# Biosynthesis of Rubbellins in _Ramularia collo-cysni_--Genetic Basis and Pathway Proposition
## 3 Discussion
### Comparison of Rubellin and Chrysophanol Toxicity  
The phytotoxicity of chrysophanol **7**, an intermediate in the biosynthesis of rubellins, was compared with that of rubellin D **4** (Figure 5). Chrysophanol **7** exhibited significantly lower phytotoxic activity than rubellin D **4** when infiltrated in leaves of _A. thaliana_. In the proposed pathway, rubellins, including rubellin D **4**, resulted primarily from the dimerisation of chrysophanol **7** (Figure 6). Dimerisation and the atomic rearrangement associated with it can profoundly alter the biological activity of a molecule. For instance, jesterone, an anti-oomycete epoxyquinone SM produced by the endophytic fungus _Pestalotiopsis jestereri_[51], inhibits the growth and development of cancer cells but a synthetic dimer of jesterone exhibits up to 100 times greater activity against the same cancer lines [52]. Similar findings were previously reported for SM structurally related to rubellins as the activity of nataloe-emodin was compared with that of cladofulvin. Cladofulvin, a dimer of nataloe-emodin, exhibited up to 320 times higher cytotoxic activity than nataloe-emodin when tested against different animal cell lines [23]. The cytotoxic and antibiotic activities of chrysophanol **7** were previously compared with that of rubellins and ureadinorbellins, another chrysophanol dimer intermediate in the biosynthesis of rubellins. At a concentration of 10 uM, chrysophanol **7** and rubellin D **4** exhibited a similar level of cytotoxicity against the HT29 and J774A.1 lines, but interestingly, only rubellin D **4** showed cytotoxicity against the HIG-82 line [44]. Rubellin D **4** and ureadinorbellins also exhibited antibiotic activity against several strains of _Streptococcus aureus_, including methicillin-resistant _S. aureus_ (MRSA), whereas chrysophanol **7** did not appear to affect bacterial growth [44]. Taken together, these results suggest that the dimerisation step in the biosynthesis of rubellinsalters the biological activity of the SM and may be critical to considerably increase phytotoxic activity. The role of rubellins in RLS disease development being currently unknown, the _rccM_ gene, which is a candidate for the dimerisation step, would be a good target for gene silencing. Deletion of _rccM_ would result in the accumulation of the easily identified compound chrysophanol 7, which can be used as a control to confirm successful rubellins biosynthesis disruption. Furthermore, _rccM_ deletion would also result in decreased phytotoxicity, therefore, allowing for a better understanding of the role played by rubellins in the host-pathogen interactions.  
Figure 6: Proposed biosynthetic pathway of rubellins A–F (**1–6**) and 14-dehydro rubellin D **9**. RccG—nonreducing, iterative type I polyketide synthase (PKS). PKS domains: ACP—acyl carrier protein; PT—product template; AT—acyl transferase; KS—keto synthase; SAT—starter unit acyl carrier protein transacylas; RccF—\(\beta\)-lactamase; RccH—decarboxylase; Rec—short-chain reductase; RecM—cytochrome P450; RccD—FAD-dependent monooxygenase/hydroxylase; RecL—Baeyer-Villiger monooxidase; RecB—dehydratase.