The Plasmodiophora brassicae Golgi-localized UPF0016 protein PbGDT1 mediates calcium but not manganese transport in yeast and Nicotiana benthamiana.

Manganese and calcium homeostasis and signalling, in eukaryotic organisms, are regulated through membrane located pumps, channels and exchangers, including the Mn2+/Ca2+ uncharacterized protein family 0016 (UPF0016). Here we show that Plasmodiophora brassicae PbGDT1 is a member of the UPF0016 and an ortholog of Saccharomyces cerevisiae Gdt1p (GCR Dependent Translation Factor 1) protein involved in manganese homeostasis as well as the calcium mediated stress response in yeast. PbGDT1 complemented the ScGdt1p and ScPMR1 (Ca2+ ATPase) double null mutant under elevated calcium stress but not under elevated manganese conditions. In both yeast and Nicotiana benthamiana, PbGDT1 localizes to the Golgi apparatus, with additional ER association in N. benthamiana. Expression of PbGDT1 in N. benthamiana, suppresses BAX-triggered cell death, further highlighting the importance of calcium homeostasis in maintaining cell physiology and integrity in a stress environment.

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.

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.

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.

Identification of Plasmodiophora brassicae effectors - A challenging goal.

Clubroot is an economically important disease affecting Brassica plants worldwide. Plasmodiophora brassicae is the protist pathogen associated with the disease, and its soil-borne obligate parasitic nature has impeded studies related to its biology and the mechanisms involved in its infection of the plant host. The identification of effector proteins is key to understanding how the pathogen manipulates the plant's immune response and the genes involved in resistance. After more than 140 years studying clubroot and P. brassicae, very little is known about the effectors playing key roles in the infection process and subsequent disease progression. Here we analyze the information available for identified effectors and suggest several features of effector genes that can be used in the search for others. Based on the information presented in this review, we propose a comprehensive bioinformatics pipeline for effector identification and provide a list of the bioinformatics tools available for such.

Transcriptome analysis of response to Plasmodiophora brassicae infection in the Arabidopsis shoot and root.

BACKGROUND: Clubroot is an important disease caused by the obligate parasite Plasmodiophora brassicae that infects the Brassicaceae. As a soil-borne pathogen, P. brassicae induces the generation of abnormal tissue in the root, resulting in the formation of galls. Root infection negatively affects the uptake of water and nutrients in host plants, severely reducing their growth and productivity. Many studies have emphasized the molecular and physiological effects of the clubroot disease on root tissues. The aim of the present study is to better understand the effect of P. brassicae on the transcriptome of both shoot and root tissues of Arabidopsis thaliana. RESULTS: Transcriptome profiling using RNA-seq was performed on both shoot and root tissues at 17, 20 and 24 days post inoculation (dpi) of A. thaliana, a model plant host for P. brassicae. The number of differentially expressed genes (DEGs) between infected and uninfected samples was larger in shoot than in root. In both shoot and root, more genes were differentially regulated at 24 dpi than the two earlier time points. Genes that were highly regulated in response to infection in both shoot and root primarily were involved in the metabolism of cell wall compounds, lipids, and shikimate pathway metabolites. Among hormone-related pathways, several jasmonic acid biosynthesis genes were upregulated in both shoot and root tissue. Genes encoding enzymes involved in cell wall modification, biosynthesis of sucrose and starch, and several classes of transcription factors were generally differently regulated in shoot and root. CONCLUSIONS: These results highlight the similarities and differences in the transcriptomic response of above- and below-ground tissues of the model host Arabidopsis following P. brassicae infection. The main transcriptomic changes in root metabolism during clubroot disease progression were identified. An overview of DEGs in the shoot underlined the physiological changes in above-ground tissues following pathogen establishment and disease progression. This study provides insights into host tissue-specific molecular responses to clubroot development and may have applications in the development of clubroot markers for more effective breeding strategies.

Differential transcript accumulation and subcellular localization of Arabidopsis ribosomal proteins.

