Infection symptoms and Lm hyphal growth in cotyledons
Among the seedlings inoculated and grown in parallel with those used for RNA-Seq, Westar showed higher infection ratings than 74-44 BL at 14 dpi (Fig. 1B). In separate experiments, however, the appearance and size of lesions, as well as the distance from the inoculation wound to lesion edge, were similar between the two cultivars at 7 dpi, while the area colonized by Lm hyphae and the distance from the inoculation site to the most distal hyphal tips were greater in Westar (Fig. 1C and E). By 10 and 14 dpi, all of the measurements had become greater in Westar than 74-44 BL (Fig. 1D and E).
RNA-sequencing
For each treatment, 14.1-17.8 million paired-end reads were produced per library. When reads were considered singly, an average of 33.5 ± 2.0, 21.3 ± 0.06, 33.0 ± 2.0 and 28.2 ± 1.8 million reads were mapped to the B. napus genome from mock‑inoculated Westar, Lm‑inoculated Westar, mock-inoculated 74-44 BL and Lm‑inoculated 74-44 BL, respectively. In comparison, an average of 10.1 ± 1.2, 5.8 ± 0.6, 0.07 ± 0.002 and 0.07 ± 0.010 reads were mapped to the L. maculans genome from Lm-inoculated Westar, inoculated 74-44 BL, mock-inoculated Westar and mock-inoculated 74-44 BL, respectively. A higher percentage of reads mapped to the L. maculans genome in inoculated Westar as compared to inoculated 74-44 BL (Fig. 2A). Principle component analysis (PCA) indicated that the treatments grouped tightly together in terms of their alignment to the B. napus genome (Fig. 2B).
Gene expression in L. maculans
When the criteria of adjusted p-value ≤ 0.05 and log2 fold change ≥ 2 in expression were applied, there were only 16 differentially expressed Lm genes between inoculated Westar and 74-44 BL. Three DEGs were more highly expressed in inoculated 74-44 BL as compared to inoculated Westar (Table 1) while thirteen DEGs showed the reverse trend (Table 2).
The Lm DEGs were all expressed at low levels. When all Lm genes were considered, there were eight genes with basemean expression values over 10,000 (ranging from 14,346 to 40,534). Three genes had expression values between 1,000 and 9,999, 85 between 100 and 999 and 152 between 50 and 99. There were a total of 12,119 genes with non-zero expression values. The most highly expressed DEG had a basemean of 40.
The three DEGs upregulated in Lm inoculated 74-44 BL had sequence similarities to genes encoding a short chain dehydrogenase/reductase, a pyoverdine biosynthesis and a hypothetical protein, respectively. Pyoverdine is a siderophore biosynthesized by Pseudomonads [28]. Zwiers et al. [29] found a gene encoding an ABC-transporter with a pyoverdine biosynthesis motif in the fungus Mycosphaerella graminicola; ABC-transporters can play a role in virulence of fungal pathogens towards host plants [30, 31]. It is unclear what role, if any, these upregulated genes played during the infection of 74-44BL by Lm.
Gene expression in B. napus
Genes upregulated in inoculated 74-44 BL
There were 908 DEGs upregulated in inoculated 74-44 BL, relative to inoculated Westar, but not differentially expressed between any other pairs of treatments. Two DEGs showed basemean expression levels over 10,000, six had basemeans between 5000 and 9999, and 65 had basemeans between 4,999 and 1,000.
Five DEGs with similarities to peptidases were among those with the highest scores. Indeed, the three DEGs with the highest scores were all putative peptidases. The DEG with the highest score is BnaA01g17570D, which has InterPro domains suggesting it is a cysteine peptidase belonging to family C1, sub-family C1A, papain family. Another DEG with a high score is BnaA09g52180D, a putative cysteine peptidase. The legumain peptidase C13 (BnaA01g04000D), also known as a vacuole processing enzyme (VPE), and BnaC02g00130D which has similarity to a protease involved in the degradation of Rubisco, were also upregulated. Additionally, numerous chlorophyll A-B binding proteins showed very high basemeans and were more highly expressed in inoculated 74-44 BL than inoculated Westar.
An ATPase of AAA-type, with protein BLAST similarity to RuBisCO activase, was also differentially expressed. The protein BLAST results also indicate that this DEG is potentially involved in endoplasmic reticulum (ER) to Golgi membrane budding.
Glycoside hydrolases, including a beta-galactosidase (BnaA04g04110D) and an alpha-1,6-glucosidases, pullulanase-type (BnaA10g25820D) are differentially expressed, with very small adjusted p-values. A putative lactate/malate dehydrogenase (BnaC02g00740D) is also differentially expressed, albeit with a less significant adjusted p-value and higher basemean expression than BnaA04g04110D or BnaA10g25820D. The DEG BnaA03g11710D, with a thiazole biosynthetic enzyme InterPro domain, also has protein sequence similarity to a ribulose-1,5-biphoshate synthetase. Table 3 and Fig. 3 summarize the putative functions of DEGs that are more highly expressed in inoculated 74-44 BL as compared to inoculated Westar.
