Nemo Peeters

Genome-Wide Analysis of Arabidopsis Pentatricopeptide Repeat Proteins Reveals Their Essential Role in Organelle Biogenesis

The complete sequence of the Arabidopsis thaliana genome revealed thousands of previously unsuspected genes, many of which cannot be ascribed even putative functions. One of the largest and most enigmatic gene families discovered in this way is characterized by tandem arrays of pentatricopeptide repeats (PPRs). We describe a detailed bioinformatic analysis of 441 members of the Arabidopsis PPR family plus genomic and genetic data on the expression (microarray data), localization (green fluorescent protein and red fluorescent protein fusions), and general function (insertion mutants and RNA binding assays) of many family members. The basic picture that arises from these studies is that PPR proteins play constitutive, often essential roles in mitochondria and chloroplasts, probably via binding to organellar transcripts. These results confirm, but massively extend, the very sparse observations previously obtained from detailed characterization of individual mutants in other organisms.

The PPR motif – a TPR-related motif prevalent in plant organellar proteins

Genome sequencing projects in eukaryotes are revealing thousands of new genes of unknown function, many of which fall into gene families. We discovered one such family while systematically screening predicted Arabidopsis proteins for those likely to be targeted to mitochondria or chloroplasts. This large gene family (almost 200 genes in the 70% of the Arabidopsis genome sequenced so far) is characterized by the presence of tandem arrays of a degenerate 35-amino-acid repeat (Fig. 1). The same family has been identified independently on the basis of other criteria by Aubourg et al.1 Two-thirds of these Arabidopsis proteins are predicted to be targeted to either mitochondria or chloroplasts (N. Peeters and I.D. Small, unpublished). None of them have been characterized in any way to our knowledge, but a few related sequences in other organisms have been studied. The maize gene crp1 (Ref. 2) is a member of the same family, and similar repeats are found in PET309 from Saccharomyces cerevisiae3 and cya-5 from Neurospora crassa4. All three genes encode proteins involved in some way in processing or translation, or both, of particular organellar mRNAs (Ref. 2). None of these three proteins show obvious sequence similarity to each other or to the Arabidopsis proteins outside the zone of repeats. The repeat structure in these proteins appears to have been initially overlooked, although Fisk et al.2 state in a note added in proof that these proteins contain TPR (tetratricopeptide) motifs. In fact, although the 35-amino-acid repeats do resemble TPR motifs, they have significant and characteristic differences. To distinguish them from TPR motifs, we propose to call them PPR (pentatricopeptide) motifs. Using BLAST (Ref. 5) searches on the non-redundant GenBank peptide database, we built up a list of more than 100 sequences likely to contain PPR motifs and then used the MEME program6 on this set of sequences to define a profile corresponding to the PPR motif (Fig. 1). This profile was then used by MOTIFSEARCH [part of the Genetics Computer Group (GCG) Wisconsin Package, Version 10.0, Madison, WI, USA] to screen the SwissProt/TrEMBL, PIR and GenPept databases. A total of 213 sequences were found with a combined probability score of less than 1024 and containing at least one pair of motifs in tandem (the latter criterion being a very stringent requirement). The number of motifs per peptide ranges from 2 to 26, with a mean of 9.1. CRP1, Pet309p and CYA-5 were found in this search, along with a few other human or yeast proteins of unknown function (Table 1) and a saltinducible protein from tobacco8. The vast majority of the other sequences are from Arabidopsis. There are numerous expressed sequence tags (ESTs) from related genes from several plant species, including rice, so this gene family is likely to be widespread in higher plants (ESTencoded peptides were not included in the set searched with MOTIFSEARCH). No prokaryotic proteins were found to possess tandem copies of this motif and none of the known TPR-containing proteins were revealed via this search. A similar search was carried out starting with known TPR-motif proteins (taken from Ref. 9), using MEME to determine a profile corresponding to the TPR motif (Fig. 1). A search using this profile with MOTIFSEARCH and the same criteria as for the PPR search (i.e. PROTEIN SEQUENCE MOTIFS TIBS 25 – FEBRUARY 2000

Physical interaction between RRS1-R, a protein conferring resistance to bacterial wilt, and PopP2, a type III effector targeted to the plant nucleus

