Resistance in plants against biotic stress through forward and reverse genetics

植物的抗性可以是定性的（定义为没有疾病），也可以是定量的（定义为疾病严重程度降低），但两者的区别并不总是明显的。通过对小麦/大麦赤霉病和马铃薯晚疫病的研究，我们已经证明其抗性主要是由代谢物引起的。接种后，病原体产生与微生物相关的分子模式，这些模式被宿主受体识别，从而触发下游基因调节耐药基因，这些耐药基因生物合成抗微生物和/或沉积的代谢物，以强制细胞壁遏制病原体的初始感染。代谢途径中的一些转录因子和枢纽基因似乎控制了大量的抗性，这是基于瞬时基因沉默的证明。商品品种也含有这些基因的代谢途径特定等级，但其中一个或几个可能是无功能的。这些缺失的链接基因可以被这里确定的抗性基因所取代，以恢复它们繁殖失去的代谢物的能力，从而产生抗性。我们推测，通过构建一些重要的效应基因，调节特定的代谢途径，可以显著提高商品易感品种对病原体胁迫的抗性。本研究将对几个抗晚疫病的马铃薯基因型和一个对晚疫病敏感的赤褐色伯班克品种进行RNA测序和代谢产物分析。具有高折叠变化的候选代谢物将用于鉴定候选生物合成和调控基因。通过对具有抗性和非功能性的候选基因进行沉默，以证明其抗性效果。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。生成原生质体，将抗性基因构建的CRISPR-Cas9系统开发并导入原生质体，用本文鉴定的功能基因替代非功能基因，再生成愈伤组织，基因验证后再生成植株。这个顺基因（性相容植物之间的基因转移）赤褐色伯班克品种将在预期的位置进行基因替换检查。在温室和田间条件下证明顺基因品种的抗性优于非顺基因品种，并注册为新品种并商品化。赤褐色伯班克马铃薯占北美马铃薯产量的40%以上，预计所有原始生产者都将使用原基因赤褐色伯班克品种。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.