
==== Front
Commun Biol
Commun Biol
Communications Biology
2399-3642
Nature Publishing Group UK London

39294271
6881
10.1038/s42003-024-06881-4
Article
Puccinia triticina avirulence protein AvrLr21 directly interacts with wheat resistance protein Lr21 to activate wheat immune response
http://orcid.org/0009-0001-8899-995X
Shen Songsong 1
Wang Fei 1
Cui Zhongchi 1
Yuan Shitao 1
Meng Linshuo 1
Liu Daqun 1
http://orcid.org/0000-0001-9288-869X
Ma Lisong malisong@hebau.edu.cn

12
http://orcid.org/0000-0003-2414-1919
Wang Haiyan ndwanghaiyan@163.com

1
1 https://ror.org/009fw8j44 grid.274504.0 0000 0001 2291 4530 Technological Innovation Center for Biological Control of Crop Diseases and Insect Pests of Hebei Province, College of Plant Protection, Hebei Agricultural University, Baoding, Hebei China
2 https://ror.org/009fw8j44 grid.274504.0 0000 0001 2291 4530 The State Key Laboratory of North China Crop Improvement and Regulation, College of Horticulture, Hebei Agricultural University, Baoding, China
18 9 2024
18 9 2024
2024
7 117018 2 2024
11 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Wheat leaf rust, caused by Puccinia triticina (Pt), remains a constant threat to wheat production worldwide. Deployment of race-specific leaf rust (Lr) resistance genes in wheat provides effective protection against leaf rust, but often leads to selective pressures that drive the rapid emergence of new virulent Pt isolates in nature. However, the molecular mechanisms underlying the evasion of Lr-delivered resistance by leaf rust remain largely unknown. Here, we identify an avirulence gene AvrLr21 in Pt that triggers Lr21-dependent immune responses. BSMV (Barley stripe mosaic virus)-mediated host-induced gene silencing assay shows that silencing AvrLr21 compromises Lr21-mediated immunity. AvrLr21 interacts directly with Lr21 protein to induce a hypersensitive response in tobacco leaves. The evolved Lr21-breaking Pt isolates can suppress Lr21-mediated immunity. Our data provide a basis for studying the molecular determinants in Pt-wheat incompatible interaction and monitoring natural Pt populations to prioritize the deployment of Lr resistance genes in the field.

Wheat leaf rust Puccinia triticina (Pt) avirulence protein AvrLr21, which directly interacts with its cognate resistance protein Lr21, triggers Lr21-dependent immune responses in wheat, aiding in understanding Pt-wheat interactions

Subject terms

Effectors in plant pathology
Plant evolution
https://doi.org/10.13039/501100001809 National Natural Science Foundation of China (National Science Foundation of China) 31501623 Wang Haiyan issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

The exponentially growing world population demands increasing annual cereal production. However, wheat, the second most cultivated cereal, is under constant threat from fungal pathogens that constitute critical concerns for future food security1. Leaf rust, caused by Puccinia triticina, is one of the agronomically important wheat fungal diseases that dramatically constrains wheat production worldwide2. The introgression of leaf rust resistance (Lr) genes into commercial wheat cultivars is the most effective and sustainable approach to providing protection of wheat against leaf rust3,4. 83 Lr genes have been identified in common wheat, but only a limited number of Lr genes have been cloned, including Lr1, Lr9, Lr10, Lr13, Lr14a, Lr21, Lr22a, Lr34, Lr42, Lr47, and Lr675–9. Most Lr genes confer race-specific resistance against Pt isolates carrying the cognate avirulence genes following the gene-for-gene hypothesis10. Recently, the first avirulence gene AvrLr15 in Pt that can trigger Lr15-dependent immune response in wheat was identified, and overcoming Lr15-mediated resistance in wheat was caused by a deletion and point mutations of amino acid sequence of AvrLr15 in the Lr15-breaking Pt races11.

Leaf rust resistance gene Lr21 that was introgressed into bread wheat from Iranian Aegilops tauschii was the first cloned leaf rust resistance gene, encoding the typical nucleotide-binding leucine-rich repeat protein12–14. Recently, Lr21-mediated complete seedling resistance and partial adult plant resistance in field conditions were found to be associated with higher expression of Lr21 at the seedling stage and lower expression at the adult plants, indicating that the expression dynamics of the resistance gene of the host plant at different developmental stages can determine the degree of immune response15.

Owing to the co-evolutionary arms race between Pt pathogens and wheat, new virulent Pt isolates with the ability to overcome Lr-mediated resistance are continuously emerging16–18, which often results in the ineffectiveness of wheat resistance delivered by Lr genes after a few years of cultivation of wheat cultivars with specific Lr genes19. For example, hard red spring wheat cultivars carrying the combinations of Lr2a, Lr10, Lr13, Lr16, Lr21, Lr23, and Lr34 genes were released in 2004 in the North American regions, but virulent Pt isolates against Lr21 was first found in North America in 201020,21. Therefore, the rapid emergence of Lr-breaking Pt isolates demands the identification of more Pt avirulence (Avr) genes to study the molecular basis of Pt-wheat compatible and incompatible interactions, which will aid us in understanding the molecular mechanisms underlying Avr protein recognition by Lr proteins and Avr evasion by R gene and guiding the selective cultivation of Pt-resistant wheat cultivars in different wheat-growing regions.

Here, we identify an avirulence gene AvrLr21 with the ability to trigger its cognate resistance gene Lr21-dependent immunity in wheat. Functional analyses of the interaction between AvrLr21 and Lr21 uncover that AvrLr21 physically interacts with Lr21 protein and the evolved Lr21-breaking Pt isolates are able to inhibit Lr21-mediated immunity in the presence of AvrLr21. These findings can deepen our understanding of the molecular interaction in Pt-wheat incompatible interactions and expand the molecular markers used to monitor natural Pt populations in the field.

