, but not affected the callose deposition. Interestingly, the effect on disease resistance enhanced by Bacillus spp. strains against bacterial wilt showed similar responses to fluorescent signals in AtGSL5-GFP at 2 week-post-inoculation. The fluorescent increasing PMB05 strain reduced the disease severity, the fluorescent reducing strains accelerate the disease severity. On the other hand, PMB05 exhibited lowest inhibitory effect among the strains with inhibitory zone against Ralstonia solanacearum on nutrient agar plate. Taken together, the PopW-induced fluorescent model in AtGSL5-GFP transgenic plant can be used to screen plant immunity intensifying microbes for disease control of bacterial wilt.

並列關鍵字

Plant-ferredoxin like protein (PFLP) ； Bacillus amyloliquefaciens ； plant immunity ； callose ； glucan synthase-like 5(GSL5)

參考文獻

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