# Biosynthesis of Rubbellins in _Ramularia collo-cysni_--Genetic Basis and Pathway Proposition
## 3 Discussion
### Identification of the Candidate Rubellin Biosynthetic Candidate Cluster  
Through the combined in silico genome walking and BLAST analyses carried out in the present study, a more comprehensive candidate gene cluster for rubellin biosynthesis in _R. collo-cygni_ was identified. The putative rubellin biosynthetic cluster appears to involve the main cluster organised around the core PKS gene, _rccG_, and two miniclusters located elsewhere in the _R. collo-cygni_ genome and organised around the _rccL_ and _rccD_ genes. This organisation is reminiscent of that of other biosynthetic gene clusters responsible for the biosynthesis of SMs structurally related to rubellins. Dothstromin, an anthraquinone-derived SM produced by _Dothstroma setpostporum_, a pathogen of pine trees, is synthesised by a cluster comprising six small clusters spread on chromosome 12 and a single gene located on chromosome 11 [45]. Similarly, the organisation of the cladofulvin biosynthetic cluster resembles that of the candidate identified in this study, as it consists of the main cluster comprising most of the gene required for cladofulvin biosynthesis located around the core PKS gene _claG_ supplemented by two miniclusters, each containing a single gene [23]. Furthermore, all the genes present in the cladofulvin biosynthetic cluster have a homologue in the candidate cluster in _R. collo-cygni_, be it in the main cluster or in the _rccL_ minicluster (Figure 3). The _rccL_ minicluster consists of two genes, including the _rccL_ BVMO. BVMOs are enzymes that catalyse the formation of an ester from a ketone through the integration of an oxygen atom adjacent to the carbonyl group. Although BVMO activity is relatively common in fungi [46], only a limited number of BVMOs has been characterised in fungal genomes. _R. collo-cygni rccL_ exhibits homology with several BVMOs that have been identified in gene clusters responsible for the biosynthesis of SMs structurally related to rubellins. [PERSON] et al. [42] reported that the BVMO _mdpL_ was involved in the biosynthesis of monodictyphenone. Similarly, _rccL_ also exhibit homology with _claL_ and _agnL3_, the BVMOs involved in the biosynthesis of cladofulvin and agnestins in C. _fulvum_ and _P. divaricatus_, respectively [23; 25]. The _rccD_ minicluster, which does not comprise homologues to the cladofulvin biosynthetic genes, likely consists of a single monooxygenase (_rccD_) as the organisation of the condensation, adenylation and peptidyl carrier protein domains of the putative NRPS identified in this minicluster suggests this protein is nonfunctional [26]. Furthermore, this NRPS and the MFS transporter located near _rccD_ showed no expression based on the previously published RNA-seq study [43]. The full complement of genes involved in the candidate gene cluster was further defined via a gene expression approach. Ten genes located on the main _rccG_ cluster, both genes on the _rccL_ minicluster as well as the _rccD_ gene on the _rccD_ minicluster, appear to be coregulated (Figure 4). The transcript levels of these genes were highest during the asymptomatic and early symptoms formation stages of infection (5 and 7 dpi). [PERSON] et al. [26] reported similar findings for several PKSs, NRPSs, and hybrids PKS-NRPS in _R. collo-cygni_, suggesting that several SMs may be synthesised and involved in the early stages of disease development. These results contrast with those observed for the dothstromin biosynthetic cluster in _D. setpostporum_ as all the genes present in that cluster exhibit their highest expression level during the mid and late stages of disease development, i.e., during lesions formation and sporulation [47]. Similarly, the genes responsible for cladofulvin biosynthesis are downregulated during the biotrophic growth of _C. fulvum_ in planta [48]. Dothstromin is a known virulence factor that mediates disease severity, whereas _C. fulvum_ lines overproducing cladofulvin showed larger lesions compared with the wild type but reduced sporulation.