Molecular genetics of plant hormone signaling

寄生杂草威胁着非洲大约1亿人的生计。这些杂草中最有害的是Striga hermonthica（Striga），它侵扰了撒哈拉以南40%的农田，是非洲大陆减贫的最大障碍。目前，防治独脚金最常用的方法是手工除草，这种方法耗时、劳动密集，而且妇女和儿童从事的工作不成比例。不幸的是，由于Striga造成的大部分作物损害发生在它出现之前，手工除草是无效的。进一步加剧这种情况的是，独脚金种子在土壤中存活长达20年，因此清除田地非常困难，严重的虫害往往导致田地被遗弃。育种方法是目前最经济的寄生杂草控制方法，然而，尚未获得对独脚金的长期抗性。这些挑战需要创新的方法来消除利用其生命周期中的弱点的Striga。为此，独脚金，需要一个小分子，独脚金内酯（SL），发芽。SL从宿主植物根部发出，导致独脚金发芽并侵染宿主。由于Striga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Striga“自杀”种子萌发。 开发干扰独脚金发芽的化合物是基于对该物种SL受体的机械理解。在过去的资助周期中，我的团队确定了Striga中的关键SL受体，并显示了一个被命名为ShHTL7的受体对Striga发芽至关重要。在这个资助周期中，我们将使用遗传和结构信息的组合来了解ShHTL7如何感知SL。我们以前的工作表明，这种受体是高度敏感的皮摩尔水平的SL在植物中（体内），但这种敏感性并没有反映在试管中（体外）。ShHTL7超敏反应的性质在生物化学上是有趣的，因为ShHTL7具有大的配体结合结构域（LBD），原则上，该配体结合结构域应该降低而不是增加其对SL的亲和力。为了调和体内与体外观察，我们已经开发了三种方法。首先，我们将使用“反向遗传学”的方法，以确定关键氨基酸的LBD，有助于SL的敏感性。其次，我们将使用无偏的“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可能有助于SL敏感性的任何ShHTL7伴侣蛋白。最后，我们将建立一个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寄生植物SL受体的理解，而且还将指导新的干扰独脚金发芽的先导化合物的开发。这反过来可能对遏制非洲的独脚金侵扰具有长期效用。

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.