植物賀爾蒙吲哚乙酸參與茭白結筍機制之探討

Abstract

茭白，為禾本科（Gramineae）菰屬（Zizania）多年生宿根性草本植物。茭白黑穗菌，為黑穗菌（smut fungi）的一員，感染茭白後，導致茭白植株無法正常結穗，並在莖基部產生膨大的菌癭，經由農民留種無性繁殖，成為鮮嫩可口的茭白筍。茭白的膨大結筍，普遍認為是因植物賀爾蒙的改變，進而誘發組織膨大所致，唯其機制仍不甚清楚。本研究的目的，在探討茭白黑穗菌生合成的吲哚乙酸（IAA）是否為茭白結筍的重要因子。首先，根據真菌IAA生合成路徑相關基因資訊，利用生物資訊學的方式，於茭白黑穗菌基因體資料庫中進行分析比對，鑑定IAA主要生合成路徑「IPA pathway」及次要生合成路徑「TAM pathway」生合成相關基因。其中，發現茭白黑穗菌Ueipdc（indole-pyruvate decarboxylase）基因，為IPA pathway中間步驟單一調控基因，故本研究以IPA pathway及Ueipdc基因為主要研究對象。為了進一步探討茭白黑穗菌IAA的生合成，利用split-marker突變技術及基因槍轉殖法進行Ueipdc基因的剔除，分析突變株IAA生合成能力發現與野生型菌株並無差異，比較ΔUeipdc突變株與野生株間IAA pathway生合成之基因，發現IPA pathway Ueiad1基因及TAM pathway Uegdc3與Uenit2二基因表現有所變化，推測Ueipdc基因的突變可能促使茭白黑穗菌使用旁支路徑TAM pathway回復IAA生合成的能力，此現象值得進一步剖析驗證。針對結筍樣品轉錄體及real time qRT-PCR基因表現分析，可偵測到茭白黑穗菌IAA生合成基因於結筍初期的表現，而後期階段則觀察到茭白ZlYUCCA基因有明顯持續上升的現象。進一步地，將野生株及ΔUeipdc突變株配對接種於公株誘導結筍，結果顯示突變株感染及誘導結筍能力皆與野生株相似，無明顯差別。綜合本研究結果顯示，茭白黑穗菌具有多重路徑進行IAA的生合成，茭白黑穗菌合成的IAA可能在茭白結筍並非扮演主要角色，未來可能需建構不同路徑的多重突變株，方能更明確闡明茭白黑穗菌IAA與茭白結筍的關係。

Zizania latifolia is a perennial herbaceous wild rice species and belongs to Gramineae. Ustilago esculenta is a member of smut fungi, which infects Zizania latifolia, blocks ear production and induces gall formation at the basal part of the stem. The galls are edible with good flavor and texture and infected plants have been vegetatively propagated by farmers and become a favorable vegetable, so-called Jiaobai or water bamboo. Gall formation depends on the interaction between Z. latifolia and U. esculenta and phytohormone such as IAA has been implied to involve in the process. However, the detailed mechanisms are largely unknown. In this study, we aim to determine whether IAA produced by U. esculenta plays a major role in gall formation. According to the literature described IAA biosynthetic pathway in fungi, we conducted bioinformatic search to identify candidate genes in U. esculenta genome database and the putative homologues involved in IPA pathway and TAM pathway were identified. The Ueipdc gene, a single copy gene involved in the conversion of indole-3-pyruvic acid to indole-3-acetaldehyde in the IPA pathway, was selected for further characterization. To study IAA biosynthesis in U. esculenta, we generated Ueipdc knockout strains by split-maker strategy and biolistic transformation. However, ΔUeipdc mutants retained IAA biosynthetic ability similarly like the wild type strain. Gene expression study revealed that the expression pattern of Ueiad1 in IPA pathway and Uegdc3 and Uenit2 in TAM pathway was changed in the ΔUeipdc mutants, indicating Ueipdc deletion may compensate IAA biosynthesis via alternate TAM pathway. According to transcriptomic and real time qRT-PCR studies of gall materials collected from different stages in the fields, U. esculenta genes in IPA pathway were differentially expressed at early stage and plant ZlYUCCA gene instead was upregulated in later stage of galling materials. Furthermore, the wild type strains and ΔUeipdc mutants were respectively paired and inoculated onto uninfected plants to induce gall formation. The results revealed that the infection and gall-inducing abilities of mutants remained intact like wild type strains. In conclusion, this study reveal that more than one IAA synthetic pathways are functional and used for IAA biosynthesis in U. esculenta. IAA synthesized by U. esculenta may not play a major role in gall formation. However, further experiments such as construction of mutants carrying multiple defects in these pathways will help to further clarify the relation of U. esculenta IAA and gall formation.