Results and discussion

Pt_21 triggers Lr21-dependent cell death in the leaves of wheat and N. benthamiana

Our previous studies found that an effector gene Pt_21 was significantly up-regulated at 24 hpi during Pt infecting susceptible wheat cultivar and its product interacted with wheat TaTLP1 in the apoplast to suppress wheat immunity22. To identify the avirulence gene that genetically induces wheat resistance gene-mediated resistance, the produced recombinant protein ΔspPt_21 without signal peptide using the Escherichia coli (E. coli) expression system was purified and infiltrated into the leaves of 41 near-isogenic wheat lines of Thatcher carrying Lr resistance genes. Strikingly, cell death responses were specifically observed in the leaves of TcLr21 (RL6043). In contrast, no cell death responses were observed in the leaves of Thatcher and the other 40 near-isogenic lines of Thatcher carrying individual Lr gene as well as in the leaves infiltrated with buffer (Supplementary Fig. 1). We further validated these observations by infiltrating the purified ΔspPt_21 protein into the leaves of wheat TcLr21 and cultivar Fielder carrying non-functional lr21 allele (lr21-Fielder with 1 bp deletion in exon 2)15, respectively. As we expected, pronounced cell death responses appeared in the infiltrated region of Lr21 leaves, while no cell death responses were visible in the infiltrated leaves of Fielder and Thatcher (Fig. 1a). Leaves infiltrated with buffer did not display any cell death (Fig. 1a). In addition, a gene-based molecular marker of Lr21 confirmed the presence of Lr21 in the line TcLr21 and non-functional lr21 in Fielder (Supplementary Fig. 2). Taken together, these findings indicated that purified ΔspPt_21 protein induces an Lr21-dependent cell death response, indicating that Pt_21 might be AvrLr21. Protein-mediated cell death in wheat has been used to study the function of fungal effectors. For example, the infiltration of purified effector protein SnTox3 from Parastagonospora nodorum can trigger a robust necrotic response in the leaves of wheat carrying Snn323,24. Purified AvrSr35 protein was infiltrated into the leaves of wheat with Sr35 gene to induce cell death, but not in the leaves of wheat without Sr35 gene25.Fig. 1 Pt_21 induces Lr21-mediated cell death in the leaves of wheat and N. benthamiana.

a Infiltration of the ΔspPt_21 protein into the leaves of TcLr21, lr21-Fielder carrying a non-functional lr21 with a 1 bp deletion in exon 2 and Thatcher (Tc) wheat line without any leaf rust resistance genes. SDS-PAGE analysis of the recombinant ΔspPt_21 protein was conducted. Buffer was used as a negative control. b Co-infiltration of N. benthamiana leaves with Agrobacterium expressing the labeled Lr21 and ΔspPt_21 constructs. The same leaf was examined before (left) and after (right) being decolorized with the de-staining solution.

To further confirm that Pt_21 is AvrLr21, we constructed recombinant vectors carrying pCamA: ΔspPt_21 or pCamA: Lr21 (Supplementary Fig. 3) and co-infiltrated Agrobacterium tumefaciens carrying pCamA: ΔspPt_21 or pCamA: Lr21 on N. benthamiana leaves. Surprisingly, at 4 days after infiltration, strong cell death was observed in the co-infiltrated regions of N. benthamiana leaves (Fig. 1b). Another Pt effector, PTTG_27401, served as a negative control and failed to induce cell death when co-expressed with Lr21. To further validate this observed phenotype, co-infiltration was performed in Nicotiana tabacum leaves using Agrobacterium tumefaciens carrying ΔspPt_21 or Lr21 and the development of cell death was recorded at 2, 3 and 4 days after infiltration. Visible cell death appeared in co-infiltrated areas at 2 days, increased at 3 days, and fully occupied the infiltrated region at 4 days, but no cell death was observed in the negative control regions (Supplementary Fig. 4). Fluorescence microscopy observation confirmed the expression of GFP-tagged proteins, including Pt_21-GFP, Lr21-GFP and PTTG_27401-GFP (Supplementary Fig. 5). These results indicated that Pt_21 has an avirulence activity that induces an Lr21-dependent immune response in tobacco leaves. Previous studies showed that co-expression of wheat stem rust avirulence genes, such as AvrSr3525,26, AvrSr5027, and AvrSr2728, with its corresponding resistance gene in tobacco, triggers cell death.

Pt_21 is the avirulence gene AvrLr21

To further confirm that Pt_21 is the cognate avirulence gene of Lr21, we generated two BSMV constructs to knock down the expression of Pt_21 (Supplementary Fig. 6). The wheat PDS gene (TaPDS) served as a positive control to examine the silencing efficiency, and the empty BSMV:γ vector was used as a negative control. 10 days after the inoculation of BSMV by rubbing, the visible photobleaching phenotype was observed in BSMV:TaPDS-inoculated TcLr21 wheat leaves, whereas typical BSMV viral symptoms with mild chlorosis appeared in all leaves inoculated with BSMV:γ and BSMV:Pt_21. Next, the leaves of BSMV-inoculated TcLr21 wheat plants were challenged with urediniospores of Pt avirulent isolate PHNT. 14 days after inoculation of Pt, significantly increased rust pustules were observed in the leaves inoculated with BSMV: Pt_21 (Fig. 2a) compared to the leaves of TcLr21 without BSMV inoculation and the leaves of TcLr21 inoculated with BSMV:γ. The expression level of Pt_21 was significantly decreased in BSMV:Pt_21-treated TcLr21 plants compared with BSMV:γ-inoculated TcLr21 wheat at 24, 48 and 120 hpi (Fig. 2b). In addition, histological observation of Pt infection found that the hyphal length, infection areas, hyphal branching number and overall infection area of Pt at 24, 48 and 120 hpi were significantly increased following the silencing of Pt_21 by HIGS in TcLr21(Fig. 2c and Supplementary Fig. 7a–c). The areas of cell death and H2O2 accumulation were significantly lower at 120 hpi in BSMV:Pt_21 plants than those observed in BSMV:γ-inoculated and non-inoculated leaves (Fig. 2d and Supplementary Fig. 7d). Collectively, these findings indicate that BSMV-mediated silencing of Pt_21 is accurate, enabling us to conclude that Pt_21 is the avirulence gene AvrLr21. BSMV (Barley stripe mosaic virus)-mediated host-induced gene silencing assay has been widely used to study the functional role of wheat rust effectors during rust pathogen infection. For example, BSMV-mediated HIGS of the Pst_12806 effector from wheat stem rust Pst demonstrated that Pst_12806 is indispensable for Pst-wheat compatible interaction. Silencing of PstGSRE1 using BSMV-mediated HIGS impaired the virulence of Pst in wheat29–33.Fig. 2 Pt_21 effector is the avirulence protein AvrLr21 triggering Lr21-dependent wheat immune response.

