Page 196 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

[O182] ALLELIC DIVERSITY AND EVOLUTIONARY PATTERNS OF TOXB GENE IN PYRENOPHORA
TRITICI-REPENTIS AND RELATED SPECIES: A GLOBAL PERSPECTIVE. Mohamed Hafez , Ryan
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Gourlie , Megan McDonald ; Melissa Telfer , Marcelo A. Carmona , Francisco J. Sautua , Caroline S.
Moffat , Paula M. Moolhuijzen , Pao Theen See , and Reem Aboukhaddour . Agriculture and Agri-Food
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Canada, Lethbridge Research and Development Centre, Lethbridge, AB, Canada; School of
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Biosciences, University of Birmingham, Institute of Microbiology and Infection, Edgbaston,
Birmingham, UK; Cátedra de Fitopatología, Facultad de Agronomía, Universidad de Buenos Aires,
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Buenos Aires, Argentina; and Centre for Crop and Disease Management, School of Molecular and Life
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Sciences, Curtin University, Australia
Correspondence to: reem.aboukhaddour@agr.gc.ca

Pyrenophora tritici-repentis, known for causing tan spot, exerts significant damage as a foliar pathogen on
wheat globally. It produces several necrotrophic effectors, among which ToxB is the chlorosis-inducing
effector predominant in the fertile crescent and surrounding regions. So far, research on ToxB has been
sporadic. In this study, we investigated the allelic diversity and evolutionary patterns of ToxB gene in P.
tritici-repentis in a global collection representing different races and geographic regions. To gain a
comprehensive insights into ToxB evolution, we examined their presence and variability in P. tritici-
repentis and other related fungal species. ToxB was found to be geographically limited to specific regions
in North Africa and the Fertile Crescent. The presence of the toxb gene (the ToxB homolog) was
exclusive to isolates of races 3 and 4, primarily identified in Canada and Syria. Notably, the presence of
toxb was also observed for the first time in the barley net blotch pathogen Pyrenophora teres.
Evolutionary analysis of ToxB/toxb genes indicated selection pressure characterized by functional loss,
duplication events, and diverse mutations. A notable finding was the discovery of a ∼5.6-kb Copia-like
retrotransposon, referred to as Copia-1_Ptr, inserted into the toxb gene of a race 3 isolate. This insertion
led to the impairment of ToxB's function, marking a unique case of effector gene disruption caused by a
transposable element in P. tritici-repentis. Exploration of ToxB-like protein distribution in other
ascomycetes revealed their presence in 19 additional species, including the Leotiomycetes class,
marking the first report of ToxB-like proteins in this class. The presence/absence pattern of ToxB-like
proteins challenged species relatedness as indicated by a phylogenetic analysis, suggesting a past
horizontal gene transfer event during the evolution of the ToxB gene. The correlation between selection
pressure, fitness, and the isoform of secreted effectors underscores the significance of studying allelic
variations in effector-encoding genes on a global scale.

[O183] ENDOGENOUS RUST PEPTIDES FROM PUTATIVE SHORT OPEN READING FRAMES
IDENTIFIED USING PEPTIDOMICS AND DE NOVO SEQUENCING STRATEGIES. Christof
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Rampitsch , Slavica Djuric-Ciganovic , Zhen Yao , and Mark Lubberts . Morden Research and
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Development Centre, Agriculture and Agri-Food Canada, 101, Rte 100, Morden MB, Canada R6M 1Y5;
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and Summerland Research and Development Centre, Agriculture and Agri-Food Canada, Hwy 97,
Summerland BC, Canada V0H 1Z0
Correspondence to: christ.rampitsch@agr.gc.ca

Puccinia triticina (Pt) is an obligate fungal parasite that causes leaf rust on wheat. This disease occurs
annually and potentially results in large yield losses. Host-pathogen communication at the protein level
has been well-studied in this and similar pathosystems, but the potential roles of peptides (smaller than
10 kDa) has not been examined. Small peptides, transcribed from short open reading frames, have been
reported from model fungal species. This research investigates the role(s) of such peptides in the wheat-
rust interaction, using top-down LC-MS analyses to detect novel peptides. For the LC-MS approach we
evaluated several approaches, including N-terminal enrichment and C4 RP-HPLC, before settling on
SEC-HPLC. Enriched peptides were left intact or digested with trypsin and then analyzed by LC-MS in a
high-resolution Orbitrap mass spectrometer. Automated de novo sequencing, which does not require any
databases, was used to obtain candidate peptide sequences and these were queried against wheat and
rust genomic sequences to eliminate fragments resulting from protein turnover or breakdown. To find
potential short open reading frames, peptides were mapped back on to the genomic sequence of Pt,
while accounting for both codon redundancy, all reading frames and potential de novo sequencing errors
(e.g. AB to BA reversals). We have found several candidate peptides with no significant homology to the

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