Plasmodiophora brassicae effector PbPE23 induces necrotic responses in both host and non-host plants.

Plasmodiophora brassicae is an obligate biotroph that causes clubroot disease in cruciferous plants, including canola and Arabidopsis. In contrast to most known bacterial, oomycete and fungal pathogens that colonize at the host apoplastic space, the protist P. brassicae establishes an intracellular colonization within various types of root cells and secretes a plethora of effector proteins to distinct cellular compartments favourable for survival and growth of the pathogen during pathogenesis. Identification and functional characterization of P. brassicae effectors has been hampered by the limited understanding of this unique pathosystem. Here, we report a P. brassicae effector, PbPE23, containing a Ser/Thr kinase domain, that induces necrosis after heterologous expression by leaf infiltration in both host and non-host plants. While PbPE23 is an active kinase, the kinase activity itself is not required for triggering the necrosis in plants. PbPE23 shows a nucleocytoplasmic localization in Nicotiana benthamiana and its N-terminal 25TPdPAQKQ32 sequence, resembling the contiguous hydrophilic TPAP motif and Q-rich region in many Nep1-like proteins (NLPs) from plant-associated microbes, is required for the induction of necrosis. Further, transcript profiling of PbPE23 reveals its high expression at the transition stages from primary to secondary infection, suggesting its potential involvement in the development of clubroot disease.

Transcriptome Analysis Identifies Plasmodiophora brassicae Secondary Infection Effector Candidates.

Plasmodiophora brassicae (Wor.) is an obligate intracellular plant pathogen affecting Brassicas worldwide. Identification of effector proteins is key to understanding the interaction between P. brassicae and its susceptible host plants. To date, there is very little information available on putative effector proteins secreted by P. brassicae during a secondary infection of susceptible host plants, resulting in root gall production. A bioinformatics pipeline approach to RNA-Seq data from Arabidopsis thaliana (L.) Heynh. root tissues at 17, 20, and 24 d postinoculation (dpi) identified 32 small secreted P. brassicae proteins (SSPbPs) that were highly expressed over this secondary infection time frame. Functional signal peptides were confirmed for 31 of the SSPbPs, supporting the accuracy of the pipeline designed to identify secreted proteins. Expression profiles at 0, 2, 5, 7, 14, 21, and 28 dpi verified the involvement of some of the SSPbPs in secondary infection. For seven of the SSPbPs, a functional domain was identified using Blast2GO and 3D structure analysis and domain functionality was confirmed for SSPbP22, a kinase localized to the cytoplasm and nucleus.

Structural analysis and molecular docking of potential ligands with chorismate synthase of Listeria monocytogenes: a novel antibacterial drug target.

Listeriosis, in particular that caused by Listeria monocytogenes, is a major foodborne pathogen, and its control is becoming difficult because of widespread emergence of drug resistance strains. Chorismate synthase (CS), an essential enzyme of shikimate pathway present only in bacteria, fungi, plant and some apicomplexan parasites, is a validated potential antimicrobial drug target. Antimicrobial development through the elucidation of essential structural features of the CS of L. monocytogenes (LmCS), identification and prioritization of potential lead compounds targeted against LmCS were done. Structure-based virtual screening and docking studies were performed using Autodock tools to retrieve potential candidates with high affinity binding against LmCS model from several ligand repositories. The potency of binding was also checked with other structurally similar CS from Streptococcus pneumoniae (SpCS) and Mycobacterium tuberculosis (MtCS). The sequence and structural studies revealed LmCS was similar to be other CS structures (1Q1L, 1QXO, 1R52, 1R53, 1SQ1, 1UMO, 1UMF, 1ZTB, 2011, 2012, 4ECD and 2G85) with each monomer presenting ß-α-ß sandwich topology with a central helical core. Molecular docking studies and ADME/Tox results revealed that ZINC03803450 and ZINC20149031 were most potent molecules binding into the active site of LmCS. Other two ligands ZINC13387711-and ZINC16052528 showed a strong binding affinity score against all three structures (LmCS, SpCS and MtCS) and bind to LmCS with the predicted inhibition constant (K(i)) values of 22.94 nM and 35.84 nM, respectively. A reported benzofuran-3[2H]-one analog CHEMBL135212 with good ADME/Tox properties and experimental IC50 (nM) value of 7000 nM with SpCS could also be considered as a potential inhibitor of LmCS, as compared to previously reported 41 benzofuran-3[2H]-one analogs against SpCS. This information will assist in discovering those compounds that may act as potent CS inhibitors. Further experimental studies and evaluation of structure-activity relationship could help in the development of potential inhibitors against listeriosis, as well as antibacterial chemotherapy.

Endomembrane-Targeting Plasmodiophora brassicae Effectors Modulate PAMP Triggered Immune Responses in Plants.

Plasmodiophora brassicae is a devastating obligate, intracellular, biotrophic pathogen that causes clubroot disease in crucifer plants. Disease progression is regulated by effector proteins secreted by P. brassicae. Twelve P. brassicae putative effectors (PbPEs), expressed at various stages of disease development [0, 2, 5, 7, 14, 21, and 28 days post inoculation (DPI)] in Arabidopsis and localizing to the plant endomembrane system, were studied for their roles in pathogenesis. Of the 12 PbPEs, seven showed an inhibitory effect on programmed cell death (PCD) as triggered by the PCD inducers, PiINF1 (Phytophthora infestans Infestin 1) and PiNPP1 (P. infestans necrosis causing protein). Showing the strongest level of PCD suppression, PbPE15, a member of the 2-oxoglutarate (2OG) and Fe (II)-dependent oxygenase superfamily and with gene expression during later stages of infection, appears to have a role in tumorigenesis as well as defense signaling in plants. PbPE13 produced an enhanced PiINF1-induced PCD response. Transient expression, in Nicotiana benthamiana leaves of these PbPEs minus the signal peptide (SP) (Δsp PbPEGFPs), showed localization to the endomembrane system, targeting the endoplasmic reticulum (ER), Golgi bodies and nucleo-cytoplasm, suggesting roles in manipulating plant cell secretion and vesicle trafficking. Δsp PbPE13GFP localized to plasma membrane (PM) lipid rafts with an association to plasmodesmata, suggesting a role at the cell-to-cell communication junction. Membrane relocalization of Δsp PbPE13GFP, triggered by flagellin N-terminus of Pseudomonas aeruginosa (flg22 - known to elicit a PAMP triggered immune response in plants), supports its involvement in raft-mediated immune signaling. This study is an important step in deciphering P. brassicae effector roles in the disruption of plant immunity to clubroot disease.

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.