a BSMV-mediated host gene silencing of Pt_21 impairs Lr21-mediated resistance against Pt isolate PHNT that is avirulent on TcLr21 plant. The fourth leaves of TcLr21 wheat at 10 days after infection with BSMV:γ, BSMV:TaPDS, BSMV:Pt_21 were inoculated with urediospores. BSMV:γ-and BSMV:TaPDS-inoculated Thatcher and mock leaves treated with buffer were used as positive and negative control, respectively. At 14 days after urediospore inoculation, representative pictures were taken. b Relative expression levels of Pt_21 in Pt_21-silenced plants and negative control BSMV:γ plants at 24, 48, and 120 hpi after urediospore inoculation. The y-axis indicates the amounts of Pt_21 transcript normalized to the GAPDH gene. The x-axis indicates sampling times. Data are means ± standard errors (SE) of three independent experiments. Differences between Pt_21-silenced plants and control plants were assessed using Student’s t-tests (*p < 0.05). c Histological observations of hypha development and host cell death in BSMV-mediated HIGS of Pt_21 wheat leaves after inoculation with Pt PHNT at 24, 48, and 120 hpi. AP appresorium, SV substomatal vesicle, IH infection hypha, HMC haustorial mother cell, SH secondary hypha, NC necrotic cell, U urediospore. Scale bar, 50 μm. d H2O2 accumulation at infection sites was detected by staining with DAB and viewed under differential interference contrast microscopy. Scale bar, 20 µm.

AvrLr21 directly interacts with Lr21

AvrLr21 with the ability to induce the wheat Lr21-dependent cell death in planta suggests that the recognition specificity between AvrLr21 and Lr21 is likely mediated by direct interaction. To examine the interaction between AvrLr21 and Lr21, a yeast two-hybrid (Y2H) experiment was conducted using pGBKT7:ΔspAvrLr21 and pGADT7:Lr21 constructs (Supplementary Fig. 8). As shown in Fig. 3, the yeast Y2HGold transformant co-expressing pGBKT7:ΔspAvrLr21 and pGADT7:Lr21 was able to grow on the selective-HAWL medium and produced blue coloration, which was similar to the positive control ΔspTaPR1 and ΔspTaTLP1. In contrast, yeast co-transformed with Lr21 and the empty bait or ΔspAvrLr21 and the empty prey vector was unable to grow on the selective plates and failed to produce blue coloration (Fig. 3a). To further validate the interaction, a co-immunoprecipitation (Co-IP) assay was conducted. Figure 3 showed that recombinant Lr21-GFP proteins were co-purified with ΔspAvrLr21-GST protein using GST beads (IP), confirming that ΔspAvrLr21 directly interacts with Lr21 (Fig. 3b). In addition, RFP-tagged Lr21 and GFP-tagged ΔspAvrLr21 colocalized in the nucleus and cytosol of N. benthamiana leaves (Fig. 3c), implying that the direct interaction between ΔspAvrLr21 and Lr21 likely occurs in the plant nucleus and cytosol. Our results were consistent with previous studies. It has been documented that the rust avirulence protein is directly recognized by its corresponding resistance protein. The wheat stem rust avirulence protein AvrSr35 interacted directly with the Sr35 to induce Sr35 resistosome assembly25,26, and AvrSr50 interacted directly with the Sr50 protein in Y2H assay27. The flax rust avirulence protein AvrL567 specifically interacted with the corresponding R proteins in yeast34.Fig. 3 AvrLr21 directly interacts with Lr21.

a Y2H analysis of the direct interaction between Lr21 and ΔspAvrLr21. Growth of yeast strains coexpressing ΔspAvrLr21 fused to the GAL4 DNA binding domain (BD) with Lr21 fused to the GAL4 activation domain (AD) on control media lacking leucine and tryptophan (SD-WL) or selective media lacking histidine and adenine supplemented with X-α-Gal (SD-HAWL-X-α-Gal). ΔspTaPR1 (BD) and ΔspTaTLP1 (AD) were used as positive controls. A ten-fold serial dilution is shown. b Confirmation of the interaction between ΔspAvrLr21 and Lr21 by Co-IP assays. Western blots of total proteins transiently expressing the marked constructs and proteins eluted from GST beads were detected using the anti-GFP or anti-GST antibody. The protein marker was labeled on the left. c Co-expressing GFP-tagged ΔspAvrLr21 and RFP-tagged Lr21 in the leaves of N. benthamiana, showing that ΔspAvrLr21 and Lr21 co-localize in plant nucleus and cytosol. The white arrows point to the nucleus. Scale bar, 50 μm.

Dissecting the molecular mechanisms underlying Lr21-breaking Pt isolates

The natural Lr21-breaking Pt isolates have been reported in America and China35. To understand the evolutionary mechanism underlying the emerged Pt isolates with the ability to overcome Lr21-mediated resistance, PCR amplification of the AvrLr21 genes from 6 natural Lr21-virulent isolates was conducted, and the resulting PCR products were sequenced. Surprisingly, all sequences of AvrLr21 genes cloned from Lr21-overcoming isolates are identical to the sequences of the AvrLr21 gene from the Lr21-avirulent isolate (Supplementary Fig. 9). We quantified the relative expression of AvrLr21 gene in our available Lr21-avirulent and -breaking isolates. The expression patterns of AvrLr21 in Lr21-avirulent and -breaking isolates at different time points upon infection of TcLr21 wheat line were similar, as evidenced by the observation that AvrLr21 was significantly induced and peaked at 72 hpi and subsequently decreased from 96 to 144 hpi. Notably, we found the expression level of AvrLr21 in Lr21-avirulent isolate both at 48 and 72 hpi was 2-fold higher than that in Lr21-virulent isolates, suggesting that expression variations of AvrLr21 at specific infection stage of Pt are likely attributed to the evasion of Lr21-mediated resistance in Lr21-breaking isolates (Supplementary Fig. 10). However, the protomer sequences of AvrLr21 in Lr21-breaking and Lr21-avirulent isolates are exactly same (Supplementary Fig. 11). Gain of virulence function through altering the expression level of avirulence genes by fungal pathogens has been reported. Wheat stem rust Puccinia graminis f. sp. tritici (Pgt) AvrSr27 can avoid the recognition by Sr27 gene via deletion, copy number and expression level variations at the AvrSr27 locus detected in the field Sr27-breaking Pst isolates28.

