Cloning of the beet cyst nematode resistance gene Hs4 from the wild beet Patellifolia procumbens

Plant parasitic nematodes are important pests in crop production worldwide. Therefore, breeding nematode resistant crops is an important aim nowadays. The beet cyst nematode Heterodera schachtii causes severe losses in sugar beet production. The only source of resistance are sugar beet wild relatives from the genus Patellifolia. Sugar beet lines carrying a translocation stemming from chromosome 1 of P. procumbens are resistant to beet cyst nematode. The aim of this study was to clone the beet cyst nematode resistance gene Hs4. In this study, I characterized the translocation segment in the resistant translocation line TR520. I then identified the translocation breakpoint to the precision of a single base pair. Finally, I identified a putative resistance gene. I functionally characterized my candidate gene by two approaches, CRISPR-Cas mediated knock-out and overexpression studies. To characterize the translocation segment, I first generated a de-novo assembly of the resistant translocation line TR520. Then, using whole genome sequencing reads from P. procumbens, I identified the translocation specific scaffolds. Finally, two super scaffolds for the whole translocation segment, super scaffold 1 and super scaffold 2 were generated which are 1.109 and 2.121 Mb in size, respectively. I identified the translocation breakpoint in the following way. I inspected mate-pair read libraries for TR520 and found a set of reads joining super scaffold 2 with a scaffold from chromosome 9 of the sugar beet genome. Therefore, I concluded that the breakpoint lies between these two scaffolds. I amplified the breakpoint by PCR, followed by Sanger sequencing, and I could identify the exact nucleotide position at which the sequence of chromosome 9 from sugar beet ended and the translocation sequence started. To find the resistance gene, I used whole genome sequencing data from two resistant translocation lines, TR520 and TR363, and one γ-irradiated susceptible translocation line TR659. I then identified the translocation regions that are present in the resistant lines but absent in the susceptible line. I found three regions, encompassing a total of 229 kb, and referred to them as “critical regions”. The next step was to identify the putative resistance gene. Using transcriptome data from the roots of the resistant plant after infection, I could narrow down the search to 19 gene-models within the critical regions. None of the genes in the critical regions was annotated as resistance gene analogue. Considering the role of proteases in plant-defence mechanisms, I chose a rhomboid-like protease encoding gene as a putative Hs4, which is predicted to be bound to the endoplasmic reticulum. To functionally characterize my candidate gene, I performed CRISPR-Cas mediated knockout in hairy roots of a resistant translocation variety and overexpression study in hairy roots of a susceptible sugar beet line. Knock-out of my candidate gene resulted in complete loss of resistance, while overexpression led to complete resistance. This study has revealed the Hs4 gene, encoding a rhomboid-like protease and conferring complete resistance to beet cyst nematodes. This gene is the first protease which alone causes resistance to a pest. Thus, it constitutes a previously unknown mechanism of plants to fight plant parasitic nematodes.


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