Natural variation in the VELVET gene bcvel1 affects virulence and light-dependent differentiation in Botrytis cinerea

Abstract

Botrytis cinerea is an aggressive plant pathogen causing gray mold disease on various plant species. In this study, we identified the genetic origin for significantly differing phenotypes of the two sequenced B. cinerea isolates, B05.10 and T4, with regard to light-dependent differentiation, oxalic acid (OA) formation and virulence. By conducting a map-based cloning approach we identified a single nucleotide polymorphism (SNP) in an open reading frame encoding a VELVET gene (bcvel1). The SNP in isolate T4 results in a truncated protein that is predominantly found in the cytosol in contrast to the full-length protein of isolate B05.10 that accumulates in the nuclei. Deletion of the full-length gene in B05.10 resulted in the T4 phenotype, namely light-independent conidiation, loss of sclerotial development and oxalic acid production, and reduced virulence on several host plants. These findings indicate that the identified SNP represents a loss-of-function mutation of bcvel1. In accordance, the expression of the B05.10 copy in T4 rescued the wild-type/B05.10 phenotype. BcVEL1 is crucial for full virulence as deletion mutants are significantly hampered in killing and decomposing plant tissues. However, the production of the two best known secondary metabolites, the phytotoxins botcinic acid and botrydial, are not affected by the deletion of bcvel1 indicating that other factors are responsible for reduced virulence. Genome-wide expression analyses of B05.10- and Δbcvel1-infected plant material revealed a number of genes differentially expressed in the mutant: while several protease- encoding genes are under-expressed in Δbcvel1 compared to the wild type, the group of over-expressed genes is enriched for genes encoding sugar, amino acid and ammonium transporters and glycoside hydrolases reflecting the response of Δbcvel1 mutants to nutrient starvation conditions.

Figure 1. Identification of the VELVET gene bcvel1 as candidate for the sclerotia gene locus.

(A) Genetic and physical maps of the locus linked to sclerotia formation (Bcscl). The genetic map (on the left; units in centiMorgans, cM) was obtained by analyzing the progeny from a cross between strains T4 and 32. The ability to form sclerotia in 68 ascospore isolates was tested by three independent experiments. The segregation data indicated that the Bcscl marker is genetically linked with the microsatellite markers (Bc335, Bc342, Bc389, Bc246) of the genetic group 15. The design of additional markers (Bc427, Bc413, Bc433, Bc420 and Bc423) allowed locating Bcscl between markers Bc246 and Bc427 (for details see Materials and Methods). This 19.5 cM region corresponds to 115 kb of the supercontig 2.1 of the T4 genome sequence that comprises bcvel1 (shaded in black on the right; see also Figure S1). (B) Comparison of bcvel1 sequences derived from wild-type isolates B05.10 and T4. A SNP (base pair 621 of the coding region) in T4 results in a stop codon. (C) Expression of bcvel1 in B05.10 and T4. Strains were grown for 3 days on solid complete medium covered with a cellophane overlay in light-dark (LD) conditions or in continuous darkness (DD). Infected plant material (48 hpi on primary leaves of P. vulgaris) was used to detect the transcript levels in planta (IP). rRNA is shown as loading control. (D) Subcellular localization of GFP-tagged BcVEL1 proteins derived from B05.10 and T4 sequences in a B05.10 genomic background. Conidia of the strains were incubated in Gamborgs B5+2% glucose on microscope slides in light-dark (LD) conditions for 18 h (12 h darkness+6 h light). Nuclei were visualized using the fluorescent dye Hoechst 33342. Scale bars represent 5 µm.

Figure 2. The four VELVET proteins of B. cinerea.

(A) Phylogenetic tree of VELVET proteins of B. cinerea, A. nidulans and F. fujikuroi. Sequence alignment and tree construction were performed using the bioinformatics program at Phylogeny.fr (http://www.phylogeny.fr/). Analysed sequences are: B. cinerea BcVEL1^B05.10 (HE977589), BcVEL2^B05.10 (HE977591), BcVEL3^B05.10 (HE977592), BcVEL4^B05.10 (HE977594), A. nidulans VeA (AAD42946), VelB (ABQ17967), VelC (ABQ17968), VosA (ABI51618), F. fujikuroi VEL1 (CBE54373), VEL2 (CBK25977), and VEL3 (L. Studt and B. Tudzynski, unpublished data). The sequence alignment of BcVEL1 with other homologous protein sequences is shown in Fig. S2A. (B) Expression of the four VELVET-like genes during different stages of the life cycle of B. cinerea. Wild-type strain B05.10 was incubated on complete medium covered with cellophane overlays for the indicated time periods in continuous light (LL) and light-dark (LD) for induction of conidiation and in continuous darkness (DD) for induction of sclerotia formation (non-pigmented sclerotia initials were present after 6 d of incubation). Infected plant material (primary leaves of P. vulgaris, harvested 2 dpi and 4 dpi) was used to detect the transcripts in planta (IP). rRNA is shown as loading control. (C) Domain architecture of the four identified VELVET-like proteins of B. cinerea. Putative nuclear localization signals (NLS) are shown as green bars, leucine-rich nuclear export signals (NES) as blue bars, and potential PEST domains (proline (P), glutamate (E), serine (S) and threonine (T)-rich) are indicated as orange bars.

Figure 3. Effects of the different bcvel1 mutations on light-dependent differentiation and oxalic acid (OA) formation.