In addition, we found that pre-inoculation of Lr21-breaking Pt isolate THTT on TcLr21 leaves can completely inhibit AvrLr21-induced cell death in Tc·Lr21 leaves at 1d and 10d after infiltration of purified ΔspAvrLr21 protein (Fig. 4 and Supplementary Fig. 12). In contrast, ΔspAvrLr21 induces strong cell death in the leaves of TcLr21 without THTT inoculation as well as in leaves with Lr21-avirulent PHNT inoculation (Fig. 4 and Supplementary Fig. 12). Histological observation of the Pt hyphae at 24 hpi and 48 hpi indicated the successful inoculation of Pt isolates on wheat leaves (Supplementary Fig. 13). These observations indicated that Lr21-breaking Pt isolate evolved unknown mechanisms to suppress Lr21-mediated resistance, likely utilizing the secreted effector proteins. Previous studies have reported that fungal pathogens can employ secreted effectors to suppress plant resistance gene-delivered resistance. For example, xylem-invading fungus Fusarium oxysporum f.sp. lycopersici effector Avr1 can suppress tomato resistance genes I-2- and I-3-mediated immunity36. The effector AvrLm4-7 of Leptosphaeria maculans causing stem canker in oilseed rape can inhibit oilseed resistance genes Rlm3-, Rlm5- and Rlm9-mediated resistance37,38. In the future, we need to dissect the molecular mechanism underlying the inhibition of AvrLr21-induced cell death by Lr21-breaking Pt isolates.Fig. 4 Lr21-breaking Pt isolates can inhibit AvrLr21-triggered cell death in the leaves of TcLr21.

Lr21-breaking Pt isolate THTT can specifically inhibit AvrLr21-induced cell death in the leaves of wheat line TcLr21. The leaves of TcLr21 were inoculated with urediospores of PHNT and THTT, respectively. At 12 h post-inoculation, the inoculated leaves were infiltrated with purified ΔspAvrLr21 protein. Representative pictures were taken at 1 day and 10 days after infiltration. The non-inoculated leaves and leaves inoculated with PHNT served as negative controls. PHNT (+): avirulent on TcLr21 wheat, THTT (+): virulent on TcLr21 wheat.

Methods

Plant materials, Pt race and sample collection

All near-isogenic lines of Thatcher, susceptible wheat Chinese Spring and tobacco are preserved in the Laboratory of Wheat Leaf Rust, Hebei Agricultural University. Thatcher line without any leaf rust resistance genes is susceptible to all leaf rust pathogens. The Fielder line contains the non-functional lr21 allele (lr21-Fielder with 1 bp deletion in exon 2). Second seedling leaves inoculated with PHNT (isolate 07-10-426-1) or distilled water (control) for RNA extraction were harvested at 0, 24, 48, 72, 96, 120, and 144 hpi. All samples were immediately frozen in liquid nitrogen and stored at −80 °C. Each treatment included three independent biological replicates.

RNA isolation and quantitative PCR

Total RNA was extracted using the TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Beijing) according to the manufacturer’s instructions. Contaminating DNA was digested with amplification-grade DNase I (TaKaRa, Beijing) and cDNA was synthesized using the SuperScript II reverse transcriptase, oligo (dT)-18, and RNase Inhibitor (TaKaRa, Beijing). qPCR was performed with the LightCycler 96 (Roche, Shanghai). Expression of Pt_21 was investigated, and the β-actin gene was used to calibrate the expression level of the query genes, as previously described (Supplementary Table S1). Quantification of the target gene was assessed by relative standard curves. The 2−ΔΔCT method was employed to quantify the relative gene expression levels. Statistical analysis was conducted using Microsoft Excel to calculate mean values and standard errors (SE). The statistical significance of differences was calculated using Student’s t test by the SPSS 21.0 (IBM SPSS Statistic) to obtain the P value. Data were shown as mean ± SE of three biological replicates from one representative experiment. Significant differences between treatments and controls or between time-course points were represented by asterisk (**p < 0.01, *p < 0.05).

Expression and purification of the fusion protein

Pt_21 was individually cloned into pGEX-6p-3 (with GST tag) and expressed in E. coli. The recombinant plasmids were transformed into E. coli BL21 (DE3) chemically competent cells. The production of protein was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG, Mei5bio, Beijing). A GST-Agarose Label Kit (TRAN, Beijing) was used to bind GST-tagged protein following the manufacturer’s instructions. The purified protein products were separated by 15% SDS-PAGE and visualized using Coomassie Blue (Mei5bio, Beijing) staining.

Infiltration of wheat near-isogenic lines

We infiltrated the leaves of 21-day-old seedlings of all available 41 near-isogenic lines of Thatcher with ΔspPt_21 protein. This protein was diluted to 0.1 mg/mL in 1X PBS buffer (137 mM NaCl, 2.7 Mm KCl, 10 Mm Na2HPO4,and 2 Mm KH2PO4) and infiltrated into the abaxial side of the first leaf using a 1 ml needleless syringe. Buffer was infiltrated into a second leaf to test if the cell death was caused by a non-specific protein. The purified ΔspPt_21 protein was infiltrated into the leaves of TcLr21, and infiltrated seedlings were transferred to a growth chamber maintained at 22 °C with a photoperiod of 16 h. Cell death was evaluated at 24 h after infiltration. All these experiments were repeated three times. Genomic DNA of TcLr21 (RL6043) was extracted by a previously published method21.