Arabidopsis cytoplasmic ribosomes are an assembly of four rRNAs and 81 ribosomal proteins (RPs). With only a single molecule of each RP incorporated into any given ribosome, an adequate level of each RP in the nucleolus is a prerequisite for efficient ribosome biogenesis. Using Genevestigator (microarray data analysis tool), we have studied transcript levels of 192 of the 254 Arabidopsis RP genes, as well as the sub-cellular localization of each of five two-member RP families, to identify the extent to which these two processes contribute to the nucleolar pool of RPs available for ribosome biogenesis. While transcript levels from different RP genes show up to a 300-fold difference across the RP population, this difference is drastically reduced to ∼8-fold when considering RP gene families. Under various stimuli, while the transcript level for most RP genes remains unchanged some show a significantly increased or decreased level. Subcellular localization studies in tobacco not only showed differential targeting of RPs to the cytoplasm, nucleus and nucleolus, but also differential nucleolar import rates. This degree of variation in gene regulation and subcellular localization of RPs hints at the possibility of extra-ribosomal functions for some RP isoforms.

Charge versus sequence for nuclear/nucleolar localization of plant ribosomal proteins.

Ribosomal subunit assembly in the nucleolus is dependent on efficient targeting of ribosomal proteins (RPs) from the cytoplasm into the nucleus and nucleolus. Nuclear/nucleolar localization of a protein is generally mediated by one or more specific stretches of basic amino acids-nuclear/nucleolar localization signals (NLSs/NoLSs). Arabidopsis thaliana RPL23aA has eight putative NLSs/NoLSs (pNLSs/NoLSs). Here we mutated all eight NLS/NoLSs individually and in groups and showed, via transient expression in tobacco cells that nucleolar localization of RPL23aA was disrupted by mutation of various combinations of five or more pNLSs/NoLSs. Mutation of all eight pNLSs/NoLSs, a 50 % reduction in total basic charge of RPL23aA, resulted in a complete disruption of nucleolar localization, however, the protein can still localize to the nucleus. As no individual or specific combination of NoLSs was absolutely required for nucleolar localization, we suggest that nucleolar localization/retention of RPL23aA is dependent on the overall basic charge. In addition to the optimal basic charge conferred by these NoLSs, nucleolar localization/retention of RPL23aA also required a C-terminal putative 26S rRNA binding site. In contrast, in the RPs RPS8A and RPL15A, mutation of just two and three N-terminal pNLSs, respectively, disrupted both nuclear and nucleolar localization of these two RPs, indicating differential signal requirements for nuclear and nucleolar localization of the three Arabidopsis RPs RPL23aA, RPL15A and RPS8A.

Sequence context for transcription and translation of the Arabidopsis RPL23aA and RPL23aB paralogs.

The 80S cytoplasmic ribosome is responsible for translating the transcriptome into the proteome. Demand for ribosome production depends on growth rate, and both the ribosomal RNA (rRNA) and ribosomal protein (RP) components must respond coordinately and rapidly to positive and negative growth stimuli to prevent deleterious effects of excess or insufficient subunits. The 81 RPs of the Arabidopsis 80S ribosome are encoded by multigene families that often exhibit overlapping patterns of transcript accumulation; however, only one isoform of each RP family (with the exception of a small number of acidic RPs) assembles into a single ribosome. Here we dissected the regulatory regions (RRs) of both members of the RPL23a family (RPL23aA and RPL23aB) to identify salient cis-acting elements involved in transcriptional, posttranscriptional, and translational regulation of expression. Full length and truncated RRs of RPL23a paralogs were cloned upstream of a GUS reporter gene and expressed in Arabidopsis transgenic plants. High level expression in mitotically active tissues, driven by RPL23aA and RPL23aB RRs, required TATA-box, telo-box, and site II motif elements. First and second introns were found to play a minor role in posttranscriptional regulation of paralogs, and conserved transcript features (e.g., UTR base composition) may be involved in enhancing translational efficiency. Overall, our results indicate that RPL23a expression is governed by a complex network of multiple regulatory layers.

A role for the anaphase promoting complex in hormone regulation.

To increase our knowledge of anaphase promoting complex (APC/C) function during plant development, we characterized an Arabidopsis thaliana T-DNA-insertion line where the T-DNA fell within the 5' regulatory region of the APC10 gene. The insert disrupted endogenous expression, resulting in overexpression of APC10 mRNA from the T-DNA- internal CaMV 35S promoter, and increased APC10 protein. Overexpression of APC10 produced phenotypes resembling those of known auxin and ethylene mutants, and increased expression of two tested auxin-regulated genes, small auxin up RNA (SAUR) 15 and SAUR24. Taken together, our data suggests that elevated APC10 likely mimics auxin and ethylene sensitive phenotypes, expanding our understanding of proteolytic processes in hormone regulation of plant development.

Transcript profiling demonstrates absence of dosage compensation in Arabidopsis following loss of a single RPL23a paralog.

