The Ralstonia solanacearum csp22 peptide,but not flagellin‐derived peptides,is perceived by plants from the Solanaceae family

Ralstonia solanacearum, the causal agent of bacterial wilt disease, is considered one of the most destructive bacterial pathogens due to its lethality, unusually wide host range, persistence and broad geographical distribution. In spite of the extensive research on plant immunity over the last years, the perception of molecular patterns from R. solanacearum that activate immunity in plants is still poorly understood, which hinders the development of strategies to generate resistance against bacterial wilt disease. The perception of a conserved peptide of bacterial flagellin, flg22, is regarded as paradigm of plant perception of invading bacteria; however, no elicitor activity has been detected for R. solanacearum flg22. Recent reports have shown that other epitopes from flagellin are able to elicit immune responses in specific species from the Solanaceae family, yet our results show that these plants do not perceive any epitope from R. solanacearum flagellin. Searching for elicitor peptides from R. solanacearum, we found several protein sequences similar to the consensus of the elicitor peptide csp22, reported to elicit immunity in specific Solanaceae plants. A R. solanacearum csp22 peptide (csp22^Rsol) was indeed able to trigger immune responses in Nicotiana benthamiana and tomato, but not in Arabidopsis thaliana. Additionally, csp22^Rsol treatment conferred increased resistance to R. solanacearum in tomato. Transgenic A. thaliana plants expressing the tomato csp22 receptor (SlCORE) gained the ability to respond to csp22^Rsol and became more resistant to R. solanacearum infection. Our results shed light on the mechanisms for perception of R. solanacearum by plants, paving the way for improving current approaches to generate resistance against R. solanacearum.     

Atomic model of CPV reveals the mechanism used by this single-shelled virus to economically carry out functions conserved in multishelled reoviruses

Unlike the multishelled viruses in the Reoviridae, cytoplasmic polyhedrosis virus (CPV) is single shelled, yet stable and fully capable of carrying out functions conserved within Reoviridae. Here, we report a 3.1 ? resolution cryo electron microscopy structure of CPV and derive its atomic model, consisting of 60 turret proteins (TPs), 120 each of capsid shell proteins (CSPs) and large protrusion proteins (LPPs). Two unique segments of CSP contribute to CPV's stability: an inserted protrusion domain interacting with neighboring proteins, and an N-anchor tying up CSPs together through strong interactions such as β sheet augmentation. Without the need to interact with outer shell proteins, LPP retains only the N-terminal two-third region containing a conserved helix-barrel core and interacts exclusively with CSP. TP is also simplified, containing only domains involved in RNA capping. Our results illustrate how CPV proteins have evolved in a coordinative manner to economically carry out their conserved functions.     

A family of cold shock proteins in Bacillus subtilis is essential for cellular growth and for efficient protein synthesis at optimal and low temperatures

Like other bacteria, Bacillus subtilis possesses a family of homologous small acidic proteins (CspB, CspC and CspD, identity > 70%) that are strongly induced in response to cold shock. We show that deletion of cspC or cspD genes did not result in a detectable phenotype; in contrast, csp double mutants exhibited severe reduction in cellular growth at 15°C as well as at 37°C, including impairment of survival during the stationary phase. Two-dimensional gel analysis showed that protein synthesis was deregulated in csp double mutants and that the loss of one or two CSPs led to an increase in the synthesis of the remaining CSP(s) at 37°C and after cold shock, suggesting that CSPs down-regulate production of members from this protein family. A cspB/C/D triple mutant (64BCDbt) could only be generated in the presence of cspB in trans on a plasmid that was not lost, in spite of lack of antibiotic pressure, indicating that a minimum of one csp gene is essential for viability of B . subtilis . After cold shock, synthesis of CspB in 64BCDbt was drastically lower than in wild-type cells accompanied by cessation in growth and strong reduction in general protein synthesis. As CspB, CspC and CspD are shown to bind to RNA in a co-operative and interactive manner, CSPs are suggested to function as RNA chaperones facilitating the initiation of translation under optimal and low temperatures.     

Identification and characterization of five cspA homologous genes from Myxococcus xanthus.

