番茄萎凋病菌鈣調磷酸酶之下游標靶 CRZ1基因功能之探討

Abstract

番茄是世界上廣泛種植的蔬菜之一。番茄萎凋病乃由尖鐮孢菌Fusarium oxysporum f. sp. lycopersici引起，是重要的植物真菌性病害之一，可造成嚴重的經濟損失。番茄萎凋病通常發生在潮濕和高溫的夏季，常見的病徵為葉片黃化、維管束褐變及植株萎凋。Crz1 是一種鋅指轉錄因子，在許多真菌物種中為鈣調磷酸酶(calcineurin)的重要下游標靶。在鈣調磷酸酶迅號路徑中，鈣調磷酸酶將Crz1去磷酸化，使Crz1得以進入細胞核來調控下游基因，例如與真菌發育，細胞壁完整性和離子平衡有關的基因表達。在實驗室前人的研究中Hou et al. (2020)，鈣調磷酸酶已被證實與番茄萎凋菌的菌絲生長、厚膜孢子生合成和毒力有關；然而，Crz1在番茄萎凋病菌中的功能尚未被闡述。透過胺基酸序列比對，找到了兩個可能為Crz1的同源基因FOXG_00040及FOXG_05246，並利用同源重組的技術獲得了兩者的突變株。前人研究顯示，Crz1會透過特定的motifs (PxIxIT、LxVP)與鈣調磷酸酶相互作用。此研究在FOXG_00040上發現PxIxIT及LxVP，而FOXG_05246則無。此外，在鈣離子的刺激下，FOXG_00040聚集於細胞核，而FOXG_05246則無，因此推測FOXG_00040為CRZ1同源基因，並命名為FolCRZ1。∆Folcrz1突變株對得克利呈現敏感性、但對 calcofluor white 則呈現抗性。∆FOXG_05246突變株於氧化壓力下較野生株生長稍微緩慢。盆栽試驗顯示∆FOXG_05246突變株的毒力明顯下降，然而∆Folcrz1突變株的毒力則與野生株相似。此外∆Folcrz1突變株在添加鈣離子的環境下比野生株有更高的耐受性。綜上所述，這些結果表明 FolCrz1 的功能可能迥異於其它真菌。

Tomato is one of the widely grown vegetables worldwide. Tomato Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici (Fol) is one of the most important plant diseases in the world accompanied by severe economic loss. Generally, it occurs in summer when moisture and temperature are high. Infected plants show yellowing leaves, browning vascular tissues and wilting. Crz1, a downstream target of calcineurin in multiple fungi, is a zinc finger transcription factor. In calcium-calcineurin signaling pathway, calcineurin acts through the Crz1 to differentially regulate the expression of downstream targets, such as genes involved in fungal development, cell wall integrity and ion homeostasis. In our previous study, Hou et al. (2020) demonstrated that calcineurin is required for hyphal growth, chlamydospore formation and virulence in Fol. However, the functions of Crz1 in Fol have not yet been characterized. In this study, we identified two loci (FOXG_00040 and FOXG_05246) as potential orthologs of yeast Crz1. Studies have shown that Crz1 interacts with calcineurin through specific motifs (PxIxIT, LxVP) that are found in FOXG_00040 but not FOXG_05246. Furthermore, subcellular localization of FOXG_00040, but not FOXG_05246, was localized to the nucleus in response to calcium stimuli. Therefore, it is speculated that FOXG_00040 is the yeast CRZ1 ortholog and thereby named as FolCRZ1. ΔFolcrz1 mutant showed sensitivity to tebuconazole, but tolerance to calcofluor white as compared with the wild-type. ΔFOXG_05246 mutant showed sensitivity to oxidative stresses. Interestingly, pathogenicity assays revealed that the virulence of ΔFOXG_05246 mutant, but not ΔFolcrz1 mutant was attenuated as compared with the wild type. In addition, ΔFolcrz1 mutant showed tolerance to calcium stress. Taken together, these results demonstrated that the functions of FolCrz1 may be distinct from other fungi.