Genome-Wide Identification and Expression Analysis of Chitinase Genes in Watermelon under Abiotic Stimuli and Fusarium oxysporum Infection

Abstract

Chitinases, which catalyze the hydrolysis of chitin, the primary components of fungal cell walls, play key roles in defense responses, symbiotic associations, plant growth, and stress tolerance. In this study, 23 chitinase genes were identified in watermelon (Citrullus lanatus [Thunb.]) and classified into five classes through homology search and phylogenetic analysis. The genes with similar exon-intron structures and conserved domains were clustered into the same class. The putative cis-elements involved in the responses to phytohormone, stress, and plant development were identified in their promoter regions. A tissue-specific expression analysis showed that the ClChi genes were primarily expressed in the roots (52.17%), leaves (26.09%), and flowers (34.78%). Moreover, qRT-PCR results indicate that ClChis play multifaceted roles in the interaction between plant/environment. More ClChi members were induced by Race 2 of Fusarium oxysporum f. sp. niveum, and eight genes were expressed at higher levels on the seventh day after inoculation with Races 1 and 2, suggesting that these genes play a key role in the resistance of watermelon to Fusarium wilt. Collectively, these results improve knowledge of the chitinase gene family in watermelon species and help to elucidate the roles played by chitinases in the responses of watermelon to various stresses.

Keywords: Fusarium oxysporum; abiotic stresses; chitinase; expression analyses; genome-wide identification; watermelon.

Figure 1

(a) Distribution of 23 ClChi genes on the watermelon chromosomes. The scale ruler on the left side shows the physical distance (Mb) of the chromosomes. The relative positions of the ClChis are marked on the chromosomes. (b) Syntenic relationships among the Arabidopsis, cucumber, and watermelon chitinase genes are indicated in different colors; Red: Arabidopsis vs. Arabidopsis; Green: watermelon vs. watermelon; Yellow: cucumber vs. cucumber; Blue: watermelon vs. cucumber; Dark blue: Arabidopsis vs. watermelon; Purple: Arabidopsis vs. cucumber.

Figure 2

The unrooted phylogenetic tree of chitinase genes was generated based on the amino acid sequences of Arabidopsis, cucumber, and watermelon by the neighbor-joining (NJ) method using MEGA 7.0. The chitinase members were categorized into five clades and labeled as I, II, III, IV, and V. The chitinase members of species were color-coded: At, Arabidopsis (yellow); Cs, cucumber (green); Cl, watermelon (red).

Figure 3

Multiple alignments of the GH18 chitinase Class III and Class V and the GH19 chitinase subfamily of the ClChis sequences. Orange and red lines over sequences indicate signal peptides and their catalytic domains. Blue box: Glycosyl hydrolases family 18 (GH18) active site signature PS01095. Black box: chitin binding domain signature PS00026. Red box: Chitinases family 19 signature 1, PS00773. Green box: Chitinases family 19 signature 2, PS00774. The position of the conserved serine (S), threonine (T), and tyrosine (Y) predicted to be the phosphorylation sites are indicated by the black triangles.

Figure 4

Phylogenetic relationships, conserved motifs, and gene structures of the watermelon chitinases. (a) Phylogenetic tree of 23 watermelon chitinase proteins. (b) Distribution of the conserved motifs in the watermelon chitinases. (c) Gene structure of the predicted ClChi genes. Yellow boxes and black lines represent exons and introns, respectively.

Figure 5

Frequency of the occurrence of cis-acting elements upstream of the promoter sequences of chitinases under the functions. Hormone: ABRE, abscisic acid-responsive element; AuxRR-core and TGA-element, auxin-responsive element; TCA-element, salicylic acid-responsive element; CGTCA-motif and TGACG-motif, MeJA-responsive elements; GARE-motif and P-box, gibberellin-responsive elements; Stress: ARE, involved in the anaerobic induction; LTR, low-temperature-responsive element; MBS and TC-rich repeats, involved in defense and stress responsiveness; Development: CAT-box, circadian, GCN4_motif, HD-Zip I, MSA-like, motif I, and RY element, involved in meristem, circadian-controlled differentiation of the palisade mesophyll, expression of the endosperm, differentiation of the palisade mesophyll cells, cell cycle regulation, root-specific regulation, and seed-specific regulation, respectively. MeJA, methyl jasmonate.

Figure 6

The relative levels of expression of the ClChi genes in different tissues. Root (R); Stem (S); Leaf (L); Tendril (T); Female flower (FF); Male flower (MF). All the data points are the means ± SE (n = 3).

Figure 7

Heatmap of the expression of the ClChi genes in ‘M08’ under drought (a), salt (b), and low-temperature (c) stress. Red and green correspond to strong and weak expression of the ClChi genes, respectively. The plant samples at 0 dpt and 0 hpt were considered to be controls. dpt, days post-treatment; hpt, hours post-treatment.

Figure 8

Heatmap of the levels of expression of the ClChi genes under ABA, ETH, MeJA, and SA hormone treatments. (a) Levels of expression of ClChis under ABA stress visualized as a heat map (Left). Detailed patterns of expression of the ClChis under ABA stress (Right). (b) Levels of expression of the ClChis under ETH stress visualized as a heat map (Left). Detailed patterns of expression of ClChis under ETH stress (Right). (c) Levels of expression of the ClChis under MeJA and SA stress visualized as a heat map. The plant samples at 0 dpt and 0 hpt were considered to be controls. ABA, abscisic acid; ETH, ethylene; MeJA, methyl jasmonate; SA, salicylic acid.

Figure 8

Heatmap of the levels of expression of the ClChi genes under ABA, ETH, MeJA, and SA hormone treatments. (a) Levels of expression of ClChis under ABA stress visualized as a heat map (Left). Detailed patterns of expression of the ClChis under ABA stress (Right). (b) Levels of expression of the ClChis under ETH stress visualized as a heat map (Left). Detailed patterns of expression of ClChis under ETH stress (Right). (c) Levels of expression of the ClChis under MeJA and SA stress visualized as a heat map. The plant samples at 0 dpt and 0 hpt were considered to be controls. ABA, abscisic acid; ETH, ethylene; MeJA, methyl jasmonate; SA, salicylic acid.

Figure 9

(a) Heatmap of the expressed ClChi genes in the root tissue of ‘M08’ after infection with the causal agent of Fusarium wilt. The gene clusters were generated using the average linkage clustering method. (b) Detailed patterns of expression of ClChi1, 2, 5, 8, 10, 13, 17, and 23 infected with Fon R1 and Fon R2 at 7 dpt. Fon, Fusarium oxysporum f. sp. niveum; R1, race 1; R2, race 2.