Agrobacterium tumefaciens infiltration assays and confocal microscopy

The recombinant vectors pCamA:Lr21, pCamA:ΔspPt_21 and pCamA:PTTG_27401 were transformed into Agrobacterium tumefaciens strain GV3101 (Pyaest, Wuhan). Agrobacterium cultures containing the Lr21 and ΔspPt_21 or Lr21 and PTTG_27401 expression vectors were grown overnight at 28 °C in LB medium with appropriate antibiotic selections. The cells were pelleted and resuspended in MMAi buffer (5 g/L MS salts, 20 g/L sucrose, 10 mM MES, 200 μΜ AS) to an optical density (OD600) of 0.6, followed by incubation at room temperature for 1 hour. Cultures were infiltrated into leaves of 4-week-old tobacco with a 1 mL needleless syringe. Symptoms were monitored and recorded from 2 to 4 days after infiltration. Three independent biological replicates were conducted for each experiment. Protein expression was visualized using fluorescence microscopy with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

Y2H validation

Primers were designed to amplify the coding regions of AvrLr21 and Lr21 (Supplementary Table S1). The recombinant vectors pGBKT7:ΔspAvrLr21 and pGADT7:Lr21 were constructed. The Matchmaker Gold Y2H System was used to verify the interaction between ΔspAvrLr21 (bait) and Lr21 (prey). The bait and prey plasmids were co-transformed into yeast strain Y2HGold (Pyaest, Wuhan) according to the manufacturer’s instructions. Transformed yeast cells were assayed for growth on synthetic dropout SD/-Trp-Leu plates and SD/-Trp-Leu-His-Ade plates containing X-α-Galactosidase (X-α-Gal) and Aureobasidin A (AbA) (Pyaest, Wuhan).

Co-IP assay

Agrobacterium strain GV3101 carrying the recombinant vector pCamA:Lr21 was infiltrated into the leaves of 4-week-old N. benthamiana. At 48 h post agroinfiltration, total protein was extracted using extraction buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 5 mM DTT, 100×Protease Inhibitor Cocktail, For Bacterial Cell, with EDTA (Songon, Shanghai). pGEX-6p-3:ΔspAvrLr21 was induced by IPTG with a final concentration of 0.5 mmol/L. For Co-IP assays, a mixture of 2 mL ΔspAvrLr21 and Lr21 containing 50 μl GST agarose beads were incubated at 4 °C for 4 h with gentle shaking. The precipitated proteins were washed five times with washing buffer. The precipitated proteins and crude proteins (input) were detected by immunoblotting with 5 μL anti-GST antibody (Solarbio, Beijing, K000478P) and 5 μL anti-GFP antibody (Solarbio, Beijing, K009323P) (The dilution ratio is 1:1000).

Co-localization assays

Red fluorescent fusion constructs were produced by cloning the coding sequence of Lr21 into the vector pGWB454. GFP fluorescent fusion constructs were produced by cloning the coding sequence of ΔspAvrLr21 into the vector pCamA (Supplementary Table S1). Agrobacterium strains GV3101 carrying pGWB454:Lr21 and pCamA:ΔspAvrLr21 were resuspended to a final OD600 of 0.8 and mixed in equal proportions immediately prior to infiltrations. Infiltrations were performed on 4-5-week-old of N. benthamiana plants. Fluorescence in leaves of N. benthamiana was monitored at 48 h after agroinfiltration, and then imaged directly using a confocal laser scanning microscopy with an excitation wavelength of 488 nm and emission of 540 nm (Olympus FluoView FV1000).

Sequence analysis of AvrLr21

AvrLr21 was amplificated from Pt race PHNT using 2×Es Taq MasterMix (CWBIO, Beijing). Sanger sequencing was used for sequencing of the AvrLr21 gene. The molecular weight of AvrLr21 protein was predicted by ProtParam (https://web.expasy.org/protparam/), and the localization of AvrLr21 protein was predicted by EffectorP-fungi 3.0 (http://effectorp.csiro.au/). The signal peptide of AvrLr21 was identified using SignalP 5.0 (http://www.cbs.dtu.dk/services/SignalP/). All primers used in this study are listed in Supplementary Table S1.

Host-induced gene silencing (HIGS)

To confirm the specificity of the silenced fragments, cDNA fragments of AvrLr21 with no or lower similarity to other genes were selected to prevent off-target effects. Next, the selected cDNA fragments, containing NotI and PacI restriction sites (Supplementary Table S1), were obtained by reverse transcription PCR and inserted into the empty barley stripe mosaic virus (BSMV) vector to generate BSMV:Pt_21. The wheat phytoene desaturase gene (PDS) was inserted into the empty BSMV vector to generate BSMV:TaPDS as a positive control for examining the silencing efficiency. BSMV:γ was used as a negative control. In vitro transcription was performed using the mMESSAGE mMACHINE Kit High Yield Capped RNA Transcription Kit (Ambion) with linearized plasmid as a template according to the manufacturer’s protocol. Then, 240 μl of the BSMV mixture (10 μl of linearized product, 90 μl of DEPC water, and 120 μl of GKP Buffer) was applied to the fully expanded leaves of 2-leave stage TcLr21 wheat plants by rubbing according to the method described previously. Leaf rust urediniospores were inoculated on the fourth and fifth leaves of TcLr21 and the susceptible wheat cultivar Thatcher. Wheat leaves were inoculated with sterile water as a negative control. 10 days after virus inoculation, the fresh urediniospores of PHNT were inoculated on the virus-inoculated leaves. The inoculated leaves were sampled at 24 h, 48 h, and 120 h after inoculation to examine the silencing efficiency of AvrLr21 using RT-qPCR and for histological observations. These experiments were repeated three times.

Histological observations of fungal growth and host responses

Auto-fluorescence of fungus-penetrated mesophyll cells was observed in necrotic areas by epifluorescence microscopy (excitation filter, 488 nm; dichromic mirror, 510 nm; and barrier filter, 520 nm). The 3,3-diaminobenzidine (DAB, Solarbio, Beijing) staining was conducted to detect H2O2 accumulation and viewed following the protocols described previously39. A minimum of 50 infection sites were examined on each of five randomly selected leaf segments for each treatment. The hyphal length, the branching of hyphae and H2O2 accumulation were observed with the Nikon Ti2-LAPP Ti2 Laser Application System (Nikon Corporation).

