Plants have evolved multifaceted defence mechanisms to resist pathogen infection. Production of the pathogenesis-related (PR) proteins in response to pathogen attack has been implicated in plant disease resistance specialized in systemic-acquired resistance (SAR). Our earlier studies have reported that a full length TaLr35PR5 gene, encoding a protein exhibiting amino acid and structural similarity to a sweet protein thaumatin, was isolated from wheat near-isogenic line TcLr35. The present study aims to understand the function of TaLr35PR5 gene in Lr35-mediated adult resistance to Puccinia triticina. We determined that the TaLr35PR5 protein contained a functional secretion peptide by utilizing the yeast signal sequence trap system. Using a heterologous expression assay on onion epidermal cells we found that TaLr35PR5 protein was secreted into the apoplast of onion cell. Expression of TaLr35PR5 was significantly reduced in BSMV-induced gene silenced wheat plants, and pathology test on these silenced plants revealed that Lr35-mediated resistance phenotype was obviously altered, indicating that Lr35-mediated resistance was compromised. All these findings strongly suggest that TaLr35PR5 is involved in Lr35-mediated adult wheat defense in response to leaf rust attack.

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 H₂O₂ 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. In host-pathogen interactions, novel proteins that are specifically involved in pathological response are collectively referred to as “pathogenesis-related proteins” (PRs). PR proteins encoded by plant defense genes play key roles in plant defense responses, particularly in systemic-acquired resistance. However, their molecular roles in the response of wheat to fungal infection are typically not well characterized. PR5 family proteins are referred to as thaumatin-like proteins (TLPs), are able to rapidly accumulate to high levels in response to biotic or abiotic stress and exhibit antifungal activity in various plant species. Thus, investigation of PR5 proteins is crucial for understanding the mechanism of wheat defense against Pt and searching for new ways to control the wheat leaf rust disease.

TaLr35PR5 gene was obtained from the gDNA and cDNA of TcLr35 wheat. It was induced by Puccinia triticina, ABA and SA, but TaLr35PR5 was induced earlier and its expression level was higher in the incompatible interaction than that in the compatible interaction. In addition, the accumulations of TaLr35PR5 increased stably and showed significant peak challenged by P. triticina at different growth and development periods of TcLr35 wheat while it maintained similar level and changed little in mock inoculated. Western blottings were conducted to confirm that TaLr35PR5 be induced by P. triticina infection at the protein expression level. Similar to the expression patterns of TaLr35PR5 at RNA levels, the accumulations of TaLr35PR5 protein were weak in the seedling stage, then increased to the peak and kept constant levels at the mature stage which is consistent with the expression feature of Lr35 gene as an adult plant resistance gene.

Pathogenesis-related (PR) protein-5, is involved in host defense system against both biotic and abiotic stresses as well as the regulation of physiological processes in numerous plant species. Our earlier studies have reported the isolation of a full-length TaLr19TLP1 gene (516 bp, GenBank accession No. KJ764822) from wheat infected by leaf rust. Quantitative real-time polymerase chain reaction analyses revealed that TaLr19TLP1 transcript was significantly induced and upregulated during incompatible interaction, while a relatively low level of the transcript was detected during compatible interaction. In the current study, we demonstrate that the accumulation of TaLr19TLP1 transcript is significantly different in tested wheat organs. TaLr19TLP1 was induced by salicylic acid (SA), methyl jasmonic (MeJA), ethephon (ETH) and abscisic acid (ABA). The transcripts of TaLr19TLP1 accumulated at higher levels following pretreatment with SA, MeJA and ABA prior to infection with P. triticina. A slight induction was observed in ETH pretreated seedlings compared with the treatment without inoculation. In addition, TaLr19TLP1 was found to be predominately localized to extracellular spaces of onion epidermal cell. Knocking down the expression of TaLr19TLP1 through virus-induced gene silencing reduced wheat resistance against leaf rust pathogen. These results suggested that TaLr19TLP1 mediated disease resistance in wheat exposed to leaf rust pathogen.