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. 2023 Dec 23;11(2):uhad276.
doi: 10.1093/hr/uhad276. eCollection 2024 Feb.

ABA-CsABI5-CsCalS11 module upregulates Callose deposition of citrus infected with Candidatus Liberibacter asiaticus

Lixiao Yao  1 , Xingru Guo  1 , Juan Su  1 , Qingwen Zhang  1 , Mengyao Lian  1 , Hao Xue  1 , Qiang Li  1 , Yongrui He  1 , Xiuping Zou  1 , Zhen Song  1 , Shanchun Chen  1
Affiliations

ABA-CsABI5-CsCalS11 module upregulates Callose deposition of citrus infected with Candidatus Liberibacter asiaticus

Lixiao Yao et al. Hortic Res. .

Abstract

Huanglongbing (HLB) primarily caused by Candidatus Liberibacter asiaticus (CLas) has been threatening citrus production globally. Under HLB conditions, an excessive accumulation of the polysaccharide callose in citrus phloem occurs, leading to phloem blockage and starch accumulation in leaves. The callose production is controlled by callose synthases (CalS), which have multiple members within plants. However, the knowledge of callose production in the citrus upon infection with CLas is limited. In this study, we firstly identified 11 CalSs in the Citrus sinensis genome through bioinformatics and found the expression pattern of CsCalS11 exhibited a positive correlation with callose deposition in CLas-infected leaves (correlation coefficient of 0.77, P ≤ 0.05). Knockdown of CsCalS11 resulted in a reduction of callose deposition and starch accumulation in CLas-infected citrus. Interestingly, we observed significantly higher concentrations of abscisic acid (ABA) in HLB-infected citrus leaves compared to uninfected ones. Furthermore, the expressions of CsABI5, CsPYR, and CsSnRK2 in the ABA pathway substantially increased in citrus leaves upon CLas infection. Additionally, the expression of CsCalS11 was significantly upregulated in citrus leaves following the application of exogenous ABA. We confirmed that CsABI5, a pivotal component of the ABA signaling pathway, regulates CsCalS11 expression by binding to its promoter using yeast one-hybrid assay, dual luciferase assay, and transient expression in citrus leaves. In conclusion, our findings strongly suggest that the CsABI5-CsCalS11 module plays a crucial role in regulating callose deposition through the ABA signaling pathway during CLas infection. The results also revealed new function of the ABA signaling pathway in plants under biotic stress.

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Conflict of interest statement

The authors have no conflict of interest to declare.

