感染日日春葉片黃化病植物菌質體之日日春以生長素處理後其病害發展之研究

Abstract

植物菌質體 (phytoplasma) 可感染多種經濟作物，並於感病後常造成枝條增生 (proliferation)、矮化 (dwarfism)、簇葉 (witches’ broom) 等病徵，造成農業重大損失。近來已發現植物菌質體之致病蛋白 TENGU 與病徵之誘發相關，且 TENGU 之表現亦使生長素之相關基因如 Aux/IAAs、 SAURs (small Auxin-up RNAs)、GH3s 之表現量下降。又生長素 (auxin) 已證實與植物防禦之相關荷爾蒙如水楊酸 (salicylic acid)、茉莉酸 (jasmonic acid)、乙烯 (ethylene) 互有交互作用，進而調控植物之抗病、感病路徑。另一方面，將感染植物菌質體之日日春枝條，培養於含有高量生長素之培養基中，也可有效使枝條病徵減緩，同時植物體內之菌量也有明顯減少，顯見生長素在植物與植物菌質體交互作用上或在植物之抗性表現上扮演重要角色。本研究以日日春葉片黃化病(periwinkle leaf yellowing, PLY) 植物菌質體為病原、日日春為寄主植物進行研究，試圖了解生長素、植物寄主與植物菌質體三者間交互作用之機制，以期找出對抗植物菌質體病害之新策略。為了釐清前人研究中，生長素訊息傳導相關基因表現量之下降，是否導致植物罹病後對生長素之感受性改變，首先，在本研究中檢查了此類基因於感病後之表現量變化，結果發現生長素訊息傳導相關基因 IAA9、IAA14、IAA14-2、IAA19 於罹病後表現下降，而 IAA8、SAUR5 表現上升，又 IAA3、IAA4、IAA12 之表現量無顯著變化。然而我們亦發現其中僅 IAA3、IAA4、IAA12、IAA19、SAUR5 受人工合成生長素 NAA (napthaleneacetic acid) 之誘導，而僅有 SAUR5 之表現於罹病株中較健株為低，故推論植物菌質體之感染會干擾寄主體內生長素訊息傳導相關基因表現；同時也發現在以生長素處理之罹病株中， IAAs 等基因受誘導之表現情形較健株為高，而 SAUR5 在罹病株中被誘導之表現則與健株相比稍低，即無法被誘導之情形。本研究進一步觀察植物罹病後，若直接處理生長素是否可造成病徵減緩，故於植物嫁接 PLY 植物菌質體兩周後，以噴灑方式處理高濃度之 NAA (25 ppm)，定時觀察植株病害發展。結果發現以 NAA 處理後之 PLY 罹病株，可使未顯現病徵之枝條維持健康，而且在處理 NAA 之罹病株上同時也發現出現較多之健康枝條，於其中無法針測到植物菌質體之存在，但在原已顯現病徵之發病部位的花部病徵有加速發展之情形，而且其病枝含有較高之菌量；此外，在觀察以 NAA 處裡後之罹病株病枝中水楊酸、茉莉酸、乙烯相關基因表現量時，亦發現水楊酸相關之 PR1 及 Pr1b 等系統性抗病相關基因表現受抑制，而於誘導性抗病相關基因之表現中，LOX2 在處理 NAA 前後無差別， AOC 於處理後表現量未上升，而 ACO4 於處理後表現量上升。將 PLY 植物菌質體嫁接至以生長素做前處理之日日春後，發現其較未處理生長素之植株不易罹病，顯見生長素有助於植株健康部位之抗性提升。前人研究顯示胼胝體 (callose) 之累積已被發現與植物限制病原擴散有關，因此本研究亦對處理 NAA 是否造成植株胼胝體沉積之變化進行觀察，結果發現處理 NAA 後，健株有胼胝體之產生，如此或可將處理 NAA 後罹病株含有較多建康枝條之現象，推論乃導因於植物菌質體在移至新枝條之情形受阻。本研究對植物與植物菌質體間之交互作用做進一步之了解，並試圖提出生長素對於植物及植物菌質體間之交互作用影響之可能模式。

Phytoplasmas, wall-less obligate bacterial pathogens, cause more than one thousand diseases in hundreds of economical crops. Because their disease symptoms are often associated with plant developments, phytohormone imbalance caused by the pathogen was proposed to be the main cause of the symptoms. Recently, a symptom inducing effector, TENGU, was found to suppress expressions of several auxin-related genes. Symptoms caused by phytoplasma can be relieved by treatment of high concentration of auxin in an in vitro culture condition. Therefore, in this study, we aimed to realize detail mechanisms of auxin associated defense responses in periwinkles against periwinkle leaf yellowing (PLY) phytoplasma. Consisting with previous finding, expressions of several auxin-related genes were down-regulated after PLY-phytoplasma infection. However, genes with no change and with up-regulated expressions were also observed. Surprisingly, genes examined generally more strongly responded to auxin treatment with SAUR5 the lone gene showing reduced sensitivity to auxin in diseased plants. After NAA treatment, symptom developments were accelerated in the inoculated shoots while more healthy shoots with no phytoplasma detected were observed. The accelerated symptom developments were associated with early accumulations of phytoplasmas, and suppression of peak induction on Pr1 and Pr1b. Genes encoding potential JA and ET biosynthesis key enzymes were also analyzed and no conclusive results for involvement of JA biosynthesis in the accelerated symptoms were observed, while a strong induction of ACO4, a key ET biosynthesis gene, was only found in diseased shoots treated with NAA. After auxin pre-treatment, periwinkles were more resistant to phytoplasma infection, showing that auxin promote the resistance to phytoplasma in healthy part. Since callose deposition is a basal defense to prevent pathogen spreading, callose deposition were examined and an increased level of callose deposition was observed in healthy plants treated with auxin. The association of reduction in disease rates in callose containing healthy shoots may infer that it may help preventing phytoplasma infection. The study provided clues of interactions of plant-microbe. Scheme of the hypothetic working model among auxin, host plant and phytoplasma was proposed in this study.