A clubroot pathogen effector targets cruciferous cysteine proteases to suppress plant immunity.

Plant pathogen effector proteins are key to pathogen virulence. In susceptible host Brassicas, the clubroot pathogen, Plasmodiophora brassicae, induces the production of nutrient-sink root galls, at the site of infection. Among a list of 32 P. brassiae effector candidates previously reported by our group, we identified SSPbP53 as a putative apoplastic cystatin-like protein highly expressed during the secondary infection. Here we found that SSPbP53 encoding gene is conserved among several P. brassicae pathotypes and that SSPbP53 is an apoplastic protein able to directly interact with and inhibit cruciferous papain-like cysteine proteases (PLCPs), specifically Arabidopsis XYLEM CYSTEINE PEPTIDASE 1 (AtXCP1). The severity of clubroot disease is greatly reduced in the Arabidopsis xcp1 null mutant (AtΔxcp1) after infection with P. brassicae resting spores, indicating that the interaction of P. brassicae SSPbP53 with XCP1 is important to clubroot susceptibility. SSPbP53 is the first cystatin-like effector identified and characterized for a plant pathogenic protist.

Comparative Transcriptome Analysis of Rutabaga (Brassica napus) Cultivars Indicates Activation of Salicylic Acid and Ethylene-Mediated Defenses in Response to Plasmodiophora brassicae.

Clubroot, caused by Plasmodiophora brassicae Woronin, is an important soilborne disease of Brassica napus L. and other crucifers. To improve understanding of the mechanisms of resistance and pathogenesis in the clubroot pathosystem, the rutabaga (B. napus subsp. rapifera Metzg) cultivars 'Wilhelmsburger' (resistant) and 'Laurentian' (susceptible) were inoculated with P. brassicae pathotype 3A and their transcriptomes were analyzed at 7, 14, and 21 days after inoculation (dai) by RNA sequencing (RNA-seq). Thousands of transcripts with significant changes in expression were identified in each host at each time-point in inoculated vs. non-inoculated plants. Molecular responses at 7 and 14 dai supported clear differences in the clubroot response mechanisms of the two genotypes. Both the resistant and the susceptible cultivars activated receptor-like protein (RLP) genes, resistance (R) genes, and genes involved in salicylic acid (SA) signaling as clubroot defense mechanisms. In addition, genes related to calcium signaling and genes encoding leucine-rich repeat (LRR) receptor kinases, the respiratory burst oxidase homolog (RBOH) protein, and transcription factors such as WRKYs, ethylene responsive factors, and basic leucine zippers (bZIPs), appeared to be upregulated in 'Wilhelmsburger' to restrict P. brassicae development. Some of these genes are essential components of molecular defenses, including ethylene (ET) signaling and the oxidative burst. Our study highlights the importance of activation of genes associated with SA- and ET-mediated responses in the resistant cultivar. A set of candidate genes showing contrasting patterns of expression between the resistant and susceptible cultivars was identified and includes potential targets for further study and validation through approaches such as gene editing.

Most Plasmodiophora brassicae Populations in Single Canola Root Galls from Alberta Fields are Mixtures of Multiple Strains.

Clubroot, caused by Plasmodiophora brassicae, is an important disease on canola in Alberta, Canada. The pathogen is grouped into pathotypes according to their virulence reaction on differential hosts. Multiple pathotypes or strains are known exist in one field, one plant, or even one gall. This study was conducted with the objective of testing the prevalence of the coexistence of multiple strains in a single gall. In all, 79 canola clubroot galls were collected from 22 fields across Northern Alberta in 2018. Genomic DNA extracted from these single galls was analyzed using RNase H-dependent PCR (rhPCR). The rhPCR primers were designed to amplify a partial sequence of a dimorphic gene, with one primer pair specific to one sequence and the other primer pair specific to the alternative sequence. The amplification of both sequences from DNA obtained from a single gall would indicate that it contains two different P. brassicae strains. The rhPCR analyses indicated that the P. brassicae populations in 50 of the 79 galls consisted of more than one strain. This result emphasizes the need for cautious interpretation of results when a single-gall population is subject to pathotyping or being used as inoculum in plant pathology research. It also confirms that the maintenance of pathotype diversity within single root galls is a common occurrence which has implications for the durability, and stewardship, of single-gene host resistance.

Infection of Plasmodiophora brassicae changes the fungal endophyte community of tumourous stem mustard roots as revealed by high-throughput sequencing and culture-dependent methods.

Diverse fungal endophytes live in plants and are shaped by some abiotic and biotic stresses. Plant disease as particular biotic stress possibly gives an impact on the communities of fungal endophytes. In this study, clubroot disease caused by an obligate biotroph protist, Plasmodiophora brassicae, was considered to analyze its influence on the fungal endophyte community using an internal transcribed spacer (ITS) through high-throughput sequencing and culture-dependent methods. The results showed that the diversity of the endophyte community in the healthy roots was much higher than the clubroots. Ascomycota was the dominant group of endophytes (Phoma, Mortierella, Penicillium, etc.) in the healthy roots while P. brassicae was the dominant taxon in the clubroots. Hierarchical clustering, principal component analysis (PCA), principal coordinates analysis (PCoA) and analysis of similarities (ANOSIM) indicated significant differences between the endophyte communities in the healthy roots and clubroots. Linear discriminant analysis effect size (LefSe) analysis showed that the dominant genera could be regarded as potential biomarkers. The endophyte community in the healthy roots had a more complex network compared with the clubroots. Also, many plant pathogenic Fusarium were isolated from the clubroots by the culture-dependent method. The outcome of this study illustrates that P. brassicae infection may change the fungal endophyte community associated with the roots of tumourous stem mustard and facilitates the entry of soil pathogen into the roots.