Inoculation of Lr21-breaking Pt isolate to inhibit cell death

Inoculation of the Lr21-breaking isolate THTT and the Lr21-avirulent isolate PHNT on the leaves of TcLr21 at the three-leaf stage were conducted following the methods previously described40. At 12 h after inoculation and moisturizing, the purified ΔspAvrLr21 protein with a concentration of 0.1 mg/mL with 1X PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4) was infiltrated into the leaves of TcLr21 using a needleless syringe, and the infiltrated seedlings were transferred to a growth chamber and maintained at 22 °C with a photoperiod of 16 h. Development of cell death around the infiltration regions was observed, and representative pictures were photographed at 1 day and 10 days after protein infiltration. The experiment was repeated three times with similar results.

Statistics and reproducibility

Different statistical tests were conducted for different experiments. One-way ANOVA analysis was conducted with the Turkey’s post-hoc test using SPSS to determine the significance of differences between treatments or samples. Sample sizes and the number of replicates for each experiment are indicated in relevant experiments.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information

Description of Additional Supplementary Files

Supplemental Data

Reporting Summary

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-024-06881-4.

Acknowledgements

We thank Prof. R.A. McIntosh (Plant Breeding Institute, University of Sydney) for review and English editing of the manuscript; Prof. M. Hossein Borhan (Agriculture and Agri-Food Canada), for providing the gateway vectors; Prof. Zhimin Hao (Hebei Agricultural University) for providing the pGEX-6p-3 plasmids. This study was funded by the State Key Laboratory of North China Crop Improvement and Regulation (NCCIR2023ZZ-10), the National Natural Science Foundation of China (32172384, 31501623), Chunhui Talent Program of the Natural Science Foundation of Hebei (C2022204116) and the Hundred Talents Program for the introduction of high-level overseas talents in Hebei Province (E2020100004).

Author contributions

H.Y.W., F.W., and S.T.Y. conceived the project and designed the experiments. S.S.S., F.W., Z.C.C., and L.S.M. performed the experiments and interpreted the results. F.W., S.S.S., Z.C.C., and L.S.M. analyzed the data. H.Y.W., S.S.S, and L.M. wrote and revised the manuscript. L. M., H.Y.W., and D.Q.L. supervised this experiment.

Peer review

Peer review information

Communications Biology thanks Anindya BANDYOPADHYAY, Ahmet Caglar Ozketen, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Dr. Leena Tripathi and Dr. Ophelia Bu.

Data availability

The gene sequence of AvrLr21 is available in the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) with accession number OK033362.1. Source data underlying graphs can be obtained in Supplementary Data.

Competing interests

The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Songsong Shen, Fei Wang, Zhongchi Cui, Shitao Yuan.
==== Refs
References