RRS1-R confers broad-spectrum resistance to several strains of the causal agent of bacterial wilt, Ralstonia solanacearum. Although genetically defined as recessive, this R gene encodes a protein whose structure combines the TIR-NBS-LRR domains found in several R proteins and a WRKY motif characteristic of some plant transcriptional factors and behaves as a dominant gene in transgenic susceptible plants. Here we show that PopP2, a R. solanacearum type III effector, which belongs to the YopJ/AvrRxv protein family, is the avirulence protein recognized by RRS1-R. Furthermore, an interaction between PopP2 and both RRS1-R and RRS1-S, present in the resistant Nd-1 and susceptible Col-5 Arabidopsis thaliana ecotypes, respectively, was detected by using the yeast split-ubiquitin two-hybrid system. This interaction, which required the full-length R protein, was not observed between the RRS1 proteins and PopP1, another member of the YopJ/AvrRxv family present in strain GMI1000 and that confers avirulence in Petunia. We further demonstrate that both the Avr protein and the RRS1 proteins colocalize in the nucleus and that the nuclear localization of the RRS1 proteins are dependent on the presence of PopP2.

Interfamily transfer of a plant pattern-recognition receptor confers broad-spectrum bacterial resistance

Plant diseases cause massive losses in agriculture. Increasing the natural defenses of plants may reduce the impact of phytopathogens on agricultural productivity. Pattern-recognition receptors (PRRs) detect microbes by recognizing conserved pathogen-associated molecular patterns (PAMPs). Although the overall importance of PAMP-triggered immunity for plant defense is established, it has not been used to confer disease resistance in crops. We report that activity of a PRR is retained after its transfer between two plant families. Expression of EFR (ref. 4), a PRR from the cruciferous plant Arabidopsis thaliana, confers responsiveness to bacterial elongation factor Tu in the solanaceous plants Nicotiana benthamiana and tomato (Solanum lycopersicum), making them more resistant to a range of phytopathogenic bacteria from different genera. Our results in controlled laboratory conditions suggest that heterologous expression of PAMP recognition systems could be used to engineer broad-spectrum disease resistance to important bacterial pathogens, potentially enabling more durable and sustainable resistance in the field.

Dual targeting to mitochondria and chloroplasts.

Plant cells contain two organelles originally derived from endosymbiotic bacteria: mitochondria and plastids. Their endosymbiotic origin explains why these organelles contain their own DNA, nonetheless only a few dozens of genes are actually encoded by these genomes. Many of the other genes originally present have been transferred to the nuclear genome of the host, the product of their expression being targeted back to the corresponding organelle. Although targeting of proteins to mitochondria and chloroplasts is generally highly specific, an increasing number of examples have been discovered where the same protein is imported into both organelles. The object of this review is to compare and discuss these examples in order to try and identify common features of dual-targeted proteins. The study helps throw some light on the factors determining organelle targeting specificity, and suggests that dual-targeted proteins may well be far more common than once thought.

Ralstonia solanacearum requires F-box-like domain-containing type III effectors to promote disease on several host plants

The phytopathogenic bacterium Ralstonia solanacearum encodes a family of seven type III secretion system (T3SS) effectors that contain both a leucine-rich repeat and an F-box domain. This structure is reminiscent of a class of typical eukaryotic proteins called F-box proteins. The latter, together with Skp1 and Cullin1 subunits, constitute the SCF-type E3 ubiquitin ligase complex and control specific protein ubiquitinylation. In the eukaryotic cell, depending on the nature of the polyubiquitin chain, the ubiquitin-tagged proteins either see their properties modified or are doomed for degradation by the 26S proteasome. This pathway is essential to many developmental processes in plants, ranging from hormone signaling and flower development to stress responses. Here, we show that these previously undescribed T3SS effectors are putative bacterial F-box proteins capable of interacting with a subset of the 19 different Arabidopsis Skp1-like proteins like bona fide Arabidopsis F-box proteins. A R. solanacearum strain in which all of the seven GALA effector genes have been deleted or mutated was no longer pathogenic on Arabidopsis and less virulent on tomato. Furthermore, we found that GALA7 is a host-specificity factor, required for disease on Medicago truncatula plants. Our results indicate that the GALA T3SS effectors are essential to R. solanacearum to control disease. Because the F-box domain is essential to the virulence function of GALA7, we hypothesize that these effectors act by hijacking their host SCF-type E3 ubiquitin ligases to interfere with their host ubiquitin/proteasome pathway to promote disease.