Escherichia coli contains a large CspA family consisting of nine homologues, in which four are cold-shock inducible and one is stationary-phase inducible. Here, we demonstrate that Myxococcus xanthus possesses at least five CspA homologues, CspA to CspE. Hydrophobic residues forming a hydrophobic core, and aromatic residues, which are included in functional motifs RNP-1 and RNP-2 involved in binding to RNA and ssDNA, are well conserved. These facts suggest that M. xanthus CspA homologues have a similar structure and function as E. coli CspA. However, in contrast to the E. coli CspA family, the expression of M. xanthus csp genes as judged by primer extension analysis is not significantly regulated by temperature changes, except for cspB of which expression was reduced to less than 10% upon heat shock at 42 degrees C. Such constitutive expression of the csp genes may be important for M. xanthus, a soil-dwelling bacterium, to survive under conditions of exposure to various environmental changes in nature.     

CSP41, a sequence-specific chloroplast mRNA binding protein, is an endoribonuclease.

Correct 3' processing of chloroplast precursor mRNAs (pre-mRNAs) requires a stem-loop structure within the 3' untranslated region. In spinach, a stable 3' stem-loop-protein complex has been shown to form in vitro between petD pre-mRNA, encoding subunit IV of the cytochrome b6/f complex, and chloroplast proteins. This complex contains three chloroplast stem-loop binding proteins (CSPs), namely, CSP29, CSP41, and CSP55. Here, we report the purification of CSP41 and cloning of the csp41 gene and show that CSP41 is encoded by a single nuclear gene. Characterization of bacterially expressed CSP41 demonstrates that this protein binds specifically to the 3' stem-loop structure and a downstream AU-rich element of petD pre-mRNA and that its binding affinity is enhanced by associating with CSP55. Our data also show that CSP41 has substantial nonspecific endoribonuclease activity. These data suggest that CSP41 could be involved in 3' processing of petD pre-mRNA and/or in RNA degradation. The fact that different reaction conditions favor RNA binding over ribonuclease activities suggests a possible mode of in vivo regulation.     

Inhibition of competence development, horizontal gene transfer and virulence in Streptococcus pneumoniae by a modified competence stimulating peptide

Competence stimulating peptide (CSP) is a 17-amino acid peptide pheromone secreted by Streptococcus pneumoniae. Upon binding of CSP to its membrane-associated receptor kinase ComD, a cascade of signaling events is initiated, leading to activation of the competence regulon by the response regulator ComE. Genes encoding proteins that are involved in DNA uptake and transformation, as well as virulence, are upregulated. Previous studies have shown that disruption of key components in the competence regulon inhibits DNA transformation and attenuates virulence. Thus, synthetic analogues that competitively inhibit CSPs may serve as attractive drugs to control pneumococcal infection and to reduce horizontal gene transfer during infection. We performed amino acid substitutions on conserved amino acid residues of CSP1 in an effort to disable DNA transformation and to attenuate the virulence of S. pneumoniae. One of the mutated peptides, CSP1-E1A, inhibited development of competence in DNA transformation by outcompeting CSP1 in time and concentration-dependent manners. CSP1-E1A reduced the expression of pneumococcal virulence factors choline binding protein D (CbpD) and autolysin A (LytA) in vitro, and significantly reduced mouse mortality after lung infection. Furthermore, CSP1-E1A attenuated the acquisition of an antibiotic resistance gene and a capsule gene in vivo. Finally, we demonstrated that the strategy of using a peptide inhibitor is applicable to other CSP subtype, including CSP2. CSP1-E1A and CSP2-E1A were able to cross inhibit the induction of competence and DNA transformation in pneumococcal strains with incompatible ComD subtypes. These results demonstrate the applicability of generating competitive analogues of CSPs as drugs to control horizontal transfer of antibiotic resistance and virulence genes, and to attenuate virulence during infection by S. pneumoniae.     

