Cloning and Functional Analysis of BobHLH34 Gene in Cabbage that Interacts with XopR from Xanthomonas

doi:10.16420/j.issn.0513-353x.2021-1274

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (2): 319-330.doi: 10.16420/j.issn.0513-353x.2021-1274

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Cloning and Functional Analysis of BobHLH34 Gene in Cabbage that Interacts with XopR from Xanthomonas

HAN Rui¹^,², ZHONG Xionghui², CHEN Denghui¹^,², CUI Jian², YUE Xiangqing¹^,², XIE Jianming¹^,^*(), KANG Jungen²^,^*()

1. Colleage of Horticulture，Gansu Agricultural University，Lanzhou 730070，China
2. Key Laboratory of Biology and Genetic Improvement of Horticultural Crops，Ministry of Agriculture and Rural Affairs，Beijing Vegetable Research Center，Beijing Academy of Agriculture and Forestry Sciences，Beijing 100097，China

Received:2022-09-16 Revised:2022-11-01 Online:2023-02-25 Published:2023-03-06
Contact: *（E-mail：xiejianminggs@126.com，kangjungen@nercv.org)

Abstract

Abstract:

Xanthomonas campestris pv. campestris（Xcc）is the causal agent of black rot of Brassicaceae crops. The cabbage protein BobHLH34 was found to be interacted with the effector XopR through the yeast two-hybrid system. The CDS of BobHLH34 was 996 bp encoding peptides with 331 amino acids. It has a conserved domain（basic/helix1-loop-helix2）. Phylogenetic tree analysis showed that bHLH34 proteins from Brassica cretica had the highest homology with BobHLH34 protein. The subcellular localization results showed that BobHLH34 was located in the nucleus. The qRT-PCR results showed that the expression of BobHLH34 was increased significantly after Xcc inoculation of cabbage leaves. The expression reached the peak at 16 h，which was 7.7 times that of the control. Vacuum infiltration of cabbage seedlings with the BobHLH34-PCVA/PCVB vector effectively down-regulated the expression of BobHLH34 gene，which resulted in susceptibility to Xcc. Taken together，BobHLH34 could positively regulate cabbage defense to black rot.

Key words: cabbage, Xanthomonas campestris pv. campestris, BobHLH34 transcription factor, VIGS, XopR

CLC Number:

S635

HAN Rui, ZHONG Xionghui, CHEN Denghui, CUI Jian, YUE Xiangqing, XIE Jianming, KANG Jungen. Cloning and Functional Analysis of BobHLH34 Gene in Cabbage that Interacts with XopR from Xanthomonas[J]. Acta Horticulturae Sinica, 2023, 50(2): 319-330.

Figures/Tables 9

Table 1 Primers used in this study

引物名称 Primer name	序列（5′-3′） Sequence
XopR-AD-F	GCCATG GAGGCCAGTGAATTCATGTATCAATCAAACGAAGA
XopR-AD-R	CAGCTCGAGCTCGATGGATCCAGCAGCAAAAGAGCAGTTTTG
GAPDH-F	CAGGTTTGGAATTGTCGAGG
GAPDH-R	GAGCTGTGGAAGCACCTTTC
CE-bhlh34-F	TGGCGCGCCACTAGTGGATCCATGTATCAATCAAACGAAGA
CE-bhlh34-R-no-TGA	CCCGGGAGCGGTACCCTCGAGTCAAGCAGCAAAAGAGCAGT
BobHLH34-qRT-F2	GCTCAAGAAAGAGGGGACGA
BobHLH34-qRT-R2	GTTTACGACCCGGATTGCAT
BobHLH34-PVCA-F	GCCTAAGCGGCTAGCGGTACCCTGCCCTTTGCTTCGATCAA
BobHLH34-PVCA-R	TAGGCTAGCGAGCTCAGATCTAAACTTGCTCCTGTTCCGTC

Fig. 1 Amplification of full-length open reading frame of BobHLH34 using cDNA Marker：DL2000.

Fig. 2 Comparative alignment analysis of BobHLH34 protein and homologous proteins in different species

Fig. 3 Phylogenetic analysis of the bHLH34 proteins in different species

Fig. 4 The transient expression of BobHLH34 gene in Nicotiana benthamiana leaf epidermal cells A-C：BobHLH34-PYBA1132 recombinant expression vector showed green fluorescence in the nucleus；D-F：PYBA1132 showed green fluorescence in the whole cells.

Fig. 5 The interaction between BobHLH34 and XopR was verified in yeast-two hybrid system

Fig. 6 The interaction between BobHLH34 and XopR was detected by bimolecular fluorescence complementary analysis

Fig. 7 Transcription analysis of BobHLH34 over time after inoculation with Xcc in cabbage **P ≤ 0.01.

Fig. 8 Sensitivity analysis of BobHLH34 silenced cabbage plants to Xcc A，B：Cabbage seedlings were infected with Agrobacterium containing empty vectors（PCVA/PCVB）or silenced vectors（BobHLH34-PCVA/PCVB）. Leaves were sampled on the three weeks after vacuum infiltration for detection of susceptiblity to Xcc. C：Cabbage seedlings were infected with Agrobacterium containing silenced vectors（PDS-PCVA/PCVB），and the photobleached phenotype was observed in cabbage plants. D：Analysis of BobHLH34 expression by qRT-PCR in silenced plants. E：The lesion area was measured 8-12 days after Xcc inoculation.* P ≤ 0.05，**P ≤ 0.01.

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Cloning and Functional Analysis of BobHLH34 Gene in Cabbage that Interacts with XopR from Xanthomonas

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