1. Fisher MC Emerging fungal threats to animal, plant and ecosystem health Nature 2013 484 7393
Fisher, M. C. et al. Emerging fungal threats to animal, plant and ecosystem health. Nature 484, 7393 (2013).
2. Dinh HX Singh D Periyannan S Park RF Pourkheirandish M Molecular genetics of leaf rust resistance in wheat and barley Theor. Appl. Genet. 2020 133 2035 2050 10.1007/s00122-020-03570-8 32128617
Dinh, H. X., Singh, D., Periyannan, S., Park, R. F. & Pourkheirandish, M. Molecular genetics of leaf rust resistance in wheat and barley. Theor. Appl. Genet. 133, 2035–2050 (2020).32128617
3. Ellis JG Lagudah ES Spielmeyer W Dodds PN The past, present and future of breeding rust resistant wheat Front. Plant Sci. 2014 5 641 10.3389/fpls.2014.00641 25505474
Ellis, J. G., Lagudah, E. S., Spielmeyer, W. & Dodds, P. N. The past, present and future of breeding rust resistant wheat. Front. Plant Sci. 5, 641 (2014).25505474
4. Liu F Exome association analysis sheds light onto leaf rust (Puccinia triticina) resistance genes currently used in wheat breeding (Triticum aestivum L.) Plant Biotechnol. J. 2020 18 1396 1408 10.1111/pbi.13303 31782598
Liu, F. et al. Exome association analysis sheds light onto leaf rust (Puccinia triticina) resistance genes currently used in wheat breeding (Triticum aestivum L.). Plant Biotechnol. J. 18, 1396–1408 (2020).31782598
5. Kumar K An update on resistance genes and their use in the development of leaf rust resistant cultivars in wheat Front. Genet. 2022 13 816057 10.3389/fgene.2022.816057 35432483
Kumar, K. et al. An update on resistance genes and their use in the development of leaf rust resistant cultivars in wheat. Front. Genet. 13, 816057 (2022).35432483
6. Li HN Cloning of the wheat leaf rust resistance gene Lr47 introgressed from Aegilops speltoides Nat. Commun. 2023 14 6072 10.1038/s41467-023-41833-2 37770474
Li, H. N. et al. Cloning of the wheat leaf rust resistance gene Lr47 introgressed from Aegilops speltoides. Nat. Commun. 14, 6072 (2023).37770474
7. Lin GF Cloning of the broadly effective wheat leaf rust resistance gene Lr42 transferred from Aegilops tauschii Nat. Commun. 2022 13 3044 10.1038/s41467-022-30784-9 35650212
Lin, G. F. et al. Cloning of the broadly effective wheat leaf rust resistance gene Lr42 transferred from Aegilops tauschii. Nat. Commun. 13, 3044 (2022).35650212
8. Hewitt T Wheat leaf rust resistance gene Lr13 is a specific Ne2 allele for hybrid necrosis Mol. Plant. 2021 14 1025 1028 10.1016/j.molp.2021.05.010 33965633
Hewitt, T. et al. Wheat leaf rust resistance gene Lr13 is a specific Ne2 allele for hybrid necrosis. Mol. Plant. 14, 1025–1028 (2021).33965633
9. Yan XC High-temperature wheat leaf rust resistance gene Lr13 exhibits pleiotropic effects on hybrid necrosis Mol. Plant. 2021 14 1029 1032 10.1016/j.molp.2021.05.009 33965635
Yan, X. C. et al. High-temperature wheat leaf rust resistance gene Lr13 exhibits pleiotropic effects on hybrid necrosis. Mol. Plant. 14, 1029–1032 (2021).33965635
10. Bolton MD Kolmer JA Garvin DF Pathogen profile Wheat leaf rust caused by Puccinia triticina Mol. Plant Pathol. 2008 9 563 575 10.1111/j.1364-3703.2008.00487.x 19018988
Bolton, M. D., Kolmer, J. A. & Garvin, D. F. Pathogen profile Wheat leaf rust caused by Puccinia triticina. Mol. Plant Pathol. 9, 563–575 (2008).19018988
11. Cui ZC Evasion of wheat resistance gene Lr15 recognition by the leaf rust fungus is attributed to the coincidence of natural mutations and deletion in AvrLr15 gene Mol. Plant Pathol. 2024 25 e13490 10.1111/mpp.13490 38952297
Cui, Z. C. et al. Evasion of wheat resistance gene Lr15 recognition by the leaf rust fungus is attributed to the coincidence of natural mutations and deletion in AvrLr15 gene. Mol. Plant Pathol. 25, e13490 (2024).38952297
12. Huang L Evolution of new disease specificity at a simple resistance locus in a crop–weed complex: reconstitution of the Lr21 gene in wheat Genetics 2009 182 595 602 10.1534/genetics.108.099614 19364806
Huang, L. et al. Evolution of new disease specificity at a simple resistance locus in a crop–weed complex: reconstitution of the Lr21 gene in wheat. Genetics 182, 595–602 (2009).19364806
13. Huang L Map-based cloning of leaf rust resistance gene Lr21 from the large and polyploid genome of bread wheat Genetics 2003 164 655 664 10.1093/genetics/164.2.655 12807786
Huang, L. et al. Map-based cloning of leaf rust resistance gene Lr21 from the large and polyploid genome of bread wheat. Genetics 164, 655–664 (2003).12807786
14. Rowland GG Kerber ER Telocentric mapping in hexaploid wheat of genes for leaf rust resistance and other characters derived from Aegilops squarrosa Can. J. Genet. Cytol. 1974 16 137 144 10.1139/g74-013
Rowland, G. G. & Kerber, E. R. Telocentric mapping in hexaploid wheat of genes for leaf rust resistance and other characters derived from Aegilops squarrosa. Can. J. Genet. Cytol. 16, 137–144 (1974).
15. Naz AA Lr21 diversity unveils footprints of wheat evolution and its new role in broad-spectrum leaf rust resistance Plant Cell Environ. 2021 10 1151 1158
Naz, A. A. et al. Lr21 diversity unveils footprints of wheat evolution and its new role in broad-spectrum leaf rust resistance. Plant Cell Environ. 10, 1151–1158 (2021).
16. Zhao J Kang ZS Fighting wheat rusts in China: a look back and into the future Phytopathol. Res. 2023 5 6 10.1186/s42483-023-00159-z
Zhao, J. & Kang, Z. S. Fighting wheat rusts in China: a look back and into the future. Phytopathol. Res. 5, 6 (2023).
17. Figlan S Breeding wheat for durable leaf rust resistance in southern Africa: variability, distribution, current control strategies, challenges and future prospects Front. Plant Sci. 2020 11 549 10.3389/fpls.2020.00549 32499800
Figlan, S. et al. Breeding wheat for durable leaf rust resistance in southern Africa: variability, distribution, current control strategies, challenges and future prospects. Front. Plant Sci. 11, 549 (2020).32499800
18. Maor R Shirasu K The arms race continues: battle strategies between plants and fungal pathogens Curr. Opin. Microbiol. 2005 8 399 404 10.1016/j.mib.2005.06.008 15996507
Maor, R. & Shirasu, K. The arms race continues: battle strategies between plants and fungal pathogens. Curr. Opin. Microbiol. 8, 399–404 (2005).15996507
19. McCallum BD A review of wheat leaf rust research and the development of resistant cultivars in Canada Can. J. Plant. Pathol. 2016 38 1 18 10.1080/07060661.2016.1145598
McCallum, B. D. et al. A review of wheat leaf rust research and the development of resistant cultivars in Canada. Can. J. Plant. Pathol. 38, 1–18 (2016).
20. Oelke, L. M. & Komer, J. A. Genetics of leaf rust resistance in spring wheat cultivars alsen and norm. Genet. Resist. 95, 773–778 (2005).
21. Kolmer JA Long DL Hughes ME Physiologic specialization of Puccinia triticina on wheat in the United States in 2010 Plant Dis. 2012 96 1216 1221 10.1094/PDIS-01-12-0048-SR 30727073
