Fine mapping of Rcr1 and analyses of its effect on transcriptome patterns during infection by Plasmodiophora brassicae

Mingguang Chu 0 Tao Song 0 Kevin C Falk Xingguo Zhang Xunjia Liu Adrian Chang Rachid Lahlali Linda McGregor Bruce D Gossen Gary Peng Fengqun Yu 0 Equal contributors Department of Agriculture and Agri-Food Canada (AAFC), Saskatoon Research Centre , 107 Science Place, Saskatoon, Saskatchewan S7N 0X2 , Canada Background: The protist Plasmodiophora brassicae is a biotrophic soil-borne pathogen that causes clubroot on Brassica crops worldwide. Clubroot disease is a serious threat to the 8 M ha of canola (Brassica napus) grown annually in western Canada. While host resistance is the key to clubroot management, sources of resistance are limited. Results: To identify new sources of clubroot resistance (CR), we fine mapped a CR gene (Rcr1) from B. rapa ssp. chinensis to the region between 24.26 Mb and 24.50 Mb on the linkage group A03, with several closely linked markers identified. Transcriptome analysis was conducted using RNA sequencing on a segregating F1 population inoculated with P. brassicae, with 2,212 differentially expressed genes (DEGs) identified between plants carrying and not carrying Rcr1. Functional annotation of these DEGs showed that several defense-related biological processes, including signaling and metabolism of jasmonate and ethylene, defensive deposition of callose and biosynthesis of indole-containing compounds, were up-regulated significantly in plants carrying Rcr1 while genes involved in salicylic acid metabolic and signaling pathways were generally not elevated. Several DEGs involved in metabolism potentially related to clubroot symptom development, including auxin biosynthesis and cell growth/development, showed significantly lower expression in plants carrying Rcr1. Conclusion: The CR gene Rcr1 and closely linked markers will be highly useful for breeding new resistant canola cultivars. The identification of DEGs between inoculated plants carrying and not carrying Rcr1 is an important step towards understanding of specific metabolic/signaling pathways in clubroot resistance mediated by Rcr1. This information may help judicious use of CR genes with complementary resistance mechanisms for durable clubroot resistance. - Background Clubroot, caused by the biotrophic protist Plasmodiophora brassicae Woronin, is one of the most serious diseases of Brassica crops worldwide [1]. In western Canada, clubroot disease has become a major threat to the production of canola (Brassica napus L) [2], where more than 8 M ha of canola crops are grown annually [3]. The pathogen is able to survive for up to 20 years in soil [4] and many conventional disease-management measures, including cultural techniques and application of fungicides, are not effective [3,5,6]. Genetic resistance is the most effective and economical approach to clubroot management on canola. European fodder turnips (Brassica rapa L. ssp. rapifera) are the major source of clubroot-resistance (CR) genes, which have been introduced into other Brassica crops including oilseed rape (B. napus), rutabaga (B. napus L. ssp. napobrassica) and Chinese cabbage (B. rapa L. ssp. chinensis) [7-11]. Since 2009, several resistant (R) canola cultivars have been released in Canada, and all of them carry a single dominant CR gene. The source and genetic information are not revealed for these CR genes [12]. The durability of these clubroot R cultivars remains unknown in western Canada, but resistance conferred by a single gene is generally not durable. Breakdown of clubroot resistance has been reported on Chinese cabbage [13] and oilseed rape [14,15]. A resistant canola cultivar showed substantially increased clubroot severity after being exposed to pathotype 3 of P. brassicae after only two cycles under controlled conditions [16]. Rotation or pyramiding of CR genes with different mechanisms of resistance may be used to increase the durability of clubroot resistance if a diverse group of CR genes can be identified and their resistance mechanisms characterized. Our prior work evaluated 955 Brassica accessions and identified a range of CR candidates from B. rapa, B. nigra and B. oleracea [17]. Most of the known CR genes have been identified from B. rapa, with eight loci reported previously: Crr1, Crr2, Crr3, Crr4, CRa, CRb, CRc and CRk [18-22]. CRa and Crr1 have been isolated recently [23,24]. Another CR gene, RPB1, was identified from Arabidopsis thaliana ecotype Tsu-0 [25], but there has been no further report on its orthlogs in other Arabidopsis ecotypes. A new CR gene (Rpb1) was identified recently from the cv. Flower Nabana (FN) of pak choy (B. rapa ssp. chinensis) via rough mapping [26]. Rpb1 is identical to Rcr1described in this paper, and the name change was to avoid potential confusion with the RPB1 from Arabidopsis. There has been little information on molecular mechanisms associated with any of the CR genes reported. In A. thaliana, host metabolism was altered by P. brassicae infection; transcriptome studies based on microarray analysis showed that genes encoding enzymes involved in carbohydrate metabolism were upregulated in root tissues of the susceptible (S) Col-0 ecotype [27,28], but not in moderately resistant (MR) ecotypes which appeared to reduce or delay pathogen-triggered metabolic diversion and cell enlargement or proliferation in the host [29]. Reduced trehalose and arginine metabolism were also reported with the partially resistant A. thaliana ecotype Bur-0 when compared with that in a susceptible ecotype [30,31]. Secondary metabolism, including flavonoids, may also contribute to formation of characteristic club symptoms in Arabidopsis, and inhibition of oxoglutaric aciddependent dioxygenases reduced club development [32]. Treatment with the phytohormone salicylic acid or biofungicides reduced clubroot development on A. thaliana and B. napus via activation of several defense-related pathways in the hosts [33-36]. However, there is no information on molecular mechanisms of clubroot resistance in Brassica species based on transcriptome analysis. RNA sequencing (RNA-seq) has been employed recently to elucidate resistance mechanisms involved in plant-pathogen interactions including Sclerotinia homoeocarpea-creeping bentgrass [37] and Phytophthora infestans-potato tuber [38]. In the present study, we intended to: 1) identify and characterize the CR gene from a highly resistant pak choy cultivar using genetic mapping; 2) develop molecular markers closely linked to this CR gene to facilitate marker-assisted selection (MAS) at the young seedling stage; and 3) analyze the global transcriptome profile associated with the CR gene based on RNA-seq. We examined differential gene expression between R and S F1 plants, and the result provided important insights into the molecular mechanisms of clubroot resistance. This work also sets the first step toward the development of canola germplasm using CR genes with potentially different modes of action against clubroot. Results The clubroot resistance in c (...truncated)