PlWRKY65影响芍药叶片响应细极链格孢菌侵染的防御通路分析

doi:10.16420/j.issn.0513-353x.2025-0312

摘要/Abstract

摘要： 由细极链格孢菌（Alternaria tenuissima）引发的叶斑病是芍药田间生产中的主要叶部病害。前期研究表明PlWRKY65可增强芍药对细极链格孢菌侵染的抗病性。本研究中以芍药‘大富贵’为供试材料，利用基因沉默（virus-induced gene silencing，VIGS）技术沉默PlWRKY65后再接种致病菌A. tenuissima，观测叶片发病进程及细胞超敏反应；取接种后0、12、48和96 h叶片，进行转录组测序分析，以未沉默PlWRKY65的叶片为对照。结果表明：PlWRKY65沉默组叶片的发病时间（48 h）较对照明显提前，12 h超敏反应差异最大；在致病菌侵染芍药叶片0、12、48和96 h后，PlWRKY65沉默组与对照组之间分别产生3 487、2 792、2 712、2 388个差异表达基因（differentially expressed gene，DEG）；这些DEG主要反映了细胞壁、植物激素信号转导及花青素生物合成代谢中相关基因表达水平的差异。致病菌侵染后，沉默组叶片中木质素、果胶、纤维素、半纤维素等细胞壁成分含量显著降低，而花青素含量的变化无规律。综上，PlWRKY65通过介导细胞壁代谢而增强芍药抵御病原菌侵染的能力。

关键词: 芍药, 细极链格孢菌, 转录因子, PlWRKY65, 转录组, 抗病通路

Abstract:

Leaf spot disease caused by Alternaria tenuissima is one of the main leaf diseases in the field production of herbaceous peony. Previous study has shown that PlWRKY65 could enhance the resistance to A. tenuissima infection in peony. With Paeonia lactiflora‘Dafugui’，virus-induced gene silencing（VIGS）technology was used to silence PlWRKY65 and then peony leaves were inoculated with the pathogen A. tenuissima in this study. Next the infection phenotype and hypersensitive response（HR） were observed. The leaves at 0，12，48 and 96 h after inoculation were sampled for transcriptome sequencing analysis，with the PlWRKY65-non-silenced plants as control. The results exhibited that the disease symptoms in the PlWRKY65-silenced group appeared at 48 h，earlier than those in the control；meanwhile the evident HR symptom appeared at 12 h in the control，ahead of the PlWRKY65-silenced group. RNA-seq results showed that at 0，12，48 and 96 h after pathogen infection，a total of 3 487，2 792，2 712 and 2 388 differentially expressed genes（DEGs）were respectively produced between the PlWRKY65- silenced group and the control. These DEGs mainly reflected the differences in the expression level of the cell wall-related genes，plant hormone signal transduction and anthocyanin biosynthesis metabolism. Compared with the control，the content of lignin，pectin，cellulose and hemicellulose-the four main components of cell wall-in the PlWRKY65-silenced group decreased significantly after pathogen infection，whereas the content of anthocyanin displayed irregular pattern. In conclusion，PlWRKY65 could enhance the resistance to A. tenuissima via mediating cell wall metabolism.

Key words: Paeonia lactiflora, Alternaria tenuissima, transcription factor, PlWRKY65, transcriptome, resistance pathway

朱永芳, 姜玉蕾, 郭先锋. PlWRKY65影响芍药叶片响应细极链格孢菌侵染的防御通路分析[J]. 园艺学报, 2026, 53(3): 709-722.

ZHU Yongfang, JIANG Yulei, GUO Xianfeng. Analysis of Defense Pathways Affected by PlWRKY65 in Herbaceous Peony Leaves in Response to Alternaria tenuissima[J]. Acta Horticulturae Sinica, 2026, 53(3): 709-722.