Translation of nucleus-encoded messages in plants is conducted by the cytoplasmic ribosome, an enzyme that is comprised of two RNA/protein subunits. In Arabidopsis thaliana, the 81 different ribosomal proteins (r-proteins) of the cytosolic ribosome belong to gene families with multiple expressed members. Given that ribosomes generally contain only one copy of each r-protein, regulatory mechanisms must exist to ensure their stoichiometric accumulation. These mechanisms must be dynamic, allowing for adjustments to ribosome biogenesis to fulfill biological requirements for protein synthesis during development, and following stress induction of global changes in gene expression. In this study, we investigated whether r-protein paralogs are feedback regulated at the transcript level by obtaining a T-DNA knockout of one member, RPL23aB, from the two-member RPL23a family. Expression of the lone functional paralog in this line, RPL23aA, was compared to the expression of both paralogs in wildtype plants under non-stressed, low temperature-, and high light stresses. RPL23aA expression was not upregulated in RPL23aB knockouts to compensate for paralog-loss, and consequently knockouts showed reduced total abundance of RPL23a transcripts. However, no phenotype developed in RPL23aB knockouts, suggesting that this paralog is dispensable under experimental conditions examined, or that compensation by RPL23aA may occur post-transcriptionally. Patterns of RPL23aA and RPL23aB transcript accumulation in wildtype plants suggest that paralogs respond coordinately to developmental and stress stimuli.

Arabidopsis ribosomal proteins RPL23aA and RPL23aB are differentially targeted to the nucleolus and are disparately required for normal development.

Protein synthesis is catalyzed by the ribosome, a two-subunit enzyme comprised of four ribosomal RNAs and, in Arabidopsis (Arabidopsis thaliana), 81 ribosomal proteins (r-proteins). Plant r-protein genes exist as families of multiple expressed members, yet only one r-protein from each family is incorporated into any given ribosome, suggesting that many r-protein genes may be functionally redundant or development/tissue/stress specific. Here, we characterized the localization and gene-silencing phenotypes of a large subunit r-protein family, RPL23a, containing two expressed genes (RPL23aA and RPL23aB). Live cell imaging of RPL23aA and RPL23aB in tobacco with a C-terminal fluorescent-protein tag demonstrated that both isoforms accumulated in the nucleolus; however, only RPL23aA was targeted to the nucleolus with an N-terminal fluorescent protein tag, suggesting divergence in targeting efficiency of localization signals. Independent knockdowns of endogenous RPL23aA and RPL23aB transcript levels using RNA interference determined that an RPL23aB knockdown did not alter plant growth or development. Conversely, a knockdown of RPL23aA produced a pleiotropic phenotype characterized by growth retardation, irregular leaf and root morphology, abnormal phyllotaxy and vasculature, and loss of apical dominance. Comparison to other mutants suggests that the phenotype results from reduced ribosome biogenesis, and we postulate a link between biogenesis, microRNA-target degradation, and maintenance of auxin homeostasis. An additional RNA interference construct that coordinately silenced both RPL23aA and RPL23aB demonstrated that this family is essential for viability.

The two ribosomal protein L23A genes are differentially transcribed in Arabidopsis thaliana.

Arabidopsis thaliana ribosomal protein (r-protein) L23A (RPL23A) is a member of the conserved L23/L25 family of primary ribosomal RNA (rRNA) binding proteins. The 2 AtRPL23A isoforms, RPL23A-1 and RPL23A-2, are 94% identical at the amino acid level, yet RPL23A-1 and RPL23A-2 share only approximately 40-50% primary sequence identity within the 5' regulatory regions. While the RPL23A-1 and -2 5' regulatory regions share many similar predicted motifs, the arrangement and number of these motifs differs between the 2 genes. Differences in regulation between RPL23A-1 and -2 have been investigated via reverse transcription-PCR (RT-PCR) expression profiles. Overall, transcript abundance for RPL23A-1 and -2 varied slightly in specific tissues and under some abiotic stresses. The highest transcript abundance for both RPL23A genes was detected in mitotically active tissues such as bud, flower and elongating carpel, as well as in root and stem while the lowest transcript levels were found in mature leaf and bract. Hormone-treated seedlings showed increased RPL23A-1 and -2 transcript levels following IAA and BAP treatment while ABA treatment resulted in a transient lowering of transcript levels. Expression patterns differed between RPL23A-1 and -2 in cold-, wound-, and copper-stressed seedlings. In all tissues examined, RPL23A-2 transcript levels were consistently lower than those of RPL23A-1. This report shows differential transcriptional regulation of the 2 RPL23A genes, which should no longer be identified as "housekeeping" genes.

