A thaumatin-like effector protein suppresses the rust resistance of wheat and promotes the pathogenicity of Puccinia triticina by targeting TaRCA

Introduction

Naturally, plants are continuously threatened by various biotic and abiotic stresses, and the most common biotic stresses include phytopathogenic bacteria, viruses, and fungi, etc. However, plants have developed a sophisticated multilayered immune system to protect themselves against attacks from potential pathogens (Dodds & Rathjen, 2010; Jones et al., 2024). When a pathogen infects a host plant, plants first significantly upregulate the expression of pathogenesis-related (PR) genes to initiate the first line of defense (Fisher et al., 2012; Dos & Franco, 2023). Currently, 19 classes of PR proteins have been discovered based on structural similarity and functional activity (Dos & Franco, 2023; Li et al., 2023), among which the sweet-tasting thaumatin homologs, thaumatin-like proteins (TLPs) isolated from Thaumatococcus danielli, belong to the PR-5 family (Liu et al., 2019; Nawrot et al., 2021). Research indicates that TLPs play a crucial role in plant responses to both biotic and abiotic stresses. For example, the stable expression of TaTLP1 in wheat enhanced resistance to Pt and common root rot (Cui et al., 2021). AnTLP13 from Ammopiptanthus nanus localizes in the apoplast and overexpression of AnTLP13 in tobacco enhanced its tolerance to low-temperature stress (Liu et al., 2023). Wheat TaLr35PR5 is involved in the Lr35-mediated defense response of adult wheat against leaf rust disease (Zhang et al., 2018). Silencing of GhTLP19 rendered cotton more sensitive to drought and Verticillium dahlia, whereas overexpressing it in transgenic Arabidopsis enhanced drought tolerance (Li et al., 2020). TLP proteins purified from bananas trigger antifungal activity by inducing membrane disruption and cell wall disintegration in fungi (Jiao et al., 2018). Currently, there is limited research on the function of TLPs in fungi. The effector protein PTTG_04779 in wheat leaf rust fungus contains a thaumatin domain and has been identified as a candidate protein for AvrLr19, which can inhibit BAX-induced programmed cell death in tobacco cells (Cui et al., 2023). However, the impact of TLP proteins in fungi on their pathogenicity has not been reported. Therefore, investigating the role of TLPs in the fungal pathogenic process is of significant importance for elucidating the pathogenic mechanism of the pathogen.

The chloroplast plays a pivotal role in oxygenic photosynthesis and primary metabolism, which are important targets in the intricate virulence strategies of many pathogens. Recently, the chloroplast have been recognized as crucial hubs of immune signaling, serving as a fundamental component in the integration and decoding of environmental signals (Breen et al., 2022). Chloroplast-to-nucleus retrograde signaling is essential for the efficient function and assembly of photosynthetic apparatus (Chan et al., 2016). Likewise, alterations in the growth or metabolic conditions of the chloroplast lead to significant modifications in the patterns of gene expression in the nucleus (Koussevitzky et al., 2007), indicating the crucial role of the chloroplast as an environmental sensor that modulates various environmental cues for transcriptional regulation of particular nuclear genes (Li & Kim, 2022). Chloroplasts are involved in the production of secondary metabolites and defense compounds and they are also the sites of reactive oxygen species (ROS) production, calcium oscillations, and also the production of phytohormones such as salicylic acid (SA), and jasmonic acid (JA) that are key components of plant defense mechanisms against biotrophic pathogens (SA) and necrotrophic pathogens (JA) (Serrano et al., 2016; Kretschmer et al., 2019; Kuzniak & Kopczewski, 2020; Littlejohn et al., 2021; Yokochi et al., 2021; Bittner et al., 2022). The ability of chloroplasts to synthesize phytohormones and a wide variety of secondary metabolites, in combination with retrograde and reactive oxygen signaling, allows exceptional flexibility in both sensing and responding to biotic stressors. These processes therefore provide a plethora of chances for pathogens to develop mechanisms to directly or indirectly target ‘chloroplast immunity’ (Littlejohn et al., 2021). During host-pathogen interactions, various pathogens such as bacteria, oomycetes, and fungi secrete effector proteins that target and interfere with the functions of the chloroplast (de Torres et al., 2015; Xu et al., 2019; Irieda & Takano, 2021; Liu et al., 2021; Savage et al., 2021). For instance, a Puccinia striiformis f. sp. tritici (Pst) effector protein Pst_12806 translocates into the chloroplast where it interacts with the C-terminal Rieske domain of wheat TaISp protein, reducing the expression of defense-related genes, callose deposition and production of ROS, thereby promoting Pst infection (Xu et al., 2019). Furthermore, two other Pst effectors Pst_4 and Pst_5 also suppress the host defense response by disrupting the sorting of the chloroplast protein TaISp, thereby restricting host ROS and enhancing Pst pathogenicity (Wang et al., 2021). Nevertheless, the precise mechanisms by which effectors specifically target chloroplasts to regulate the host's defense responses have not been fully understood in Puccinia triticina (Pt).

Pt is an obligate biotrophic fungus, relies on living host tissues for growth and reproduction. During the infection period, Pt produces a highly sophisticated structure haustorium, which is essential for the absorption of nutrients and secretion of a repertoire of effector proteins, through which it can inhibit and regulate various mechanisms of the host immune responses (Voegele et al., 2001; Jones et al., 2024). However, only a few of the identified Pt effector proteins has been successfully cloned and functionally characterized such as Pt18906 (Qi et al., 2020), Pt13024 (Qi et al., 2022) and Pt_21 (Wang et al., 2023). PTTG_08198 was found to accelerate the progress of cell death and promoted the accumulation of reactive oxygen species (ROS) (Zhao et al., 2020). Pt13024 suppresses PCD and triggers ROS accumulation and callose deposition (Qi et al., 2022). Pt_21 suppresses host defense responses by directly targeting wheat TaTLP1 and inhibiting its antifungal activity (Wang et al., 2023). However, the research on the pathogenic mechanism of wheat leaf rust fungus is still in the initial stage, and the role of more effector proteins in Pt pathogenesis needs to be further explored.

In this study, we identified a Pt effector protein Pt9029 containing a thaumatin domain and a transit peptide. Pt9029 expression was upregulated at early stages during the infection process, and it inhibited programmed cell death (PCD) induced by BAX and INF1, indicating that it can inhibit host defense mechanisms. Overexpressing Pt9029 through the bacterial type III secretion system in TcLr2b suppressed the immune response. Silencing Pt9029 using host-induced gene silencing (HIGS) technology in TcLr2b resulted in smaller lesion sizes and reduced disease severity on plants. Overexpression of Pt9029 in Fielder inhibited the expression of TaPR1 and TaPR2, increased the expression of TaPAL and TaSOD, decreased the accumulation of H₂O₂, and increased the pathogenicity of Pt. Pt9029 was found to interact with TaRCA in chloroplasts. TaRCA positively regulates the resistance of wheat to leaf rust in TcLr2b. Pt9029 exerts its influence on the function of TaRCA when translocated into the chloroplasts, leading to consequential effects on Rubisco enzyme activity within plants, ultimately attenuating their resistance to diseases. These findings are significant because they demonstrate the critical role the effector Pt9029 plays in pathogenic invasions.