A Non-classical Secretory Protein ncSP35 of Candidatus Phytoplasma Ziziphi Inhibits Hypersensitive Response and Plant Growth and Development

doi:10.13304/j.nykjdb.2025.0529

Abstract

Abstract:

Jujube witches’ broom （JWB）， caused by Candidatus Phytoplasma ziziphi， is currently the most devastating jujube disease. A novel non-classical secretory protein， ncSP35， was identified from the JWB phytoplasma through bioinformatics analysis combined with an Escherichia coli-based alkaline phosphatase reporter system. Transient expression of ncSP35 in Nicotiana benthamiana using a potato virus X-based expression vector demonstrated that the protein suppressed Bax-triggered hypersensitive response （HR） and hydrogen peroxide accumulation， and interfered with plant growth and development. Deletion and site-directed mutagenesis analyses revealed that the central region （amino acid residues 37~71） and the polar amino acid cluster HKKENK?2 of ncSP35 were critical for HR suppression， while the C-terminal 18 amino acid residues were essential for interfering with plant growth and development. Subcellular localization analysis showed that ncSP35 formed abundant aggregates in the cytoplasm and inhibited the nuclear localization of histone H2B， causing H2B to also form aggregates in the cytoplasm. Yeast two-hybrid， co-immunoprecipitation and luciferase complementation assays confirmed the specific interaction between ncSP35 and jujube ZjH2B. Above results provided new molecular insights for JWB pathogenesis.

Key words: candidatusphytoplasma ziziphi, non-classical secretory protein, hypersensitive response, plant growth and development, histone 2B, protein-protein interaction

摘要：

枣疯病由枣植原体（Candidatus Phytoplasma ziziphi）引起，是当前最具毁灭性的枣树病害。通过生物信息学分析结合大肠杆菌碱性磷酸酯酶报告系统，从枣植原体中鉴定了一个新的非典型分泌蛋白ncSP35；利用马铃薯X病毒载体在本氏烟中瞬时表达ncSP35，证明其可抑制Bax激发的超敏反应（hypersensitive response， HR）及H₂O₂积累，并能干扰植物生长发育；缺失和定点突变分析表明，ncSP35中间区域（第37~71位氨基酸残基）及极性氨基酸簇HKKENK⁸²是其抑制HR的重要功能区，而C-末端18个氨基酸残基为干扰植株生长发育的必需元件。蛋白亚细胞定位分析显示，ncSP35在细胞质中产生大量凝聚体，并干扰组蛋白H2B的核定位，导致H2B在胞质中形成凝聚体。酵母双杂交、免疫共沉淀和荧光素酶互补试验证实，ncSP35与枣ZjH2B蛋白存在相互作用。研究结果为揭示枣疯病致病机理提供了新的分子视角。

关键词: 枣植原体, 非典型分泌蛋白, 超敏反应, 植物生长发育, 组蛋白H2B, 蛋白互作

CLC Number:

S665.1

Yuqian JIN, Ziheng FU, Xiaoyu GAO, Weimin LI. A Non-classical Secretory Protein ncSP35 of Candidatus Phytoplasma Ziziphi Inhibits Hypersensitive Response and Plant Growth and Development[J]. Journal of Agricultural Science and Technology, 2025, 27(11): 164-173.

金玉倩, 傅子恒, 高小语, 李为民. 枣植原体分泌蛋白ncSP35抑制超敏反应及植物生长发育[J]. 中国农业科技导报, 2025, 27(11): 164-173.

