Genome-Wide Identification and Expression of Chitinase Class I Genes in Garlic (Allium sativum L.) Cultivars Resistant and Susceptible to Fusarium proliferatum

Abstract

Vegetables of the Allium genus are prone to infection by Fusarium fungi. Chitinases of the GH19 family are pathogenesis-related proteins inhibiting fungal growth through the hydrolysis of cell wall chitin; however, the information on garlic (Allium sativum L.) chitinases is limited. In the present study, we identified seven class I chitinase genes, AsCHI1-7, in the A. sativum cv. Ershuizao genome, which may have a conserved function in the garlic defense against Fusarium attack. The AsCHI1-7 promoters contained jasmonic acid-, salicylic acid-, gibberellins-, abscisic acid-, auxin-, ethylene-, and stress-responsive elements associated with defense against pathogens. The expression of AsCHI2, AsCHI3, and AsCHI7 genes was constitutive in Fusarium-resistant and -susceptible garlic cultivars and was mostly induced at the early stage of F. proliferatum infection. In roots, AsCHI2 and AsCHI3 mRNA levels were increased in the susceptible and decreased in the resistant cultivar, whereas in cloves, AsCHI7 and AsCHI5 expression was decreased in the susceptible but increased in the resistant plants, suggesting that these genes are involved in the garlic response to Fusarium proliferatum attack. Our results provide insights into the role of chitinases in garlic and may be useful for breeding programs to increase the resistance of Allium crops to Fusarium infections.

Keywords: Fusarium spp.; GH19 family; biotic stress; class I chitinases; garlic Allium sativum L.; gene expression; gene structure.

Figure 1

Structural and phylogenetic analysis of garlic chitinases. (A) Evolutionary relationship and exon–intron structures of class I chitinases from Allium sativum cv. Ershuizao (AsCHI1–7) and Arabidopsis thaliana (AT3G12500). The unrooted dendrogram is based on amino acid sequences. Analysis was performed using the maximum likelihood method. Percentages of replicate trees in which the associated sequences clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. (B) Sequence alignment and functionally important sites of class I chitinases from A. sativum cv. Ershuizao and A. thaliana. The regions with 50–100% identity are shaded. The chitin-binding domain (CBD1) and glycoside hydrolase domain (GH19) are indicated by red and blue frames, respectively. CBD1 signature PS00026 (Cx(4,5)-CSx(2)-GxCGx(4)-[FYW]-C) is underlined in green, chitinase 19_1 signature PS00773 (Cx(4,5)-FY-[ST]-x(3)-[FY]-[LIVMF]-xAx(3)-[YF]-x(2)-F-[GSA])—in violet, and chitinase 19_2 signature PS00774 ([LIVM]-[GSA]-Fx-[STAG](2)-[LIVMFY]-W-[FY]-W-[LIVM])—in orange.

Figure 2

Distribution of conserved motifs in AsCHI proteins of A. sativum cv. Ershuizao. Analysis was performed using Multiple Em for Motif Elicitation (MEME) 5.3.0. The length of each box corresponds to that of the motif.

Figure 3

Heatmap of AsCHI1–7 mRNA expression in Allium sativum cv. Ershuizao tissues. Gene transcription was analyzed in roots, bulbs (1, 2, 3, 4, 5, 6, and 7 correspond to 192-, 197-, 202-, 207-, 212-, 217-, 222-, and 227-day-old bulbs), pseudo (ps) stems, leaves, buds, flowers, and sprouts. Colors red to green indicate a gene expression gradient from low to high.

Figure 4

Symptoms of Fusarium rot disease in infection-resistant (Sarmat) and -susceptible (Strelets) garlic cultivars. Cloves were soaked in the suspension of F. proliferatum (Fp) conidia for 5 min and analyzed after 24 and 96 h; control cloves were treated with distilled water.

Figure 5

Expression of the AsCHI genes in roots, stems, and cloves of Allium sativum cv. Sarmat and Strelets after 24 and 96 h of Fusarium proliferatum infection. The data were normalized to GAPDH and UBQ mRNA levels and presented as the mean ± SE (n = 3) compared to control taken as 1. * p < 0.05 compared to uninfected control.

Figure 6

Expression of the AsCHI genes in roots, stems, and cloves of Allium sativum cv. Sarmat and Strelets: in control (C) and treated (T; after 24 and 96 h of Fusarium proliferatum infection) plants. The data were normalized to mRNA levels of GAPDH and UBQ genes and presented as the mean ± SE (n = 3).