Proto-oncogenes in a eukaryotic unicellular organism play essential roles in plasmodial growth in host cells.

BACKGROUND: The eukaryotic unicellular protist Plasmodiophora brassicae is an endocellular parasite of cruciferous plants. In host cortical cells, this protist develops a unicellular structure that is termed the plasmodium. The plasmodium is actually a multinucleated cell, which subsequently splits and forms resting spores. The mechanism for the growth of this endocellular parasite in host cell is unclear. RESULTS: Here, combining de novo genome sequence and transcriptome analysis of strain ZJ-1, we identified top five significant enriched KEGG pathways of differentially expressed genes (DEGs), namely translation, cell growth and death, cell communication, cell motility and cancers. We detected 171 proto-oncogenes from the genome of P. brassicae that were implicated in cancer-related pathways, of which 46 were differential expression genes. Three predicted proto-oncogenes (Pb-Raf1, Pb-Raf2, and Pb-MYB), which showed homology to the human proto-oncogenes Raf and MYB, were specifically activated during the plasmodial growth in host cortical cells, demonstrating their involvement in the multinucleate development stage of the unicellular protist organism. Gene networks involved in the tumorigenic-related signaling transduction pathways and the activation of 12 core genes were identified. Inhibition of phosphoinositol-3-kinase relieved the clubroot symptom and significantly suppressed the development process of plasmodia. CONCLUSIONS: Proto-oncogene-related regulatory mechanisms play an important role in the plasmodial growth of P. brassicae.

Differential expression of miRNAs in Brassica napus root following infection with Plasmodiophora brassicae.

Canola (oilseed rape, Brassica napus L.) is susceptible to infection by the biotrophic protist Plasmodiophora brassicae, the causal agent of clubroot. To understand the roles of microRNAs (miRNAs) during the post-transcriptional regulation of disease initiation and progression, we have characterized the changes in miRNA expression profiles in canola roots during clubroot disease development and have compared these to uninfected roots. Two different stages of clubroot development were targeted in this miRNA profiling study: an early time of 10-dpi for disease initiation and a later 20-dpi, by which time the pathogen had colonized the roots (as evident by visible gall formation and histological observations). P. brassicae responsive miRNAs were identified and validated by qRT-PCR of miRNAs and the subsequent validation of the target mRNAs through starBase degradome analysis, and through 5' RLM-RACE. This study identifies putative miRNA-regulated genes with roles during clubroot disease initiation and development. Putative target genes identified in this study included: transcription factors (TFs), hormone-related genes, as well as genes associated with plant stress response regulation such as cytokinin, auxin/ethylene response elements. The results of our study may assist in elucidating the role of miRNAs in post-transcriptional regulation of target genes during disease development and may contribute to the development of strategies to engineer durable resistance to this important phytopathogen.

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.

Acquisition and transmission of Peanut clump virus by Polymyxa graminis on cereal species.

The objective of this study was to investigate the specificity of the interactions between Polymyxa graminis, Peanut clump virus (PCV), and cereals, particularly the acquisition and the transmission of the virus by three P. graminis formae speciales. A new strategy has been developed: it involves using sugarcane as the common host for both the virus and its vector in order to produce the viruliferous zoospores of P. graminis f. sp. subtropicalis, temperata, and tropicalis that were then inoculated on cereal species. This experiment enabled the role of P. graminis f. sp. tropicalis and subtropicalis zoospores in PCV transmission to be demonstrated. The efficiency of this transmission was shown to vary, depending on the P. graminis special forms. Interestingly, the high transmission of the PCV isolate from Burkina Faso by an isolate of P. graminis f. sp. tropicalis from Niger on pearl millet suggests that there is a coevolution mechanism in this pathosystem. The study also provides evidence that the host plant species in which Polymyxa zoospores are produced could affect the infectivity of the vector. Finally, using Polymyxa quantitation by quantitative reverse-transcription polymerase chain reaction and in situ observations of the virus, the study demonstrates the independence of the development of PCV and its vector in the host plants.

In vitro dual culture of Polymyxa betae in Agrobacterium rhizogenes transformed sugar beet hairy roots in liquid media.

Polymyxa betae is a soil-borne protist and an obligate parasite of sugar beet that transmits the beet necrotic yellow vein virus. Sugar beet hairy roots, transformed by Agrobacterium rhizogenes, were inoculated with surface-sterilized root fragments infected by P. betae. After 10 wk in a liquid medium, typical structures of P. betae were observed in this in vitro system. This first in vitro culture of P. betae in liquid medium will contribute to a better understanding of this protist's biology through providing a way to conserve and produce purified isolates of the protist.

Indicator organisms for assessing sanitization during composting of plant wastes.

The potential for using plant pathogens and seeds as indicator organisms for assessing sanitization of plant wastes during composting was tested in bench-scale flask and large-scale systems. Plasmodiophora brassicae was unsuitable due to high temperature tolerance in dry to moist composts, and detection of viable inoculum post-composting using bioassay plants not corresponding with that using TaqMan® PCR, possibly due to preservation of nucleic acids at elevated temperatures. Several other plant pathogens (Sclerotinia sclerotiorum, Microdochium nivale, Phytophthora cinnamomi and Phytophthora nicotianae) were unsuitable due their low temperature tolerance. Fusarium oxysporum f.sp. cepae and f.sp. radicis-lycopersici chlamydospores and tomato seeds were suitable indicators due to their moderate temperature tolerance and ease of viability testing post-composting. Abutilon seeds were more tolerant than tomato seeds of compost temperatures ≥52°C but more prone to degradation at lower temperatures and therefore less suitable as indicators. Relationships between compost temperature during exposures of 2-10 days and subsequent viability of the above chlamydospores or seeds enabled the sanitizing effect of composting processes to be predicted within 2-6 days. Plant waste type (woody or vegetable) had a small but significant effect on the relationship for tomato seeds but not for F. oxysporum chlamydospores.

Molecular characterization of a serine protease Pro1 from Plasmodiophora brassicae that stimulates resting spore germination.

Clubroot, caused by Plasmodiophora brassicae, is one of the most serious diseases of cultivated cruciferous crops in the world. However, the basis for pathogenicity in P. brassicae is not well understood. In this study, a serine protease gene (PRO1) was cloned from P. brassicae and its molecular characteristics were investigated. Southern analysis and specific polymerase chain reaction (PCR) amplification indicated that PRO1 is a single-copy gene present in a broad range of P. brassicae pathotypes. Northern analysis revealed that the expression of PRO1 was induced during plant infection, and that the quantity of transcript fluctuated according to the stage of pathogenesis. Amino acid sequence analysis suggested that the encoded protein (Pro1) belongs to the S28 family of proteases, with a predicted signal peptide and a theoretical molecular mass of 49.4 kDa. The open reading frame (ORF) of PRO1 was transferred into Pichia pastoris and Pro1 was heterologously produced. Pro1 showed proteolytic activity on skimmed milk and N-succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin, and the activity could be inhibited by serine protease inhibitors and the chelating agent ethylenediaminetetraacetic acid. The optimal temperature of Pro1 was 25 degrees C, and it exhibited high activity at pH 6.0-6.4. These values coincide with the temperature and pH conditions favourable for P. brassicae resting spore germination in the field. When Pro1 was used to treat canola root exudates, it enhanced the stimulating effect of the root exudates on P. brassicae resting spore germination, indicating that Pro1 may play a role during clubroot pathogenesis by stimulating resting spore germination through its proteolytic activity.