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. 2023 Jan 6:13:1067695.
doi: 10.3389/fpls.2022.1067695. eCollection 2022.

MePAL6 regulates lignin accumulation to shape cassava resistance against two-spotted spider mite

Xiaowen Yao  1   2 , Xiao Liang  1   2 , Qing Chen  1   2 , Ying Liu  1   2 , Chunling Wu  1   2 , Mufeng Wu  1   2 , Jun Shui  1   2 , Yang Qiao  1   2 , Yao Zhang  1   2 , Yue Geng  1   2
Affiliations

MePAL6 regulates lignin accumulation to shape cassava resistance against two-spotted spider mite

Xiaowen Yao et al. Front Plant Sci. .

Abstract

Introduction: The two-spotted spider mite (TSSM) is a devastating pest of cassava production in China. Lignin is considered as an important defensive barrier against pests and diseases, several genes participate in lignin biosynthesis, however, how these genes modulate lignin accumulation in cassava and shape TSSM-resistance is largely unknown.

Methods: To fill this knowledge gap, while under TSSM infestation, the cassava lignin biosynthesis related genes were subjected to expression pattern analysis followed by family identification, and genes with significant induction were used for further function exploration.

Results: Most genes involved in lignin biosynthesis were up-regulated when the mite-resistant cassava cultivars were infested by TSSM, noticeably, the MePAL gene presented the most vigorous induction among these genes. Therefore, we paid more attention to dissect the function of MePAL gene during cassava-TSSM interaction. Gene family identification showed that there are 6 MePAL members identified in cassava genome, further phylogenetic analysis, gene duplication, cis-elements and conserved motif prediction speculated that these genes may probably contribute to biotic stress responses in cassava. The transcription profile of the 6 MePAL genes in TSSM-resistant cassava cultivar SC9 indicated a universal up-regulation pattern. To further elucidate the potential correlation between MePAL expression and TSSM-resistance, the most strongly induced gene MePAL6 were silenced using virus-induced gene silencing (VIGS) assay, we found that silencing of MePAL6 in SC9 not only simultaneously suppressed the expression of other lignin biosynthesis genes such as 4-coumarate--CoA ligase (4CL), hydroxycinnamoyltransferase (HCT) and cinnamoyl-CoA reductase (CCR), but also resulted in decrease of lignin content. Ultimately, the suppression of MePAL6 in SC9 can lead to significant deterioration of TSSM-resistance.

Discussion: This study accurately identified MePAL6 as critical genes in conferring cassava resistance to TSSM, which could be considered as promising marker gene for evaluating cassava resistance to insect pest.

Keywords: PAL gene family; VIGS; cassava; lignin biosynthesis; mite-resistance; two-spotted spider mite.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Influence on lignin biosynthesis pathway while TSSM-resistant and TSSM-susceptible cassava cultivars were infested by TSSM. (A) The potential schematic diagram of lignin biosynthesis pathway in cassava. The biosynthesis genes, the intermediate products and the final different forms of lignin products were marked with blue letters, white frames and yellow frames, respectively; (B) The mite damage indexes and the corresponding resistance levels of the tested cassava cultivars; (C) Changes in transcription of ten lignin biosynthesis genes; (D) Changes in activity of four enzymes involved in lignin biosynthesis pathway; (E) Changes in lignin content. Different letters above standard error bars indicate significant differences based on ANOVA followed by Tukey’s HSD multiple comparison test (p < 0.05) within the same time point.
Figure 2
Figure 2
The chromosomal locations and phylogeny of MePALs. (A) Chromosomal locations of the six MePAL genes. The chromosomes were presented as narrow rectangles, and color bars within the rectangles denoted the M. esculenta chromosome density. Scale bars on the left indicated the chromosome lengths (Mb); (B) Phylogenetic tree of the 6 MePAL proteins. The tree was constructed by using MEGA X based on the full-length amino acid sequences from M. esculenta (Me) (marked with stars), R. communis (Rc) (marked with circles), H brasiliensis (Hb) (marked with triangles) and A thaliana (At) (marked with boxes). All nodes had significant bootstrap support based on 1,000 replicates. The tree was constructed with cut-off value of 50%. Genes that distributed in the same clusters were shadowed with different colors.
Figure 3
Figure 3
Conserved motifs, gene structures, Cis-acting element and gene duplicate of the MePAL genes. (A) MEME analysis revealed the conserved motifs of the MePAL proteins. The colored boxes at the bottom denoted 8 motifs; (B) Structures of the six MePAL genes. The yellow boxes, black lines, and green boxes represented exon, intron, and UTR (untranslated region), respectively; (C) Prediction of cis-acting elements and visualization with Tbtools, the colored boxes at the bottom indicated the predicted elements; (D) Circos diagram of the MePAL duplication pairs in M. esculenta. The outer boxes indicated the gene density of each chromosomes, and the interior orange and grey curves indicated the collinearity relationships among MePAL genes and all the genes in the chromosomes, respectively; (E) Collinearity analysis between M. esculenta and A thaliana, M. esculenta and H brasiliensis, M. esculenta and R. communis. The interior red curves indicated the collinearity relationships of MePAL genes between two plant species, while the grey curves indicated the collinearity relationships of all the genes in the chromosomes between two plant species, respectively.
Figure 4
Figure 4
Transcription level of six MePAL genes in SC9-(R) after TSSM infestation. Different letters above standard error bars indicate significant differences based on ANOVA followed by Tukey’s HSD multiple comparison test (p < 0.05) within the same time point.
Figure 5
Figure 5
Effects of MePAL6 silencing on lignin biosynthesis pathway and TSSM-resistance performance of cassava. (A) Transcription changes of six MePAL genes (MePAL1-MePAL6) in MePAL6-silenced cassava lines while under TSSM infestation; (B) Transcription changes of downstream lignin biosynthesis genes (Me4CL, MeCCR and MeHCT) in MePAL6-silenced cassava lines while under TSSM infestation, the simplified lignin biosynthesis pathway was presented on the left, and the investigated downstream genes were labeled; (C) Enzyme activity changes of PAL, 4CL, CCR and HCT in MePAL6-silenced cassava lines while under TSSM infestation. Different letters above standard error bars indicate significant differences based on ANOVA followed by Tukey’s HSD multiple comparison test (p < 0.05) within the same time point.
Figure 6
Figure 6
Performance of MePAL6 silencing lines against TSSM infestation. (A) The TSSM infestation symptom of MePAL6 silencing lines and negative controls. The “zoom in” areas of plants after mite infestation on 0, 1, 4 days were indicated by red dashed boxes. (B) Identification of mite damage index after 4 dpi in MePAL6 silencing lines and negative controls. (C) Changes in lignin content in MePAL6-silenced cassava lines while under TSSM infestation. Different letters above standard error bars indicate significant differences based on ANOVA followed by Tukey’s HSD multiple comparison test (p < 0.05) within the same time point.
Figure 7
Figure 7
Potential mechanism of MePAL6 regulates lignin accumulation and shapes cassava resistance to two-spotted spider mite.
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