Figures/Tables 7

Table 1 Genes， recombinant constructs and primers involved in this study

基因/载体Gene/construct

引物名称

Primer name

引物序列

Primer sequence （5’-3’）

ncSP35

ZjH2B

pET-ncSP35-phoA

pGR-ncSP35

pGR-SP35^Δ2-11

pGR-SP35^Δ90-107

pCam-ncSP35-GFP

pNubG-H2B

pCub-ncSP35

pCam-H2B-HA

pCLuc-ncSP35

pNLuc-ZjH2B

35-F

35-R

H2B-F

H2B-R

phoA-35-F

phoA-35-R

PVX-35-F

PVX-35-R

PVX-35^Δ2-11-F

PVX-35-R

PVX-35-F

PVX-35^Δ90-107-R

35-GFP-F

35-GFP-R

RR3-H2B-F

RR3-H2B-R

BT3-35-F

BT3-35-FR

H2B-HA-F

H2B-HA-R

CLuc-35-F

CLuc-35-R

NLuc-H2B-F

NLuc-H2B-R

ATGAATAATTTTGCTTTAGGTTTTA

TTAATATTCTTGTTGATTATGATGAAT

ATGGCACCCAAGGCTGAGAA

ATTCGAGCTGGTCACCCTAGGC

AAGGAGATATACATATGAATAATTTTGCTTTAGGTTTTA

CAAGCTTATCGGCGGTCGACATATTCTTGTTGATTATGATGAAT

AGCACCAGCTAGCATCGATATGAATAATTTTGCTTTAGGTTTTA

TTAAAAGAAACCAAAAGTTTTTTTC

AGCACCAGCTAGCATCGATATGAGTCTTTTTAATTTATTTGTCA

---

CAAGCTTATCGGCGGTCGAC TGGTTTGGATCAACAACATT

GACGAGCTCGGGTACCATGAATAATTTTGCTTTAGGTTTTA

TGGTGTCGACTCTAGAATATTCTTGTTGATTATGATGAAT

CAGAGTGGCCATTACGGCCATGGCACCCAAGGCTGAGAA

TCGAGAGGCCGAGGCGGCCCCTCAAGAGCTGGTAAATTTAGTCA

TGTAATGGCCATTACGGCCATGAATAATTTTGCTTTAGGTTTTA

GCAGATGGCCGAGGCGGCCCCATATTCTTGTTGATTATGATGAAT

GGACTCTTGACCATGGAT ATGGCACCCAAGGCTGAGAA

ATTCGAGCTGGTCACCCTAGGCATAATCCGGCACATCATAAGGGTAAGAGCT

GGTAAATTTAGTCATCCCGGGGCGGTACCAATAATTTTGCTTTAGGTTTTA

CTGCAGGTCGACTTAATATTCTTGTTGATTATGATGAAT

TCGGTACCCGGGATCC ATGGCACCCAAGGCTGAGAA

ACGAGATCTGGTCGAC AGAGCTGGTAAATTTAGTCA

Fig. 1 Verification of ncSP35 extracellular secretionA： Schematic diagram of prokaryotic expression for phoA and ncSP35； B： Analysis of extracellular secretion of ncSP35-mphoA using the E. coli-based alkaline phosphatase reporter system

Fig. 2 ncSP35 suppressed Bax-triggered HR and H2O2 accumulation in N. benthamianaA： Schematic diagrams of the PVX recombinant plasmids； B： Phenotype of ncSP35 inhibiting Bax-triggered hypersensitive response； C： DAB staining.1—GFP；2—ncSP35；3—Buffer；4—GFP+Bax；5—ncSP35+Bax；6—Buffer+Bax

Fig. 3 Function analysis of the ncSP35 mutants in HR suppressionA： Three-dimensional structure of ncSP35 and spatial location of the corresponding mutants； B： Diagram of the ncSP35 mutants； C： Statistical analysis of the ncSP35 mutants in HR suppression； D： Typical HR suppression phenotypes of the ncSP35 mutants in N. benthamiana （after 5 d）

Fig. 4 Developmental phenotypes of N. benthamiana induced by the ncSP35

Fig. 5 Subcellular localizations of ncSP35 and its mutants in N. benthamiana （Scale bar=25 μm）

Fig. 6 ncSP35 protein interacted with ZjH2BA： Validation of the interaction between ncSP35 and ZjH2B via yeast two-hybrid； yeast cells co-transformed with Cub-APP/NubG-Fe65 was used as a positive control， and those co-transformed with Cub-ncSP35/NubG or Cub/NubG-H2B were as negative controls， SD-2 indicates the medium lacking leucine and tryptophan， while SD-4 represents the medium lacking leucine， tryptophan， histidine and adenine； B： Luciferase complementation imaging assay of the interaction between ncSP35 and ZjH2B，1—nLUC-H2B+ncSP35-cLUC，2—nLUC-H2B+cLUC，3—nLUC+ncSP35-cLUC； C： Co-IP analysis of the interaction between ncSP35 and ZjH2B

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