Potential mechanisms of quantitative resistance to Leptosphaeria maculans (blackleg) on cotyledons of canola (Brassica napus)

Abstract Background: Blackleg disease, caused by Leptosphaeria maculans (Lm), can lead to significant losses of canola/rapeseed crops. Growing resistant canola cultivars can be an effective and environmentally friendly way to manage blackleg. Major resistance genes may stop infection, but can also be rapidly overcome by shifts in pathogen population towards virulence. Thus, using race-nonspecific or quantitative resistance (QR) is of interest because it is potentially more durable. However, the mechanisms and genes underlying QR are mostly unknown. In this study, we explored QR in “74‑44 BL”, a Canadian canola cultivar carrying a moderate level of race nonspecific resistance, based on cotyledon inoculation (Supple. Fig.1) . The susceptible cultivar “Westar” was used as a control. Lesions developed more slowly on 74-44BL than on Westar. We used RNA-Seq to identify genes and gene functions putatively involved in the QR. Results: Relative to inoculated Westar, some of the B. napus genes that were differentially expressed strongly in inoculated 74-44 BL included those putatively involved in programmed cell death (PCD), reactive oxygen species (ROS) generation, signal transduction and/or intracellular endomembrane transport. Examples included genes annotated as a Bax inhibitor 1, a development/cell death (DCD) domain containing proteinases and peptidases, all of which could play a role in PCD and a zinc-finger Sec23/Sec24 and five small GTPases likely involved in endoplasmic reticulum (ER) to Golgi vesicle traffic and/or signal transduction. Further experiments, however, did not confirm changes in genomic DNA degradation, a potential marker for PCD, between the two cultivars. In addition, infection progression in cotyledons was not altered by applying protease inhibitors directly to cotyledons. Additional testing was done using green fluorescent protein (GFP)-tagged Lm for cotyledon colonization as well as ROS production, in relation to the lesion development. The results showed that ROS production occurred beyond the area colonized by Lm hyphae in 74-44 BL.Conclusions: ROS may also be involved in signal transduction and/or intracellular endomembrane transport. These results provide a starting point for a better understanding of the mechanisms behind QR against Lm in canola and developing new host-resistance strategies for management of blackleg.