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. 2024 Feb 15;22(1):100357. doi: 10.1016/j.jgeb.2024.100357

Foliar application of plant-derived peptides decreases the severity of leaf rust (Puccinia triticina) infection in bread wheat (Triticum aestivum L.)

Urbashi Panthi a, Brent McCallum a, Igor Kovalchuk b, Christof Rampitsch a, Ana Badea c, Zhen Yao a, Andriy Bilichak a,⁎
PMCID: PMC10903759  PMID: 38494271

Abstract

Background

Screening and developing novel antifungal agents with minimal environmental impact are needed to maintain and increase crop production, which is constantly threatened by various pathogens. Small peptides with antimicrobial and antifungal activities have been known to play an important role in plant defense both at the pathogen level by suppressing its growth and proliferation as well as at the host level through activation or priming of the plant’s immune system for a faster, more robust response against fungi. Rust fungi (Pucciniales) are plant pathogens that can infect key crops and overcome resistance genes introduced in elite wheat cultivars.

Results

We performed an in vitro screening of 18 peptides predominantly of plant origin with antifungal or antimicrobial activity for their ability to inhibit leaf rust (Puccinia triticina, CCDS-96-14-1 isolate) urediniospore germination. Nine peptides demonstrated significant fungicidal properties compared to the control. Foliar application of the top three candidates, β-purothionin, Purothionin-α2 and Defensin-2, decreased the severity of leaf rust infection in wheat (Triticum aestivum L.) seedlings. Additionally, increased pathogen resistance was paralleled by elevated expression of defense-related genes.

Conclusions

Identified antifungal peptides could potentially be engineered in the wheat genome to provide an alternative source of genetic resistance to leaf rust.

Keywords: Wheat, Triticum aestivum L., Leaf rust, Puccinia triticina, Antifungal peptides

1. Background

Fungal pathogens constantly threaten crop health and food security throughout the world. Due to their flexible genomes, fungi can quickly adapt to environmental conditions, resulting in new fungicide-resistant1 and virulent strains. Some of the most common fungal pathogens that threaten cereal cultivation include Fusarium head blight (FHB), three rusts (stem, leaf and stripe) and leaf spotting pathogens.2 Breeding for broad-spectrum disease resistance is the ultimate goal for achieving resistance against several pathogens in a single crop.

An alternative approach could be engineering crops to express peptides with antifungal/antimicrobial peptides (AFP/AMP, respectively)3, 4 or their foliar application to control pathogen infection.5 AMPs are characterized by high stability, a lack of cytotoxic effects on mammals and plants,6 and a low risk of resistance development by fungal pathogens.7 They are synthesized by a wide range of organisms,8, 9 including plants, and are known to have a broad spectrum of antibacterial, antifungal and antiviral activities.10 Most AMPs are ribosomally encoded. AMPs can also be synthesized by nonribosomal peptide synthases (NRPSs), although primarily in bacteria (e.g., Actinomycetes and Bacilli).11

AMPs can act through different mechanisms on target cells. For instance, cationic AMPs physically interact with negatively charged microbial membranes12 and potentially are aided by the components of the membranes inside the cell. Based on the interaction with the plasma membranes, AMPs are classified as membrane-disruptive or nondisruptive.13 Membrane-disruptive or lytic peptides cause pore formation in the membranes, loss of biophysical properties and killing of a cell.14, 15 Such peptides often demonstrate high cytotoxic properties to different cell types.16 On the other hand, cell-penetrating peptides (CPPs)17 act through a nonlytic mechanism through the disruption of homeostasis once inside the cell (e.g., signaling cascades, induction of reactive oxygen species, DNA damage, etc.).16, 18

Plants express AMPs constitutively both in storage and reproductive organs, although the peptides could also be induced locally or systematically during the defense response.19 They are classified into families based on their amino acid sequence, number and position of the cysteine residues and function.20 Several AMPs show inhibitory activity against mycotoxin-producing fungi from the Fusarium genus,21 some of the most destructive crop pathogens.