Kolmer, J. A., Long, D. L. & Hughes, M. E. Physiologic specialization of Puccinia triticina on wheat in the United States in 2010. Plant Dis. 96, 1216–1221 (2012).30727073
22. Wang F Puccinia triticina effector protein Pt_21 interacts with wheat thaumatin-like protein TaTLP1 to inhibit its antifungal activity and suppress wheat apoplast immunity Crop J. 2023 11 1431 1440 10.1016/j.cj.2023.04.006
Wang, F. et al. Puccinia triticina effector protein Pt_21 interacts with wheat thaumatin-like protein TaTLP1 to inhibit its antifungal activity and suppress wheat apoplast immunity. Crop J. 11, 1431–1440 (2023).
23. Sung YC PR1-mediated defence via C-terminal peptide release is targeted by a fungal pathogen effector N. Phytol. 2021 229 3467 3480 10.1111/nph.17128
Sung, Y. C. et al. PR1-mediated defence via C-terminal peptide release is targeted by a fungal pathogen effector. N. Phytol. 229, 3467–3480 (2021).
24. Outram MA The crystal structure of SnTox3 from the necrotrophic fungus Parastagonospora nodorum reveals a unique effector fold and provides insight into Snn3 recognition and pro-domain protease processing of fungal effectors N. Phytol. 2021 231 2282 2296 10.1111/nph.17516
Outram, M. A. et al. The crystal structure of SnTox3 from the necrotrophic fungus Parastagonospora nodorum reveals a unique effector fold and provides insight into Snn3 recognition and pro-domain protease processing of fungal effectors. N. Phytol. 231, 2282–2296 (2021).
25. Salcedo A Variation in the AvrSr35 gene determines Sr35 resistance against wheat stem rust race Ug99 Plant Sci. 2017 358 1604 1606
Salcedo, A. et al. Variation in the AvrSr35 gene determines Sr35 resistance against wheat stem rust race Ug99. Plant Sci. 358, 1604–1606 (2017).
26. Zhao YB Pathogen effector AvrSr35 triggers Sr35 resistosome assembly via a direct recognition mechanism Sci. Adv. 2022 8 eabq5108 10.1126/sciadv.abq5108 36083908
Zhao, Y. B. et al. Pathogen effector AvrSr35 triggers Sr35 resistosome assembly via a direct recognition mechanism. Sci. Adv. 8, eabq5108 (2022).36083908
27. Chen JP Loss of AvrSr50 by somatic exchange in stem rust leads to virulence for Sr50 resistance in wheat Plant Sci. 2017 358 1607 1610
Chen, J. P. et al. Loss of AvrSr50 by somatic exchange in stem rust leads to virulence for Sr50 resistance in wheat. Plant Sci. 358, 1607–1610 (2017).
28. Upadhyaya NM Genomics accelerated isolation of a new stem rust avirulence gene–wheat resistance gene pair Nat. Plants 2021 7 1220 1228 10.1038/s41477-021-00971-5 34294906
Upadhyaya, N. M. et al. Genomics accelerated isolation of a new stem rust avirulence gene–wheat resistance gene pair. Nat. Plants 7, 1220–1228 (2021).34294906
29. Panwar V McCallum B Bakkeren G Host-induced gene silencing of wheat leaf rust fungus Puccinia triticina pathogenicity genes mediated by the Barley stripe mosaic virus Plant Mol. Biol. 2013 81 595 608 10.1007/s11103-013-0022-7 23417582
Panwar, V., McCallum, B. & Bakkeren, G. Host-induced gene silencing of wheat leaf rust fungus Puccinia triticina pathogenicity genes mediated by the Barley stripe mosaic virus. Plant Mol. Biol. 81, 595–608 (2013).23417582
30. Bai XX A candidate effector protein PstCFEM1 contributes to virulence of stripe rust fungus and impairs wheat immunity Stress Biol. 2022 2 12 10.1007/s44154-022-00042-5 37676556
Bai, X. X. et al. A candidate effector protein PstCFEM1 contributes to virulence of stripe rust fungus and impairs wheat immunity. Stress Biol. 2, 12 (2022).37676556
31. Wang YQ A stripe rust fungal effector PstSIE1 targets TaSGT1 to facilitate pathogen infection Plant J. 2022 112 1413 1428 10.1111/tpj.16019 36308427
Wang, Y. Q. et al. A stripe rust fungal effector PstSIE1 targets TaSGT1 to facilitate pathogen infection. Plant J. 112, 1413–1428 (2022).36308427
32. Xu Q An effector protein of the wheat stripe rust fungus targets chloroplasts and suppresses chloroplast function Nat. Commun. 2019 10 5571 10.1038/s41467-019-13487-6 31804478
Xu, Q. et al. An effector protein of the wheat stripe rust fungus targets chloroplasts and suppresses chloroplast function. Nat. Commun. 10, 5571 (2019).31804478
33. Qi T Stripe rust effector PstGSRE1 disrupts nuclear localization of ROS-promoting transcription factor TaLOL2 to defeat ROS-induced defense in wheat Mol. Plant. 2019 12 1624 1638 10.1016/j.molp.2019.09.010 31606466
Qi, T. et al. Stripe rust effector PstGSRE1 disrupts nuclear localization of ROS-promoting transcription factor TaLOL2 to defeat ROS-induced defense in wheat. Mol. Plant. 12, 1624–1638 (2019).31606466
34. Dodds PN Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes PNAS 2006 103 8888 8893 10.1073/pnas.0602577103 16731621
Dodds, P. N. et al. Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes. PNAS 103, 8888–8893 (2006).16731621
35. Zhang L Zhang LY Meng QF Yan HF Liu DQ Virulence and molecular genetic diversity, variation, and evolution of the Puccinia triticina population in Hebei Province of China from 2001 to 2010 Plant Sci. 2023 14 1095677
Zhang, L., Zhang, L. Y., Meng, Q. F., Yan, H. F. & Liu, D. Q. Virulence and molecular genetic diversity, variation, and evolution of the Puccinia triticina population in Hebei Province of China from 2001 to 2010. Plant Sci. 14, 1095677 (2023).
36. Houterman PM Cornelissen BJC Rep M Suppression of plant resistance gene-based immunityby a fungal effector PLoS Pathog. 2008 5 e1000061 10.1371/journal.ppat.1000061
Houterman, P. M., Cornelissen, B. J. C. & Rep, M. Suppression of plant resistance gene-based immunityby a fungal effector. PLoS Pathog. 5, e1000061 (2008).
37. Plissonneau C A game of hide and seek between avirulence genes AvrLm4-7 and AvrLm3 in Leptosphaeria maculans N. Phytol. 2016 209 1613 1624 10.1111/nph.13736
Plissonneau, C. et al. A game of hide and seek between avirulence genes AvrLm4-7 and AvrLm3 in Leptosphaeria maculans. N. Phytol. 209, 1613–1624 (2016).
38. Ghanbarnia K Leptosphaeria maculans AvrLm9: a new player in the game of hide and seek with AvrLm4-7 Mol. Plant Pathol. 2018 19 1754 1764 10.1111/mpp.12658 29330918
Ghanbarnia, K. et al. Leptosphaeria maculans AvrLm9: a new player in the game of hide and seek with AvrLm4-7. Mol. Plant Pathol. 19, 1754–1764 (2018).29330918
39. Wang F TaTLP1 interacts with TaPR1 to contribute to wheat defense responses to leaf rust fungus PLoS Genet. 2020 16 e1008713 10.1371/journal.pgen.1008713 32658889
Wang, F. et al. TaTLP1 interacts with TaPR1 to contribute to wheat defense responses to leaf rust fungus. PLoS Genet. 16, e1008713 (2020).32658889
40. Roelfs AP Martell LB Uredospore dispersal from a point source within a wheat canopy Phytopathology 1984 74 1262 1267 10.1094/Phyto-74-1262
Roelfs, A. P. & Martell, L. B. Uredospore dispersal from a point source within a wheat canopy. Phytopathology 74, 1262–1267 (1984).
