Analysis of Defense Pathways Affected by PlWRKY65 in Herbaceous Peony Leaves in Response to Alternaria tenuissima

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

Acta Horticulturae Sinica ›› 2026, Vol. 53 ›› Issue (3): 709-722.doi: 10.16420/j.issn.0513-353x.2025-0312

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Analysis of Defense Pathways Affected by PlWRKY65 in Herbaceous Peony Leaves in Response to Alternaria tenuissima

ZHU Yongfang, JIANG Yulei, GUO Xianfeng^*()

College of Forestry，Shandong Agricultural University，Tai’an，Shandong 271018，China

Received:2025-06-12 Revised:2025-11-19 Online:2026-03-25 Published:2026-03-20
Contact: GUO Xianfeng

Abstract

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

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.

Figures/Tables 7

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

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

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

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

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

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

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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Analysis of Defense Pathways Affected by PlWRKY65 in Herbaceous Peony Leaves in Response to Alternaria tenuissima

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