Rust fungi (Pucciniales) are widespread plant pathogens that infect important crops and cause significant yield loss if uncontrolled.22 Breeders must constantly look for new sources of resistance genes due to fast-evolving rust pathogens.22, 23 Wheat leaf rust caused by the obligate biotrophic fungus Puccinia triticina (Pt) can result in a significant annual loss in crop production. Durable leaf rust protection can be achieved by pyramiding leaf rust resistance genes (Lr), such as Lr34, Lr2a, and Lr67. At the same time, Pt populations have developed virulence to many of the resistance genes used in Canadian wheat cultivars (e.g., Lr1, Lr10, Lr12, Lr13 and Lr14a).24 Therefore, searching for new ways to control wheat leaf rust disease is critical.

A recent review on antirust peptides (ARPs) summarized findings from sixteen papers demonstrating 35 ARPs active against 11 different rust species.25 Three of these reports showed antifungal properties of the Penicillium antifungal protein (PAF) as well as wheat peptides Thaumatin-like protein 1 (TaTLP1) and Puroindoline-a/b (Pina/Pinb) against wheat stripe and leaf rust fungi (P. striiformis f. sp. tritici and P. triticina, respectively).26, 27, 28 Leaf rust is an obligate biotroph that grows and propagates exclusively on living tissues. Therefore, examining the antifungal activity of secreted peptides in vitro remains challenging. As an initial test to screen for the potential ARP of selected peptides in protecting wheat against Pt, we conducted a growth inhibition test and selected the most promising candidates. We add to the knowledge of the use of ARPs for crop protection and demonstrate the use of selected peptides in reducing the severity of Pt infection in wheat seedlings through foliar application.

2. Results

Seventeen synthetic peptides (Fig. 1 and Table S1) of plant origin and one from the yellow-spotted longicorn beetle Psacothea hilaris were selected to evaluate antirust properties using an in vitro experiment. The peptides represented different families, including alliumin, thionins, defensins, cyclic peptides, hairpin-like peptides, cysteine-rich, knottin-type and hevein. The peptides were chemically synthesized (CanPeptide, QC, Canada) with a purity above 90 %.

Fig. 1.

Fig. 1

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Effect of selected peptides on germination of leaf rust (P. triticina, isolate CCDS-96-14-1) urediniospores on 2 % solid agar medium. (A) Representative images of the leaf rust urediniospores germinated on agar medium either without (Control) or with peptides (100 µg/ml). Photos were taken 4 h after urediniospore germination at room temperature. The scale bar is 2 mm. (B) Average spore viability on solid agar media supplemented with different peptides at 100 µg/ml. At least five measurements were used to calculate the mean values, and error bars represent the standard error. * - p < 0.05 compared to the control (unpaired Student’s t test) (C) Length distribution of tested peptides and predicted isoelectric points (pI - color gradient for isoelectric point).

2.1. Evaluation of the antirust properties of selected peptides in an in vitro assay

Leaf rust (CCDS isolate) urediniospores were germinated on a 2 % water-based agar medium supplemented with selected peptides at a final concentration of 100 µg/ml. Germination of urediniospores was evaluated four hours post-treatment and compared to the control treatment without peptides (Fig. 1A). Nine peptides demonstrated significant reductions in the proportion of germinated urediniospores compared to the control (p <0.05, unpaired Student’s t test, Fig. 1B).

We selected β-purothionin and Purothionin-α2 peptides that demonstrated the highest inhibition of urediniospore germination activity (germination rate, 1.31 % ± 0.81 and 8.35 % ± 1.74), a peptide with average activity, Defensin-2 (19.43 % ± 3.88), and one peptide with minimal inhibitory activity, WAMP-1a (87.63 % ± 0.90), for further evaluation. We tested the dose-dependent response of rust germ tube elongation for the first three peptides and observed the concentration-dependent inhibition of urediniospore germination in vitro (Fig. 2).

Fig. 2.

Fig. 2

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Dose–response effect of selected peptides on germination of leaf rust (Puccinia triticina, CCDS isolate) urediniospores on 2 % solid agar medium. (A) Representative images of the leaf rust urediniospores germinated on agar medium either without (Control) or with selected peptides at three different concentrations. The WAMP-1a peptide at 100 µg/ml was used as a peptide control. Photos were taken at 4 h after urediniospore application to the media at room temperature. The scale bar is 2 mm. (B) Dose–response graph of germinated urediniospores on solid agar media supplemented with selected peptides at three different concentrations – 25, 50, and 100 µg/ml. At least five measurements were used to calculate the mean values, and error bars represent the standard error. (C) Biological activity of selected peptides. IC50 – inhibitory concentration 50, MIC - minimal inhibitory concentration.