Arabidopsis thaliana mutants reveal a role for CSP41a and CSP41b, two ribosome-associated endonucleases, in chloroplast ribosomal RNA metabolism

A proteomic analysis of Chlamydomonas reinhardtii 70S ribosomes identified two proteins, RAP38 and RAP41, which associate in stoichiometric amounts with intact ribosomes. In this work we show results that suggest the Arabidopsis thaliana homologs, CSP41b and CSP41a, participate in ribosomal RNA metabolism. Csp41a-1 and csp41b-1 single mutants show little phenotype, while the loss of both proteins is lethal. Plants homozygous for the csp41b-1 mutation and heterozygous for the csp41a-1 mutation (csp41b-1/csp41a-1*) fail to accumulate CSP41b and show a marked reduction in the levels of CSP41a. These mutants have reduced chlorophyll content, grow slower and over-accumulate 23S precursor rRNAs compared to their wild-type (WT) siblings, whereas other rRNAs or mRNAs are unaffected. Chloroplast polysome assembly is reduced in csp41b-1/csp41a-1* mutants, which also contain increased amounts of pre-ribosomal particles compared to mature 70S ribosomes. Our results also indicate that CSP41b associates with pre-ribosomal particles in vivo. In vitro, the pattern of 23S precursors and mature rRNAs is altered upon incubation with recombinant CSP41a and CSP41b. Taken together, these results suggest that CSP41a and CSP41b have a role in chloroplast ribosomal RNA metabolism, most likely acting in the final steps of 23S rRNA maturation.     

Single-stranded nucleic acid binding in Arabidopsis thaliana cold shock protein is cold shock domain dependent

Cold shock proteins (CSPs) are ancient nucleic acid-binding proteins and well conserved from bacteria to animals as well as plants. In prokaryotes, CSPs possess a single cold shock domain (CSD) while animal CSPs, flanked by N- and C-terminal domains, are commonly named Y-box proteins. Interestingly, the plants CSPs contain auxiliary C-terminal domains in addition to their N-terminal CSD. The CSPs have been shown to play important role in development and stress adaptation in various plant species. The objective of this study was to find out the possible nucleic acid-binding affinities of whole CSP as well as independent domains, so that role of each individual domain may be revealed in Arabidopsis thaliana, the model plant species. The structure of CSP 3 protein from A. thaliana was modeled by homology-based approach and docking was done with different nucleic acid types.     

The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants

Innate immunity is based on the recognition of pathogen-associated molecular patterns (PAMPs). Here, we show that elongation factor Tu (EF-Tu), the most abundant bacterial protein, acts as a PAMP in Arabidopsis thaliana and other Brassicaceae. EF-Tu is highly conserved in all bacteria and is known to be N-acetylated in Escherichia coli. Arabidopsis plants specifically recognize the N terminus of the protein, and an N-acetylated peptide comprising the first 18 amino acids, termed elf18, is fully active as inducer of defense responses. The shorter peptide, elf12, comprising the acetyl group and the first 12 N-terminal amino acids, is inactive as elicitor but acts as a specific antagonist for EF-Tu-related elicitors. In leaves of Arabidopsis plants, elf18 induces an oxidative burst and biosynthesis of ethylene, and it triggers resistance to subsequent infection with pathogenic bacteria.     

Solution structure of the PABC domain from wheat poly (A)-binding protein: an insight into RNA metabolic and translational control in plants

In animals, the PABC domain from poly (A)-binding protein recruits proteins containing a specific interacting motif (PAM-2) to the mRNP complex. These proteins include Paip1, Paip2, and eukaryotic release factor 3 (eRF3), all of which regulate PABP function in translation. The following reports the solution structure of PABC from Triticum avestium (wheat) poly (A)-binding protein determined by NMR spectroscopy. Wheat PABC (wPABC) is an alpha-helical protein domain, which displays a fold highly similar to the human PABC domain and contains a PAM-2 peptide binding site. Through a bioinformatics search, several plant proteins containing a PAM-2 site were identified including the early response to dehydration protein (ERD-15), which was previously shown to regulate PABP-dependent translation. The plant PAM-2 proteins contain a variety of conserved sequences including a PABP-interacting 1 motif (PAM-1), RNA binding domains, an SMR endonuclease domain, and a poly (A)-nuclease regulatory domain, all of which suggest a function in either translation or mRNA metabolism. The proteins identified are well conserved throughout plant species but have no sequence homologues in metazoans. We show that wPABC binds to the plant PAM-2 motif with high affinity through a conserved mechanism. Overall, our results suggest that plant species have evolved a distinct regulatory mechanism involving novel PABP binding partners.