The Botrytis cinerea phytotoxin botcinic acid requires two polyketide synthases for production and has a redundant role in virulence with botrydial

Abstract

The grey mould fungus Botrytis cinerea produces two major phytotoxins, the sesquiterpene botrydial, for which the biosynthesis gene cluster has been characterized previously, and the polyketide botcinic acid. We have identified two polyketide synthase (PKS) encoding genes, BcPKS6 and BcPKS9, that are up-regulated during tomato leaf infection. Gene inactivation and analysis of the secondary metabolite spectra of several independent mutants demonstrated that both BcPKS6 and BcPKS9 are key enzymes for botcinic acid biosynthesis. We showed that BcPKS6 and BcPKS9 genes, renamed BcBOA6 and BcBO9 (for B. cinerea botcinic acid biosynthesis), are located at different genomic loci, each being adjacent to other putative botcinic acid biosynthetic genes, named BcBOA1 to BcBOA17. Putative orthologues of BcBOA genes are present in the closely related fungus Sclerotinia sclerotiorum, but the cluster organization is not conserved between the two species. As for the botrydial biosynthesis genes, the expression of BcBOA genes is co-regulated by the Gα subunit BCG1 during both in vitro and in planta growth. The loss of botcinic acid production does not affect virulence on bean and tomato leaves. However, double mutants that do not produce botcinic acid or botrydial (bcpks6Δbcbot2Δ) exhibit markedly reduced virulence. Hence, a redundant role of botrydial and botcinic acid in the virulence of B. cinerea has been demonstrated.

Figure 1

Chemical structures of the compounds secreted by Botrytis cinerea. The quantities isolated from the wild‐type (WT) strain and the mutants are indicated in Table 1.

Figure 2

Expression of the polyketide synthase (PKS)‐encoding genes revealed by reverse‐Northern analysis. Polymerase chain reaction (PCR) fragments of BcPKS genes predicted by Kroken et al. (2003) and other Botrytis cinerea genes were spotted onto Nylon membranes. BcActA and EF1b are the actin‐ and elongation factor‐encoding genes used as constitutively expressed genes (Gioti et al., 2006). BcBOT genes are the botrydial biosynthesis genes described in Pinedo et al. (2008). All gene and primer information is given in Table S1. The B05.10 strain was cultivated on grape juice medium and inoculated on tomato leaves. Three days post‐inoculation, total RNA was extracted, labelled with ³²P and hybridized to the membranes.

Figure 3

Domain organization of the polyketide synthases BcBOA6 (BcPKS6) and BcBOA9 (BcPKS9), and BcBOA5 (OxR), an enzyme presumably acting in concert with the polyketide synthase BcBOA6. Protein enzymatic domains are as follows: AT, acyl transferase; DH, dehydratase; ER, enoyl reductase; KR, ketoreductase; KS, β‐ketoacyl synthase; MT, methyltransferase; PP, phosphopantetheine attachment site. Domains were predicted by Protein Families (PFAM) and National Center for Biotechnology Information (NCBI) domain search (see Table 2).

Figure 4

The putative botcinic acid biosynthetic genes (BcBOA) are separated into at least two clusters in the Botrytis cinerea wild‐type (WT) strain B05.10. Black arrows indicate genes that might be involved in botcinic acid biosynthesis in B. cinerea. Dark grey arrows in Sclerotinia sclerotiorum (strain 1980; Sclerotinia sclerotiorum Database, http://www.broadinstitute.org/annotation/genome/sclerotinia_sclerotiorum/MultiHome.html) indicate the genes that are the homologues of the B. cinerea BcBOA genes. Sclerotinia sclerotiorum genes that are probably not related to secondary metabolism and the botcinic acid cluster are indicated by light grey arrows. Grey boxes at the ends of the B. cinerea clusters indicate AT‐rich regions exhibiting AT contents from 70% to 90%. For more details on sequence assembly, see Experimental procedures; for predicted gene functions, see Table 2.

Figure 5

The expression of putative botcininc acid biosynthetic genes is co‐regulated and dependent on the presence of the α subunit BCG1 of a heterotrimeric G protein. (A) Expression of BcBOA genes in submerged culture. No hybridization signals for BcBOA14, BcBOA15 and BcBOA16 were detected (data not shown). (B) Expression of botrydial biosynthesis genes (BcBOT) in submerged culture. (C) Expression of several BcBOA and BcBOT genes in planta (48 h post‐inoculation). For cultivation conditions, see Experimental procedures. BcActA encoding actin and rRNA were used as loading controls.

Figure 6

Growth and virulence of botcinic acid‐deficient mutants, botrydial‐deficient mutant and mutants deficient for the production of both toxins. (A) Growth on minimal medium: growth diameters were measured at 4 days post‐inoculation (dpi). (B) Virulence on bean leaves: plugs of young mycelium were inoculated on young leaves and lesion diameters were measured at 4 dpi. (C) Photographs of infected bean leaves taken at 4 dpi. The numbers indicate the strains as follows: B05.10 wild‐type strain (1), pks6Δ‐1 (2), pks6Δ‐4 (3), pks6Δ‐5 (4), pks9Δ‐3 (5), pks9Δ‐14 (6), pks9Δ‐16 (7), bcbot2Δ (8), bcbot2Δ pks6Δ−1 (9), bcbot2Δ pks6Δ‐2 (10), bcbot2Δ pks6Δ‐9 (11), bcbot2Δ pks6Δ‐14 (12).