Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Apr 15;25(8):4353.
doi: 10.3390/ijms25084353.

Molecular Mechanism of Resistance to Alternaria alternata Apple Pathotype in Apple by Alternative Splicing of Transcription Factor MdMYB6-like

Xianqi Zeng  1 , Chao Wu  1 , Lulu Zhang  1 , Liming Lan  1 , Weihong Fu  1 , Sanhong Wang  1
Affiliations

Molecular Mechanism of Resistance to Alternaria alternata Apple Pathotype in Apple by Alternative Splicing of Transcription Factor MdMYB6-like

Xianqi Zeng et al. Int J Mol Sci. .

Abstract

As a fruit tree with great economic value, apple is widely cultivated in China. However, apple leaf spot disease causes significant damage to apple quality and economic value. In our study, we found that MdMYB6-like is a transcription factor without auto-activation activity and with three alternative spliced variants. Among them, MdMYB6-like-β responded positively to the pathogen infection. Overexpression of MdMYB6-like-β increased the lignin content of leaves and improved the pathogenic resistance of apple flesh callus. In addition, all three alternative spliced variants of MdMYB6-like could bind to the promoter of MdBGLU H. Therefore, we believe that MdMYB6-like plays an important role in the infection process of the pathogen and lays a solid foundation for breeding disease-resistant cultivars of apple in the future.

Keywords: Alternaria alternata apple pathotype; R2R3-MYB; alternative splicing; apple; biotic stress.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Cloning and structure analysis of MdMYB6-like and its novel variants: (a) the three alternative spliced variants of MdMYB6-like confirmed by RT-PCR using ‘Starking Delicious’ cDNA; (b) diagrammatic representation of the genomic and protein structure of the three alternative spliced variants. The red triangle indicates a premature termination codon (PTC); (c) amino acid sequence analysis of the three alternative spliced variants.
Figure 2
Figure 2
Amino acid sequence alignment and phylogenetic tree construction of MdMYB6-like-α, MdMYB6-like-β, MdMYB6-like-γ and other highly homologous MYB TFs. (a) Amino acid sequence comparison between MdMYB6-like-α, MdMYB6-like-β, MdMYB6-like-γ and ten R2R3-MYB TFs. The following amino acid sequences were retrieved from the GenBank database: MdMYB6-like (sequence ID: XP_017192216.1), MrMYB6 (sequence ID: KAB1209240.1), PaMYB4-like (sequence ID: XP_021824312.1), PbMYB6-like (sequence ID: XP_018502946.2), PbMYB8 (sequence ID: XP_009357784.1), PdMYB4 (sequence ID: BBG94536.1), PpMYB18 (sequence ID: ALO81021.1), PsMYB18 (sequence ID: QGQ60117.1), RcMYB6 (sequence ID: XP_024196862.1), ZjMYB8-like (sequence ID: XP_048318055.2). The red and orange boxes represent the R1 and R2 structural domains, respectively. (b) Phylogenetic tree of MdMYB6-like-α, MdMYB6-like-β, MdMYB6-like-γ and their homologs in different plant species. The tree is drawn proportionally. Numbers next to the node are bootstrap values from 1000 replications. (c) Protein structures of three alternative spliced variants generated with SMART.
Figure 3
Figure 3
The expression levels after infection by AAAP and identification of transcription factor properties of MdMYB6-like and its alternative spliced variants: (a) differential expression of MdMYB6-like and its alternative spliced variants in leaves after different times (0, 12, 24, 36, 72 h) of infection. Error bars represent the SDs from three biological replicates. Lowercase letters represent significant differences at p < 0.05 (Tukey’s HSD test). (b) Yeast two-hybrid assays of auto-activation activity identification of MdMYB6-like and its alternative spliced variants. Co-transformation of empty pGADT7 and pGBKT7 vector was used as a negative control, and co-transformation of pGBKT7-p53 and pGADT7-T vector was used as a positive control. Each co-transformation was diluted ten times. (c) Subcellular localization of GFP-MdMYB6-like-α, GFP-MdMYB6-like-β and GFP- MdMYB6-like-γ in leaf cells of N. benthamiana. The constructed plasmids for the assay were transferred into Agrobacterium tumefaciens GV3101 and transiently transfected into N. benthamiana leaves. DAPI was used to stain the nucleus prior to observations of GFP fluorescence. GFP, DAPI, bright-field, and merged images are presented. All bars = 100 μm.
Figure 4
Figure 4
Effects of transient overexpression and stable overexpression on apple materials. (a) RT-qPCR analysis of MdMYB6-like and its alternative spliced variants in the transient overexpression of ‘Gala’ apple leaves. Error bars represent the SDs from three biological replicates. The level of significance is indicated by *, denoting significant differences at p < 0.05, The level of significance is indicated by ****, denoting significant differences at p < 0.0001; (b) lignin content analysis of ‘Gala’ apple leaves following transient overexpression of the three alternatively spliced variants. Error bars represent the SDs from three biological replicates. Lowercase letters represent significant differences at p < 0.05; (c) wild-type, overexpressing empty pCAMBIA2300 vector and overexpressing pCAMBIA2300-MdMYB6-like-β apple flesh callus were placed into 1.5 mL centrifuge tubes for GUS staining, respectively. Blue color means that the overexpression vector was successfully transferred into apple flesh callus; (d) the inoculation AAAP experiment on normal-growing apple flesh callus. The white round spots represent the area of fungal infection after 5 d and indicate the amount of mycelia.
Figure 5
Figure 5
MdMYB6-like regulation of other genes. Yeast one-hybrid assays were conducted on SD-Leu medium, supplemented with appropriate AbA concentrations (0 or 100 ng/mL) to verify DNA–protein interactions, with each co-transformation diluted 10 times. Transformation of empty pGADT7 vector with promoter of the MdBGLU H was used as a negative control.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Arnold M., Gramza-Michalowska A. Recent Development on the Chemical Composition and Phenolic Extraction Methods of Apple (Malus domestica)—A Review. Food Bioprocess Technol. 2023;10:1–42. doi: 10.1007/s11947-023-03208-9. - DOI
    1. Liu B., Zhang Y., He D., Li Y. Identification of Apple Leaf Diseases Based on Deep Convolutional Neural Networks. Symmetry. 2017;10:11. doi: 10.3390/sym10010011. - DOI
    1. Abe K., Iwanami H., Kotoda N., Moriya S., Takahashi S. Evaluation of apple genotypes and Malus species for resistance to Alternaria blotch caused by Alternaria alternata apple pathotype using detached-leaf method. Plant Breed. 2010;129:208–218. doi: 10.1111/j.1439-0523.2009.01672.x. - DOI
    1. Qin L., Zhao L., Wu C., Qu S., Wang S. Identification of microRNA transcriptome in apple response to Alternaria alternata infection and evidence that miR390 is negative regulator of defense response. Sci. Hortic. 2021;289:110435. doi: 10.1016/j.scienta.2021.110435. - DOI
    1. Saito A., Nakazawa N., Suzuki M. Selection of mutants resistant to Alternaria blotch from in vitro-cultured apple shoots irradiated with X- and γ-rays. J. Plant Physiol. 2001;158:391–400. doi: 10.1078/0176-1617-00235. - DOI

Supplementary concepts

Grants and funding

This research received no external funding.

LinkOut - more resources

-