The role of myosins in targeting of chitin synthases to apical growth regions during growth and infection by Ustilago maydis

植物病原菌是对人类的一种重要威胁，它们被认为是造成全球粮食和经济作物收割前重大损失的原因。在这些害虫中有丝状和二相性真菌，它们通过其基本单位，即所谓的菌丝的顶端生长进入植物。菌丝的侵袭性生长依赖于生长顶端新细胞壁的形成。因此，了解壁合成酶，包括它们针对生长区域的作用，是植物病理学中的一项重要任务，它有望识别杀菌剂的新靶点(Debono和Gordee 1994，ANN)。Rev.Microbiol。48,471)。真菌生长的一个重要步骤是将几丁质合成酶等壁合成酶输送到膨大的顶端。这被认为依赖于为细胞机器提供轨迹的细胞骨架纤维，也就是所谓的分子马达。这些马达沿着细胞骨架行走，以便将它们的货物带到不断增长的菌丝尖端(斯坦伯格，2007，真核细胞，6,351，自己的贡献由*表示)。支持这一概念的证据主要来自使用细胞骨架的特定抑制剂的研究，它们表明细胞骨架对菌丝生长和致病性是必不可少的。一种类型的分子马达，肌球蛋白，被认为在真菌中特别重要。然而，到目前为止，只有一种肌球蛋白被证明是植物致病所必需的(Weber等人。2003年，植物细胞，15,2826*)，并且没有证据表明真菌肌球蛋白马达在将酶极地输送到菌丝顶端中所起的作用。在所研究的所有病原真菌中，有一类真菌肌球蛋白是真菌所特有的，对植物侵染是必不可少的，包括尖孢镰刀菌(Fusum Oxporum)(马德里等人)。2003年，Mol Microbiol.，47,257)，Colletotrichum graminicola(Werner等.2007年，摩尔植物微生物互动。20,1555*)和玉米黑粉菌(Weber等人)。2006年，植物细胞，18,225*)。有趣的是，所有的XVII类肌球蛋白都包含一个保守的肌球蛋白运动结构域和一个高度保守的几丁质合成酶结构域。这一区域组织表明，这些肌球蛋白通过其自身运动域的活动将自己带到菌丝顶端，在那里参与构建细胞壁。虽然这是一个有吸引力的模型，但没有实验证据证明这种角色定向机制存在。在这个项目中，我们将阐明几丁质合成酶被输送到菌丝顶端的机制。我们将利用植物病原菌Ustilago maydis，这是研究细胞骨架在真菌致病中作用的最好的模型系统之一。我们先前证明了4个几丁质合成酶定位于菌丝顶端(包括MCS1)，其中3个参与了该病原菌的毒力(Weber等人。2006年，植物细胞，18,225*)。在对大量突变蛋白的广泛分析中，我们将分析(1)肌球蛋白马达结构域和V类几丁质合成酶结构域对极性传递和毒力的需求，(2)将研究其他三个肌球蛋白马达(Myo1，Myo2，Myo5)在针对极性几丁质合成酶Chs5，Chs6，Chs7(可能还有Mcs1)的顶端靶向中的作用，以及(3)将尝试分离Mcs1结合的壳小体，以确定与Mcs1结合的其他蛋白因子，这些蛋白因子可能在植物侵染中同样重要。该项目的预期结果将为甲壳素合成酶靶向菌丝顶端的机制提供重要而新颖的见解。因此，它将对真菌研究的各个方面产生根本的影响。这个项目将对了解真菌的致病性具有特别重要的意义，它将为有效控制人类病原体和植物病虫害的机制提供关键的见解。

Plant pathogens are an important threat to mankind, and they are thought to be responsible for a significant preharvest loss to food and cash crops of the global production. Among these pest are filamentous and dimorphic fungi that enter the plant by apical growth of their basic unit, the so-called hypha. Invasive growth of the hypha depends on the formation of new cell wall at the growing tip. Thus, the understanding wall-synthesizing enzymes, including their targeting to the growth region, is an important task in phytopathology, which promises the identification of novel targets for fungicides (Debono and Gordee 1994, Ann. Rev. Microbiol. 48, 471). An essential step in fungal growth is the delivery of wall-synthesizing enzymes, such as chitin synthases to the expanding apex. This is thought to be depending on fibres of the cytoskeleton that provide tracks for cellular machines, the so-called molecular motors. These motors walk along the the cytoskeleton in order to take their cargo to the growing hyphal tip (overview in Steinberg, 2007, Eukaryot Cell., 6, 351, own contribution indicated by*). Supporting evidence for this concept is mainly coming from studies using specific inhibitors of the cytoskeleton and they demonstrate that the cytoskeleton is essential for hyphal growth and pathogenicity. One type of molecular motors, the myosins, are thought to be of particular importance in fungi. However, so far only one myosin was shown to be required for plant pathogenicity (Weber et al. 2003, Plant Cell, 15, 2826*), and no evidence exists for the assumed role of fungal myosin motors in polar delivery of enzymes to the hyphal tip. One class of fungal myosins is fungal specific and essential for plant infection in all pathogenic fungi investigated, including Fusarium oxysporum (Madrid et al. 2003, Mol Microbiol., 47, 257), Colletotrichum graminicola (Werner et al. 2007, Mol Plant Microbe Interact. 20, 1555*) and Ustilago maydis (Weber et al. 2006, Plant Cell, 18, 225*). Interestingly, all class XVII myosins contain both a conserved myosin motor domain and an even more highly conserved chitin synthase domain. This domain organisation suggests that these myosins take themself to the hyphal apex by the activity of its own motor domain, where take part in building the cell wall. Although this is an attractive model, no experimental evidence for such a role targeting mechanism exists. In this project we are going to elucidate the mechanism by which chitin synthases are delivered to the hyphal tip. We will make use of the plant pathogenic fungus Ustilago maydis, which is one of the best established model systems for studying the role of the cytoskeleton in fungal pathogenicity. We previously demonstrated that 4 chitin synthases localize to the hyphal tip (including Mcs1) and that 3 of them are involved in virulence of this pathogen (Weber et al. 2006, Plant Cell, 18, 225*). In an extensive analysis of numerous mutant proteins expressed in U. maydis we will analyse (1) the requirement of the myosin motor domain and the class V chitin synthase domain for polar delivery and virulence, (2) will investigate the role of the other three myosin motors (Myo1, Myo2, Myo5) in apical targeting of the polar chitin synthases Chs5, Chs6, Chs7 (and maybe also Mcs1), and (3) will attempt to isolate Mcs1-bound chitosomes in order to identify other protein factors that are co-delivered and that might be equally important in plant infection. The expected outcome of this project will give important and novel insights into the mechanism of chitin synthase targeting to the hyphal tip. It will therefore be of fundamental interest to all aspects of fungal research. This project will of particular importance for understanding fungal pathogenicity and will it promises key insight into a mechanism that might be used for the effective control of human pathogens and plant pests.