Plants rely on innate immunity and the transmission of systemic signals from pathogen infection sites to resist pathogen invasion (Dangl and Jones, 2001; Chisholm et al., 2006; Jones and Dangl, 2006). Pathogens have conserved pathogen-associated molecular patterns (PAMPs), such as flagellin, that lead to PAMP-triggered immunity (PTI). Pathogens can secrete effector proteins targeted to plant apoplasts or delivered inside the host cytoplasm to suppress PTI and contribute to successful infection. Plants have an internal immune receptor-disease resistance gene (R-gene) encoding a nucleotide-binding leucine-rich repeat (NB-LRR) protein to recognize effectors and induce defense responses. These responses include programmed cell death (PCD) and reactive oxygen species (ROS) bursts and are known as effector-triggered immunity (ETI). Plasmodiophora brassicae is a well-adapted pathogen of Brassica but PTI and ETI are not well-characterized in the host-P. brassicae pathosystem (Pérez-López et al., 2018).

P. brassicae is an intracellular obligate biotrophic plant pathogen responsible for clubroot disease in the Brassicaceae (Kageyama and Asano, 2009). The life cycle of P. brassicae has three stages. In the soil survival stage, resting spores germinate to primary zoospores under optimal conditions. In the primary infection stage, primary zoospores colonize root hairs and form primary plasmodia and cleave into zoosporangia. Secondary zoospores released from zoosporangia penetrate the cortical tissues. The secondary infection develops into secondary plasmodia, which proliferate and produce characteristic swollen gall or club-shaped plant roots (Kageyama and Asano, 2009; Feng et al., 2012). The infection stages are critical periods for the host to recognize the pathogen and establish an immune defense system (McDonald et al., 2014). However, the recognition mechanism and interaction between P. brassicae and hosts harboring clubroot resistance genes is unknown.

To facilitate host plant colonization many pathogens secrete proteins and other molecules, collectively known as effectors, in the hosts to disable the plant defenses (Hogenhout et al., 2009). Many effectors have been characterized in bacteria, fungi, oomycetes and nematodes using agroinfiltration of Nicotiana benthamiana (Pitino et al., 2016). The development of bioinformatics and ultra-deep sequencing of plant pathogenic microbes has enabled genome-wide putative effectors genes to be cataloged based on special motifs (Huang et al., 2005; Liu et al., 2014). The basic criteria used to identify candidate secreted effector proteins include proteins with a signal peptide (within the initial 60 amino acids at the N-terminus), no trans-membrane domains and a size of 300 to 450 amino acids (Pérez-López et al., 2020). Many predicted effectors in each pathogen can be identified using bioinformatics analysis but assessing the functions of the candidate effectors is challenging. In the combined secretome of Plasmopara viticola, 51 predicted effectors with the RXLR motif were identified and 23 fully suppressed programmed cell death that was elicited by cell death-inducing proteins in N. benthamiana (Ling et al., 2015; Xiang et al., 2016). In fungi, 18 of 30 randomly selected putative effectors of Ustilaginoidea virens suppressed the Burkholderia glumae-triggered hypersensitive reaction (HR) at different levels in N. benthamiana (Zhang et al., 2014). In the cereal cyst nematode, Heterodera avenae, 95 candidate effector genes were evaluated and 78 effectors suppressed BT-PCD in N. benthamiana leaves (Lin et al., 2016).

A limited number of P. brassicae effectors have been functionally characterized based on the genome of sequences (Rolfe et al., 2016). A total of 553 secreted proteins in P. brassicae isolate e3 were predicted by bioinformatics analysis. A secretory methyltransferase can methylate salicylic acid and alter host susceptibility to P. brassicae (Schwelm et al., 2015). During primary infection, 23 of 33 secretory proteins suppressed mouse Bcl-2-associated X protein (BAX)-induced cell death and two effectors could induce cell death in N. benthamiana (Chen et al., 2019). And endomembrane-targeting P. brassicae effectors were reported to modulate PAMP and triggered immune responses in plants (Hossain et al., 2021). However, the other putative effectors at different infection stages and isolates have not been identified and characterized.

In this study, secreted protein genes were initially screened and predicted by bioinformatics software. A total of 63 candidate effectors of P. brassicae were selected and cloned. The capacities of these secretory proteins to induce or suppress cell death were assessed. The results of these experiments helped to reveal the pathogenic mechanism of P. brassicae and its interactions with host plants.

