Genome-wide transcriptome reveals mechanisms underlying Rlm1-mediated blackleg resistance on canola.

Genetic resistance to blackleg (Leptosphaeria maculans, Lm) of canola (Brassica napus, Bn) has been extensively studied, but the mechanisms underlying the host-pathogen interaction are still not well understood. Here, a comparative transcriptome analysis was performed on a resistant doubled haploid Bn line carrying the resistance gene Rlm1 following inoculation with a virulent (avrLm1) or avirulent (AvrLm1) Lm isolate on cotyledons. A total of 6999 and 3015 differentially expressed genes (DEGs) were identified, respectively, in inoculated local tissues with compatible (susceptible) and incompatible (resistant) interactions. Functional enrichment analysis found several biological processes, including protein targeting to membrane, ribosome and negative regulation of programmed cell death, were over-represented exclusively among up-regulated DEGs in the resistant reaction, whereas significant enrichment of salicylic acid (SA) and jasmonic acid (JA) pathways observed for down-regulated DEGs occurred only in the susceptible reaction. A heat-map analysis showed that both biosynthesis and signaling of SA and JA were induced more significantly in the resistant reaction, implying that a threshold level of SA and JA signaling is required for the activation of Rlm1-mediated resistance. Co-expression network analysis revealed close correlation of a gene module with the resistance, involving DEGs regulating pathogen-associated molecular pattern recognition, JA signaling and transcriptional reprogramming. Substantially fewer DEGs were identified in mock-inoculated (control) cotyledons, relative to those in inoculated local tissues, including those involved in SA pathways potentially contributing to systemic acquired resistance (SAR). Pre-inoculation of cotyledon with either an avirulent or virulent Lm isolate, however, failed to induce SAR on remote tissues of same plant despite elevated SA and PR1 protein. This study provides insights into the molecular mechanism of Rlm1-mediated resistance to blackleg.

Fine mapping of Brassica napus blackleg resistance gene Rlm1 through bulked segregant RNA sequencing.

The fungal pathogen Leptosphaeria maculans causes blackleg disease on canola and rapeseed (Brassica napus) in many parts of the world. A B. napus cultivar, 'Quinta', has been widely used for the classification of L. maculans into pathogenicity groups. In this study, we confirmed the presence of Rlm1 in a DH line (DH24288) derived from B. napus cultivar 'Quinta'. Rlm1 was located on chromosome A07, between 13.07 to 22.11 Mb, using a BC1 population made from crosses of F1 plants of DH16516 (a susceptible line) x DH24288 with bulked segregant RNA Sequencing (BSR-Seq). Rlm1 was further fine mapped in a 100 kb region from 19.92 to 20.03 Mb in the BC1 population consisting of 1247 plants and a F2 population consisting of 3000 plants using SNP markers identified from BSR-Seq through Kompetitive Allele-Specific PCR (KASP). A potential resistance gene, BnA07G27460D, was identified in this Rlm1 region. BnA07G27460D encodes a serine/threonine dual specificity protein kinase, catalytic domain and is homologous to STN7 in predicted genes of B. rapa and B. oleracea, and A. thaliana. Robust SNP markers associated with Rlm1 were developed, which can assist in introgression of Rlm1 and confirm the presence of Rlm1 gene in canola breeding programs.

Fine Mapping of a Clubroot Resistance Gene in Chinese Cabbage Using SNP Markers Identified from Bulked Segregant RNA Sequencing.

Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola (Brassica napus) in western Canada and worldwide. In this study, a clubroot resistance gene (Rcr2) was identified and fine mapped in Chinese cabbage cv. "Jazz" using single-nucleotide polymorphisms (SNP) markers identified from bulked segregant RNA sequencing (BSR-Seq) and molecular markers were developed for use in marker assisted selection. In total, 203.9 million raw reads were generated from one pooled resistant (R) and one pooled susceptible (S) sample, and >173,000 polymorphic SNP sites were identified between the R and S samples. One significant peak was observed between 22 and 26 Mb of chromosome A03, which had been predicted by BSR-Seq to contain the causal gene Rcr2. There were 490 polymorphic SNP sites identified in the region. A segregating population consisting of 675 plants was analyzed with 15 SNP sites in the region using the Kompetitive Allele Specific PCR method, and Rcr2 was fine mapped between two SNP markers, SNP_A03_32 and SNP_A03_67 with 0.1 and 0.3 cM from Rcr2, respectively. Five SNP markers co-segregated with Rcr2 in this region. Variants were identified in 14 of 36 genes annotated in the Rcr2 target region. The numbers of poly variants differed among the genes. Four genes encode TIR-NBS-LRR proteins and two of them Bra019410 and Bra019413, had high numbers of polymorphic variants and so are the most likely candidates of Rcr2.

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

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.

Identification and immunogold localization of a novel bromegrass (Bromus inermis Leyss) peroxisome channel protein induced by ABA, cold and drought stresses, and late embryogenesis.

A cDNA (BG-15) was isolated through differential screening of a cDNA library made from an ABA-treated bromegrass (Bromus inermis Leyss) suspension cell culture. The 819 bp pair cDNA encoded a 174 amino acid polypeptide with a calculated molecular mass of 18.08 kD and isolectric point of 7.50. The deduced amino acid sequences for the cDNA were 29.5% and 32.6% homologous to the known amino acid-selective channel proteins of the chloroplastic outer membrane in pea and barley, but were highly homologous (55.6% to 83.2%) to the putative membrane channel proteins from rice and Arabidopsis. Immunogold localization demonstrated that the channel protein encoded by this cDNA was present on the peroxisome membrane. High stringency southern analysis revealed that 1 to 2 copies of the peroxisomal channel protein (PCP) genes were present in the bromegrass genome. Northern and Western blots revealed that the PCP gene was responsive to both cold and drought stresses, and was rapidly induced by ABA (75 microM). The transcript of the PCP gene also accumulated during late embryogenesis, but declined rapidly during germination. Data taken together, responsiveness of the PCP to cold and drought stresses, and accumulation during late embryogenesis suggest this novel peroxisomal channel protein is associated with sugar and fatty acid metabolism through fatty acid import or succinate export from peroxisome during desiccation tolerance and energy metabolism.

A lipid transfer protein gene BG-14 is differentially regulated by abiotic stress, ABA, anisomycin, and sphingosine in bromegrass (Bromus inermis).

The objective was to investigate the expression of a lipid transfer protein gene (LTP) both in bromegrass (Bromus inermis) cells and seedlings after exposure to abiotic stresses, abscisic acid (ABA), anisomycin, and sphingosine. A full-length cDNA clone BG-14 isolated from bromegrass suspension cell culture encodes a polypeptide of 124 amino acids with typical LTP characteristics, such as a conserved arrangement of cysteine residues. During active stages of cold acclimation LTP expression was up-regulated, whereas at the final stage of cold acclimation LTP transcript level declined to pre-acclimation level. A severe drought stress induced the LTP gene; yet, LTP expression doubled 3 d after re-hydration. Both temperature and heat shock duration influence LTP induction; however temperature is the primary factor. Treatment with NaCl stimulated accumulation of LTP mRNA within 15 min and the transcripts remained at elevated levels for the duration of the salinity stress. Most interestingly, Northern blots showed LTP was rapidly induced not only by ABA, but also by anisomycin and sphingosine in suspension cell cultures. Of the three chemicals, ABA induced the most rapid and highest response in LTP expression as well as highest freezing tolerance, whereas sphingosine was the least active for both LTP expression and freezing tolerance.