Exploring Mechanisms of Quantitative Resistance to Leptosphaeria maculans (Blackleg) in the Cotyledons of Canola (Brassica napus) Based on Transcriptomic and Microscopic Analyses

Abstract

<jats:p>Using resistant cultivars is a common approach to managing blackleg of canola/rapeseed caused by Leptosphaeria maculans (Lm). Quantitative resistance (QR), as opposed to major-gene resistance, is of interest because it is generally more durable, due to its multi-genetic basis. However, the mechanisms and genes underlying QR are mostly unknown. In this study, potential QR modes of action in “74‑44 BL” was explored. This Canadian canola cultivar showed moderate but consistent race-nonspecific resistance at the cotyledon and adult-plant stages. A susceptible cultivar, “Westar”, was used as a control. After inoculation, the lesions developed more slowly on the cotyledons of 74-44 BL than those of Westar. We used RNA sequencing (-RNA-seq) to identify genes and their functions, putatively related to this resistance, and found that genes involved in programmed cell death (PCD), reactive oxygen species (ROS), signal transduction or intracellular endomembrane transport were most differentially expressed. ROS production was assessed in relation to Lm hyphal growth and lesion size; it occurred beyond the tissue colonized by Lm in 74-44 BL and appeared to trigger rapid cell death, limiting cotyledon colonization by Lm. In contrast, Lm grew more rapidly in Westar, often catching up with the ring of ROS and surpassing lesion boundaries. It appears that QR in 74-44 BL cotyledons is associated with limited colonization by Lm possibly mediated via ROS. The RNA-seq data also showed a link between ROS, signal transduction, and endomembrane vesicle trafficking, as well as PCD in the resistance. These results provide a starting point for a better understanding of the mechanisms behind QR against Lm in canola.</jats:p>

Comparison of non-subjective relative fungal biomass measurements to quantify the Leptosphaeria maculans—Brassica napus interaction

“…However, visual scoring of lesions on cotyledons or leaves may not provide an accurate measure of pathogen load. In this and other studies, microscopic images of cotyledon colonisation show abundant hyphae invading tissue beyond the visual boundary of the lesion (Huang et al [ 31 ], Hubbard et al [ 43 ]). In turn, hyphal colonisation can be significantly smaller than the areas stained for ROS (Reaction Oxygen Species involved in Programmed Cell Death) such as hydrogen peroxide using 3,3-diaminobenzidine (DAB) (Hubbard et al [ 43 ]).…”

“…In this and other studies, microscopic images of cotyledon colonisation show abundant hyphae invading tissue beyond the visual boundary of the lesion (Huang et al [ 31 ], Hubbard et al [ 43 ]). In turn, hyphal colonisation can be significantly smaller than the areas stained for ROS (Reaction Oxygen Species involved in Programmed Cell Death) such as hydrogen peroxide using 3,3-diaminobenzidine (DAB) (Hubbard et al [ 43 ]). The described assays could be used in conjunction with standard visual scoring techniques to provide more precise differentiation of host responses, particularly in the identification of novel sources of genetic resistance that act in the leaf during infection.…”

Breeding and management of major resistance genes to stem canker/blackleg in Brassica crops

“…Resistance gene analog prediction can also be compared or applied to transcriptomic data (e.g. Lowe et al 2014;Becker et al 2017Becker et al , 2019Hubbard et al 2020;Song et al 2021) and applied across accessions with known resistance responses and large databases such as BnPIR. This latter approach can be particularly useful, considering the high diversity in terms of gene content between accessions, to assess the conservation of a particular candidate resistance gene analog (Song et al 2020).…”

Status and advances in mining for blackleg (Leptosphaeria maculans) quantitative resistance (QR) in oilseed rape (Brassica napus)

“…For QR, the role of a diverse set of genes has been proposed. In a recent transcriptome profiling experiment, Hubbard et al ( 2020 ) found genes controlling programmed cell death (PCD), reactive oxygen species (ROS) and intracellular endomembrane transport as critical regulators of QR. The ROS burst was coincident with inhibited fungal growth and rapid cell death.…”