Resistance in plants against biotic stress through forward and reverse genetics

植物的抗性可以是定性的，定义为没有病害，也可以是定量的，定义为降低病害严重程度，但区别并不总是明显的。基于小麦/大麦赤霉病和马铃薯晚疫病，我们已经证明了抗性主要是由于代谢产物。接种后，病原体产生由宿主受体识别的微生物相关分子模式，其触发下游基因以调节抗性基因，所述抗性基因生物合成抗微生物和/或沉积的代谢物以强制细胞壁容纳病原体以进行初始感染。代谢途径中的一些转录因子和枢纽基因似乎控制着大量的抗性，如基于瞬时基因沉默所证明的。商业栽培品种也含有这些基因的代谢途径特异性层次，但一个或几个可能是无功能的。这些缺失的连接基因可以被这里鉴定的抗性基因所取代，以恢复它们复制丢失的代谢物的能力，从而恢复抗性。我们推测，在商业感病品种中，通过抑制一些显著的效应基因，调节特定的代谢途径，可以显着提高对病原菌胁迫的抗性。在本提案中，将对几种抗性马铃薯基因型和一种对晚疫病敏感的栽培品种Russet伯班克进行RNA测序和代谢物分析。具有高倍数变化的候选代谢物将用于鉴定候选生物合成和调控基因。将在赤褐色伯班克中有抗性功能而无抗性功能的候选基因沉默以证明抗性效应。这些基因或DNA片段将基于基因组编辑工具在Russet伯班克被替换。将产生原生质体，开发抗性基因的CRISPR-Cas9系统构建体并将其引入原生质体以用本文鉴定的功能基因替换非功能基因，再生到愈伤组织中，基因被验证并再生到植物中。将检查该顺基因（性相容植物之间的基因转移）Russet伯班克栽培品种在预期位置的基因置换。在温室和大田条件下证明了顺基因的抗病性优于非顺基因品种，登记为新品种并进行了商品化。Russet伯班克占北美马铃薯产量的40%以上，所有原始生产者都将使用顺基因Russet伯班克品种。这里开发的技术可以用于改善其他植物病原体系统。

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.