Molecular genetics of plant hormone signaling

寄生杂草威胁着非洲大约1亿自给农民。这些杂草中最有害的是斯特里加（Striga hermonthica），它在撒哈拉以南40%的农田中肆虐，是非洲大陆减轻贫困的最大障碍。目前，控制斯特里加最常见的方法是手工除草，这是一项耗时、劳动密集的工作，而且由妇女和儿童进行的工作比例过高。不幸的是，由于斯特里加对作物造成的大部分损害在其出现之前就发生了，因此手工除草是无效的。使情况更加复杂的是，Striga种子在土壤中可存活长达20年，因此清除田地极其困难，严重的虫害往往导致放弃田地。育种方法是目前控制寄生杂草最经济的方法，然而，对斯特里加的长期抗性尚未达到。这些挑战需要创新的方法来根除Striga在其生命周期中的弱点。为此，Striga需要一种小分子，Strigolactone （SL）来发芽。SLs从寄主植物的根部发出，使Striga发芽并感染寄主。由于斯特里加是一种专性寄生虫，研究人员试图开发廉价稳定的SL类似物，在没有宿主的情况下刺激斯特里加“自杀”种子发芽。干扰Striga萌发的化合物的发育是基于对该物种SL受体的机制理解。在过去的资助周期中，我的团队鉴定了Striga中关键的SL受体，并发现其中一个被命名为ShHTL7的受体对Striga的萌发至关重要。在这个资助周期中，我们将结合遗传和结构信息来了解ShHTL7如何感知SL。我们之前的工作表明，该受体对植物（体内）皮摩尔水平的SL高度敏感，但这种敏感性在试管（体外）中没有反映出来。ShHTL7超敏性的性质在生物化学上是有趣的，因为ShHTL7有一个大的配体结合域（LBD），原则上应该降低而不是增加其对SL的亲和力。为了调和体内和体外观察，我们开发了三种方法。首先，我们将使用“反向遗传学”方法来鉴定LBD中有助于SL敏感性的关键氨基酸。其次，我们将使用一种无偏倚的“正向遗传学”方法来鉴定ShHTL7 LBD中的关键氨基酸。第二种方法还将鉴定任何可能对SL敏感性有贡献的ShHTL7伴侣蛋白。最后，我们将开发一种基于酵母的高通量系统来检测Striga SL受体。收集到的基本信息不仅将启发我们对这种独特的超敏感寄生植物SL受体的理解，而且还将指导开发新的干扰Striga萌发的先导化合物。反过来，这可能对遏制非洲斯特里格菌的侵扰具有长期效用。

Parasitic weeds threaten approximately 100 million subsistence framers in Africa. The most pernicious of these weeds, Striga hermonthica (Striga), infests 40% of the sub-Saharan farmland and is the biggest impediment to poverty alleviation on the continent. Presently, the most common method of Striga control is hand weeding, which is time consuming, labour-intensive and disproportionately carried out by women and children. Unfortunately, since much of the crop damage inflicted by Striga occurs before it surfaces, hand weeding is ineffective. Further compounding the situation is Striga seeds remain viable in the soil for up to 20 years, thus purging fields is extremely difficult with severe infestations often resulting in abandonment of the field. Breeding approaches are presently the most economically sought method of parasitic weed control, however, long-term resistance to Striga has not been attained. These challenges necessitates innovative approaches to eradicate Striga that exploit weaknesses in its lifecycle. To this end, Striga, requires a small molecule, Strigolactone (SL), to germinate. SLs emanate from host plant roots causing Striga to germinate and infest the host. Because Striga is an obligate parasite, researchers have tried to develop cheap stable SL analogs that stimulate Striga "suicide" seed germination in the absence of the host.    The development of compounds that perturb Striga germination is predicated on mechanistically understanding SL receptors in this species. Over the past grant cycle, my group identified the key SL receptors in Striga and showed one, designated ShHTL7, is essential to Striga germination. In this grant cycle, we will use a combination of genetic and structural information to understand how ShHTL7 perceives SL. Our previous work shows this receptor is highly sensitive to picomolar levels of SLs in the plant (in vivo) but this sensitivity is not reflected in the test tube (in vitro). The nature of the ShHTL7 hypersensitivity is biochemically intriguing because ShHTL7 has a large ligand binding domain (LBD) that should, in principle, decrease not increase its affinity to SL. To reconcile in vivo with in vitro observations we have developed three approaches. First, we will use a  "reverse genetics" approach to identify key amino acids in the LBD that contribute to SL sensitivity. Second, we will use an unbiased "forward genetics" approach to also identify key amino acids in the ShHTL7 LBD. This second approach will also identify any ShHTL7 partner proteins that may contribute to SL sensitivity. Finally, we will develop a yeast based high throughput system to assay Striga SL receptors. The basic information gleaned will not only enlighten our understanding of this uniquely hypersensitive parasitic plant SL receptor but will also direct the development of new lead compounds that perturb Striga germination. This in turn, may have long term utility in curbing Striga infestations in Africa.