(A) Conidiation and sclerotia formation. The strains were incubated on solid complete medium (CM) at 20°C in light-dark (LD) conditions for conidiation and in continuous darkness (DD) for sclerotia formation. The few sclerotia formed by the mutant T4+bcvel1 are indicated by white arrows. (B) OA secretion. Acidification of the culture medium was monitored on solid CM pH 7.5 supplemented with 0.1% bromothymolblue (BTB, pH indicator). The color change from green to yellow indicates acidification (pH<6.0). The expression of bcoahA encoding the OA-forming enzyme oxaloacetate hydrolase was detected by northern blot analysis. For that, strains were grown for three days on solid CM with pH 5.0 or pH 6.5 covered with cellophane overlays. rRNA is shown as loading control.

Figure 4. Impact of BcVEL1 on asexual and sexual reproduction and conidial germination.

(A) Numbers of conidia (per Petri dish) produced by the strains in light-dark (LD) conditions and in continuous darkness (DD), respectively. Data represent mean values of the results from three Petri dishes per strain and condition. (B) Numbers of sclerotia (per Petri dish) produced by the strains in continuous darkness. Data represent mean values of the results from three Petri dishes per strain and condition. (C) Apothecia derived from crossing of sclerotia of strain SAS405 (MAT1-2) with microconidia of mutant B05.10:Δbcvel1 (MAT1-1). Picture was taken after 14 weeks of joining sclerotia and microconidia. (D) Germination on hydrophobic surfaces. Conidia were suspended in water and incubated for 24 h on polypropylene. The experiments were done in triplicates, each time hundred conidia were counted. Short germ tubes do not exceed the length of the conidia while long germ tubes do. Scale bar represents 20 µm.

Figure 5. Virulence of strains carrying the different bcvel1 mutations.

(A) Penetration of plant cells by germ tubes on onion epidermal strips (1 dpi). Conidia and hyphae on the surface were stained with lactophenol blue. Sites of penetration are indicated by black arrows. Scale bars represent 20 µm. (B) Complete infection cycle on primary leaves of P. vulgaris (2 to 7 dpi). Plants were incubated in humid conditions under natural illumination at room temperature. In this experiment, lesions of bcvel1 loss-of-function mutants were able to spread (SL, spreading lesions). (C) A further virulence assay on primary leaves of P. vulgaris (only 7 dpi). In this experiment, infection by bcvel1 loss-of-function mutants stopped in the primary stage (NSL, non-spreading lesions), while infection by the wild type B05.10 and complemented mutants was not affected.

Figure 6. Impact of BcVEL1 on invasive growth.

(A) Subcellular localization of BcVEL1-GFP (in the B05.10 genomic background) during infection of onion epidermal layers (24 hpi). The localization is shown in hyphae that are growing on the top of the cells (left panel) or growing invasively (middle and right panel); arrows indicate sites of penetration. (B) Expression of known virulence factors in B05.10 and Δbcvel1 in planta. Primary leaves of P. vulgaris were inoculated with conidial suspensions and harvested 48 hpi (primary lesions, prior to lesion spreading). Northern blots were hybridized with probes of bcoahA (oxalic acid biosynthesis), bcbot1 (botrydial biosynthesis), bcboa3 (botcinic acid biosynthesis), bcspl1 (cerato-platanin family protein) and bcxyn11A (endo-beta-1,4-xylanase) coding sequences. rRNA is shown as loading control. (C) Close-up views of lesions caused by B05.10 and Δbcvel1 on P. vulgaris. Outcome of the Δbcvel1-plant interaction is variable: infection stops in primary stage (NSL, non-spreading lesion) or proceeds slowly (SL, spreading lesion). (D) Trypan blue staining of spreading lesions (52 hpi). Fungal hyphae and dead plants cells are stained blue. The right panels show higher magnification of the edges of the lesions. Only few dead plant cells were observed around Δbcvel1 lesions, while massive staining of plant cells in the range of the wild-type lesions indicates the complete maceration of the tissue.

Figure 7. Temporal H2O2 accumulation in spreading (SL) and non-spreading (NSL) lesions.

WT:B05.10- and Δbcvel1-infected P. vulgaris leaves were stained with DAB (3,3′-diaminobenzidine) that is oxidized by hydrogen peroxide in the presence of peroxidases to give a dark-brown color. Non-spreading lesions of Δbcvel1 are surrounded by distinct brown rings (lower panels, pictures correspond to non-stained lesions in Fig. 6C). Scale bars represent 2 mm.

Figure 8. Disease symptoms on other plant tissues.

All plant tissues were inoculated with conidial suspensions of strains WT:B05.10 and Δbcvel1 (two independent mutants, Δ5 and Δ8). Surfaces of apple, cucumber and grape berries were wounded with a needle prior to inoculation. Pictures of infected apple fruit were taken 5 and 8 dpi, of infected grape berries 3, 6 and 9 dpi, cucumber 7 dpi, and of the infected primrose flower 2 and 4 dpi. Leaves of young kohlrabi and lettuce plants were detached prior to inoculation while leaves of Arabidopsis thaliana Col-0 plants were separated 3 dpi. M, mock-treated.

Figure 9. Analysis of the BcVEL1-dependent expression profile during infection.

Primary leaves of living P. vulgaris plants were inoculated with many droplets of conidial suspensions of the wild-type strain B05.10 and the Δbcvel1 deletion mutant, respectively. Infected plant material (from four biological replicates) was sampled at 48 hpi before infections started to spread. RNA was extracted, labeled and hybridized to B. cinerea microarrays. The comparative analysis of the array data revealed 227 genes that were under- and 419 genes that were over-expressed in the Δbcvel1 mutant (for details see Tables S1, S2; Fig. S9). Expression profile of some arbitrarily chosen genes was confirmed by northern blot analyses.