Thaumatin-like proteins (TLPs), which are defined as pathogenesis-related protein family 5 (PR5) members, are common plant proteins involved in defense responses and confer antifungal activity against many plant pathogens. Our earlier studies have reported that the TaTLP1 gene was isolated from wheat and proved to be involved in wheat defense in response to leaf rust attack. The present study aims to identify the interacting proteins of TaTLP1 and characterize the role of the interaction between wheat and Puccinia triticina ( Pt ). Pull-down experiments designed to isolate the molecular target of TaTLP1 in tobacco resulted in the identification of TaPR1, a pathogenesis-related protein of family 1, and the interaction between TaTLP1 and TaPR1 was confirmed by yeast two-hybrid experiments (Y2H), bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation (Co-IP). In vitro , TaTLP1 and TaPR1 together increased antifungal activity against Pt . In vivo , the disease resistance phenotype, histological observations of fungal growth and host responses, and accumulation of H 2 O 2 in TaTLP1-TaPR1 in co-silenced plants indicated that co-silencing significantly enhanced wheat susceptibility compared to single knockdown TaTLP1 or TaPR1 plants. The accumulation of reactive oxygen species (ROS) was significantly reduced in co-silenced plants compared to controls during Pt infection, which suggested that the TaTLP1-TaPR1 interaction positively modulates wheat resistance to Pt in an ROS-dependent manner. Our findings provide new insights for understanding the roles of two different PRs, TaTLP1 and TaPR1, in wheat resistance to leaf rust.

Pathogenesis-related (PR) proteins play important roles in plant defense response and systemic acquired resistance (SAR). PR1 has antifungal activity against many plant pathogens. In our previous study, RNA sequencing (RNA-seq) was conducted on resistant wheat line TcLr19 and sensitive wheat cultivar Chinese Spring inoculated with Puccinia triticina (Pt) race PHNT. In this study, seven salicylic acid (SA)-induced TaPR1 genes involved in plant disease resistance were found in the RNA-seq library. Quantitative PCR (qPCR) results showed that TaPR1-4 was most induced by Pt among these seven TaPR1 genes in the incompatible interaction. Yeast two-hybrid (Y2H) results showed that TaPR1-4 interacted with TaTLP1 via the αIV helix. Protein-mediated phenotyping assays in vivo and antifungal activity in vitro demonstrated that wheat leaves infiltrated with pure TaPR1-4 protein developed significantly less disease compared to control leaves. This effect was correlated with a strong increase in defense gene expression, and resistance activity was dependent on the CAPE1 motif located in the C-terminal region of TaPR1-4. These findings increase current knowledge regarding the interaction of TaPR1 and TaTLP1 and provide new insights on the role of TaPR1 protein in the resistance of wheat to Pt.

Background: Wheat leaf rust caused by Puccinia triticina (Pt) remains one of the most destructive diseases of common wheat worldwide. Cultivating resistant cultivars is an effective way to control this disease, but race-specific resistance can be overcome quickly due to the rapid evolution of Pt populations. The critical to control wheat leaf rust is to understand the pathogenicity mechanisms of Pt. Results: In this study, the spores of the Pt race PHNT (wheat leaf rust resistance gene Lr19-avirulent isolate) were mutagenized with ethyl methanesulfonate (EMS) and two Pt Lr19-virulent mutants named M1 and M2 were isolated, suggesting that they carry mutations affecting the Lr19-specific avirulent factor. RNA sequencing was performed on samples collected from the wheat cultivars Chinese Spring and TcLr19 that were infected by wild-type (WT) PHNT and the two Lr19-virulent mutant isolates at 14 days post-inoculation (dpi). The assembled transcriptome data were compared to the reference genome “Pt 1-1 BBBD Race 1.” A total of 216 differentially expressed genes (DEGs) were found from three different sample comparisons including M1-vs-WT, M2-vs-WT, and M1-vs-M2. Of these DEGs, 29 common DEGs were shared between M1-vs-WT and M2-vs-WT comparisons. Among the 216 DEGs encoding proteins, 30 were predicted to be effector candidates. Then 6 effector candidates (PTTG_27844, PTTG_05290, PTTG_27401, PTTG_27224, PTTG_26282, PTTG_25521) were verified that these genes were differentially expressed during Pt infection by quantitative real-time PCR (qRT-PCR) and were validated on tobacco, and the results showed that PTTG_27401 could inhibit progress of cell death (PCD) induced by BAX.Conclusions: Our results showed that a large number of genes participate in the interaction between Pt and TcLr19, which will provide valuable resources for the identification and targeting of AvrLr19 effector candidates and pathogenesis-related genes. Furthermore, our analyses are of great significance to reveal the pathogenesis of Pt.

IntroductionWheat leaf rust caused by Puccinia triticina (Pt) remains one of the most destructive diseases of common wheat worldwide. Understanding the pathogenicity mechanisms of Pt is important to control wheat leaf rust.MethodsThe urediniospores of Pt race PHNT (wheat leaf rust resistance gene Lr19-avirulent isolate) were mutagenized with ethyl methanesulfonate (EMS), and two Lr19-virulent mutants named M1 and M2 were isolated. RNA sequencing was performed on samples collected from wheat cultivars Chinese Spring and TcLr19 infected with wild-type (WT) PHNT, M1, and M2 isolates at 14 days post-inoculation (dpi), respectively. Screening AvrLr19 candidates by quantitative reverse transcription PCR (qPCR) and Agrobacterium-mediated transient assays in Nicotiana benthamiana.Results560 genes with single nucleotide polymorphisms (SNPs) and insertions or deletions (Indels) from non-differentially expressed genes were identified. Among them, 10 secreted proteins were screened based on their fragments per kilobase of exon model per million mapped reads (FPKM) values in the database. qPCR results showed that the expression profiles of 7 secreted proteins including PTTG_27471, PTTG_12441, PTTG_28324, PTTG_26499, PTTG_06910, PTTG_26516, and PTTG_03570 among 10 secreted proteins in mutants were significantly different with that in wild-type isolate after infection wheat TcLr19 and might be related to the recognition between Lr19 and AvrLr19. In addition, a total of 216 differentially expressed genes (DEGs) were obtained from three different sample comparisons including M1-vs-WT, M2-vs-WT, and M1-vs-M2. Among 216 DEGs, 15 were predicted to be secreted proteins. One secreted protein named PTTG_04779 could inhibit programmed progress of cell death (PCD) induced by apoptosis-controlling genes B-cell lymphoma-2 associated X protein (BAX) on Nicotiana benthamiana, indicating that it might play a virulence function in plant. Taken together, total 8 secreted proteins, PTTG_04779, PTTG_27471, PTTG_12441, PTTG_28324, PTTG_26499, PTTG_06910, PTTG_26516, PTTG_03570 are identified as AvrLr19 candidates.DiscussionOur results showed that a large number of genes participate in the interaction between Pt and TcLr19, which will provide valuable resources for the identification of AvrLr19 candidates and pathogenesis-related genes.