A high- and low-temperature inducible Arabidopsis thaliana HSP101 promoter located in a nonautonomous mutator-like element.

Transcriptional activity of a 573-bp fragment of HSP101 (At1g74310) incorporated into a Mutator-like element (MULE) transposon was investigated in Arabidopsis thaliana Columbia. Sequence identity between the HSP101-MULE arrangement and a continuous segment of the original HSP101 promoter, 5' UTR exon, and open reading frame (ORF) was high (87%) but lower in the 5' UTR intron (69%). Collectively, the HSP101 ORF, the MULE 5' terminal inverted repeat (TIR), and the 1.3 kb immediately upstream of the TIR is located on chromosome IV, and we refer to it as HSP101B. Located within the HSP101B promoter, upstream of 2 heat shock elements (HSEs), are 4 COR15a-like low-temperature response elements (LTREs). The HSP101B ORF was transcribed in the leaves and influorescences of high-temperature stress (HTS) treated Arabidopsis thaliana but not in low-temperature stress (LTS) and control plants. Transiently transformed Arabidopsis seedlings, as well as stable transformed lines of Linum usitatissimum (flax) and Brassica napus (canola) containing a HSP101B promoter:GUS construct, showed either LTS-, or LTS- and HTS-, induced beta-glucuronidase expression. Results from PCR amplifications of HpaII- and MspI-digested Arabidopsis genomic DNA suggest that endogenous expression of HSP101B may be downregulated by partial methylation of the HSP101B sequence between the TIRs of the associated MULE.

High temperature stress of Brassica napus during flowering reduces micro- and megagametophyte fertility, induces fruit abortion, and disrupts seed production.

High temperature stress (HTS), during flowering, decreases seed production in many plants. To determine the effect of a moderate HTS on flowering, fruit and seed set in Brassica napus, plants were exposed to a HTS (8/16 h dark/light, 18 degrees C night, ramped at 2 degrees C h-1, over 6 h, to 35 degrees C for 4 h, ramped at 2 degrees C h-1 back to 23 degrees C for 6 h) for 1 or 2 weeks after the initiation of flowering. Although flowering on the HTS-treated plants, during both the 1 week and 2 week HTS treatments, was equal to that of control-grown plants, fruit and seed development, as well as seed weight, were significantly reduced. Under HTS, flowers either developed into seedless, parthenocarpic fruit or aborted on the stem. At the cessation of the HTS, plants compensated for the lack of fruit and seed production by increasing the number of lateral inflorescences produced. During the HTS, pollen viability and germinability were slightly reduced. In vitro pollen tube growth at 35 degrees C, from both control pollen and pollen developed under a HTS, appeared abnormal, however, in vivo tube growth to the micropyle appeared normal. Reciprocal pollination of HTS or control pistils with HTS or control pollen indicated that the combined effects of HTS on both micro- and megagametophytes was required to knock out fruit and seed development. Expression profiles for a subset of HEAT SHOCK PROTEINs (HSP101, HSP70, HSP17.6) showed that both micro- and megagametophytes were thermosensitive despite HTS-induced expression from these genes.

Isolation and characterization of a Brassica napus cDNA corresponding to a B-class floral development gene.

B-class floral homeotic genes are required for the proper formation and identity of petals and stamens in dicot flowers. A partial cDNA clone encoding a B-class gene, BnAP3 (Brassica napus APETALA3), was isolated from a B. napus cDNA library derived from young inflorescence meristems. The 5' region of the cDNA was retrieved by RACE. The deduced amino acid sequence of the full-length clone exhibited high similarity to APETALA3 of Arabidopsis thaliana and functionally homologous proteins from other species. 5' RACE and Southern analysis suggests that BnAP3 has multiple alleles in B. napus. Expression analysis assayed by RT-PCR shows that BnAP3 is expressed in floral tissues, as well as non-floral tissues such as root and bract. Transformation of wild-type A. thaliana and B. napus plants with BnAP3 under the control of a promoter specific to reproductive organs converts carpels to stamens, while the expression of this construct in A. thaliana plants mutant for AP3 restores the development of third-whorl stamens in addition to directing a carpel to stamen conversion in the fourth whorl.