GO term enrichment analysis of these 908 DEGs was consistent with the results presented in Table 3 and Fig. 3 in which many of GO terms with the lowest FDR were related to photosynthesis and light responses. Furthermore, three GO terms were linked to hydrogen peroxide. While none of the enriched GO terms suggested peptidase activities, the GO term with the second lowest FDR was associated with cysteine biosynthesis (Table 5). This is consistent with the putative cysteine peptidase activity of BnaA01g17570D (Table 3).
Genes upregulated in inoculated Westar
A total of 640 DEGs were more highly expressed in inoculated Westar as compared to inoculated 74-44 BL, but not differentially expressed when any other pair of treatments were compared. The expressions of these DEGs ranged from a basemean of 3,410 to 1.25, with only 11 DEGs showing basemeans over 1,000. Twenty eight DEGs had basemeans between 500 and 999, while 73 had basemeans between 100 and 499. The remaining 527 DEGs had basemeans under 100.
The DEG with the highest score, BnaC09g20030D, showed similarity to a Bax inhibitor-1. BnaCnng58090D, a DEG with a basemean of 2,354, is similar to a development/cell death domain (DCD). BnaC08g42820D is a DEG similar to a heat shock protein 70. BnaA04g06220D and BnaA09g26960D have similarities to Sec23/Sec24 and Sec61/SecY, respectively. Sec23 and sec24 are part of the coat protein II (COPII) complex, involved in ER to Golgi vesicle transport [32]. Five DEGs, BnaA08g26550D, BnaA06g05280D, BnaC06g24690D, BnaA07g09950D and BnaCnng06680D appeared similar to small GTPases. These DEGs have basemeans ranging from 972 to 3,100. Table 4 and Fig. 3 summarize these DEGs.
GO terms related to the ER, ER stress, vesicle transport and the cellular endomembrane system were enriched. None of the enriched GO terms, however, were associated with PCD. One enriched GO term was related to response to hydrogen peroxide (Table 6). BnaCnng58090D is not associated with any GO terms.
Hydrogen peroxide in cotyledons
RNA-seq results suggested that ROS, such as hydrogen peroxide, may play a role in the QR to Lm carried by 74-44 BL. To validate this finding, DAB staining was used to quantify the area of ROS production surrounding the infection site.
Hydrogen peroxide at seven days post inoculation
The size of visible lesion, area of hyphal colonization and area with ROS detection in cotyledons varied, depending on the cultivar and parameter measured. In inoculated Westar, the area colonized by hyphae (as visualized by GFP fluorescence) and area staining positive for hydrogen peroxide were both larger than the area of necrotic lesions, while the former two parameters were not different from each other (Fig. 4A). In contrast, the lesion size and area colonized by GFP-tagged Lm hyphae did not differ in 74-44 BL, whereas the area with ROS staining was bigger than that of former two. As with the results in Fig. 1, the lesion size did not differ between Westar and 74-44 BL at 7 dpi, while the area colonized by Lm hyphae was substantially greater in Westar. The area with ROS staining did not differ between the cultivars at 7 dpi either (Fig. 4).
Hydrogen peroxide time series experiment
When examined over time post inoculation, most of the parameters measured tended to increase over time. Westar and 74-44 BL responded differently to the Lm infection. In Westar, the lesions were consistently smaller than either the area colonized by Lm hyphae or the area with ROS staining (Tukey adjusted p ≤ 0.05) (Fig. 5). In 74-44 BL, however, the area stained for hydrogen peroxide was larger than that occupied by fungal hyphae or visible lesions (Tukey adjusted p ≤ 0.05), and the area of pathogen colonization was either smaller than (11 dpi) or not different (5, 7 and 9 dpi) from the size of lesion (Tukey adjusted p ≤ 0.05; Fig. 5).
Genomic DNA degradation as an indicator of programmed cell death
Because the RNA-seq results suggested that PCD could play a role in QR to Lm in 74-44 BL, we examined degradation of genomic DNA as a proxy for PCD. No difference in genomic DNA degradation was apparent between any of the treatments by either agarose gel electrophoresis or Experion 12K (Fig. 6).
Impact of protease inhibitors on Lm infection of cotyledons
Results from the RNA-seq experiments led us to hypothesize that proteases could contribute to 74-44 BL QR to Lm. We attempted to test this hypothesis by treating cotyledons with several protease inhibitors. The direct application of protease inhibitors to surface of Westar or 74-44 BL cotyledons did not have a significant impact on either the lesion size or the area colonized by Lm hyphae within a given cultivar. However, the latter was consistently greater in Westar than in 74-44 BL cotyledons, regardless of the protease inhibitor used (Fig. 7).