β-purothionin demonstrated the highest activity (IC50 = 28.4 µg/ml, MIC = 43.5 µg/ml, Fig. 2C), followed by Defensin (IC50 = 46.5 µg/ml, MIC = 227.6 µg/ml) and Purothionin-α2 (IC50 = 82.2 µg/ml, MIC = 197.7 µg/ml). Similar to previous reports (reviewed in,25 the inhibitory effect of selected peptides on rust urediniospore germination was observed at a concentration above 25 µg/ml.

2.2. Foliar application of selected ARPs decreases severity of leaf rust infection in wheat, cv. Fielder

We further evaluated the potential of these three peptides to increase wheat seedling resistance to leaf rust following foliar application. Peptides were diluted in 0.5 % MSO surfactant and applied to plants at the two-leaves stage (cv. Fielder) 24 h prior to and 24 h postinoculation with leaf rust. The severity of infection was scored 14 days after inoculation with water, and 0.5 % MSO-treated plants were used as mock-treatment controls (Fig. 3A). WAMP-1a was a control peptide with minimal antifungal activity (Figure S1). The severity of the infection was examined visually, and the amount of Pt gDNA in relation to wheat gDNA was quantified using qPCR. Three studied peptides (β-purothionin, Defensin-2, and Purothionin-α2) significantly decreased the proportion of Pt gDNA to wheat gDNA compared to the untreated and MSO-treated infected controls (p <0.05, unpaired Student’s t test, Fig. 3B). At the same time, we also observed a prominent, but to a lower extent, decrease in infection for plants treated with just 0.5 % MSO (p <0.05, unpaired Student’s t test). A stock MSO solution composed of at least 70 % methylated soybean seed oil provides enhanced penetration and droplet adhesion without cuticle disruption. The MSO surfactant (0.5 %) was used previously in solutions with stable antimicrobial peptides (SAMPs) to control Citrus Huanglongbing (HLB), which is caused by the vector-transmitted phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) in citrus Huang et al., 2021;118.29 It is possible that foliar-applied MSO could interfere with urediniospore germination on the leaf surface, decreasing the severity of infection. Nevertheless, the treatment groups with peptide in 0.5 % MSO solution demonstrated significantly lower pathogen accumulation than the MSO-alone plants (p < 0.05, unpaired Student’s t test).

Fig. 3.

Fig. 3

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Evaluation of wheat (cv. Fielder) susceptibility to leaf rust (Puccinia triticina, isolate CCDS) following foliar application of selected peptides. (A) Representative images of leaf rust infection either in the presence or absence of peptide application at 14 days after infection (DAI). Peptide solutions (100 µg/ml in 0.5 % methylated seed oil) were applied to wheat seedlings (two-leaves stage) 24 hr before inoculation with rust urediniospores and 24 hr postinfection. (B) qPCR quantification of rust gDNA in infected leaves treated either with aqueous 0.5 % MSO solution or peptides in 0.5 % MSO. The bar plots represent the mean values of the relative normalized expression calculated from at least four plants per experimental group with two technical replicates. The error bars represent the standard error of the mean. The Pt- and wheat-specific primers/probes were designed to amplify single copy genes PtRTP1 and TaPINb-D1b, respectively. ** p < 0.05 compared to the MSO group and * p < 0.05 compared to the control group (unpaired Student’s t test).

2.3. Selective upregulation of the expression of defense-related genes in peptide-treated plants

Endogenous elicitors, such as active peptides produced following a pathogen attack, can induce an immune response in plants.30 Therefore, we decided to test the expression of the NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (TaNPR1), ENHANCED DISEASE SUSCEPTIBILITY 1 (TaEDS1), and PATHOGENESIS-RELATED GENE 1 (TaPR1) following foliar application of selected peptides.

Similar to the rust inoculation experiment, the leaves of the wheat seedlings (two-leaves stage, cv. Fielder) were sprayed with the selected set of peptides with nontreated and mock-treated plants used as the controls. Twenty-four hours post-treatment, the plants were mock-treated with soltrol (oil used for urediniospore suspension) and following oil evaporation the leaves were sprayed again with the peptides. The leaf tissue was collected 24 h after the second peptides application, RNA was isolated, and gene expression was quantified for three subgenomes of TaNPR1, subgenome A of TaEDS1, and subgenomes B and D of the TaPR1 homeologs using qPCR (Fig. 4).