Mimicking the Host Regulation of Salicylic Acid: A Virulence Strategy by the Clubroot Pathogen Plasmodiophora brassicae.

The plant hormone salicylic acid (SA) plays a critical role in defense against biotrophic pathogens such as Plasmodiophora brassicae, which is an obligate pathogen of crucifer species and the causal agent of clubroot disease of canola (Brassica napus). P. brassicae encodes a protein, predicted to be secreted, with very limited homology to benzoic acid (BA)/SA-methyltransferase, designated PbBSMT. PbBSMT has a SA- and an indole-3-acetic acid-binding domain, which are also present in Arabidopsis thaliana BSMT1 (AtBSMT1) and, like AtBSMT1, has been shown to methylate BA and SA. In support of the hypothesis that P. brassicae uses PbBSMT to overcome SA-mediated defenses by converting SA into inactive methyl salicylate (MeSA), here, we show that PbBSMT suppresses local defense and provide evidence that PbBSMT is much more effective than AtBSMT1 at suppressing the levels of SA and its associated effects. Basal SA levels in Arabidopsis plants that constitutively overexpress PbBSMT compared with those in Arabidopsis wild-type Col-0 (WT) were reduced approximately 80% versus only a 50% reduction in plants overexpressing AtBSMT1. PbBSMT-overexpressing plants were more susceptible to P. brassicae than WT plants; they also were partially compromised in nonhost resistance to Albugo candida. In contrast, AtBSMT1-overexpressing plants were not more susceptible than WT to either P. brassicae or A. candida. Furthermore, transgenic Arabidopsis and tobacco plants overexpressing PbBSMT exhibited increased susceptibility to virulent Pseudomonas syringae pv. tomato DC3000 (DC3000) and virulent Pseudomonas syringae pv. tabaci, respectively. Gene-mediated resistance to DC3000/AvrRpt2 and tobacco mosaic virus (TMV) was also compromised in Arabidopsis and Nicotiana tabacum 'Xanthi-nc' plants overexpressing PbBSMT, respectively. Transient expression of PbBSMT or AtBSMT1 in lower leaves of N. tabacum Xanthi-nc resulted in systemic acquired resistance (SAR)-like enhanced resistance to TMV in the distal systemic leaves. Chimeric grafting experiments revealed that, similar to SAR, the development of a PbBSMT-mediated SAR-like phenotype was also dependent on the MeSA esterase activity of NtSABP2 in the systemic leaves. Collectively, these results strongly suggest that PbBSMT is a novel effector, which is secreted by P. brassicae into its host plant to deplete pathogen-induced SA accumulation.

Live cell imaging of Plasmodiophora brassicae-host plant interactions based on a two-step axenic culture system.

Plasmodiophora brassicae, a parasitic protist, induces club-shaped tumor-like growth of host Brassicas roots and hypocotyls after infection. Due to its soil-borne nature and intracellular, biotrophic parasitism the infection biology and early pathogenesis remains in doubt. In this study, we have established a new protocol, based on a two-step axenic culture of P. brassicae with its host tissues, for easy and in planta observation of cellular interactions between P. brassicae and host plants: first, coculture of P. brassicae with infected canola root tissues, on growth-medium plates, enables the propagation of P. brassicae that serves as pure inoculum for pathogenicity assays, and second, the pure inoculum is subsequently used for pathogenicity tests on both canola and Arabidopsis seedlings grown on medium plates in Petri dishes. During the first axenic culture, we established a staining protocol by which the pathogen was fluorescently labeled with Nile red and calcofluor white, thus allowing in planta observation of pathogen development. In the pathogenicity assays, our results showed that axenic cultures of P. brassicae, in calli, remains fully virulent and completes its life cycle in both canola and Arabidopsis roots grown in Petri dishes. Combining visualization of fluorescent probe-labeled P. brassicae structures with fluorescent protein tagging of Arabidopsis cellular components, further revealed dynamic responses of host cells at the early stages of P. brassicae infection. Thus, established protocols for in planta detection of P. brassicae structures and the live cell imaging of P. brassicae-Arabidopsis interactions provide a novel strategy for improving our detailed knowledge of P. brassicae infection in host tissues.

Gall formation in clubroot-infected Arabidopsis results from an increase in existing meristematic activities of the host but is not essential for the completion of the pathogen life cycle.

Plasmodiophora brassicae (clubroot) infection leads to reprogramming of host development resulting in the formation of characteristic galls. In this work we explored the cellular events that underly gall formation in Arabidopsis thaliana with the help of molecular markers of cell division (CYCB1:GUS) and meristematic activity (ANT:GUS). Our results show that gall development involved the amplification of existing meristematic activities within the vascular cambium (VC) and phloem parenchyma (PP) cells in the region of the hypocotyl. Additionally we found that the increase in VC activity and prolonged maintenance of cambial-derived cells in a meristematic state was crucial for gall formation; disruption of the VC activity significantly decreased the gall size. Gall formation also perturbed vascular development with a significant reduction in xylem and increase in PP in infected plants. This situation was reflected in a decrease in transcripts of key factors promoting xylogenesis (VND6, VND7 and MYB46) and an increase in those promoting phloem formation and function (APL, SUC2). Finally we show, using the cell cycle inhibitor ICK1/KRP1 and a cle41 mutant with altered regulation of cambial stem cell maintenance and differentiation, that a decrease in gall formation did not prevent pathogen development. This finding demonstrates that although gall formation is a typical symptom of the disease and influences numbers of spores produced, it is not required for completion of the pathogen life cycle. Together, these results provide an insight into the relationship of the cellular events that accompany Plasmodiophora infection and their role in disease progression.