Page 171 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

[O133b] ASCOPHYLLUM NODOSUM-DERIVED FUCOIDAN INDUCES FLOWERING BY
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REGULATING THE MIR156-MEDIATED AGE PATHWAY IN ARABIDOPSIS. Ramin Bahmani , Pramod
Rathor , and Balakrishnan Prithiviraj . Marine Bio-Products Research Laboratory, Department of Plant,
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Food and Environmental Sciences, Faculty of Agriculture, Dalhousie University, Nova Scotia, B2N5E3,
Canada
Correspondence to: bprithiviraj@dal.ca

Flowering, the change from vegetative development to the reproductive phase, represents a crucial and
intricate stage in the life cycle of plants, which is tightly controlled by both internal and external influences.
In this study, we investigated the effect of Ascophyllum nodosum extract (ANE) on the flowering time of
Arabidopsis. We found that a 0.1% concentration of ANE induced flowering in Arabidopsis, accompanied
by the upregulation of key flowering time genes: FT (FLOWERING LOCUS T), SOC1 (SUPPRESSOR OF
OVEREXPRESSION OF CONSTANS 1), and LFY (LEAFY). Further investigation showed that ANE
specifically promotes flowering through the MIR156-mediated age pathway. ANE treatment resulted in the
repression of negative regulator genes, MIR156, while simultaneously enhancing the expression of
positive regulator genes, including SPLs and MIR172. This, in turn, led to the downregulation of AP2-like
genes, which are known floral repressors. It is worth noting that ANE did not alleviate the late flowering
phenotype of MIR156-overexpressing plants and spl mutants. Furthermore, ANE-derived fucoidan mimics
the function of sugars in regulating MIR156, closely mirroring the effects induced by ANE treatments. It
suppresses the transcript levels of MIR156 and AP2-like genes while inducing those of SPLs and
MIR172, thereby reinforcing the involvement of fucoidan in the control of flowering by ANE. In summary,
our results demonstrate that ANE induces flowering by modulating the MIR156-SPL module within the
age pathway, and this effect is mediated by fucoidan.

*[O134] THE IDENTIFICATION AND FUNCTIONAL ASSESSMENT OF PLASMODIOPHORA
BRASSICAE EFFECTORS. Emilee Storfie , Leonardo Galindo-González , Sheau-Fang. Hwang , and
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Stephen Strelkov. Department of Agricultural, Food and Nutritional Science, University of Alberta, 116 St
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& 85 Ave, Edmonton, AB T6G 2R3, Canada; and Canadian Food Inspection Agency, 1400 Merivale Rd,
Ottawa, ON K1A 0Y9, Canada
Correspondence to: strelkov@ualberta.ca

Plasmodiophora brassicae is an obligate biotrophic pathogen that causes clubroot disease on canola
(Brassica napus). To facilitate infection, the pathogen secretes effectors to manipulate the defence
response and other important biological pathways of the host. This study seeks to identify and
characterize selected effectors secreted by P. brassicae pathotype 3A to facilitate its infection of B.
napus. RNA sequencing was conducted at 7-, 14-, and 21-days post-inoculation of resistant and
susceptible rutabaga (B. napus ssp. rapifera) cultivars. Predicted effectors with non-redundant transcripts
and low covariance were identified and analyzed across time points and cultivars. Gene expression
validation was performed using NanoString technology. Two highly expressed putative effectors,
SPR01261.1 and SPQ99289.1, were selected for further investigation. The functionality of each effector’s
signal peptide, which enables their secretion, was confirmed using a yeast signal sequence trap assay.
Each effector localized to the nucleus and cytoplasm in planta when monitored via fluorescent protein
tagging in Nicotiana benthamiana. As the pathogen cannot be cultured in vitro, functional studies of P.
brassicae effectors are often labour-intensive and time-consuming. Additionally, many in silico tools
provide limited information. Bacterial and yeast heterologous protein expression systems were evaluated
and compared to determine their suitability for P. brassicae protein isolation. For the bacterial systems,
the open reading frames of SPR01261.1 and SPQ99289.1 were cloned separately into pDEST17 (T7
promoter and N-terminal 6xhistidine tag), pDEST15 (T7 promoter and N-terminal glutathione S-
transferase tag), and pMAL-c6T (tac promoter and N-terminal 6xhistidine tagged malE gene) vectors.
These constructs were then transformed into different expression strains of Escherichia coli and Vibrio
natriegens. For the yeast system, each open reading frame was cloned separately into pPICZα (AOX1
promoter, α-factor SP, and C-terminal 6xhistidine tag) and pPICZ (AOX1 promoter and C-terminal
6xhistidine tag) vectors and then transformed into Pichia pastoris. After inducing protein expression, the
expression level and solubility of each protein were confirmed by Western blotting. Once expressed and
purified, the functions of SPR01261.1 and SPQ99289.1, predicted to encode a serine carboxypeptidase
and an unknown protein with a kinase domain, respectively, were evaluated using enzyme assays.

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