Expression Characteristics and Function Analysis of CsMEKK1-1 Gene in Response to Huanglongbing in Citrus

doi:10.16420/j.issn.0513-353x.2023-0792

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (9): 2168-2182.doi: 10.16420/j.issn.0513-353x.2023-0792

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Expression Characteristics and Function Analysis of CsMEKK1-1 Gene in Response to Huanglongbing in Citrus

DONG Liting, QU Rongrong, PANG Shuwei, MO Kaiqin, CHEN Shuang, SHANG Lanyue, ZOU Xiuping^*()

Integrative Science Center of Germplasm Creation in Western China（Chongqing）Science City，Citrus Research Institute，Southwest University，Chongqing 400712，China

Received:2024-05-29 Revised:2024-08-08 Online:2024-09-25 Published:2024-09-19
Contact: ZOU Xiuping

Abstract

Abstract:

It has been shown that MAPK（mitogen-activated protein kinase）signaling pathway plays a key role in regulating citrus Huanglongbing（HLB）disease，but the key genes and mechanisms involved in this pathway need to be elucidated. Comparative transcriptome analysis of the mesophyll and midrib tissues from‘Jincheng’oranges[Citrus sinensis (L.) Osbeck]at the early stage（four months）of“CandidatusLiberibacter asiaticus”（CLas）infection revealed that differentially expressed genes（DEGs） were significantly enriched in the MAPK signaling pathway. Among these DEGs，four MEKK1 genes involved in the first cascade of the MAPK signaling pathway were up-regulated in the mesophylls，but they had no differential expression in midribs. Real-time fluorescence quantitative PCR（qRT-PCR）analysis showed that the expression of four CsMEKK1 genes were significantly different in different tissues from the HLB-susceptible‘Jincheng’orange and the HLB-tolerant‘Mafenggan’orange（Citrus hystrix）during CLas infection. CsMEKK1-1 showing the most significantly differential expression was selected for further analysis. Bioinformatics analysis showed that CsMEKK1-1 belongs to the MEKK gene family. Tobacco subcellular localization showed that CsMEKK1-1 was localized in the cell cytoplasm，nucleus and chloroplast. Expression characteristics of CsMEKK1-1 gene in response to salicylic acid（SA），jasmonic acid（JA），abscisic acid（ABA），ethylene（ETH）and CLas-flg22 in healthy‘Jincheng’leaves were analyzed by qRT-PCR. The results showed that CsMEKK1-1 expression was downregulated by SA，JA and ETH and CLas-flg22 mainly down-regulated CsMEKK1-1 expression. To evaluate CsMEKK1-1 function，MEKK1-1 overexpression and RNA interference（RNAi）vectors were constructed，and were introduced into CLas-infected‘Jincheng’branches to generate transgenic hairy roots using Agrobacterium rhizogenes-mediated citrus transformation. Quantitative PCR analysis showed that CsMEKK1-1 positively promoted CLas proliferation in hairy roots，indicating that CsMEKK1-1 is a susceptible gene for HLB disease.

Key words: Citrus, Huanglongbing, MAPK signaling pathway, CsMEKK1-1, disease-susceptible gene

DONG Liting, QU Rongrong, PANG Shuwei, MO Kaiqin, CHEN Shuang, SHANG Lanyue, ZOU Xiuping. Expression Characteristics and Function Analysis of CsMEKK1-1 Gene in Response to Huanglongbing in Citrus[J]. Acta Horticulturae Sinica, 2024, 51(9): 2168-2182.

Figures/Tables 8

Table 1 Primers used in this study

引物名称 Primer ID	引物序列（5′-3′） Primer sequence
Cs_ont_1g020870 Cs_ont_6g014270 Cs_ont_3g009990	F：GCGGCTGAATCCAACTTTCG；R：GGAGGACCTCTCCTAGCCAT F：TGCTCCACTTCAAATGAGCTT；R：CGCAGGAGAAGTCCTACCAC F：CCGAGGTCGAGGTTGAAACA；R：TCAACTTGGTCCGAGTCAGC
Cs_ont_3g032130	F：TTTGGTGTAGCGCCATGGAT；R：AACGTGTTGACCCTGGTCTC
GAPDH HLBas HLBp	F：GCTTTCCGTGTACCCACTGT；R：CTCTGACTCCGCCTTGATGG F：TCGAGCGCGTATGCAATACG；R：GCGTTATCCCGTAGAAAAAGGTAG AGACGGGTGAGTAACGCG

Fig. 1 Visualization of differentially expressed genes（DEGs）in MAPK pathway responding to CLas in‘Jincheng’oranges Red and green indicate up-regulated and down-regulated，purple indicate up-regulated and down-regulated respectively. “+P”indicates phosphorylation. The“O”after pathogen infection represents the infectious organism or organism.

Fig. 2 The relative expression levels of MEKK1 in‘Jincheng’and‘Mafenggan’infected with Huanglongbing（HLB）and uninfected（Control） * t-test，P < 0.05，n = 3.

Fig. 3 Analysis of CsMEKK1-1 gene structures（A），phylogenetic tree（B）and protein sequence（C） The numbers in B indicate the confidence levels of the nodes，while the numbers in C represent the positions of the amino acid residues. Cs：Citrus sinensis；Pt：Populus tremula；Os：Oryza sativa；At：Arabidopsis thaliana；Hb：Hevea brasiliensis；So：Syzygium odoratum；Mi：Mangifera indica；Jr：Juglans regia.

Fig. 4 Subcellular location analysis of CsMEKK1-1 in tobacco epidermal cells

Fig. 5 Expression analysis of CsMEKK1-1 induced by SA，JA，ETH，ABA in‘Jincheng’orange * t-test，P < 0.05，n = 3.

Fig. 6 Expression analysis of CsMEKK1-1 induced by CLas-flg22 in‘Jincheng’orange * t-test，P < 0.05，n = 3.

Fig. 7 Pathogen content and gene expression of CsMEKK1-1 transgenic plant‘Jincheng’infected with CLas A：GFP fluorescence screening. B：Hairy root phenotype. C and D：Identification of transgenic hairy roots by PCR（M：Marker；+：Positive plasmid；-：Negative control）；E and F：Bacterial content in midrib of leaves and roots；EV：pNmGFPer control；OE：OE-CsMEKK1-1 transgenic plants；RI：RNAi-CsMEKK1-1 transgenic plants. Three biological replicates，each sample comes from 3 or 4 roots. * indicates significant difference compared to the empty control.

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Expression Characteristics and Function Analysis of CsMEKK1-1 Gene in Response to Huanglongbing in Citrus

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