Fig. 4.

Fig. 4

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Differential gene expression of defense-related genes following foliar application of selected peptides. Peptide solutions (100 µg/ml in 0.5 % methylated seed oil) were applied to wheat seedlings (two-leaves stage) 24 hr before treatment with soltrol and 24 hr post treatment. Control and MSO group plants were also treated with soltrol. Leaf samples (cv. Fielder) were collected from plants at the two-leaves stage at 24 h post second treatment with the peptides, followed by RNA isolation and gene expression analysis using qPCR. Normalization was performed against the TaGA3PD gene. The bar graphs represent the mean values calculated for four plants per treatment group in two technical repeats. The error bars represent the standard error of the mean, with the asterisk denoting a significant difference compared to the control (p < 0.05, unpaired Student’s t test). TaNPR1 - NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1, TaEDS1 - ENHANCED DISEASE SUSCEPTIBILITY 1, TaPR1 - PATHOGENESIS-RELATED GENE 1.

The expression of the TaNPR1, sgA and TaEDS1, sgA genes was significantly upregulated in plants treated with β-purothionin and Defensin-2 compared to untreated and mock-treated controls (p <0.05, unpaired Student’s t test). Similarly, the combined TaPR1, sgB, and sgD gene expression increased in plants treated with β-purothionin and Purothionin-α2 (p <0.05, unpaired Student’s t test).

3. Discussion

Overall, the mechanism of action of ARPs remains largely unknown25), with only two ARPs (EC 3.2.1.1431 and NaD132, 33 shown to act either through chitinase activity or binding to the cell wall and permeabilizing the plasma membrane. Purothionin-α2 and β-purothionin belong to the thionin peptide family (THI, Table S1). Thionins are found exclusively in plants and are first expressed as preproproteins that are processed to produce basic peptides of approximately 5 kDa in size, with three or four disulfide bridges and two consecutive cysteine residues at positions 3 and 4.34 The purothionins and related peptides were isolated and sequenced from the endosperm of maize, barley and oat and are present in other species of the Poaceae family.35, 36 Purothionins-α2 and β were originally crystallized from the endosperm of bread wheat (Triticum aestivum L.) and were shown to have antimicrobial activity against plant phytopathogens.37 The Purothionin-α2 peptide is part of the protein encoded by the THI1.238 gene on the D subgenome (TraesCS1D02G405700, Figure S2), whereas β-purothionin originates from the A subgenome homeologue (TraesCS1A02G398200). The homeologs are expressed explicitly in grain, with the highest expression observed in the endosperm (Figure S3). In addition to grain, thionins could also be expressed in leaves, with cultivated (Hordeum vulgare L.) and wild barley species containing a large multigene family for closely related leaf thionins.36, 39, 40 Leaf thionins in barley are light-regulated, and the expression of thionin genes in etiolated barley seedlings drops drastically following illumination.41 Curiously, the genes are also stress-responsive, and watering of plants with a 1 mM ZnCl2 solution or inoculation of barley seedlings with powdery mildew (Erisyphe graminis f.sp. hordei) results in a rapid transient increase in the transcript levels of the genes.36 Overall, along with other AMPs and larger proteins (e.g., chitinases, glucanases, thaumatin-like proteins (TLPs), proteinase inhibitors, peroxidases, etc.), they belong to the PR proteins activated during pathogen attack.42 Thionins are highly toxic to different bacteria and fungi, including plant pathogens, and even small animals.36, 43, 44, 45 Therefore, it is not surprising that the three thionins tested in our study (β-purothionin, CaThi, and Purothionin-α2) were highly effective against leaf rust urediniospores in in vitro germination and in vivo assays (Fig. 1, Fig. 2, Fig. 3). The peptides contain 8 (β-purothionin and Purothionin-α2) or 9 (CaThi) cysteine residues and belong to the class 1 thionins.34, 46 Tyrosine at position 13, a phospholipid binding site, was found to be indispensable for toxicity47 and is present in all known structures of thionins with AMP activity. Two possible mechanisms were proposed for their action – withdrawal of phospholipids that disturb the membrane's fluidity with subsequent lysis55 and insertion of the peptides in the membrane and acting as water channels causing local membrane disruption.48 Although both β-purothionin and Purothionin-α2 contain tyrosine at position 13 (Table S1 and Figure S2, position 40 in the amino acid multiple alignment figure), CaThi, which originates from hot pepper Capsicum annuum, has no tyrosine at this position (Table S1), although it was also found in our study to have a strong inhibitory effect on rust urediniospore germination in vitro (Fig. 1, germination rate 22.73 % ± 7.39, p < 0.05, unpaired Student’s t test).

