Identification of Arabidopsis Phospholipase A Mutants With Increased Susceptibility to Plasmodiophora brassicae

Abstract

Clubroot, caused by the obligate parasite Plasmodiophora brassicae, is one of the most devastating diseases of canola (Brassica napus) in Canada. The identification of novel genes that contribute to clubroot resistance is important for the sustainable management of clubroot, as these genes may be used in the development of resistant canola cultivars. Phospholipase As (PLAs) play important roles in plant defense signaling and stress tolerance, and thus are attractive targets for crop breeding. However, since canola is an allopolyploid and has multiple copies of each PLA gene, it is time-consuming to test the functions of PLAs directly in this crop. In contrast, the model plant Arabidopsis thaliana has a simpler genetic background and only one copy of each PLA. Therefore, it would be reasonable and faster to validate the potential utility of PLA genes in Arabidopsis first. In this study, we identified seven homozygous atpla knockout/knockdown mutants of Arabidopsis, and tested their performance following inoculation with P. brassicae. Four mutants (pla1-iiα, pla1-iγ3, pla1-iii, ppla-iiiβ, ppla-iiiδ) developed more severe clubroot than the wild-type, suggesting increased susceptibility to P. brassicae. The homologs of these Arabidopsis PLAs (AtPLAs) in B. napus (BnPLAs) were identified through Blast searches and phylogenic analysis. Expression of the BnPLAs was subsequently examined in transcriptomic datasets generated from canola infected by P. brassicae, and promising candidates for further characterization identified.

Functions of the AP2/ERF family transcription factor AIL7 in immunity against soilborne clubroot pathogen in Arabidopsis

“…Given that seed-specific overexpression of AIL7 in Arabidopsis was previously reported to result in the up-regulation of various genes encoding putative R and PR proteins in developing siliques (Singer et al, 2021; Table S2), we began by assessing the expression of TIR-NBS-LRR genes (AT1G63860, AT5G45240, AT5G38340, AT4G36140, AT1G72950), PR thaumatin superfamily genes (AT1G18250, AT1G73620), and PR genes ( PR1, PR2, PR5 ) ( Table 1 ; Table 2 ). All of these genes have been linked to the clubroot immune response in previous studies (Adhikary et al, 2022; Fu et al, 2019; Irani et al, 2018; Jia et al, 2017; Singer et al, 2021; Zhou et al, 2022). While most of these genes did not show any significant changes in expression levels in the root tissues of AIL7 overexpression lines following either P. brassicae or mock-inoculation ( Figures 7A and 7C ), both PR1 and PR2 were significantly upregulated in the T-DNA mutant lines compared to wild-type plants following mock-inoculation ( Figure 7A ).…”

“…To examine the role of AIL7 in clubroot disease response, we first evaluated disease severity in homozygous AIL7 overexpression lines, T-DNA insertion ail7 knockout mutants, and wild-type Arabidopsis, following inoculation with P. brassicae pathotype 3H at a concentration of 1.0 x 10 5 resting spores/mL water. Plants began to show a purpling of the leaves approximately 16-18 days post-inoculation (Figure 5A and 5B), and root disease severity measurements were carried out 21 days post-inoculation ( Figure S1A ; Zhou et al, 2022). Both the overexpression and knockout mutants had significantly lower disease indexes (DI) (41.3% - 62.1% and 39.9 - 66.7%, respectively) than wild-type plants (72.71%), indicating enhanced resistance to P. brassicae ( Figure 5C ).…”

“…P. brassicae single-spore isolate 3H, classified based on the Canadian Clubroot Differential set (Strelkov et al, 2018), was used as the inoculum in this study due to the fact that it is widespread throughout canola-producing areas of Western Canada and exhibits high virulence against canola varieties lacking clubroot resistance (Hollman et al, 2021; Strelkov et al, 2018). Inoculum preparation and inoculation were carried out as described previously (Zhou et al, 2022). In brief, 5 g of frozen root galls, infected with pathotype 3H, were blended in 100 mL water and strained through eight layers of cheesecloth to remove plant tissue and other debris.…”

“…Clubroot severity was assessed 3 weeks following inoculation on a 0 – 3 scale based on Zhou et al (2022), where: 0 = no galls, healthy root system; 1 = small galls, primarily on the lateral roots; 2 = small to medium galls on both the primary and lateral roots, moderate reduction in root system size; and 3 = large galls on the primary root, substantial reduction in root system size. The severity ratings were then used to calculate a DI for each replicate using a formula described previously (Strelkov et al, 2006): DI (%) = [(n 1 × 1 + n 2 × 2 + n 3 × 3)/(N × 3)] × 100, where n 1 , n 2 and n 3 are the number of plants in each severity class and N is the total number of plants tested.…”