Ubiquitination during plant immune signaling

Plant responses to pathogens depend on the rapid and effective coordination of microbial perception and downstream signal transduction events. Detection of pathogen invasion starts by the recognition of conserved microbial molecules called pathogen-associated molecular patterns ([PAMPs][1]), mainly

Ralstonia solanacearum, a widespread bacterial plant pathogen in the post-genomic era

UNLABELLED Ralstonia solanacearum is a soil-borne bacterium causing the widespread disease known as bacterial wilt. Ralstonia solanacearum is also the causal agent of Moko disease of banana and brown rot of potato. Since the last R. solanacearum pathogen profile was published 10 years ago, studies concerning this plant pathogen have taken a genomic and post-genomic direction. This was pioneered by the first sequenced and annotated genome for a major plant bacterial pathogen and followed by many more genomes in subsequent years. All molecular features studied now have a genomic flavour. In the future, this will help in connecting the classical field of pathology and diversity studies with the gene content of specific strains. In this review, we summarize the recent research on this bacterial pathogen, including strain classification, host range, pathogenicity determinants, regulation of virulence genes, type III effector repertoire, effector-triggered immunity, plant signalling in response to R. solanacearum, as well as a review of different new pathosystems. TAXONOMY Bacteria; Proteobacteria; β subdivision; Ralstonia group; genus Ralstonia. DISEASE SYMPTOMS Ralstonia solanacearum is the agent of bacterial wilt of plants, characterized by a sudden wilt of the whole plant. Typically, stem cross-sections will ooze a slimy bacterial exudate. In the case of Moko disease of banana and brown rot of potato, there is also visible bacterial colonization of banana fruit and potato tuber. DISEASE CONTROL As a soil-borne pathogen, infected fields can rarely be reused, even after rotation with nonhost plants. The disease is controlled by the use of resistant and tolerant plant cultivars. The prevention of spread of the disease has been achieved, in some instances, by the application of strict prophylactic sanitation practices. USEFUL WEBSITES Stock centre: International Centre for Microbial Resources-French Collection for Plant-associated Bacteria CIRM-CFBP, IRHS UMR 1345 INRA-ACO-UA, 42 rue Georges Morel, 49070 Beaucouzé Cedex, France, http://www.angers-nantes.inra.fr/cfbp/. Ralstonia Genome browser: https://iant.toulouse.inra.fr/R.solanacearum. GMI1000 insertion mutant library: https://iant.toulouse.inra.fr/R.solanacearumGMI1000/GenomicResources. MaGe Genome Browser: https://www.genoscope.cns.fr/agc/microscope/mage/viewer.php?

Duplication and Quadruplication of Arabidopsis thaliana Cysteinyl- and Asparaginyl-tRNA Synthetase Genes of Organellar Origin

Abstract. Two cysteinyl-tRNA synthetases (CysRS) and four asparaginyl-tRNA synthetases (AsnRS) from Arabidopsis thaliana were characterized from genome sequence data, EST sequences, and RACE sequences. For one CysRS and one AsnRS, sequence alignments and prediction programs suggested the presence of an N-terminal organellar targeting peptide. Transient expression of these putative targeting sequences joined to jellyfish green fluorescent protein (GFP) demonstrated that both presequences can efficiently dual-target GFP to mitochondria and plastids. The other CysRS and AsnRSs lack targeting sequences and presumably aminoacylate cytosolic tRNAs. Phylogenetic analysis suggests that the four AsnRSs evolved by repeated duplication of a gene transferred from an ancestral plastid and that the CysRSs also arose by duplication of a transferred organelle gene (possibly mitochondrial). These case histories are the best examples to date of capture of organellar aminoacyl-tRNA synthetases by the cytosolic protein synthesis machinery.

Two Type III Secretion System Effectors from Ralstonia solanacearum GMI1000 Determine Host-Range Specificity on Tobacco

The model pathogen Ralstonia solanacearum GMI1000 is the causal agent of the bacterial wilt disease that attacks many solanaceous plants and other hosts but not tobacco (Nicotiana spp.). We found that two type III secretion system effector genes, avrA and popP1, are limiting the host range of strain GMI1000 on at least three tobacco species (N. tabacum, N. benthamiana, and N. glutinosa). Both effectors elicit the hypersensitive response (HR) on these tobacco species, although in different manners; AvrA is the major determinant recognized by N. tabacum and N. benthamiana, while PopP1 appears to be the major HR elicitor on N. glutinosa. Only the double inactivation of the avrA and popP1 genes allowed GMI1000 to wilt tobacco plants, thus showing that GMI1000 intrinsically possesses the functions necessary to wilt tobacco plants. A focused analysis on AvrA revealed that the first 58 N-terminal amino acids are sufficient to direct its injection into plant cells. We identified a hypervariable region in avrA, which contains variable numbers of tandem repeats (VNTR), each composed of 12 base pairs. We show that an 18-amino acid region in which the VNTR insertion occurs is an important domain involved in HR elicitation on N. benthamiana. avrA appears to be the target of various DNA insertions or mobile elements that probably allow R. solanacearum to evade the recognition and defense responses of tobacco.