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

植物病原体是对人类的重要威胁，它们被认为是全球粮食和经济作物收获前损失的主要原因。在这些有害生物中，有丝状和二型真菌，它们通过其基本单位（所谓的菌丝）的顶端生长进入植物。菌丝的侵入性生长依赖于生长顶端新细胞壁的形成。因此，理解壁合成酶，包括它们靶向生长区域，是植物病理学中的重要任务，其有望鉴定杀真菌剂的新靶标（Debono和Gordee 1994，Ann.Rev.Microbiol. 48，471）。真菌生长中的一个重要步骤是将壁合成酶，如几丁质酶输送到扩张的顶端。这被认为取决于细胞骨架的纤维，这些纤维为细胞机器（所谓的分子马达）提供轨道。这些马达沿着细胞骨架行走，以便将它们的货物带到生长的菌丝尖端（Steinberg，2007，Eukaryot Cell.，6，351，自己的贡献用 * 表示）。支持这一概念的证据主要来自使用细胞骨架特异性抑制剂的研究，它们表明细胞骨架对菌丝生长和致病性至关重要。一种类型的分子马达，肌球蛋白，被认为是特别重要的真菌。然而，到目前为止，植物致病性仅需要一种肌球蛋白（Weber等，2003，Plant Cell，15，2826*），并且没有证据表明真菌肌球蛋白马达在酶向菌丝尖端的极性递送中的假定作用。一类真菌肌球蛋白是真菌特异性的，并且对于所研究的所有病原性真菌（包括尖孢镰刀菌（Fusarium oxysporum））中的植物感染是必需的（马德里等人，2003，分子微生物学，47，257）、禾生炭疽菌（Colletotrichum graminicola）（Werner等人，2007，Mol Plant Microbe Interact. 20，1555*）和玉米黑粉菌（Weber等人，2006，Plant Cell，18，225*）。有趣的是，所有XVII类肌球蛋白都含有保守的肌球蛋白马达结构域和甚至更高度保守的几丁质合成酶结构域。这种结构域组织表明，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，但没有实验证据表明存在这样的角色定位机制。在这个项目中，我们将阐明几丁质酶传递到菌丝顶端的机制。我们将利用植物病原真菌玉米黑粉菌（Ustilago maydis），这是研究细胞骨架在真菌致病性中的作用的最佳模型系统之一。我们先前证明了4种几丁质酶定位于菌丝顶端（包括Mcs 1），并且其中3种涉及该病原体的毒力（Weber等人，2006，Plant Cell，18，225*）。在大量的突变蛋白表达的U。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的作用（Myo1，Myo2，Myo5）在极性几丁质酶Chs5，Chs6，Chs7的顶端靶向中的作用（也可能是Mcs1），以及（3）将尝试分离Mcs1结合的壳聚糖体，以鉴定共同递送的并且在植物感染中可能同样重要的其他蛋白质因子。本研究的预期成果将为深入研究几丁质合成酶作用于菌丝顶端的机制提供重要的新的思路。因此，它将是真菌研究的各个方面的根本利益。该项目对于了解真菌的致病性特别重要，并且它将有望对可能用于有效控制人类病原体和植物害虫的机制进行关键的深入了解。

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.