Yeast Two-Hybrid Technology Reveals a Novel Mechanism of Wheat Disease Resistance: How TaPIR1 "Hijacks" TaHRP1 to Suppress Chloroplast Function?-Antibody Detection Service Topic-Tekbiotech-Yeast and Phage Display CRO, Expert in Nano-body and Antibody Drug Development

Yeast Two-Hybrid Technology Reveals a Novel Mechanism of Wheat Disease Resistance: How TaPIR1 "Hijacks" TaHRP1 to Suppress Chloroplast Function?

Chloroplasts are crucial for both immunity and photosynthesis. However, the regulatory mechanisms controlling the expression of photosynthesis-associated nuclear genes (PhANGs) during plant immunity remain poorly understood. Therefore, Zhang et al., using yeast two-hybrid technology, discovered that the transcription factor E3 ubiquitin ligase (TaPIR1) negatively regulates wheat resistance to Puccinia striiformis (Pst) by degrading the transcription factor TaHRP1. They found that knockout of TaHRP1 reduced wheat resistance to Pst, while overexpression of TaHRP1 enhanced photosynthesis, ROS production, and consequently improved disease resistance. In summary, TaPIR1-mediated ubiquitination and degradation of TaHRP1 affects the expression of PhANGs, thereby reducing plant resistance to Pst.

1. Generation of Mutants Using CRISPR-Cas9: TaPIR1 is a Negative Defense Regulator in Wheat

First, leaf tissue was treated. Double knockout mutants of TaPIR1 (*tapir1-ab*) were generated. The virulent strains CYR31 and CYR34 were separately inoculated onto wild-type (WT) and mutant leaves (Figure 1a). Observation after 14 days showed that the surfaces of WT leaves were covered with spores. qRT-PCR validation (Figure 1b) revealed a highly significant difference in the Pst/wheat biomass ratio between the mutant and WT. Microscopic observation of the Pst infection structures in leaves showed that Pst development was mature in WT leaves by 24 hours (Figure 1c), with highly significant differences in hyphal length and infection area compared to the mutant (Figure 1d). Using DAB staining, large areas of reddish-brown precipitate were observed around the mutant (Figure 1e), indicating a significant increase in H?O? accumulation, which was quantitatively confirmed to be highly significant (Figure 1f). In conclusion, knockout of TaPIR1 enhances ROS accumulation, inhibits Pst infection and proliferation, thereby significantly improving wheat disease resistance.

Figure 1: TaPIR1 negatively regulates wheat resistance to Pst.

2. Validation of Protein-Protein Interaction: TaPIR1 Negatively Regulates TaHRP1

Using yeast two-hybrid technology (Figure 2a), they found that only the combination pBD-TaPIR1 + pAD-TaHRP1, along with the positive control P53 combination, grew on SD-LWHA medium and produced blue colonies, whereas empty vectors did not grow, indicating a specific interaction between the two proteins. Using bimolecular fluorescence complementation (BiFC) validation (Figure 2b), yellow fluorescence was observed in the nucleus only for the TaPIR1:cYFP + TaHRP1:nYFP combination. Using GST pull-down for in vitro functional validation (Figure 2c), TaPIR1 and TaHRP1 were found to bind directly in vitro without the need for other protein mediators. Using co-immunoprecipitation (Co-IP) validation (Figure 2d), the interaction was confirmed to occur in planta. In vitro ubiquitination validation (Figure 2e, f) demonstrated that TaPIR1 is a functional E3 ubiquitin ligase that specifically ubiquitinates TaHRP1 at lysine residues K131 and K136. Furthermore, the expression level of TaPIR1 was positively correlated with the ubiquitination level of TaHRP1.

Figure 2: Interaction and ubiquitination modification between TaPIR1 and TaHRP1.

3. Genome-Wide Target Gene Screening via DAP-seq: TaHRP1 Directly Regulates Photosynthesis-Related Genes

This study employed DAP-seq technology (Figure 3a) to screen for potential targets of TaHRP1 across the wheat genome, identifying the HBS sequence in the genome as the binding site for TaHRP1. Using the KEGG database (Figure 3b), they found that TaHRP1 target genes are enriched in the photosynthesis pathway, suggesting that TaHRP1 may influence chloroplast function by regulating these genes. Further investigation (Figure 3c, d, e, f) revealed that the N-terminus of TaHRP1 specifically binds with high affinity to the promoter regions of PhANGs containing the HBS motif.

Figure 3: Identification of specific binding between TaHRP1 and PhANGs via DAP-seq.

4. Overall Mechanistic Working Model

Figure 4: Working model of the TaPIR1-TaHRP1 cascade during Pst infection of wheat.

This result closes the regulatory loop: TaHRP1 enhances chloroplast function by activating PhANGs, promoting ROS production to activate immunity; whereas TaPIR1 degrades TaHRP1, cutting off this disease resistance pathway.

Through yeast two-hybrid technology, this study reveals for the first time a novel pathway regulating wheat stripe rust resistance via ubiquitination, establishing a complete regulatory network: ubiquitination modification (TaPIR1) → transcriptional regulation (TaHRP1) → chloroplast function (PhANGs) → ROS immune signaling.

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References

[1] Zhang, R., Wu, Y., Qu, X. et al. The RING-finger ubiquitin E3 ligase TaPIR1 targets TaHRP1 for degradation to suppress chloroplast function. Nat Commun 15, 6905 (2024).

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