New Phytol. | A new method of plant immune receptor cloning, rapidly locating immune receptors on plant membranes through RLP/RLK enrichment sequencing.

Recently, a research paper entitled "RLP / K enrichment sequencing; a new method to identify receptor ‐ like protein (RLP) and receiver ‐ like kinase (RLK) genes" was published online by new genomic Journal online.plant immune receptors are mainly composed of NBS-LRR protein and RLP or RLK protein on membrane.among them, NBS-LRR protein can recognize the effector protein (effector) secreted by pathogenic bacteria into the plant cells, thus stimulating the effector triggered immune response (ETI); while the RLP / K protein on the membrane can recognize the PAMP or extracellular effector secreted by the pathogen, and activate PTI.these two lines of defense together constitute the innate immune system of plants (Jones and Dangl, 2006).cloning these plant immune receptors can not only help people better understand the plant immune system, but also can be applied to the resistance transformation and cultivation of crops, so as to achieve effective prevention and control of crop diseases.with the rapid development of sequencing technology, in order to clone plant disease resistance genes more quickly and economically, many enrichment sequencing technologies have been developed, such as resistance gene enrichment sequencing (renseq) (jupe et al., 2013) and pacbio renseq (Witek et al., 2013) combined with three generations sequencing, 2016) and association genomics renseq (agrenseq) (Arora et al., 2019) combined with association analysis.however, these enrichment sequencing methods only focus on NBS-LRR protein.here, we developed a rapid method to identify and clone the immune receptors (RLP and RLK) on plant membrane using potato Phytophthora infestans as the model.the first step is to clone the extracellular effector of Phytophthora infestans into PVX (potato virus) by means of effector omics 10) The vectors were then screened by Agrobacterium tumefaciens mediated high-throughput screening on different wild potatoes. The wild potatoes carrying the corresponding receptor on the membrane recognized effectors and showed the phenotype of cell necrosis.in this study, the authors screened two extracellular effectors, INF1 and scr74, in which ELR of INF1 has been cloned (DU et al., 2015) and used as positive control here, while scr74 receptor is unknown. After that, two highly heterozygous diploid wild potato genotypes gig362-6 and mcd360-1 were hybridized to obtain F1 population segregation population, in which gig362-6 could recognize scr74 and mcd360-1 could recognize INF1.the response of the hybrid F1 population to scr74 and INF1 was independently separated at a ratio of 1:1, indicating that both receptors were controlled by a single dominant gene in their respective parents.here, the advantage of phenotype screening through effectors is that multiple genes can be cloned in a population.however, the traditional cloning of disease resistance genes usually calculates the segregation ratio by disease resistance phenotype. If there are multiple disease resistance genes at the same time, they need to be hybridized, self crossed or test cross to separate them. The response of F1 population to scr74 and INF1 was independent and separated by 1:1.next, based on the potato reference genome (Xu et al., 2011) and a wild potato M6 genome (leisner et al., 2018), 533 RLK genes and 444 RLP genes were predicted. In addition to the typical lrr-rlk and lrr-rlp, 70 RLK genes with wax or wax-egf domain and 38 RLK with malectin domain were predicted, There are 11 RLKs with Antifungi domain, 6 RLKs with an K repeat domain, 11 LysM RLKs, 24 l-lecrks, 103 g-lecrks, 1 c-lecrk and 22 and other types of RLKs with transmembrane structure.the authors designed and synthesized a RNA bait library. Then, two parents and F1 population were constructed according to the phenotype (scr74 response, scr74 no response, INF1 response, INF1 no response), enrichment, and sequencing.as control, ELR was successfully located on chromosome 12.meanwhile, the unknown scr74 receptor was located on chromosome 9. After that, the SNPs from RLP / kseq were transformed into molecular markers and genotyped by lightscanner. the SNP marker obtained by RLP / kseq was successfully verified, and scr74 receptor was located in a 5.99mb region of chromosome 9. then, the authors expanded the population and fine mapped the scr74 receptor using RLP / kseq and other molecular markers. finally, the BAC library of gig362-6 was established and BAC clones in the target region were screened. the scr74 receptor was located at a 43 kbp g-lecrk site. The process of RLP / kseq was completed by Dr. vivianne g.a.a. vleeshowers team from plant breeding, Wageningen ur, the Netherlands, and Dr. INGO Hein team from the James Hutton Institute in Scotland. The first author was Xiao from Wageningen University in the Netherlands Lin) is now a postdoctoral fellow of the Sainsbury laboratory, UK. the study was funded by the NWO (nwo-vidi grant 12378) of the Netherlands, the government sponsored study abroad program of China Scholarship Council and the short term scientific mission (STSM) of cost sustain. paper links: the forefront of plant science, focusing on the forefront of plant science progress, information, recruitment information release and method software sharing. for submission and recruitment, please reply "contribution" from the background, which is free of charge; for business cooperation, please contact wechat ID: zwkxqy;