Joint NSF/ERA-CAPS: Host Targets of Fungal Effectors as Keys to Durable Disease Resistance

--PI：Dan内特尔顿（爱荷华州州立大学）高级人事：罗杰·怀斯（美国农业部-农业研究所），亚当·波格丹诺夫（康奈尔大学），罗杰·英尼斯（印第安纳州大学），Fredy Altpeter（佛罗里达大学），阿达莱舍姆（爱荷华州州立大学）和杰奎琳杰克逊（塔斯基吉大学）ERA-CAPS合作者：帕特里克施韦策[莱布尼茨植物遗传学和作物研究所（IPK），德国]和皮埃特罗·斯帕努疾病造成的作物损失仍然是发达国家和发展中国家面临的最大农业挑战之一。专性真菌病原体，即病原体需要宿主才能存活，是全世界农作物生产的主要威胁。由这些病原体分泌的效应蛋白抑制宿主防御以促进营养获取和定殖。然而，这些效应器操纵这些过程的分子机制知之甚少。该项目旨在通过识别特征明确的大麦-白粉病，宿主-微生物系统中相互作用的宿主和病原体蛋白质的网络来填补这一知识空白。以前的联合NSF-BBSRC资助的研究确定了一系列由白粉病真菌Blumeria graminis f. sp. hordei，其有助于病原体毒力。这组效应子将用于使用酵母双杂交筛选来鉴定宿主靶蛋白，目的是鉴定由多个效应子靶向的蛋白质。这些宿主靶标以及关键效应物将通过RNA干扰（RNAi）沉默和细菌III型递送系统中的过表达来评估其在宿主免疫中的作用。影响宿主免疫调节的那些将在稳定的大麦转基因中使用TAL效应物核酸酶（TALEN）诱变或RNAi介导的基因沉默来进一步表征。在迄今为止鉴定和表征的真菌效应子中，预测的金属蛋白酶（BEC 1019）是毒力所需的，抑制宿主防御，并且在至少96种其他不同真菌中进化保守，包括经济上重要的植物病原体、动物病原体和自由生活的非病原体。作为概念验证，大麦将被改造成一种新发现的系统，该系统将在检测到BEC 1019活性时激活防御反应，预计该系统将赋予对多种真菌病原体的抗性。破译进化上保守的真菌效应子的分子功能和理解响应的宿主基因将阐明植物防御机制，并促进广泛适用的疾病控制策略。所有的研究目标将纳入培训和指导本科生和研究生，博士后和K-12教师的机会。与欧洲ERA-CAPS（http：//www.eracaps.org/）配套项目“小麦族物种持久抗真菌病非宿主成分的功能表征和验证（DURESTrit）"的国际研究交流将加强对学生和博士后的培训。将向中学、社区学院和1890个赠地机构中代表性不足的群体传播“大麦性状和基因遗传”教师调查研究经验，提供遗传学方面的实践培训，因为它适用于农业和人类健康。将通过一个项目网站、PLEXdb（www.plexdb.org）植物和植物病原体基因表达在线数据库以及NCBI-GEO（http：//www.ncbi.nlm.nih.gov/geo/）、Ensembl Genomes（http：//ensemblgenomes.org/）和GrainGenes（wheat.pw.usda.gov）促进公众获取项目数据。因此，该项目将促进研究，教育和传播给广大受众，同时培养新一代的农业和计算科学家。

----------------------------------------------------------------------------------------------------------------------PI: Dan Nettleton (Iowa State University) Senior Personnel: Roger Wise (USDA-ARS), Adam Bogdanove (Cornell University), Roger Innes (Indiana University), Fredy Altpeter (University of Florida), Adah Leshem (Iowa State University) and Jaquelyn Jackson (Tuskegee University)ERA-CAPS Collaborators: Patrick Schweizer [Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Germany] and Pietro Spanu (Imperial College, United Kingdom)Crop loss caused by disease remains one of the greatest agricultural challenges in both developed and developing countries. Obligate fungal pathogens, i.e., pathogens that require their host to survive, are a major threat to crop production worldwide. Effector proteins secreted by these pathogens suppress host defenses to promote nutrient acquisition and colonization. However, the molecular mechanisms by which these effectors manipulate these processes are poorly understood. This project aims to fill this knowledge gap by identifying the networks of interacting host and pathogen proteins in the well-characterized barley-powdery mildew, host-microbe system. Previous joint NSF-BBSRC funded research identified a collection of novel effectors secreted by the powdery mildew fungus, Blumeria graminis f. sp. hordei, that contribute to pathogen virulence. This group of effectors will be used to identify host target proteins, using yeast two-hybrid screens, with the goal of identifying proteins that are targeted by multiple effectors. These host targets, as well as key effectors, will be evaluated for their roles in host immunity by silencing via RNA interference (RNAi) and by overexpression in a bacterial type III delivery system. Those that impact host immune regulation will be further characterized using TAL effector nuclease (TALEN) mutagenesis or RNAi-mediated gene silencing in stable barley transgenics. Among the fungal effectors identified and characterized thus far is a predicted metalloprotease (BEC1019) that is required for virulence, suppresses host defenses and is evolutionarily conserved among at least 96 other diverse fungi, including economically important plant pathogens, animal pathogens, and free-living non-pathogens. As a proof of concept, barley will be engineered with a newly discovered system that will activate defense responses upon detection of BEC1019 activity, which is predicted to confer resistance to a wide range of fungal pathogens. Deciphering the molecular functions of evolutionarily conserved fungal effectors and understanding the host genes that respond will elucidate plant defense mechanisms and promote broadly applicable disease control strategies. All research objectives will be incorporated into training and mentoring opportunities for undergraduate and graduate students, postdocs, and K-12 teachers. International research exchanges with the European ERA-CAPS (http://www.eracaps.org/) companion project, "Functional characterization and validation of nonhost components in Triticeae species for durable resistance against fungal diseases (DURESTrit)", will enhance the training of students and postdocs. An inquiry-based Research Experience for Teachers on "Inheritance of Traits and Genes in Barley" (iTAG Barley) will be disseminated to serve underrepresented groups in secondary schools, community colleges, and 1890 land-grant institutions, providing hands-on training in genetics as it applies to agriculture and human health. Public access to project data will be fostered through a project website, the PLEXdb (www.plexdb.org) on-line database for gene expression for plants and plant pathogens, as well as NCBI-GEO (http://www.ncbi.nlm.nih.gov/geo/), Ensembl Genomes (http://ensemblgenomes.org/), and GrainGenes (wheat.pw.usda.gov). Thus, this project will promote research, education, and dissemination to a broad audience, while developing a new generation of agricultural and computational scientists.