Insights into the mechanism of Huanglongbing tolerance in the Australian finger lime (Citrus australasica)

Abstract

The Australian finger lime (Citrus australasica) is tolerant to Huanglongbing (HLB; Citrus greening). This species can be utilized to develop HLB tolerant citrus cultivars through conventional breeding and biotechnological approaches. In this report, we conducted a comprehensive analysis of transcriptomic data following a non-choice infection assay to understand the CaLas tolerance mechanisms in the finger lime. After filtering 3,768 differentially expressed genes (DEGs), 2,396 were downregulated and 1,372 were upregulated in CaLas-infected finger lime compared to CaLas-infected HLB-susceptible 'Valencia' sweet orange. Comparative analyses revealed several DEGs belonging to cell wall, β-glucanase, proteolysis, R genes, signaling, redox state, peroxidases, glutathione-S-transferase, secondary metabolites, and pathogenesis-related (PR) proteins categories. Our results indicate that the finger lime has evolved specific redox control systems to mitigate the reactive oxygen species and modulate the plant defense response. We also identified candidate genes responsible for the production of Cys-rich secretory proteins and Pathogenesis-related 1 (PR1-like) proteins that are highly upregulated in infected finger lime relative to noninfected and infected 'Valencia' sweet orange. Additionally, the anatomical analysis of phloem and stem tissues in finger lime and 'Valencia' suggested better regeneration of phloem tissues in finger lime in response to HLB infection. Analysis of callose formation following infection revealed a significant difference in the production of callose plugs between the stem phloem of CaLas+ 'Valencia' sweet orange and finger lime. Understanding the mechanism of resistance will help the scientific community design strategies to protect trees from CaLas infection and assist citrus breeders in developing durable HLB tolerant citrus varieties.

Keywords: Huanglongbing; callose deposition; citrus; host response; pathogen-related proteins; transcriptome.

Figure 1

(A) HLB infected ‘Valencia’ sweet orange in the field exhibiting the characteristic blotchy mottle pattern in the leaves. (B) Finger lime leaves from trees growing in the field with no visible disease symptom. (C) Detection of CaLas in leaf tissues of Finger lime and ‘Valencia’ trees by qPCR. Leaf samples were collected from 8-year-old field trees at the beginning of the study. (D) CaLas detection from leaf samples collected periodically from trees, side grafted with HLB infected budwood and growing in the green house. * represents the sampling time for RNAseq analysis. Different letters above the error bar indicate statistically significant differences, while the same letters signify no significant differences using the Tukey–Kramer honestly significant difference test (Tukey HSD; p <0.05).

Figure 2

(A) Volcano plot of the upregulated and downregulated DEGs. Genes with an adjusted p value of less than 0.05 found with DESeq were assigned as differentially expressed. (B) Graphical view of the most statistically significant upregulated and downregulated enriched GO terms in Finger line trees as compared to ‘Valencia’ trees. Statistically significant DEGs were analyzed using AgriGO v2 and REVIGO. (C) Differentially expressed genes as identified following MAPMAN analysis. Regulation of stress-related gene pathways by CaLas infection in the infected Finger Lime (Left). Overview of the differentially expressed genes related to the metabolic pathways in Finger lime and ‘Valencia’ sweet orange (Right). Genes that were significantly upregulated following CaLas infection are displayed in blue, and downregulated genes are displayed in red.

Figure 3

Relative transcript levels of cysteine-rich RLK (RECEPTOR-like protein kinase) as calculated by real-time PCR compared with the CaLas free ‘Valencia’. The CaLas infected samples were collected from five year old trees growing in the field and the CaLas free (control) samples were collected from trees growing in a protected greenhouse. (A–I) Relative RLK expression as detected in this study compared with CaLas free 'Valencia'. The control trees were confirmed negative for CaLas before further comparison. Data are means ± SE of twelve samples. Different letters above the error bar indicate statistically significant differences, while the same letters signify no significant differences using the Tukey–Kramer honestly significant difference test (Tukey HSD; p <0.05).

Figure 4

Relative transcript levels of CAP (Cysteine-rich secretory proteins; Antigen 5; and Pathogenesis-related 1 protein) superfamily protein is calculated by real-time PCR compared with the CaLas free ‘Valencia’. (A) CAP1, (B) CAP2, (C) CAP3 and (D) LCR69. The CaLas infected samples were collected from five years old samples growing in the field and the CaLas free (control) samples were collected from a protected greenhouse. The control samples were confirmed negative for CaLas before further comparison. Data are means ± SE of twelve samples. Different letters above the error bar indicate statistically significant differences, while the same letters signify no significant differences using the Tukey-Kramer honestly significant difference test (Tukey HSD; p <0.05).

Figure 5

(A) Inhibition capacity of Finger lime and ‘Valencia’ T1- FL (HLB+) + substrate, T2- FL (HLB+) + substrate, T3- VAL (HLB+) + substrate + E64 and T4- Val (HLB+) + substrate + E64. The inhibition capacity was compared with the HLB negative leaves. Relative transcript levels of Cysteine proteinases superfamily protein transcription factors were calculated by real-time PCR and compared with the CaLas free ‘Valencia’. The CaLas infected trees were collected from five year old trees growing in the field and the CaLas free (control) samples here instead of trees were collected from trees kept in a protected greenhouse. The control trees were confirmed negative for CaLas before further comparison. Data are means ±SE of twelve samples. (B–D) Relative gene expression of selected cysteine proteases genes in the infected finger lime compared with infected 'Valencia'. Different letters above the error bar indicate statistically significant differences, while the same letters signify no significant differences using the Tukey-Kramer honestly significant difference test (Tukey HSD; p <0.05).

Figure 6

Verification of expression levels of selected upregulated (A) or downregulated (B) DEGs in the infected Finger lime (FL-HLB) compared to infected ‘Valencia’ (Val-HLB) as determined by qPCR (2-ΔΔCt). Different letters (a, b) represent a significant difference at p ≤ 0.05 using Tukey–Kramer honestly significant difference (HSD) and error bars represent SE (n = 3). Pearson Correlation Coefficient (r)> greater than 0.5 is considered positive and strong.

Figure 7

Morphological differences between healthy and HLB-infected Finger lime and ‘Valencia’. Brightfield images of petiole and stems for each healthy and HLB-infected cultivar, Finger lime (A), ‘Valencia’ (B). Phloem Ratio (C) and Xylem Ratio (D) in Finger lime and Phloem Ratio (E) and Xylem Ratio (F) in ‘Valencia’. Bars represent standard error. NS, not significant.

Figure 8

Non-zero counts of callose plugs per 10x field image of the stem phloem of CaLas+ Finger lime (A) and ‘Valencia’ sweet orange (B) sampled after 2 years following infection. The mean callose formation count per 10x field image was significant. (C) Relative transcript levels of phloem proteins are calculated by real-time PCR and compared with the CaLas free ‘Valencia’. The CaLas infected samples were collected from five year old trees growing in the field and the CaLas free (control) samples were collected from trees kept in a protected greenhouse. The control trees were confirmed negative for CaLas before further comparison. Data are means ± SE of twelve samples (D–F). Different letters above the error bar indicate statistically significant differences, while the same letters signify no significant differences using the Tukey-Kramer honestly significant difference test (Tukey HSD; p <0.05).

Figure 9

Schematic diagram elucidating the potential mechanism of CaLas tolerance in the Finger lime, and susceptibility in ‘Valencia’ sweet orange. The figure was created in BioRender.com.