Similar to thionins, we also observed strong antirust properties of Defensin-2 originating from peas (Pisum sativum, Table S1). Defensins are a group of peptides (45–54aminoacidslong) that belong to the family of small cysteine-rich proteins (CRPs) and are highly active against an extensive range of microorganisms.8 Although divergent in sequence, the defensins are structurally similar, having β-hairpin structures stabilized by three or four disulfide bonds. Fungi secrete several defensins with AFP properties.18 Identical to the defensins tested in our study, some demonstrate activity against plant fungal pathogens and no off-target cytotoxicity to bacterial, plant or animal cells.49, 50, 51, 52

Priming the plant immune system for earlier and more robust activation of pathogen-responsive genes can significantly increase crop resistance.53 NPR1 is a master regulator of pathogen response and is involved in the initiation of systemic acquired resistance (SAR) and induced systemic resistance (ISR).54 SAR is a long-lasting, broad-spectrum plant resistance mechanism.53, 55 Similarly, the EDS1 gene acts in the salicylic acid (SA) signal transduction pathway, and the mutant is impaired in PR gene expression.56 Overexpression of the NPR1 gene enhances resistance to various biotrophic and necrotrophic pathogens in many crops, including cotton, apple, pearl millet, Brassica, carrot, tomato, tobacco, rice and wheat.57, 58, 59, 60, 61, 62, 63, 64, 65 Resistance to Fusarium head blight (FHB) was achieved in transgenic wheat lines overexpressing rye ScNPR1 (Secale cereale-NPR1).66 Curiously, we observed only significant upregulation of TaNPR1 coded on sgA for β-purothionin and Defensin-2 (over sevenfold, Fig. 4) and no response for sgB and sgD, suggesting a difference in the cis-regulatory elements in the promoter regions (not examined in this study). The TaEDS1 gene at sgA was also significantly upregulated compared to the control for plants treated with β-purothionin and Defensin-2 (over 5- and 3-fold, respectively). The highest increase in expression, although, was observed for the TaPR1 genes at sgB and sgD with over 23-fold compared to the control, indicating that reduced severity of the leaf rust infection in the peptide-treated plants could be due to a combination of the direct inhibition of the urediniospore germination on the leaf surface and upregulation of the host defense genes. Indeed, a recent report provides direct evidence for the involvement of the TaPR1 gene in protecting wheat plants against leaf rust.28 Purified TaPR1 protein significantly reduced the germination of urediniospores and the growth of germ tubes in an in vitro assay. Furthermore, virus-induced gene silencing (VIGS)-mediated downregulation of the TaPR1 gene significantly increased plant susceptibility to Pt.

The large-scale production of AMPs for field application remains too expensive.19, 67 The peptides could be expressed in heterologous expression systems, such as bacteria, yeast, filamentous fungi or plants.19, 68, 69. At the same time, preliminary screening of the antifungal properties of selected peptides could be performed in vitro using approach presented here (Figure S4), followed by strategic engineering of the peptide sequence in the crop genome using gene editing tools, such as base or prime editors. Increasing knowledge of the structure–function relationship of AFP could aid in improving their function in antifungal activity with reduced off-target toxicity on other organisms.

4. Conclusions

We performed an in vitro screening of 18 antimicrobial peptides and show that nine of them significantly inhibit leaf rust (P. triticina) urediniospores germination. β-purothionin, Defensin-2, and Purothionin-α2 reduced leaf rust infection of wheat plants when applied as a foliar spray prior- and post-pathogen inoculation. Wheat-encoded β-purothionin and Purothionin-α2 could be further engineered in the wheat genome through cis-genic or gene editing approaches to provide an alternative means of genetic resistance to the leaf rust in wheat. This research adds to the knowledge of antimicrobial peptides with potent antitrust activity.

