(64(1):10-20)Development of an Efficient Fluorescent Visualization Method for the Detection of Xanthomonas oryzae pv. oryzae Infection in Rice Plants

Abstract

為能即時觀察白葉枯病菌於剪葉接種後感染水稻的完整過程，本研究利用新型強效的ZsGFP 基因及pBBR1MCS 載體，構築可於白葉枯病病原菌Xanthomonas 中表現綠色螢光之重組質體pBRRZsGFP。接著，轉型至Escherichia coli DH5α 及Xanthomonas oryzae pv. oryzae XF89b。經基因型確認之轉型菌株 (DH5αZsGFP及XF89bZsGFP) 菌落，皆可持續地表現綠螢光蛋白質。進一步於最高分蘗期接種水稻「台農67號」(感病品種)植株，結果發現因葉綠素螢光的干擾，在接種3 d 後 (3 DAI)，勉強可在切口處觀察到XF89bZsGFP 的增殖；直至7 DAI，方能觀察XF89bZsGFP 自葉尖沿著葉脈向葉基入侵。為了降低葉綠素螢光的干擾，本研究改用「台農67 號」暗培養之白化苗作為接種宿主，結果發現在感染0.5 h，即可發現XF89bZsGFP 的增殖與入侵。在接種後1 h，接種葉的螢光達到最大的表現。但可能隨水稻宿主啟動免疫系統，壓制入侵XF89bZsGFP 的繁殖，使接種葉的螢光表現轉弱。然而，至7 DAI 接種葉的螢光表現轉強，並持續向病葉基部發展，顯示XF89bZsGFP可能突破水稻的免疫系統，沿著維管束繼續侵入、繁殖，向葉片基部蔓延感染。這是目前已知最早觀測到白葉枯病菌感染水稻植株的研究報告。同時，此系統為非破壞性取樣，可連續在同一病葉觀察病原菌於接種後之侵入、增生及病勢的發展。因此，本研究所建構之XF89bZsGFP，確實可作為即時檢測接種後病菌感染能力及感染過程的平台，亦能作為未來白葉枯病菌與水稻宿主間交互作用的檢測平台。 Detection of pathogen infection is an important step in pathogen-host interaction study. A Xanthomonas fluorescent expression plasmid, pRBBZsGFP, was constructed with a strong fluorescent gene ZsGFP and the pBBR1MCS vector for immediate detection of bacterial blight pathogen infection. The recombinant plasmid was transformed into Escherichia coli DH5α and Xanthomonas oryzae pv. oryzae XF89b, respectively. The transformants, DH5αZsGFP and XF89bZsGFP, were confirmed by PCR genotype analysis and the observation of continuously expression of green fluorescent proteins. After infection of ‘TNG67’ at the maximum tillering stage by XF89bZsGFP, the observation of ZsGFP was interfered with the fluorescence of chlorophyll. The results showed that the bacterial multiplication of XF89bZsGFP only could be observed at the incision site at 3 DAI (days after inoculation) and XF89bZsGFP colonized from the cutting site to leaf base at 7 DAI. Using dark-treated albino rice seedlings, the interference of chlorophyll fluorescence could be reduced and the multiplication and colonization of XF89bZsGFP could be detected in 0.5 h after inoculation and the maximum fluorescence was observed on the same leaf in 1 h after inoculation. However, the fluorescence was reduced in the following time course indicating that the multiplication and colonization of XF89bZsGFP might be suppressed by the endogenous immune system of rice. At 7 DAI, the stronger fluorescence was observed again on the same leaf and extended continuously to the leaf base, suggesting that the rice immune system was broken down by the XF89bZsGFP. To our knowledge, this is the first report on the observation of X. oryzae pv. oryzae in rice plants. Importantly, this is a non-destructive system for pathogen detection and the multiplication and colonization of pathogen can be directly observed on the inoculated leaf. Therefore, the XF89bZsGFP and system established in this study can be served as a platform for real-time observation of virulence and pathogenicity of X. oryzae, and can also be applied to the study of the interaction between pathogen and host