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Clubroot caused by the protist Plasmodiophora brassicaeis a major disease affecting cultivated Brassicaceae. Using a combination of quantitative trait locus (QTL) fine mapping, CRISPR-Cas9 validation, and extensive analyses of DNA sequence and methylation patterns, we revealed that the two adjacent neighboring NLR(nucleotide-binding and leucine-rich repeat) genes AT5G47260and AT5G47280cooperate in controlling broad-spectrum quantitative partial resistance to the root pathogen P. brassicaein Arabidopsisand that they are epigenetically regulated. The variation in DNA methylation is not associated with any nucleotide variation or any transposable element presence/absence variants and is stably inherited. Variations in DNA methylation at the Pb-At5.2QTL are widespread across Arabidopsisaccessions and correlate negatively with variations in expression of the two genes. Our study demonstrates that natural, stable, and transgenerationally inherited epigenetic variations can play an important role in shaping resistance to plant pathogens by modulating the expression of immune receptors.

MicroRNAs (miRNAs) are small noncoding RNAs, which modulate the abundance and spatiotemporal accumulation of target mRNAs at transcriptional and post-transcriptional levels and through that play important roles in several biological processes in plants. Here we show that in polyploid species, CRISPR/Cas9 system can be used for fine-tuning of miRNA expression, which can have broader range of applications compared to knock-out mutants. We established the complete pipeline for CRISPR-Cas9-mediated modulation of miRNA expression in potato. It consists of (1) design and assembly of dual sgRNA CRISPR/Cas9 constructs, (2) transient transfection of protoplasts following fast and efficient screening by high resolution melting analysis to select functional sgRNAs, and (3) stable transformation of potato explants with functional sgRNAs and selection of regenerated transgenic lines with desired mutations and desired miRNA abundance based on sequencing and RT-qPCR. We show that miRNA-editing using dual sgRNA approach results in different types of mutations among transgenic lines but also in different alleles of the same plant, which are target site-dependent. The most frequent were short deletions, but we also detected 1-nt insertions (T or G), deletions between two sgRNAs and larger deletions. miRNA abundance correlates with the frequency and type of introduced mutations, as more extensive mutations in more alleles result in lower miRNA abundance. Interestingly, some mutated loci can generate alternative miRNAs, now novel targets were however predicted for those. In all transgenic lines with Cas9expression, we detected mutations, suggesting high efficiency of Cas9-editing. We confirmed the miRNA-editing efficiency of our optimised approach in two different potato genotypes and three different loci.

Since its discovery as a bacterial adaptive immune system and its development for genome editing in eukaryotes, the CRISPR technology has revolutionized plant research and precision crop breeding. The CRISPR toolbox holds great promise in the production of crops with genetic disease resistance to increase agriculture resilience and reduce chemical crop protection with a strong impact on the environment and public health. In this review, we provide an extensive overview on recent breakthroughs in CRISPR technology, including the newly developed prime editing system that allows precision gene editing in plants. We present how each CRISPR tool can be selected for optimal use in accordance with its specific strengths and limitations, and illustrate how the CRISPR toolbox can foster the development of genetically pathogen-resistant crops for sustainable agriculture.