5. Methods

5.1. Selection of candidate peptides for antirust activity screening

Literature sources were examined for suitable peptide candidates with proven antifungal properties in cereals and other plant species.70 Eighteen promising candidates were synthesized (CanPeptide, QC, Canada), with most peptides having purity above 90 %, as confirmed with HPLC, mass spectrometry, and PAGE analysis (Table S1).

5.2. Evaluation of the antirust activity of selected peptides in an in vitro study

The fungicidal activity of peptides was evaluated through an in vitro experiment on the germination of leaf rust (P. triticina, isolate CCDS-96-14-1 isolate) urediniospores on 2 % solid water-based agar medium supplemented with peptides at a concentration of 100 µg/ml. Special concave slides were layered with a 2 % water-based agar solution supplemented with peptides. Urediniospores were heat shock treated at 40 °C for at least 7 min before use to promote germination. Following agar solidification, the urediniospores were applied to the medium with the brush. Slides were kept covered in Petri dishes to maintain humidity at room temperature. A nonanol solution was also applied to the filter paper placed in the lid to increase the germination rate. After 4 h, pictures were taken using a Leica MZF LIII stereomicroscope equipped with a Leica MC170 HD camera and compared to the spore germination rate without peptides. Germinated and nongerminated urediniospores were manually counted using LAS v 4.5 software to determine the proportion of germinated urediniospores (%). Three of the peptides that most strongly inhibited urediniospore germination were further evaluated in the dose-dependent response experiment with three concentrations – 25, 50 and 100 µg/ml. Each experiment was repeated at least twice, and the average germination percentage was calculated for five field views.

5.3. Assessment of leaf rust infection severity following foliar application of the peptides

To promote the adhesion of peptides to the wheat leaf surfaces, peptides were diluted in 0.5 % water-based industrial grade methylated seed oil (MSO™ concentrate with Leci-Tech, Loveland Products Canada Inc.) surfactant solution at 100 µg/ml.71. Leaves of the wheat seedlings at the two-leaves stage, cultivar Fielder (cv. Fielder), were sprayed with peptide solution (approximately 400 µl) 24 h before inoculation with leaf rust urediniospores (P. triticina, isolate CCDS-96–14-1 isolate) and 24 h post-infection. P. triticina urediniospores were heat shocked at 40 °C for 7 min and suspended in the soltrol oil for infection. Uredionospores viability was tested prior to the infection on 2 % agar plate and verified under the microscope. Infection with P. triticina urediniospores was performed using a spray applicator by randomly combining the peptide-treated and nontreated plants in groups (technical replicates). The plants were maintained in the dew chamber for 24 h to maintain humidity. Fourteen days after infection (DAI), the infection types and severity were scored using the infection scale from,72 and leaves were collected for gDNA isolation and fungal genomic DNA (gDNA) quantification using quantitative PCR (qPCR). Untreated and mock-treated plants (0.5 % MSO, cv. Fielder) were used as controls. The experiment was repeated three times (biological replicates) with nine plants per treatment group (technical replicates).

5.4. qPCR quantification of gene expression and rust gDNA in infected wheat plants

To quantify gene expression in the non-infected plants, total RNA was isolated from the leaves at the two-leaves stage seedlings treated essentially as described for the leaf rust infection experiment, but without application of the rust urediniospore. The urediniospores treatment was substituted with the soltrol oil mock used to resuspend urediniospores. RNA was isolated with a Nucleospin RNA Plant Kit (cat. # 740949, Macherey-Nagel, Germany) according to the manufacturer’s instructions. Total RNA was treated with DNase I (RNase-free, cat. # M0303L, NEB), and the absence of gDNA contamination was verified with PCR and TaGA3PD primers (Table S2). For every duplex reaction, 50 ng of RNA was combined with 1xLuna Universal Probe One-Step Reaction Mix (cat. # E3006; NEB), 400 nM of each primer pair (for the target and reference gene), 200 nM of each probe (target and reference gene, Table S2) and 1xLuna WarmStart® RT Enzyme Mix in a 20 µl final reaction volume. The probes were either 5′ FAM (6-fluorescein)- or 5ʹ HEX (hexachloro-fluorescein)-labeled and contained ZEN and Iowa Black Hole Quencher 1 (Integrated DNA Technologies, Coralville, IA, USA). qPCR was run on a CFX96 Real-Time PCR Detection System (Bio-Rad). Reverse transcription was performed at 55 °C for 10 min followed by initial denaturation at 95 °C for 1 min and 40 cycles of 95 °C for 1 min and 60 °C for 30 sec. TaGA3PD (TraesCS7A02G313100, TraesCS7B02G213300, and TraesCS7D02G309500) was used as an endogenous reference.73 At least four plants were used for analysis for every group, and reactions were run in duplicate. The average 2 − ΔΔCT was calculated using CFX manager, v. 3.1 (Bio-Rad).

