# Biosynthesis of Rubbellins in _Ramularia collo-cysni_--Genetic Basis and Pathway Proposition
## 1 Introduction  
Ramularia leaf spot (RLS) is an important disease of barley caused by the ascomycete fungus _Ramularia collo-cysni_. The disease was originally described in Europe in 1893 [1] but has become a concern in the past 20 years as it became more prevalent and widespread. The fungus is now present in many temperate regions of the world, including Australia, New Zealand [2], Argentina [3], South Africa [4], Russia, and North America [5, 6]. The disease is marked by the appearance of typical rectangular necrotic spots on leaves. In severe epidemy, symptoms can then develop on other aerial parts of the plant such as stem, awns, and grains [5]. The development of symptoms causes yield losses that can exceed 70% in extreme cases but are typically in the range of 10 to 20% [6]. Furthermore, the disease can cause quality losses as grain size, a trait used in the matlting industry to assess grain quality, can be reduced by up to 4% [6]. Controlling RLS outbreaks has become increasingly difficult because of the withdrawal in many countries of multisite chemicals for environmental reasons [7]. In addition, the fungus has now developed resistance to all major classes of single-site fungicides such as quinone outside inhibitors (QoI) [8], succinate dehydrogenase inhibitors (SDHI), and sterol demethylation inhibitors (DMI) [9, 10, 11, 12]. To date, no source of genetic resistance has been identified in barley; however, the genetic background of the host is known to play a role in disease development as varieties carrying the _mildew locus O (mlo)_ mutation, which confers resistance to powdery mildew, exhibit increased susceptibility to RLS [13]. Further supporting the role of the host genetic background in RLS development, a loss of function mutation of the _Enhanced Magnaporthe 1 (emr1)_ genein a _mlo_ background mitigates the effect of the _mlo_ mutation on RLS severity leading to decreased RLS symptoms [14]. _R. collo-cygni_ is often described as an endophyte that becomes necrotic under specific conditions leading to the development of RLS symptoms. Although the reason for the fungus transitioning from endophytic to necrotic is currently unknown, it has been linked with external stresses such as flowering or senescence [15]. Environmental conditions are also known to play an important role in RLS development as plants subjected to waterlogging exhibit increased RLS symptoms compared with plants subjected to a normal watering regime [16]. Conversely, drought has recently been shown to reduce RLS severity in the field [17]. At the cell level, the development of symptoms is thought to be associated with the induction of plant cell death through the release and action in the host of the rubellin phytotoxins (Figure 1) produced by _R. collo-cygni_. The involvement of a phytotoxin in RLS development was first hypothesised by [PERSON] and [PERSON] following the observation that plant cell death was occurring away from the fungal hyphae [18]. The identification of rubellins in infected barley leaves and the observation that rubellins are able to induce the production of the reactive oxygen species (ROS), such as superoxide (O2*) in vitro [19], further supported a putative role for the toxins in RLS development as ROS were reported at the site of symptoms development [16]. Furthermore, the breakdown of the antioxidant system, which detoxifies ROS via specialised enzymes such as catalases and superoxide dismutase, is known to occur during senescence which has also been linked with symptoms development. However, to date, the exact mode of action of these secondary metabolites (SMs) in planta remains unclear, and their role in RLS development has not yet been confirmed. Although rubellins were mostly studied in relation to the development of RLS, these toxins were first identified in _Mycosphaerella rubella_, the agent responsible for a necrotic leaf spot disease of _Angelica silvestris_[20]. Rubellins, which comprise several derivatives, are a family of anthraquinone-derived metabolites.