Abstract
The family Brassicaceae is a source of important crop species, including Brassica napus (oilseed rape), Brassica oleracea, and B. rapa, that is used globally for oil production or as a food source (e.g., pak choi or turnip). However, despite advances in recent years, including genome sequencing, a lack of established tools tailored to the study of Brassica crop species has impeded efforts to understand their molecular processes in greater detail. Here, we describe the use of a simple Agrobacterium‐mediated transient expression system adapted to B. rapa and B. napus that could facilitate study of molecular and biochemical events in these species. We also demonstrate the use of this method to characterize the N‐degron pathway of protein degradation in B. rapa. The N‐degron pathway is a subset of the ubiquitin‐proteasome system and represents a mechanism through which proteins may be targeted for degradation based on the identity of their N‐terminal amino acid residue. Interestingly, N‐degron‐mediated processes in plants have been implicated in the regulation of traits with potential agronomic importance, including the responses to pathogens and to abiotic stresses such as flooding tolerance. The stability of transiently expressed N‐degron reporter proteins in B. rapa indicates that its N‐degron pathway is highly conserved with that of Arabidopsis thaliana. These findings highlight the utility of Agrobacterium‐mediated transient expression in B. rapa and B. napus and establish a framework to investigate the N‐degron pathway and its roles in regulating agronomical traits in these species.Significance statementWe describe an Agrobacterium‐mediated transient expression system applicable to Brassica crops and demonstrate its utility by identifying the destabilizing residues of the N‐degron pathway in B. rapa. As the N‐degron pathway functions as an integrator of environmental signals, this study could facilitate efforts to improve the robustness of Brassica crops.
Highlights
The Brassica genus is a source of important crops, including B. napus, B. oleracea, and B. rapa
To validate the use of the agroinfiltration protocol, we transiently expressed N-degron reporter constructs to determine the sets of N-terminal destabilizing residues that serve as degradation signals for the PRT6 and PRT1/N-degron pathways in B. rapa
We show that seedling co-cultivation with Agrobacterium and leaf agroinfiltration allow transient expression of transgenes in B. rapa
Summary
The Brassica genus is a source of important crops, including B. napus (oilseed rape), B. oleracea (broccoli and cauliflower), and B. rapa. B. napus and B. rapa are grown globally mainly for vegetable oil derived from their seeds Both crops are widely used as fodder for livestock, while some B. rapa cultivars are grown to produce food for human consumption including pak choi and turnip. A simple floral dip method has been established for B. napus (Verma, Chinnusamy, & Bansai, 2008) While this approach facilitates the transformation procedure and the isolation of transformants, the generation time of Brassica crop species remains a bottleneck to dissect mechanisms swiftly. The development of transient expression approaches in Brassica crop species has so far been hindered by low transformation efficiency or the need for biolistic equipment, limited examples of agroinfiltration methods have emerged in recent years (Das, Naiya, Marik, Mukherjee, & Seal, 2019; Zhong et al, 2016). Were washed with autoclaved ddH2O three times and placed on 0.5x MS agar plates for 24 hr before GUS staining procedure
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