Genome-wide characterization and expression analysis of the MLO gene family sheds light on powdery mildew resistance in Lagenaria siceraria

Abstract

MLO (mildew locus O) genes play a vital role in plant disease defense system, especially powdery mildew (PM). Lagenaria siceraria is a distinct Cucurbitaceae crop, and PM is one of the most serious diseases threatening crop production and quality. Although MLOs have been exploited in many Cucurbitaceae species, genome-wide mining of MLO gene family in bottle gourd has not been surveyed yet. Here we identified 16 MLO genes in our recently assembled L. siceraria genome. A total of 343 unique MLO protein sequences from 20 species were characterized and compared to deduce a generally high level of purifying selection and the occurrence of regions related to candidate susceptibility factors in the evolutional divergence. LsMLOs were clustered in six clades containing seven conserved transmembrane domains and 10 clade-specific motifs along with deletion and variation. Three genes (LsMLO3, LsMLO6, and LsMLO13) in clade V showed high sequence identity with orthologues involved in PM susceptibility. The expression pattern of LsMLOs was tissue-specific but not cultivar-specific. Furthermore, it was indicated by qRT-PCR and RNA-seq that LsMLO3 and LsMLO13 were highly upregulated in response to PM stress. Subsequent sequence analysis revealed the structural deletion of LsMLO13 and a single nonsynonymous substitution of LsMLO3 in the PM-resistant genotype. Taken all together, it is speculated that LsMLO13 is likely a major PM susceptibility factor. The results of this study provide new insights into MLO family genes in bottle gourd and find a potential candidate S gene for PM tolerance breeding.

Keywords: Bottle gourd; Expression analysis; MLO family; Phylogenetic analysis; Powdery mildew.

Fig. 1

The information of LsMLO genes. (A) Distribution of MLO genes on the bottle gourd chromosomes. (B) Interchromosomal synteny of linked LsMLO genes.

Fig. 2

Unrooted Maximum likelihood phylogenetic tree of MLO genes in 20 species. The outer circular color strip indicated the clade classification. Red star symbol represents the specific LsMLOs. Circles with different colors represent MLOs in Cucurbit crops while squares with different colors represent MLOs in the rest species. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Direction and magnitude of natural selection on Cucurbitaceae (A) and clade V (B) MLO homologs.

Fig. 4

Conserved motifs and gene structures of LsMLO genes in the bottle gourd genome. (A): 10 Motifs are indicated by different colored boxes. (B): Green boxes indicate the exon, and lines indicate the intron. Different numbers indicate the phase of introns. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Comparative analysis of orthologous gene clusters among multiple species. (A) Green ovals on the left side represent the presence of orthologous genes while grey ovals represent the absence of orthologous genes. Different color bars on the right side indicate the number and ratio of orthologous proteins in 8 plant species. (B) Layout of the different orthologous clusters. Each edge represents the similarity between two protein sequences. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Heatmap of cis-acting regulatory elements in the promoters of LsMLOs. Different categories cis-acting regulatory elements are displayed horizontally. The value in each square indicates the number of each class of distinct cis-acting regulatory elements identified in the corresponding MLO gene promoter.

Fig. 7

Heatmaps of LsMLO genes displaying tissue-specific and cultivar-specific expression. (A) Hierarchical clustering of expressions profile of MLO genes in different tissues (flower, fruit, leaf, root, stem) of bottle gourd under normal conditions. Colour scheme was based on normalized TPM values without transformation. (B) The violin plot visualized the density and distribution of MLO gene expressions in each tissue. The value in horizontal ordinate indicated the TPM of 16 LsMLOs. (C) The expression profile of MLO genes in different bottle gourd varieties under normal conditions. Colour scheme was based on a log₂(TPM+1) transformation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

Expression profile of MLO genes in leaves of two bottle gourd cultivars (PM-S and PM-R) under PM infection. (A) The expression levels of differentially expressed genes in two bottle gourd varieties leaves under PM stress. The TPM values were showed in the square. (B) Relative expression of 16 LsMLO genes in response to PM infection in two bottle gourd genotypes at four time points. Mean ± SE of three replicates represents the significant difference with LSD test. The data followed by the different letters in the graph are significant at P ≥ 0.05.

Fig. 9

Characterization of LsMLO13 alleles from PM-R and PM-S bottle gourd genotypes. (A) Partial alignments of full length LsMLO13 amplified from cDNA from susceptible and resistant bottle gourd genotypes were sequenced. The sequences with two deletions and three single base substitutions are obtained from the resistant genotype. (B) Partial alignment of the LsMLO13 protein and other reported proteins encoded by clade V MLO S genes of several species. The regions of 2 and 5 amino acid residues deleted in the proteins of LsMLO13 are indicated by red bars. (C) Graphic display of the transmembrane structure of the predicted LsMLO13 protein. The plasma membrane is indicated by two horizontal lines. Amino acid residues highlighted in grey are predicted to be deleted in the protein of LsMLO13 in PM-R, residues highlighted in red are predicted to be substituted in the protein of LsMLO13 in PM-R. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)