The mannose-binding lectin gene FaMBL1 is involved in the resistance of unripe strawberry fruits to Colletotrichum acutatum

Abstract

The fungal pathogen Colletotrichum acutatum is the causal agent of strawberry (Fragaria × ananassa) anthracnose. Although the fungus can infect strawberry fruits at both unripe and ripe stages, the symptoms appear only on red ripe fruits. On white unripe fruits, the pathogen becomes quiescent as melanized appressoria after 24 h of interaction. Previous transcriptome analysis has indicated that a mannose-binding lectin (MBL) gene is the most up-regulated gene in 24-h-infected white strawberries, suggesting a role for this gene in the low susceptibility of unripe stages. A time course analysis of the expression of this MBL gene, named FaMBL1 (Fragaria × ananassa MBL 1a), was undertaken to monitor its expression profile in white and red fruits at early interaction times: FaMBL1 was expressed exclusively in white fruit after 24 h, when the pathogen was quiescent. Agrobacterium-mediated transient transformation was used to silence and overexpress the FaMBL1 gene in 24-h-infected white and red strawberries, respectively. FaMBL1-silenced unripe fruits showed an increase in susceptibility to C. acutatum. These 24-h-infected tissues contained subcuticular hyphae, indicating pathogen penetration and active growth. In contrast, overexpression of FaMBL1 in ripe fruits decreased susceptibility; here, 24-h-infected tissues showed a high percentage of ungerminated appressoria, suggesting that the growth of the pathogen had slowed. These data suggest that FaMBL1 plays a crucial role in the resistance of unripe strawberry fruits to C. acutatum.

Keywords: Colletotrichum acutatum; fungal quiescence; mannose binding lectin; strawberry ripening; unripe fruit resistance.

Figure 1

FaMBL1 primary structure. Amino acid sequence of FaMBL1 and blastx domain prediction analysis. The signal peptide, B‐lectin domain and PAN‐Apple domain are highlighted.

Figure 2

Time course of gene expression analysis of FaMBL1 in white and red strawberry fruits on Colletotrichum acutatum inoculation. The expression level of FaMBL1 was quantified in white and red fruits inoculated with C. acutatum (WI and RI, respectively) or mock inoculated (WH and RH), and scored at 0, 8, 16, 20 and 24 h post‐inoculation (hpi). Data were normalized to the transcript level of the housekeeping elongation factor 1α gene. Values are the means of three biological replicates.

Figure 3

Timing of T‐DNA integration in strawberry fruits. (A) Quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis of FaMBL1 expression in silenced white strawberry fruits infiltrated with Agrobacterium carrying the construct pk7:FaMBL1 (pk7:FaMBL1) or the empty vector control (pk7:00). FaMBL1 expression was scored from 1 to 3 days post‐agroinjection (DPAI). Data were normalized to the expression level of the housekeeping elongation factor 1α gene. Values are the means of three biological replicates. (B) Fluorescence microscope analysis of strawberry tissues at 1–3 DPAI carrying the plasmid pK7GWIWG2 II‐RedRoot. Transfected strawberries were observed both proximal (left) and distal (right) to the Agrobacterium infiltration point.

Figure 4

Infection of Colletotrichum acutatum in FaMBL1‐silenced white strawberry fruits. (A) Quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis of FaMBL1 expression in white wild‐type or transfected strawberries 24 h after inoculation with C. acutatum. The transcript levels of mock‐inoculated wild‐type fruits (not infected) and 24‐h C. acutatum‐inoculated fruits (wild‐type) were compared with those of mock‐silenced fruits (pK7:00) and FaMBL1‐silenced fruits (pK7:FaMBL1), both 24 h after inoculation with C. acutatum. Data were normalized to the expression level of the housekeeping elongation factor 1α gene. Values are the means of three biological replicates. (B) Disease symptom analysis in mock‐infected white fruits (not infected) (a), wild‐type white fruits (b), mock‐silenced white fruits (pk7:00) (c) and FaMBL1‐silenced white fruits (pK7:FaMBL1) (d), all (b–d) infected for 5 days with C. acutatum.

Figure 5

Histological analysis of 24‐h Colletotrichum acutatum‐infected FaMBL1‐silenced white strawberry fruits. Optical microscopy of wild‐type white fruits (A), mock‐silenced white fruits (pk7:00) (B) and silenced white fruits (pk7:FaMBL1) (C), all inoculated for 24 h with C. acutatum. Tissue slices were stained with haematoxylin and eosin. Melanized appressoria (MA) and intercellular hyphae (PH) are indicated. Bar, 10 μm.

Figure 6

Infection of Colletotrichum acutatum in FaMBL1‐overexpressing red strawberry fruits. (A) Quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis of FaMBL1 expression in red wild‐type or transfected strawberries 24 h after inoculation with C. acutatum. The transcript levels of wild‐type mock‐inoculated fruits (not infected) and 24‐h C. acutatum‐inoculated fruits (wild‐type) were compared with those of mock‐overexpressing fruits (35S:00) and FaMBL1‐overexpressing fruits (35S:FaMBL1), both at 24 hpi with C. acutatum. Data were normalized to the expression level of the housekeeping elongation factor 1α gene. Values are the means of three biological replicates. (B) Disease symptom analysis in red wild‐type mock‐infected fruits (a), red wild‐type fruits (wild‐type) (b), mock‐overexpressing red fruits (35S:00) (c) and FaMBL1‐overexpressing red fruits (35S:FaMBL1) (d), all (b–d) infected for 5 days with C. acutatum.

Figure 7

Histological analysis of 24‐h Colletotrichum acutatum‐infected FaMBL1‐overexpressing red strawberry fruits. Optical microscopy of wild‐type red fruits (A), mock‐overexpressing red fruits (35S:00) (B) and overexpressing red fruits (35S:FaMBL1) (C), all inoculated for 24 h with C. acutatum. Tissue slices were stained with haematoxylin and eosin. Melanized appressoria (MA) and intercellular hyphae (PH) are indicated. Bar, 10 μm.