Page 159 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

such as the predicted -helical transmembrane domain and positively charged flanking regions that play
a role in the targeting of OEP6 to the chloroplast outer membrane. Fusion OEP6 constructs tagged with
green fluorescent protein (GFP) will be transformed into onion epidermal cells using biolistic
bombardment and introduced into Arabidopsis mesophyll cell protoplasts using polyethylene glycol-
mediated protocols where their intracellular localization will be determined using epifluorescence and
confocal microscopy. To test the importance of secondary structures of OEP6 in its targeting, the
localization of fusion constructs containing mutations or deletions of these predicted secondary structures
in plant cells will be examined. The findings generated from this study will contribute to the overall
understanding of intracellular protein trafficking, specifically the role of protein secondary structures or
motifs with defined properties. Specific to the chloroplast, understanding the targeting and import of
proteins will have applications to both the agricultural and biotechnological industries.

*[O113] PLASTID MOLECULAR CHAPERONE HSP90C INTERACTS WITH THE SECA1 SUBUNIT OF
SEC TRANSLOCASE FOR THYLAKOID PROTEIN TRANSPORT. Adheip Monikantan Nair, Tim Jiang,
Bona Mu, and Rongmin Zhao. Department of Biological Sciences, University of Toronto Scarborough,
Toronto, ON; Department of Cell & Systems Biology, University of Toronto, Toronto, ON
Correspondence to: adheip.nair@mail.utoronto.ca

The plastid stroma-localized chaperone HSP90C plays a crucial role in maintaining optimal proteostasis
within chloroplasts and participates in protein translocation processes. While existing studies have
revealed HSP90C’s direct interaction with the Sec translocase-dependent client pre-protein PsbO1 and
the SecY1 subunit of the thylakoid membrane-bound Sec1 translocase channel system, its direct
involvement with the extrinsic homodimeric Sec translocase subunit, SecA1, remains elusive. Employing
bimolecular fluorescence complementation (BiFC) assay and other in vitro analyses, we unravelled
potential interactions between HSP90C and SecA1. Our investigation revealed dynamic interactions
between HSP90C and SecA1 at the thylakoid membrane and stroma. The thylakoid membrane
localization of this interaction was contingent upon active HSP90C ATPase activity, whereas their stromal
interaction was associated with active SecA1 ATPase activity. Furthermore, we observed a direct
interaction between these two proteins by analyzing their ATP hydrolysis activities, and their interaction
likely impacts their respective functional cycles. Additionally, using PsbO1, a model Sec translocase client
pre-protein, we studied the intricacies of HSP90C’s possible involvement in pre-protein translocation via
the Sec1 system in chloroplasts. The results suggest a complex nature of the HSP90C-SecA1 interaction,
possibly mediated by the Sec client protein. Our studies shed light on the nuanced aspects of HSP90C’s
engagement in orchestrating pre-protein translocation, and we propose a potential collaborative role of
HSP90C with SecA1 in actively facilitating pre-protein transport across the thylakoid membrane.

[O114] ADVANCING CANOLA PROTECTION: QPCR SCREENING AND MARKER DEVELOPMENT
1
FOR VERTICILLIUM STRIPE DISEASE RESISTANCE. Mohamed Samir Youssef , W. G. Dilantha
1
2
1
Fernando , Robert Duncan , Sally Vail , Isobel A. P. Parkin , and Harmeet Singh Chawla . Department
1 1
2
of Plant Science, University of Manitoba, 66 Dafoe Road, Winnipeg, MB, Canada, R3T2N2; and
2 Saskatoon Research and Development Centre, Agriculture and Agri-Food Canada, Saskatoon, SK,
Canada
Correspondence to: harmeet.chawla@umanitoba.ca

Verticillium longisporum (VL), a soil-borne vascular fungal pathogen, poses a significant threat to
cruciferous crops including canola (oilseed rape), and can cause up to 80% yield loss in severe cases. VL
gains access to plants through the roots and colonizes the vascular system, resulting in verticillium stripe
(VS) disease. This disease is an emerging threat to canola production in Canada, and its severity is
anticipated to increase with rising temperatures due to climate change and increasing inoculum load in
the fields with VL infestations. Thus, developing canola cultivars resistant to VS is critical to protect
Western Canadian producers from substantial yield losses. A unique characteristic of this pathogen is its
systemic, non-homogenous, and delayed colonization of the plant xylem, leading to an extended
symptomless latency period. Consequently, the severity of infection in the field is challenging to assess,
as symptoms become apparent only at crop maturity, and are often confused with natural senescence.
Traditional methods for assessing Verticillium disease severity in canola, such as visual scoring of
microsclerotia on harvested stubble, unsatisfactorily reflect genotypic resistance as they are heavily

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