P. triticina (Pt) gDNA was quantified in infected leaves using qPCR. Total gDNA was isolated from noninfected and infected leaves using the Warner et al. method.74 The qPCR sample reaction contained 50 ng of gDNA, 1x Luna® Universal Probe qPCR Master Mix (cat. #M3004L; NEB), 400 nM of each primer pair (specific for the Pt and wheat genomes), and 200 nM of each probe (Table S2). The Pt- and wheat-specific primers/probes were designed to amplify single copy genes PtRTP1 (Pt15 chromosome 18B, GenBank: CP110454.175 and TaPUROINDOLINE-b (TaPINb-D1b gene76 respectively. Similarly, the probes were 5′ FAM- or 5ʹ HEX-labeled, and the same cycling conditions were used, except for the initial reverse transcription step. Normalization was performed against the TaPINb-D1b gene with four independent plants/treatment groups and two technical replicates. The average 2 − ΔΔCT was calculated using CFX manager, v. 3.1 (Bio-Rad).

5.5. Estimation of MIC, IC50 and statistical analysis of the data

Calculation of the minimal inhibitory concentration (MIC) and inhibitory concentration 50 (IC50) was performed using the drc package in R.77 A significant difference between treatment groups was validated using an unpaired Student’s t test with p < 0.05.

6. Ethics approval and consent to participate

Not applicable.

7. Consent for publication

Not applicable.

8. Availability of data and materials

All data generated or analysed during this study are included in this published article and its supplementary information files.

9. Funding

This research was funded by the Manitoba Crop Alliance (grant# 20210601), the Agriculture Development Fund (grant# 20210601), the Alberta Wheat Commission (grant# 22AWC134A), the Saskatchewan Wheat Development Commission (grant# 216-211124), the Western Grains Research Foundation (grant# AGR2218) and Agriculture and Agri-Food Canada (grant# J-002616).

10. Authors’ contributions

Conceptualization, A.B.; methodology, A.B., U.P., B.M., and C.R.; formal analysis, U.P. and A.B.; writing—original draft preparation, A.B. and U.P.; visualization, A.B., Z.Y., and U.P.; supervision, A.B., I.K., B.M., C.R., and Ana B.; project administration, A.B., U.P., I.K., B.M., C.R. and Ana B.; funding acquisition, A.B., B.M., C.R., Ana B., I.K. All authors have read and agreed to the published version of the manuscript.

CRediT authorship contribution statement

Urbashi Panthi: Writing – review & editing, Writing – original draft, Methodology, Formal analysis. Brent McCallum: Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology. Igor Kovalchuk: Writing – review & editing, Writing – original draft, Project administration, Investigation. Christof Rampitsch: Writing – review & editing, Methodology, Investigation. Ana Badea: Funding acquisition, Writing – review & editing, Supervision, and Resources. Zhen Yao: Formal analysis, Data curation. Andriy Bilichak: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Acknowledgments

We want to acknowledge Agriculture and Agri-Food Canada, Manitoba Crop Alliance, Saskatchewan Wheat Development Commission, Alberta Wheat Commission, Western Grains Research Foundation, and Agriculture Development Fund for financial support. We want to thank Susan Brown, Louie Lopos, and Janeen Robinson for technical help with the in vitro spore germination experiments. We also want to thank Elsa Reimer for providing leaf rust urediniospores and guidance with plant infection assays.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jgeb.2024.100357.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary data 1
mmc1.pptx (2.7MB, pptx)
Supplementary data 2
mmc2.xlsx (22.4KB, xlsx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary data 1
mmc1.pptx (2.7MB, pptx)
Supplementary data 2
mmc2.xlsx (22.4KB, xlsx)

Data Availability Statement

All data generated or analysed during this study are included in this published article and its supplementary information files.

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