Resistance in plants against biotic stress through forward and reverse genetics

植物中的抗性可以是定性的，定义为无病，也可以是定量的，定义为降低疾病严重程度，但区别并不总是明显的。根据小麦/大麦赤霉病和马铃薯晚疫病的抗性，我们证明抗性主要是由代谢产物引起的。接种后，病原体产生被宿主受体识别的微生物相关分子模式，这些模式触发下游基因调节抗性基因，这些基因生物合成抗菌和/或沉积的代谢物，以迫使细胞壁遏制病原体对初始感染。代谢途径中的一些转录因子和HUB基因似乎控制着大量的抗性，这是基于瞬时基因沉默所证明的。商业品种也包含这些基因的代谢途径特定层级，但有一个或几个可能是无功能的。这些缺失的环节基因可以被这里确定的抗性基因取代，以恢复它们复制丢失的代谢物的能力，从而产生抗性。我们推测，通过聚合几个显著的效应基因，调节特定的代谢途径，在商业敏感品种中，可以显著提高对病原菌胁迫的抗性。在本提案中，将对几个抗病马铃薯基因型和一个对晚疫病敏感的品种Russet Burbank进行RNA测序和代谢产物分析。具有高倍数变化的候选代谢物将用于识别候选的生物合成和调控基因。在Russet Burbank中具有抗病功能和非功能的候选基因将被沉默，以证明抗性效应。这些基因或DNA片段将在Russet Burbank基于基因组编辑工具进行替换。将产生原生质体，CRISPR-Cas9系统构建抗性基因，并将其导入原生质体，用这里鉴定的功能基因取代非功能基因，再生成愈伤组织，验证基因并再生到植物中。这种顺性遗传(有性亲和植物之间的基因转移)的Russet Burbank品种将在预期的位置进行基因替换检查。在温室和大田条件下，证明了顺性抗病品种比非顺性抗病品种更具抗逆性优势，并注册为新品种并推广应用。Russet Burbank覆盖了北美生产的40%以上的马铃薯，预计所有原始生产商都将使用顺式遗传的Russet Burbank品种。这里开发的技术可用于改进其他植物病原菌系统。

Resistance in plants can be either qualitative, defined as absence of disease, or quantitative, defined as reduced disease severity, but the distinction is not always apparent. Based on wheat/barley-fusarium head blight and potato-late blight we have evidenced that the resistance is mainly due to metabolites. Following inoculation, the pathogens produce microbe associated molecular patterns that are recognized by host receptors, which trigger downstream genes to regulate resistance genes that biosynthesize metabolites that are antimicrobial and/or deposited to enforce the cell walls to contain the pathogen to initial infection. Some of the transcription factors and hub genes in the metabolic pathway appear to control significant amounts of resistance, as proved based on transient gene silencing. The commercial cultivars also contain metabolic pathway specific hierarchies of these genes but one or a few may be nonfunctional. These missing link genes can be replaced with resistance genes identified here to recover their ability to reproduce the lost metabolites, thus the resistance. We hypothesize that by pyramiding a few significant effect genes, regulating specific metabolic pathways, in a commercial susceptible cultivar the resistance against pathogen stress can be significantly increased. In the present proposal several resistant potato genotypes and a cultivar Russet Burbank susceptible to late blight will be RNA sequenced and metabolites profiled. Candidate metabolites with high fold change will be used to identify the candidate biosynthetic and regulatory genes. The candidate genes that are functional in resistant and nonfunctional in Russet Burbank will be silenced to prove the resistance effects. These gens or DNA segments will be replaced in Russet Burbank based on genome editing tools. Protoplasts will be produced, CRISPR-Cas9 system constructs of resistance genes developed and introduced to protoplasts to replace the non-functional genes with functional genes identified here, regenerated into calli, genes verified and regenerated into plants. This cisgenic (gene transfer between sexually compatible plants) Russet Burbank cultivar will be checked for gene replacement in the expected location. Superiority of cisgenic over the non-cisgenic cultivar in resistance proved under greenhouse and field conditions, registered as a new cultivar and commercialized. Russet Burbank covers more than 40% of potato produced in North America, and all original producers are expected to use cisgenic Russet Burbank cultivar. Technology developed here can be adopted to improve other plant-pathogen systems.