The homeobox BcHOX8 gene in Botrytis cinerea regulates vegetative growth and morphology

Abstract

Filamentous growth and the capacity at producing conidia are two critical aspects of most fungal life cycles, including that of many plant or animal pathogens. Here, we report on the identification of a homeobox transcription factor encoding gene that plays a role in these two particular aspects of the development of the phytopathogenic fungus Botrytis cinerea. Deletion of the BcHOX8 gene in both the B. cinerea B05-10 and T4 strains causes similar phenotypes, among which a curved, arabesque-like, hyphal growth on hydrophobic surfaces; the mutants were hence named Arabesque. Expression of the BcHOX8 gene is higher in conidia and infection cushions than in developing appressorium or mycelium. In the Arabesque mutants, colony growth rate is reduced and abnormal infection cushions are produced. Asexual reproduction is also affected with abnormal conidiophore being formed, strongly reduced conidia production and dramatic changes in conidial morphology. Finally, the mutation affects the fungus ability to efficiently colonize different host plants. Analysis of the B. cinerea genome shows that BcHOX8 is one member of a nine putative homeobox genes family. Available gene expression data suggest that these genes are functional and sequence comparisons indicate that two of them would be specific to B. cinerea and its close relative Sclerotinia sclerotiorum.

Figure 1. Deletion of the BcHOX8 gene.

(A) Schematic representation of the BcHOX8 gene replacement by the nourseothricin (NTC) resistance gene flanked by 1.5 kb of downstream and 0.95 kb of upstream sequences from the BcHOX8 locus. Distances between HindIII restriction sites are shown with dotted double arrows. The probe used for Southern analysis and the primers used for PCR analysis are shown as a black bar and arrows, respectively. (B) PCR analysis of the parental (T4/Δku80 (P80) and B05-10/Δku70 (P70)) and transformed strains (T4-T_80-1 (T_80-1) and B05-T_70-1 (T_70-1)) using three primers (shown in A) combinations: hd1 and hd2 (top), pr1 and pr2 (middle) or pr3 and pr4 (bottom). (C) Southern blotting analysis of the parental T4/Δku80 strain (P) and one transformant of each genetic background (T4-T_80-1 and B05-T_70-1) using HindIII to digest genomic DNA and the DNA probe indicated in (A) to reveal the fragments.

Figure 2. Impact of BcHOX8 deletion on the B. cinerea hyphal growth.

(A) Colonies growth kinetics on TNK, PDA and Malt medium for the two parental strains (B05-10/Δku70 (empty circle) and T4/Δku80 (empty square)) and the two respective mutant strains (B05-T_70-1 (filled circle) and T4-T_80-1 (filled square)); 10 cm dishes were used. (B and C) Microscopic observations of the colonies edges (B) and the conidium-derived hyphal growth on hydrophobic surfaces in the parental and mutant strains. A 50 µm scale bar is shown in all pictures.

Figure 3. Impact of the BcHOX8 mutation on B. cinerea development.

Microscopic observations of (A) various shapes of the Arabesque mutant conidia ranging from single (parent-like) to bi- (b: 24±6%), tri- (c: 8±3%) and multi-lobed (d–f: 20±5%) conidia, (B) conidiophores of the parental B05-10/Δku70 (left) and mutant B05-T_70-1 (right) strains and (C) infection cushions in the parent B05-10/Δku70(left) and mutant B05-T_70-1 (right) strains using 100x (C. top) and 400x (C. bottom) magnification. 10 µm, 25 µm and 50 µm scale bars are shown in panel A, B an C, respectively.

Figure 4. Impact of the BcHOX8 mutation on mycelium-derived infection.

Kinetic of green bean (top) and thale cress (bottom) leaves infection by the parental B05-10/Δku70 (circle) and the Arabesque mutant B05-T_70-1 (square) strains using 1.8 mm mycelium plugs as inoculum. In the case of green bean infection, 6 independent experiments using 6 leaves each (days 1 and 2) or 30 leaves each (days >2) were performed. In the case of thale cress infection, 3 independent experiments using 6 leaves each were performed. Monitoring of thale cress infection stopped when the necrosis zone of the parent control reached the leaves edges. Linearization of the exponential curves allowed calculation of the relative mutant growth when compared to its parent (% of the slope obtained for the parental strain). 4-days infection pictures are shown.

Figure 5. Impact of the BcHOX8 mutation on conidia-derived infection.

Kinetic of green bean leaves infection by the parental B05-10/Δku70 (circle) and the Arabesque mutant B05-T_70-1 (square) strains using conidia droplets as inoculum. Three independent experiments using 6 leaves each were performed. Linearization of the exponential curves allowed calculation of the relative mutant growth when compared to its parent (% of the slope obtained for the parental strain). A 7-days infection picture is shown. (B) Grape berries infection by the parental and Arabesque mutant strains using conidia as inoculum; a comparative 7-days infection picture is shown.

Figure 6. Expression of the BcHOX8 gene in the B05-10/Δku70 parental strain.

(A) Kinetic of B. cinerea development on Teflon membrane using conidia as starting material; germinated conidia, appressoria (triangle) and young mycelium are shown. (B) BcHOX8 expression on Teflon. RNA were collected at the four stages shown in A and used for quantitative RT-PCR analysis (3 biological repeats). (C) BcHOX8 expression in infection cushions-enriched samples. RNA were extracted from mycelium grown in liquid cultures or onto floating cellophane (enriched in infection cushions) and used for quantitative RT-PCR analysis (4 biological repeats). Standard deviations are indicated and the actin-encoding gene was used as reference. When the EF1α-encoding gene was used as reference, identical results were obtained in B and a 2-fold higher differential was observed in C.