关键词: 小麦 , 细胞自噬 , ATG18 , 白粉病

Abstract: 【Objective】 One objective of this study is to identify wheat homologues of autophagy-related gene ATG18, to analyze the sequence characteristics and 3-D structures of their encoding proteins. The other objective is to profile their expression patterns under biotic and abiotic stresses and under phytohormone applications, thus laying a foundation for elucidating their biological functions. 【Method】 EST assembly and RT-PCR were carried out to clone ATG18 cDNA sequences from RNA pool generated from the powdery mildew (Pm) causal fungus Blumeria graminis f.sp. tritici (Bgt)-challenged wheat leaves. Bioinformatics tools were employed to analyze gene exon-intron models, to predict protein domains, conserved amino acids and 3-D structures and to analyze the phylogenetic relationships among plant orthologues. Real-time fluorescence quantitative PCR was used to investigate gene expression patterns in responses to Bgt infection, treatments of various phytohormones or exposure to conditions of high salinity, drought, low temperature and darkness or nitrogen deficiency. 【Result】 Four members (TaATG18a, 18b, 18c and 18d) of the wheat ATG18 family were identified in this study. They are of great similarity and each has an ORF of 1 158 bp encoding a protein of 385 amino acids. The deduced protein sequences of TaATG18s consist of two classic WD-40 domains, one phosphatidylinositol 3-phosphate (PI3P) binding motif and the conserved amino acids at the ATG2-binding site. The four TaATG18s all have two kinds of alternatively spliced mRNA isoforms, respectively encoding the complete functional protein form and the N-terminal truncated non-functional form missing the WD-40 domain and the conserved functional residues. TaATG18a was predicted to fold into a six-blade β-propeller-like structure similar to other WD-40 proteins. The PI3P binding motif is located at the strand 4 of blade 5 and the loop connecting blades 5 and 6, and the ATG2 binding site is located at the loop connecting blades 2 and 3. Expression of TaATG18s can be upregulated by biotic stress of Bgt infection, however, the detailed regulation patterns by Bgt infection differed between wheat-Bgt compatible and incompatible reactions. In the two incompatible reactions triggered by the broad-spectrum Pm resistance gene Pm21 and the isolate-specific Pm resistance gene Pm3f, two times of transcripts accumulation of TaATG18s were observed during 0-36 hours after inoculation of Bgt, which are closely related to the progression of Bgt infection. Although TaATG18s of the moderately susceptible line Yangmai 158 also showed a bimodal Bgt-induced expression profile, the first time of transcripts accumulation had a shorter duration and a lower peak than that observed in the incompatible reaction on Pm21-carrying Yangmai 158 isogenic line. Expression of TaATG18s of the highly susceptible line Chancellor fluctuated little in response to Bgt infection. Differences were also found between the resistant line and the susceptible line as to their TaATG18s expression responding to the same exogenously applied phytohormone salicylic acid (SA) or ethylene, which showed negative effects on the resistance line but positive effects on the susceptible line. Phytohormones regulated the expression of TaATG18s not only at the transcription level, but also at the process of mRNA splicing. Expression of TaATG18s can also be upregulated by abiotic stresses of high salinity, drought, low temperature and darkness or nitrogen deficiency. 【Conclusion】 Proteins encoded by TaATG18s identified here probably participate in the wheat autophagy process through their PI3P-mediated autophagic membrane localization and the formation of ATG18-ATG2 complex. TaATG18s and their involved autophagy are implicated in the wheat immune response to Bgt infection and in the wheat responses to various abiotic stresses. Different expression responses of TaATG18s to one kind of phytohormone SA or ethylene in the resistant and the susceptible lines may partially account for the different phenotype outcomes of Pm resistance or susceptibility.

Key words: wheat (Triticum aestivum L.) , autophagy , ATG18 , powdery mildew