图/表 7

表1 本研究中所用引物序列

Table 1 Sequences of the used primers in this study

用途 Purpose	基因 Gene	正向序列（5′-3′） Forward sequence	反向序列（5′-3′） Reverse sequence
基因沉默 VIGS	PlWRKY65	GTGAGTAAGGTTACCGATTCAACCACCATGCCACCTCCTC	GAGACGCGTGAGCTCGGTACCCGAGGTGGTCCCACACCAC
内参 Internal reference	PlActin	ATGGCTGGAACAGGACTT	ACTGCTGAACGGGAAATT
实时荧光定量验证 qRT-PCR verification	PlPR2.1	CGCTTCTCAAATCTCAGGGTATC	AGAGGAGTTCGTTGGGTAAGT
	PlPR2.2	ATCTTCATCGTCGTCTTCATCT	GCCAGCAAATATACAAACATCCA
	PlPR2.3	AACGGCGATGATTCACGAGAG	ATGCTGTGTGGAGTTGTAACCTT
	PlPR2.4	GCAGAACATTCACAACGCAATT	CCAAGTCCAGGAGTCTCAAGT
	PlPR2.5	GGACAATACAACTACCAGAACTT	CAGTCTCCGACACTACAATTC
	PlPR2.6	AACGATGAATCAACGCCGATAA	CCTCCTTCTCCACAAGCATAATC
	PlPR4	AACATCATCAATGGCGGACTT	AGAGGTTGGCATATCTTTGGAAG
	PlPR5.1	CGTGTGCCGTGTTTAATCAAC	AACTATAAGCCTGAGGACATTGG
	PlPR5.2	CTTGCTTGTGATGGACTTATAGGC	TTTGATGCTGTGGGCTTTGTT
	PlPR5.3	GAGTCTGGTGGATGGCTACAA	GGCACATCACATTCAAGTCACT
	PlPR5.4	CAGATGGGAGTGTGATTTCTTG	ACTATAAGCCTGAGGACATTGG

图1 叶片接种Alternaria tenuissima后不同时期的感病表型（A）、病情指数（B）和超敏反应（C） A、B图中，鉴于首次出现病症的时间点为48 h，为使图片更加清晰，故将6、12、24 h时照片省去。A图中的黄色框线表示接种处，在其右下角辅以相应的放大图。B图中不同小写字母表示同一接种时间不同处理间差异显著（P < 0.05）。 C图中台盼蓝染的蓝色指示叶片细胞超敏反应程度

Fig. 1 Phenotype（A），disease index（B）and hypersensitive response（C）of peony leaves in each group at different time after inoculation with Alternaria tenuissima Since the disease symptom did not appear until 48 h，the phenotype or data at 6，12 and 24 h were therefore not shown in Fig. A and Fig. B to make the pictures clearer. In Fig. A，yellow rectangles indicate the inoculation sites，accompanying by the corresponding close-up views in the lower right corners. In Fig. B，different lowercase letters indicate significant difference among different treatment at the same inoculation time（P < 0.05）. In Fig. C，the blue dots stained by trypan blue indicate the degree of hypertensive response occurred in the leaf cells

图2 样品间基因表达量相关性（A）、差异表达基因数（B）及韦恩图（C）分析

Fig. 2 Correlation of gene expression（A）between samples，number of differentially expressed genes（B），and Venn diagram（C）analysis of the DEGs

图3 差异表达基因的qRT-PCR和RNA-seq结果比较

Fig. 3 Comparison of the DEG expression using qRT-PCR and RNA-seq method

图4 差异表达基因的KEGG富集分析富集因子值越大，表示富集的程度越大

Fig. 4 KEGG enrichment analysis of the differentially expressed genes The larger the enrich factor，the greater the degree of enrichment

图5 沉默PlWRKY65和空载植株在接种病原菌后其叶片中细胞壁组分含量变化

Fig. 5 Change in the content of cell wall components in the leaves of PlWRKY65-silenced and empty-vector plants after inoculation with pathogens * P < 0.05，** P < 0.01

图6 沉默PlWRKY65和空载植株在接种病原菌后其叶片中花青素含量变化 ** 表示极显著差异（P < 0.01）

Fig. 6 Change of the anthocyanin content in the leaves of PlWRKY65-silenced and empty-vector plants after inoculation with pathogens ** indicates significant difference at P < 0.01

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