Figures

Figure 1
Figure 1
The analysis of CsCalSs in Citrus sinensis. A: Chromosomal localization of CsCalSs. The scale size of the chromosome is labeled on the left. The chromosome number was denoted at the top of each chromosome. The gene location was illustrated with a line and its name was on the right side of the chromosome. Chr, chromosome. B: Phylogenetic relationships of CalS proteins from Arabidopsis thaliana and C. cinensis using MEGA. AtCalS1 (AT1G05570.1), AtCalS2 (AT2G31960.1), AtCalS3 (AT5G13000.1), AtCalS4 (AT5G36870.1), AtCalS5 (AT2G13680.1), AtCalS6 (AT3G59100.1), AtCalS7 (AT1G06490.1), AtCalS8 (AT3G14570.1), AtCalS9 (AT3G07160.2), AtCalS10 (AT2G36850.1), AtCalS11 (AT4G04970.1), AtCalS12 (AT4G03550.1), and CsCalS1–12. C: Gene structure and protein domain structure of CsCalSs. The genomic structure of CsCalSs was analyzed using TBtools: Intron was shown with a horizontal line; Extron was expressed through a box. The conserved domain and transmembrane region were analyzed using the SMART tool. Vta1 domains, FKS1 domains, glucan synthase domains, and transmembrane regions were in in different colour rectangles. D: The RT-qPCR analysis of CsCalSs in leaves of healthy and HLB-affected “Wanjincheng” (C. sinensis) in the greenhouse through 2−ΔCt analysis method. *P ≤ 0.05; **P ≤ 0.01.
Figure 2
Figure 2
The CsCalS11 expression was positively related to callose deposition and ABA. A: Callose deposition at 11 weeks and 39 weeks post-inoculation. The number of callose deposition (B) and relative expression of CsCalS11 (C) were measured in HLB-affected Citrus sinensis and healthy ones every 4 weeks. D: A positive correlation between CsCalS11 expression and callose deposition obtained with SPSS software (Pearson R = 0.772). E: The relative expression of CsCalS11 under 100 μmol·L−1 abscisic acid (ABA), 10 μmol·L−1 salicylic acid (SA), 100 μmol·L−1 methyl jasmonate (MeJA), and 10 μmol·L−1 ethephon (ETH) for 48 hours. *P ≤ 0.05.
Figure 3
Figure 3
Transient expression of CsCalS11 in citrus leaves. A: Schematic representation of the CsCalS11 overexpressing vector based on the pLGNL binary plasmid employing the In-fusion strategy. 11–3F/11–3R, 11-2F/11-2R, and 11-1F/11-1R were primer pairs for cloning fragments 3, 2, and 1 of the CsCalS11 respectively. pLGNL, 11–3F, and 11-2F include the SmaI cleavage site CCCGGG. The primer sequences from CsCalS11 are represented in capital letters and lowercase sequences from pLGNL in black letters. The expression of CsCalS11 (B) and GUS (C) and callose content (D) were quanlitified in transiently overexpressed citrus. The experiments were repeated for three times with the similar results. *: P ≤ 0.05.
Figure 4
Figure 4
Evaluation of CsCalS11-RNAi citrus strains under normal conditions (A-E) and Huanglongbing (HLB) stress (F-H). A: Four CsCalS11-RNAi citrus strains and non-transgenic control (NT). CsCalS11 relative expression (B), callose concentration (C), leaf number (D), and plant weight (E) of CsCalS11-RNAi strains (3 plantlets for each strain). F: HLB symptoms observation. Sym1 shows leaf blotch chlorosis; Sym2 shows the burst leaf vein; Unsym means asymptomatic leaves. CLas (G) and starch (H) contents in leaves of CsCalS11-RNAi seedlings affected by HLB. *: P ≤ 0.05; **: P ≤ 0.01; ***P ≤ 0.001.
Figure 5
Figure 5
The CsCalS11p was bound and activated by CsABI5 in yeast and tobacco. A: Physical interactions of CsABI5 with CsCalS11p in yeast one hybrid (Y1H) assays. The pGADT7-Rec-53 was introduced into yeast cells carrying pAbAi-P53 as a positive control. The empty vector pGADT7 was included as a negative control. Another negative control was yeast containing pGADT7-CsABI5 and pAbAi-P53. AbA200 means 200 ng·nL−1 Aureobasidin A. B: Reporter and effector constructs for dual LUC assays. LUC: firefly luciferase, REN: Renilla luciferase, 35S-P: CaMV35S promoter, 35S-T: CaMV35S terminator. CsCalS11p::LUC was constructed based on pGreenII 0800-LUC. The control effector construct was from the pLGNL plasmid. C: Dual luciferase activity assay performed by transient expression in Nicotiana benthamiana leaves. *P ≤ 0.05.
Figure 6
Figure 6
The CsCalS11 promoter (CsCalS11p) was activated under conditions of Huanglongbing (HLB) stress, exogenous abscisic acid (ABA) exposure and transient overexpression of CsABI5. A: Schematic representation of the CsCalS11p::GUS constructs T-DNA of the pGNGM1300 binary vector. B: The transgenic seedlings with CsCalS11p::GUS. C: PCR analysis with primer pair GUS-F/GUF-R using leaf DNA from (B) seedlings. D: GUS staining of leaf discs from (B) seedlings. E: The relative expression of GUS in CsCalS11p::GUS seedlings affected by HLB compared with the healthy one. F: The relative expression of GUS in CsCalS11p::GUS leaves treated with 100 μmol·L−1 ABA compared with H2O. G: GUS expression in the citrus leaves transformed with CsCalS11p::GUS through transient expression of CsABI5. NT: non-transgene seedling; P23/P57/P58: three citrus strains transformed with CsCalS11p::GUS.
Figure 7
Figure 7
The concentration of abscisic acid (ABA) and the expression of genes in its signal pathway in healthy and Huanglongbing (HLB) citrus. A: ABA concentration. B: Fold change of CsABI5, CsPYR, CsPP2C, and CsSnRK2 expression in citrus under HLB stress. *P ≤ 0.05.
Figure 8
Figure 8
A hypothesis of callose deposition via ABA-CsABI5-CsCalS11 module during infection of Candidatus Liberibacter asiaticus (CLas).
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