Identification of effector proteins is key to understanding the interaction between P. brassicae and its susceptible host plants. A bioinformatics pipeline approach enables screening of putative effectors based on the genome sequence of P. brassicae. However, it is difficult to clarify all functions of the putative effectors identified by bioinformatics. Transient expression assays in the N. benthamiana model system employing agro-infiltration have been used to identify effectors in bacteria, oomycetes, fungi and nematodes (Wang et al., 2011; Ali et al., 2015; Fang et al., 2016; Choi et al., 2017). In this study, 518 secretory proteins were screened from the genome of P. brassicae strain used in this study. The number of secretory proteins was slightly less than that (558) predicted by Arne (Schwelm et al., 2015). Although, the secretory proteins with special motif, such as RXLR, LXAR and CRN probably function as effectors, some proteins without special candidate motif were also identified in different pathogens (Kemen et al., 2005; Dodds et al., 2009; Malcova et al., 2021). Considering the special relationship between pathogen and host that P. brassicae covered by the membrane structure and parasitized in the root cells, there must be active exchange between them, including the secretary proteins. Therefore, the relative broad selection from the putative secretary proteins for function verification was necessary. In this study, 63 putative effectors were cloned based on the bioinformatics analysis of the P. brassicae genome. We found that 55 putative effectors could suppress plant immunity triggered by BAX in N. benthamiana. And 21 out of 55 could significant suppress the proliferation of Pst DC3000 (AvrRps4) in Arabidopsis. P. brassicae, an intracellular biotrophic parasite, needs to absorb nutrients from living host cells. Therefore, it secretes a large number of proteins to suppress plant immunity. This phenomenon has also been found in other biotrophic pathogens. Of the 83 putative RXLR proteins of P. viticola, 62 could suppress cell death triggered by elicitin in N. benthamiana (Xiang et al., 2016). Additionally, 78 of the 95 putative effectors of H. avenae could suppress programmed cell death triggered by BAX in N. benthamiana (Chen et al., 2013). The universal rule in the biotrophic pathogens probably benefit its propagation and colonization.

Surprisingly, two putative effectors, Pb4_102097 and Pb4_108104, which strongly expressed in the stage of rest spore maturity and release could successfully induce cell death accompanied by H₂O₂ accumulation in N. benthamiana. These secretary proteins were necessary for the life cycle of P. brassicae. The obligate pathogens try their best to hide from host’s immune-response system to hijack the nutrition and populate inside a host (Dimitrios et al., 2015). However, they are waiting an opportunity to escape from host, when their life cycle is coming to end. Pb4_102097 and Pb4_108104 showed high expressing level in the stage of rest spore maturity, which probably play an important in facility the rest spore release to the soil.

Many pathogen effectors were screened from secretary proteins identified with agroinfiltration of Nicotiana benthamiana method. The best example of plant-specific recognition is the recognition of avirulence effectors either directly or indirectly by nucleotide-binding-leucine-rich-repeat receptors (NLRs), which results in the activation of plant programmed cell death, and cessation of further pathogen growth (Whitham et al., 1994; Tang et al., 1996; Dodds et al., 2004; Rehmany et al., 2005). In B. rapa, several clubroot resistant (CR) loci have been mapped, and two resistant genes CRa, Crr1a are cloned. Both are typical resistance proteins with TIR-NBS-LRR domains (Ueno et al., 2012). Considering the interaction models between other pathogens and hosts, there must be effectors recognized by these CR genes. However, little research on the interaction mechanism between P. brassicae effectors and CR genes has been reported. Therefore, it is challenging to determine the interaction mechanism between effectors of P. brassicae and clubroot resistant genes.

Effectors delivered into host cells deregulate host immunity in a variety of subcellular compartments (Rovenich et al., 2014). The AvrPiz-t effector of Magnaporthe oryzae targets proteasome activity through interaction with the RING E3 ubiquitin ligase APIP6, leading to their mutual degradation and suppression of host immunity (Park et al., 2012). Hyaloperonospora arabidopsis directly delivers its effector to the nucleus of the host to interact with the mediator complex that controls interactions between transcription regulators and RNA polymerase (Caillaud et al., 2012). Five of the putative effectors containing the RXLR motif in this study functioned in the cytoplasm of N. benthamiana. One effector also functioned in the nucleus. Some RXLR effectors require nuclear localization to trigger or suppress cell death (Du et al., 2015; Xiang et al., 2017; Liu et al., 2018; Qi et al., 2019). Transcriptome analysis revealed a variety of genes responding to P. brassicae infection in B. rapa. These included the SWEET family responsible for glucose transporter. A large amount of nutrients are required for the propagation of P. brassicae, which is probably directly controlled by the effectors delivered into the host nucleus.

In this study, the functions of P. brassicae proteins expressed during the different infection stages in the induction and suppression of plant immunity were investigated. Only two putative effectors induced plant immunity and most of the secretory proteins suppressed plant immunity. All the effectors containing the RXLR motif functioned in the cytoplasm and the cytomembrane. These findings improved our understanding of the functions of P. brassicae effector interactions between P. brassicae and hosts. Whether the effects of these effectors in plant cell death are beneficial for P. brassicae infection and the mechanisms by which these effectors manipulate plant immunity are unclear and require further investigation.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

ZZ: analyzed the data, performed the experiments, and drafted the manuscript. SL, YY, and SL: contributed to perform the experiments. XL and ZP: conceived the study, participated in its coordination, and helped to draft the manuscript. All authors contributed to the article and approved the submitted version.

This project was supported by grants from the China Agriculture Research System (CARS-12).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.