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Brassica napus Infected with Leptosphaeria maculans.

ABSTRACT: Alternative splicing (AS) is a post-transcriptional regulatory process that enhances transcriptome diversity, thereby affecting plant growth, development, and stress responses. To identify the new transcripts and changes in the isoform-level AS landscape of rapeseed (Brassica napus) infected with the fungal pathogen Leptosphaeria maculans, we compared eight RNA-seq libraries prepared from mock-inoculated and inoculated B. napus cotyledons and stems. The AS events that occurred in stems were almost the same as those in cotyledons, with intron retention representing the most common AS pattern. We identified 1892 differentially spliced genes between inoculated and uninoculated plants. We performed a weighted gene co-expression network analysis (WGCNA) to identify eight co-expression modules and their Hub genes, which are the genes most connected with other genes within each module. There are nine Hub genes, encoding nine transcription factors, which represent key regulators of each module, including members of the NAC, WRKY, TRAF, AP2/ERF-ERF, C2H2, C2C2-GATA, HMG, bHLH, and C2C2-CO-like families. Finally, 52 and 117 alternatively spliced genes in cotyledons and stems were also differentially expressed between mock-infected and infected materials, such as HMG and C2C2-Dof; which have dual regulatory mechanisms in response to L. maculans. The splicing of the candidate genes identified in this study could be exploited to improve resistance to L. maculans.

SUBMITTER: Ma JQ

PROVIDER: S-EPMC6523698 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Project description:While our understanding of the genetics underlying the Brassica-Leptosphaeria pathosystem has advanced greatly in the last decade, differences in molecular responses due to interaction between resistance genes and host genetic background has not been studied. We applied RNAseq technology to monitor the transcriptome profiles of Brassica napus (Bn) lines carrying one of four blackleg R genes (Rlm2, Rlm3, LepR1 & LepR2) in Topas or Westar background, during the early stages of infection by a Leptosphaeria maculans (Lm) isolate carrying the corresponding Avr genes. We observed upregulation of host genes involved in hormone signalling, cell wall thickening, response to chitin and glucosinolate production in all R gene lines at 3 day after inoculation (dai) albeit having higher level of expression in LepR1 and Rlm2 than in Rlm3 and LepR2 lines. Bn-SOBIR1 (Suppressor Of BIR1-1), a receptor like kinase (RLK) that forms complex receptor like proteins (RLPs) was highly expressed in LepR1 and Rlm2 at 3 dai. In contrast Bn-SOBIR1 induction was low in Rlm3 line, which could indicate that Rlm3 may function independent of SOBIR1. Expression of Salicylic acid (SA) related defense was enhanced in LepR1 and Rlm2 at 3 dai. In contrast to SA, expression of Bn genes with homology to PDF1.2, a jasmonic acid (JA) pathway marker, were increased in all Rlm and LepR lines at 6 and 9 dai. Effect of host genetic background on induction of defense, was determined by comparison of LepR1 and LepR2 in Topas vs Westar genotype (i.e. T-LepR1 vs W-LepR1 and T-LepR2 vs W-LepR2). In both cases (regardless of R gene) overall number of defense related genes at the earliest time point (3 dai) was higher in Tops compared to Westar. SA and JA markers genes such as PR1 and PDF1.2 were more induced in Topas compared to Westar introgression lines at this time point. Even in the absence of any R gene, effect of Topas genotype in enhanced defense, was also evident by the induction of PDF1.2 that started at a low level at 3 dai and peaked at 6 and 9 dai, while no induction in Westar genotype was observed at any of these time points. Overall, variation in time and intensity of expression of genes related to defense, was clearly dependent on both R gene and the host genotype.

Project description:Using cultivar resistance against pathogens is one of the most economical and environmentally friendly methods for control of crop diseases. However, cultivar resistance can be easily rendered ineffective due to changes in pathogen populations or environments. To test the hypothesis that combining R gene-mediated resistance and quantitative resistance (QR) in one cultivar can provide more effective resistance than use of either type of resistance on its own, effectiveness of resistance in eight oilseed rape (Brassica napus) cultivars with different R genes and/or QR against Leptosphaeria maculans (phoma stem canker) was investigated in 13 different environments/sites over three growing seasons (2010/2011, 2011/2012 and 2012/2013). Cultivar Drakkar with no R genes and no QR was used as susceptible control and for sampling L. maculans populations. Isolates of L. maculans were obtained from the 13 sites in 2010/2011 to assess frequencies of avirulent alleles of different effector genes (AvrLm1, AvrLm4 or AvrLm7) corresponding to the resistance genes (Rlm1, Rlm4 or Rlm7) used in the field experiments. Results of field experiments showed that cultivars DK Cabernet (Rlm1 + QR) and Adriana (Rlm4 + QR) had significantly less severe phoma stem canker than cultivars Capitol (Rlm1) and Bilbao (Rlm4), respectively. Results of controlled environment experiments confirmed the presence of Rlm genes and/or QR in these four cultivars. Analysis of L. maculans populations from different sites showed that the mean frequencies of AvrLm1 (10%) and AvrLm4 (41%) were less than that of AvrLm7 (100%), suggesting that Rlm1 and Rlm4 gene-mediated resistances were partially rendered ineffective while Rlm7 resistance was still effective. Cultivar Excel (Rlm7 + QR) had less severe canker than cultivar Roxet (Rlm7), but the difference between them was not significant due to influence of the effective resistance gene Rlm7. For the two cultivars with only QR, Es-Astrid (QR) had less severe stem canker than NK Grandia (QR). Analysis of the relationship between severity of stem canker and weather data among the 13 sites in the three growing seasons showed that increased severity of stem canker was associated with increased rainfall during the phoma leaf spot development stage and increased temperature during the stem canker development stage. Further analysis of cultivar response to environmental factors showed that cultivars with both an Rlm gene and QR (e.g. DK Cabernet, Adriana and Excel) were less sensitive to a change in environment than cultivars with only Rlm genes (e.g. Capitol, Bilbao) or only QR (e.g. DK Grandia). These results suggest that combining R gene and QR can provide effective, stable control of phoma stem canker in different environments.

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Brassica napus Infected with Leptosphaeria maculans.

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