Loss of function of CRT1a (calreticulin) reduces plant susceptibility toVerticillium longisporumin bothArabidopsis thalianaand oilseed rape (Brassica napus)

Abstract

Brassica napus is highly susceptible towards Verticillium longisporum (Vl43) with no effective genetic resistance. It is believed that the fungus reprogrammes plant physiological processes by up-regulation of so-called susceptibility factors to establish a compatible interaction. By transcriptome analysis, we identified genes, which were activated/up-regulated in rapeseed after Vl43 infection. To test whether one of these genes is functionally involved in the infection process and loss of function would lead to decreased susceptibility, we firstly challenged KO lines of corresponding Arabidopsis orthologs with Vl43 and compared them with wild-type plants. Here, we report that the KO of AtCRT1a results in drastically reduced susceptibility of plants to Vl43. To prove crt1a mutation also decreases susceptibility in B. napus, we identified 10 mutations in a TILLING population. Three T3 mutants displayed increased resistance as compared to the wild type. To validate the results, we generated CRISPR/Cas-induced BnCRT1a mutants, challenged T2 plants with Vl43 and observed an overall reduced susceptibility in 3 out of 4 independent lines. Genotyping by allele-specific sequencing suggests a major effect of mutations in the CRT1a A-genome copy, while the C-genome copy appears to have no significant impact on plant susceptibility when challenged with Vl43. As revealed by transcript analysis, the loss of function of CRT1a results in activation of the ethylene signalling pathway, which may contribute to reduced susceptibility. Furthermore, this study demonstrates a novel strategy with great potential to improve plant disease resistance.

Targeted mutagenesis of EOD3 gene in Brassica napus L. regulates seed production

“…It has been used for genome editing in major crops (Ahmar et al, 2020; M. H. U. Khan et al, 2018). It has been efficiently utilized in rapeseed to produce the targeted mutagenesis required to improve agronomic traits, including multilocular silique, plant height and architecture, pod shatter resistance, yellow seeds, fatty acid composition, and disease resistance (Braatz et al, 2017; Hu et al, 2018; Huang et al, 2020; C. Li et al, 2018; Pröbsting et al, 2020; H. Yang, Wu, Tang, Liu, & Dai, 2017; Y. Yang et al, 2018; Zhai et al, 2019, 2020; Zheng et al, 2019).…”

Applications of CRISPR/Cas to Improve Crop Disease Resistance: Beyond Inactivation of Susceptibility Factors

“…These hypotheses can be addressed even by smaller breeding companies, because CRISPR/Cas-mediated GE is not that expensive and relatively easy to handle. Importantly, GE techniques allow a very specific KO of certain alleles, whereas a knockdown often causes off-target effects resulting in a different phenotype maybe due to lost genetic compensation ( Gao et al., 2015 ; Unniyampurath et al., 2016 ; Smith et al., 2017 ; Kumar et al., 2018 ; Kim et al., 2019 ; Pröbsting et al., 2020 ). Pathogen resistance is just one important trait that can be addressed with GE in many different crops ( Table 1 ), and CRISPR/Cas is currently also deployed to encounter citrus greening, a bacterial disease threatening sweet orange ( Ledford, 2017 ).…”

“…Such an approach was successful to identify CRT1a as a novel susceptibility factor in Brassica napus and Arabidopsis thaliana infected with Verticillium longisporum , albeit its function in endoplasmic reticulum quality control/protein folding could well have secondary effects on the functionality of other yet unknown susceptibility factors. Importantly, the plants appeared to develop normally, because only one “active” CRT1a locus was targeted, whereas other CRT1a, CRT1b, and CRT3 loci were not affected ( Pröbsting et al., 2020 ). Generally, basic research elucidating the molecular mechanisms underlying host-pathogen interactions is also a rich source for several biotechnological applications and resistance resources.…”

CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement

“…In the past eight years, there have been many reports of creating genome-edited plants with resistance to viral, bacterial, and fungal diseases in different plant species using CRISPR/Cas technology ( Zhang et al, 2021 ). These include CRISPR/Cas9 knock-outs of the following genes: mlo for plant resistance to powdery mildew in wheat ( Wang et al, 2014 ), tomato ( Nekrasov et al, 2017 ; Martínez et al, 2020 ) and grapevine ( Wan et al, 2020 ), pmr4 for plant resistance to powdery mildew in tomato (Santillán Martínez et al, 2020), 14-3-3 gene for resistance to Verticillium dahlia in cotton ( Zhang ZN et al, 2018 ), crt1a for resistance to Verticillium longisporum in both Arabidopsis thaliana and oilseed rape ( Pröbsting et al, 2020 ), OsERF922 for resistance to Magnaporthe oryzae in rice ( Wang et al, 2016 ), Clpsk1 for resistance to Fusarium oxysporum f. sp. niveum in watermelon ( Zhang et al, 2020 ), eif4e for resistance to Cucumber vein yellowing virus , Zucchini yellow mosaic virus , and Papaya ring spot mosaic virus‐W in cucumber ( Chandrasekaran et al, 2016 ), CsWRKY22 for resistance to Xanthomonas citri subsp.…”